Cerebrovascular reserve (CVR) is the dilatory response of blood vessels to match the perfusion needs of the brain. CVR is necessary for regulating oxygen and nutrient transport to maintain normal brain function. Individuals with Alzheimer’s disease (AD) often exhibit vascular pathology due to reduced perfusion. We believe that in patients with early AD, CVR can serve as a biomarker for early vascular pathology. Using breath-hold BOLD fMRI, CVR can be measured quantitatively and spatially. Older adult subjects (n = 48, age >65 years) were asked to perform normal paced breathing for 20s followed by a 15s breath-hold. This process was repeated 6 times. MRI data was processed using Python and FSL. Python was used to extract breath-hold signals from the MRI scanner. With FSL, BOLD fMRI data was corrected for motion and baseline drift. The breathing time course was used as a stimulus regressor in FSL FEAT to obtain voxel-wise maps of CVR. The CVR maps were registered to the anatomical T1 image and subsequently to the standard MNI template to obtain region-specific values of CVR. As data processing is ongoing, we have not obtained conclusive results. However, we expect to see in patients with onset of AD, CVR ability is lowered throughout regions of the brain, most notably in the frontal and parietal brain regions. Our preliminary studies in 30 older adults indicate that overall gray matter CVR in healthy older adults was 0.42±0.13%, but was 0.38±0.16% in individuals with cognitive impairment and at risk for AD. The results of this study will demonstrate the usefulness of cerebrovascular imaging to understand vascular pathology in patients with AD at an early stage, thereby allowing for treatment to begin sooner, and slowing down disease progression.

Mycobacterium tuberculosis (Mtb) is a widespread disease affecting about 33% of the human population around the world. Treatment of tuberculosis (TB) is hampered by a need for lengthy, multi-drug regimens and drug resistance requiring development of novel therapeutics. Our laboratory is interested in harnessing the host immune response to enhance Mtb killing within the macrophage. Within the host, Mtb is found within lipid-laden macrophages where lipid droplets are hypothesized to both provide nutrients as well as sequester Mtb from intracellular killing mechanisms. Hydroxycarboxylic acid receptor 2 (HCA2) is a G-protein coupled receptor (GPCR) that maintains lipid droplets after activation by its ligands nicotinic acid (NA) and beta-hydroxybutyrate (BHB). In these studies, we assess NA, BHB, and the structurally related small molecules nicotinamide (NAM), sodium butyrate (NaB) and phenylbutyrate (PBA) modulate HCA2 signaling. We have transiently transfected CHO-K1 cells with plasmids encoding human HCA2 and aequorin, the latter which oxidizes coelenterazine in a reaction that generates a luminescent signal. The effective concentration (EC50) of each small molecule as a ligand for HCA2 can then be calculated from the luminescence curve. Preliminary work in our lab suggests that PBA inhibits Mtb replication within the host macrophage, but several mechanisms are likely involved. We hypothesize that NaB, PBA, and NAM will also modulate HCA2 activity given their similar structures to NA and BHB. Future studies will also interrogate whether this panel of small molecules impacts HCA2 gene expression, lipid droplet formation and Mtb replication in macrophages.

Providing quality surgical care and anesthesia in low-resource communities has proven to be quite challenging, as many anesthetic devices are either too expensive or require electricity. Anesthetic vaporizers work by evaporating a liquid anesthetic into an air flow, which is then delivered to the patient. As the liquid anesthetic is evaporated, the temperature decreases, leading to a decrease in delivered anesthetic, which is problematic if not addressed. Current devices address this issue by encompassing the vaporizer in a large steel heat sink, but this makes the device unsuitable for transport long distances. A low-cost anesthetic vaporizer has been proposed and developed by Bioengineers Without Borders that will utilize phase change material to hold the vaporizer temperature constant, and they are seeking a predictive tool to guide design choices. A computational model of this vaporizer has been previously built utilizing laminar flow dynamics. However, this model did not sufficiently predict the dynamics of the vaporizer under various operating conditions. The model has now been converted to utilize turbulent flow dynamics, and is undergoing validation steps through comparisons with the experimental measurements of concentration output and halothane surface temperature. Additional adjustments have also been made to the laminar flow model, resulting in a more accurate model. Currently, both the turbulent and laminar models predict a decreasing temperature over time, resulting in decreasing amounts of anesthetic being delivered. Both models perform very well at high flow rates, but constantly overestimate deliveries at low flow rates. Upon validation, this model will give Bioengineers Without Borders the ability to computationally test possible design modifications to the vaporizer prior to production, saving time and resources. 

Antibodies play a crucial role in both therapeutics and diagnostics due to their high target specificity. However, a characteristic that accompanies this high specificity is the reluctance to release the bound target. This presents challenges when either the protein or target needs to purified and analyzed or the protein needs to be removed from a site of interest. Current methods to reverse binding are often toxic and require large environmental changes that are not always feasible. These environmental changes can negatively affect the ligand structure, thus hampering analysis to characterize the target ligand. Therefore, a need exists for a protein whose binding can be externally controlled in a nontoxic manner. The Thomas Lab proposes using a light-activated azobenzene linker to create a crosslinked protein whose binding affinity can be controlled by light. The protein of interest is the bacterial adhesion protein, FimH, which has two known conformations that are regulated by force. The goal is to replicate the two conformations in the absence of force by using the linker. FimH has been mutated to have two free cysteines across the two domains for crosslinking. An assay has been developed to differentiate between crosslinked and non-crosslinked proteins to determine whether the linker has bound to the free cysteines. The azobenzene linker has been crosslinked to the mutated FimH and the change in binding to the target under UV light has been characterized. If a change in binding is detected, the design of a crosslinked protein whose binding can be externally controlled by light will be successful.

Traumatic brain injury (TBI) is a pathological condition marked by chronic daytime sleepiness. This sleepiness is associated with the decrease in hypocretin neurons in the hypothalamus. Hypocretin cells are known to be sensitive to inflammation, so research on this inflammatory response is important for developing therapies to treat TBI. One type of inflammatory response to TBI is microgliosis, i.e., an increase in activated microglia, the brain’s immune cells. Depending on inflammatory cytokines released, microglial activation can be beneficial or detrimental to recovery from TBI and is therefore a potential target for TBI therapies. The overall goal of this project is to examine the time course of microglial activation in the hypothalamus using a mouse model of TBI. Mice underwent TBI or sham surgery and brains were examined 3, 7, or 15 days after surgery. Brains were sectioned coronally and numbers of microglia in the hypothalamus were quantified using immunohistochemistry. We predict that microgliosis will be most intense at either 3 or 7 days, since previous studies have shown that microglia peak around the site of injury at 5 to 7 days and then rapidly decline. Determining the extent of microglial activation across time will help to determine an appropriate time to administer therapies targeting microglia.

Bacterial endocarditis is a life-threatening disease that occurs when bacteria bind to the inner surface of cardiovascular tissue. One group of bacteria in particular, the viridans group Streptococci, cause a high percentage of cases of endocarditis. It is known that adhesion is mediated by specialized proteins on the surface of bacteria capable of binding to specific glycans expressed outside of host cells, and that binding is also dependent on shear stress from flow through allosteric regulation of the binding site. However, the exact mechanisms and resulting rates of binding are not sufficiently understood. If adhesion of the viridans group Streptococci can be experimentally tested and characterized, it could lead to the development of anti-adhesive therapies to treat or prevent diseases such as endocarditis. Current methods of testing bacterial adhesion utilize a parallel plate flow chamber to test the binding of one species of bacteria to a single glycan under one flow rate. Significant progress could be made through the construction of a microfluidic flow chamber compatible with a glycan array capable of testing bacterial adhesion to various glycans under a range of shear stresses in a single run. I have designed such a device using CAD and COMSOL fluid modeling software, and will construct it through the process of 3D stereolithography and laser-cutting. Additionally, I have conducted experiments to identify conditions that minimize non-specific adhesion, maximizing specificity for the device. Bacteria were fluorescently tagged, run through the flow chamber and across the glycan array, and then adhesion was analyzed using a microarray scanner. Data gathered from this device will help collaborators develop treatments to treat and prevent this type bacterial endocarditis. The flow chamber is also now accessible to others in the field to be used to study other clinically relevant types of bacterial adhesion.

Biological recognition proteins are widely used for a variety of biomedical applications, including in vitro diagnostics (IVD). IVD tests that use antibodies as recognition proteins, such as rapid antigen tests, are one of the fastest methods for providing point-of-care diagnosis of infectious diseases. However, many current applications of antibody-based molecular diagnostics are restricted by poor sensitivity in a sample with low target antigen concentration. One method which has shown promise for improving the sensitivity of rapid antigen tests is to pre-concentrate a target sample. We are using selection from a random library of Escherichia coli bacteria expressing the adhesion protein, FimH, to develop an activatable recognition protein for capture-and-release pre-concentration of a target antigen. FimH is a two-domain bacterial adhesion protein that exhibits conformation-dependent binding to mannose. We predict that mutations in the CDR2 and CDR3 loops of the FimH binding pocket can be used to design a protein with binding specificity for a diagnostic target while maintaining conformation-dependent affinity. In previous work, random mutations of the amino acid residues in the FimH binding pocket produced a library of FimH variants with variable binding specificity. Our results suggest that using a biomagnetic selection assay to screen this library of FimH mutants against the small molecule targets digoxigenin and cortisol enriches FimH variants with target binding specificity. Currently, we are designing and optimizing an assay, iterating on the wash conditions, conjugation strategy, and other parameters, to screen the library against protein targets such as the viral antigens influenza hemagglutinin and dengue NS1. In future work, we seek to demonstrate that a variant with binding specificity for a diagnostic target has distinct conformation-dependent affinity which can be actively regulated to capture and release a target antigen.

With the rising prevalence of drug resistant bacteria, there is a pressing need to develop novel approaches which do not rely upon antibiotics. Anti-adhesive therapies target the ability of bacteria to adhere to tissue via adhesin receptor interactions. Yet, commonly used methods for characterizing these interactions involve parallel plate flow chambers (PPFCs), and are consistently done without consideration of mass transport effects. Here, we demonstrate that measurements of adhered bacteria taken within a large adhesive spot can vary significantly based upon the location measured. A computational model of a PPFC experiment is developed and validated in order to use partial differential equations to determine how various parameters influence spatial variation in adhesion. Use of a computational model allows investigation into a wide range of parameter values which would be impractical to experimentally test. Based upon these results, analytic models are established to accurately predict how transport conditions affect adhesion measurements. Interestingly, the measurement which best demonstrates a spot’s affinity for a bacterial ligand is the difference between the concentration of bound bacteria at the start of the spot and the consistent concentration reached well into the spot, according to these models. This is validated through PPFC experiments. Additionally, we demonstrate that within a multispot array, the adhesion in the spot of interest is highly affected by the preceding spot. It is vital that experimenters recognize this issue while interpreting their data. Guidelines to help experimenters set up and interpret their data in a manner that accounts for mass transport effects are described. Ultimately these guidelines could help reduce or negate the systematic error which mass transport introduces to adhesion experiments, and lead to better anti-adhesive therapies.

As antibiotic resistance becomes a growing problem, there is a need to research alternative bacterial infection treatments without relying on antibiotics. Anti-adhesive therapy, which prevents bacteria from adhering to tissue, is an alternative solution to the antibiotic resistance problem. Flow rate (or shear stress) is one of the factors that affect bacterial adhesion in solution. Bacteria adhere to tissue in vivo where shear stress can go up to 8 Pascal, but they are unable to adhere in vitro in shear stresses above 1 Pascal. Hydrodynamic lift is the force known to push microparticles away in high flow and prevent the particles from getting close to a surface. However, there is no tool that describes how lift affects bacterial adhesion. This project aims to determine whether lift pushes the bacteria away from the wall and prevents bacteria from initiating binding in continuous flow in vitro as opposed to pulsatile flow in vivo. To do this, we developed a computational model which uses discrete finite element method to determine how lift affects initial adhesion. The model showed bacterial concentration depletes by the wall but concentrates where lift and gravity are balanced. The flow rate or wall shear stress affected where the bacteria accumulates thus influenced their adhesion. Moreover, this model has improved the mass loss problem, which the previous model faced. We validated the model by existing parallel plate flow chamber experiments with different bacteria. The validation experiments also gave some understanding regarding how lift affects the binding of different bacterial species under multiple continuous and pulsatile flow conditions. The results of this project will help researchers make informative decisions to design bacterial binding experiments by providing a tool to understand how lift and gravity affect initial bacterial adhesion.

Characterizing fundamental mechanical properties of individual molecules is essential to understanding and treating disease, because proteins have diverse responses to stimulus. The ability to predict a response enables targeted treatments. Although atomic force microscopy (AFM) and magnetic tweezers are able to measure the response of single molecules to mechanical force, it is challenging to ensure single molecules are being measured. In this project, structural DNA nanotechnology (DNA origami) is used to create a template which spaces molecules for single molecule force measurements. DNA is an excellent nanoscale building material due to its self-assembly properties, nanoscale structural precision, and the capability for precise control over placement of functionalization. By attaching functionalized DNA at specific sites and polymerizing the structure, a repeated precise interval is created for molecules of interest. This nanostructure was designed using caDNAno and canDo. Nanostructure folding time, temperature, and salt conditions were optimized using agarose gel electrophoresis. Nanostructures were imaged with transmission electron microscopy to confirm correct formation of the structure. Polymerization will be imaged using AFM, and force characteristics of the structure will be measured via AFM, to establish a base point for measuring the molecules of interest.

The city of Seattle has experienced immense urban change. Over the past 15 years, we see companies expanding, neighborhoods evolving, and populations being displaced. Recently, sociologists have discovered that neighborhoods that were traditional oases for queer life and living, including neighborhoods in Seattle, have experienced losses in the queer population. The body of research emerging on this topic largely credits this to a greater public acceptance of LGBTQ+ identities which have reduced barriers that kept queer populations from living in other areas. Though many public sources of information, news sites, blogs, and forums, propose gentrification of these neighborhoods as the force driving queer people from their homes. Between 2000 and 2012 Seattle’s traditional gayborhood, Capitol Hill, has seen a significant decrease in same-sex couples, according to the US census. With Amazon and other tech companies expanding into the Capitol Hill neighborhood and subsequent rising rents in that area, it may be the case that queer people can no longer afford to live in the gayborhood has made itself known in Seattle. In this research project I examine the motives for why queer people leave Capitol Hill through a series of interviews with LGBTQ+ people who are moving out of Capitol Hill. These interviews will provide insight into how gentrification and greater acceptance of LGBTQ+ people and other social factors may play a role in this new form of urban change.

Retinol Binding Protein (RBP) is a protein used by the body to transport Vitamin A and is often used to monitor short-term nutritional changes. Traditionally, RBP has been measured via a nephelometric immunoassay with a high sample requirement. In an attempt to reduce turnaround time and to reduce the volume of sample needed for testing, an in-house assay using liquid chromatography tandem-mass spectrometry (LC-MS/MS) technology is being developed.The method being developed is based on the Vitamin D Binding Protein assay which sets a precedent for LC-MS/MS methods targeted toward high-abundance globulins. This method consists of denaturation of RBP using heat and denaturation agents, then digested by trypsin into peptides which are detected by LC-MS/MS. Currently there are three peptides with multiple transitions being monitored for each (FSGTWYAMAK, YWGVASFLQK, and LIVHNGYC[+57]DGR).The assay is being developed and validated according to the document CLSI C-62A and the assay is currently in its pre-validation phase. Experiments done include standard curve verification and comparison to development data, simple repeatability 5x5, carryover and various troubleshooting experiments.Data analysis has exposed high control and internal standard variability in the assay which are being resolved, but progress looks promising.The RBP assay is still far from completion and further testing and troubleshooting needs to be done to reduce variability before it can enter the validation phase. Once optimized, the LC-MS/MS assay, while less automated than nephelometric platforms, should allow UW Medical Center to have better control over an in-house assay in terms of turnaround time, while also using much smaller volumes of sample. LC-MS/MS also has improved sensitivity when compared to the nephelometric assay. This increased performance may lead to assay utility with other conditions such as diabetes, liver disease and kidney disease.
 

As part of my Cultural Anthropology class I conducted an oral history interview of Tom Ficca (Colville) with my group to help document the history of Edmonds Community College’s involvement with Indigenous communities for its fiftieth anniversary. I learned that sharing culture and spirituality has played a significant role in Tom’s life and helped connect him with the college. He described his childhood as “phenomenal” and valued his connections to his community on the Colville Reservation over the past 55 years. He is a spiritual leader and a master carver. He has helped build canoes for different tribes and connected with Edmonds CC about five years ago through canoe journeys and carving projects with the Snoqualmie Tribe, where he worked as a carver and skipper. As a carver he collaborated with students and faculty in the construction of q'wəld'ali (Place of the Cooking Fire) or Cultural Kitchen on the college campus. As a skipper he has welcomed students to support and paddle on Tribal Canoe Journeys. Over the last three years Tom has been working with families that are in the streets in Anchorage Alaska where he helped set up a church and non-profit organization to provide food, water, gloves, socks, dry clothes, etc. throughout the year. Tom Ficca wishes to start a strip cedar canoe as well as moving forward on cultural projects such as canoe building, story pole carving, and plank house construction. Tom would also like to see more future work and involvement in the community and in different garden areas. All these accomplishments and other works to be done in the future are driven by Tom’s passion and commitment to culture and his spiritual side.

Within computed tomography (CT), increased image quality comes at the expense of amplified radiation exposure to patients. To reduce radiation exposure, techniques have emerged which use iterative methods to reconstruct images from limited-angle or sparse-view CT data. Superiorization is a recently proposed framework which “superiorizes” an existing reconstruction algorithm with respect to some secondary objective function. As a result, superiorization can reduce artifacts in the reconstructed image caused by sparse-view and/or limited-angle data. Within certain constraints, the superiorization method guarantees a reconstruction which fits the data equally well as the original reconstruction. More importantly, superiorization is expected to produce an image with higher quality. Existing research within superiorization has been confined to reconstructing images given data generated from monoenergetic X-ray beams. Despite the promise of this existing research, polyenergetic X-ray beams are common in clinical settings and existing methods need to be extended to handle polyenergetic data. To this end, our research goal was to extend the superiorization method to reconstruct images from polyenergetic data, using total variation (TV) and anistropic total variation (ATV) as the secondary objectives to be superiorized. In addition to successfully reconstructing images from polyenergetic data, we also demonstrate our method is capable of reconstructing images with “missing” data which is a result of sparse-view and limited-angle scans. This research further extends the potential of utilizing iterative methods in CT and thus reducing radiation exposure to patients.

Müller glia (MG) are the predominant glia of the neural retina. In fish and birds, retinal injury causes these glial cells to de-differentiate into retinal progenitor cells (RPCs), and these RPCs repair the damaged tissue by generating new neurons. This process does not happen in mammals, and so retinal damage is permanent. microRNAs (miRNAs, miRs) are small RNA molecules that regulate gene expression. Several miRNAs are expressed in the RPCs, but not in the Müller glia, and we call these RPC-miRs. This study focused on if artificially expressing the RPC-miRs would stimulate mammalian MG to regenerate new neurons in postnatal mouse retinal explants. Two leading questions were explored: Will the Müller glia treated with RPC miRNAs regenerate new neurons? What is the best time window to convert Müller glia into retinal progenitors with high efficiency? Retinas from postnatal (P) 4 up to P12 wild type or transgenic reporter mice were dissected from surrounding eye tissue and electroporated to introduce RPC artificial miRNAs (such as miR-15a, miR-17, miR-19, and miR-20a mimics) or control miRNA mimics, along with a reporter mRNA.  Explants were cultured for 3/4 or 6/7 days ex vitro (DEV), fixed, cross sectioned, stained (immunofluorescent labeling) and analyzed using confocal microscopy. The proliferation marker EdU was added to track cell proliferation. Our data showed that RPC-miRs led to an increased proliferative response in the Müller glia, indicating that the cells were adopting an RPC-like state. Moreover, the age of the explants was an important factor; the areas that were successfully electroporated became smaller in older explants and therefore fewer Müller glia can be targeted in older tissue. Our data shows that miRNAs from RPCs can promote Müller glia to adopt some RPC-like characteristics and therefore could be used to stimulate regeneration in the retina.

The large-scale configuration of the cosmos has long been a mystery to astronomers. My research project aims to shed light on this mystery. The overarching goal for my research project is to further understand the distribution of matter on 10⁵ –10⁷ light-year distances by comparing numerical simulations to actual observations of the universe. This comparison tests the current theoretical parameters of the physics governing the largest scales of the cosmos. I used simulated data from models made by Professor Matthew McQuinn and observations of quasar spectra from the Very Large Telescope (VLT). From the VLT data, I analyzed quasar spectra for absorption lines at a hydrogen electronic transition called the Lyman-alpha line. The IGM absorbs amply at this transition, creating absorption lines throughout the quasars’ spectra. I processed this data by programming Python scripts to compare flux probability distribution functions (PDFs) of the Lyman-alpha absorption in the observed quasar spectra to the simulated quasar spectra. Through this comparison, PDFs of observations fell within one standard deviation of the simulated PDFs. Although at higher redshifts (further back in cosmological time), there was considerably more variance. In order to further constrain the observational flux PDFs, I aim to eliminate systematic and statistical uncertainties that may make the observations spuriously differ from models. The salient errors include fitting precise continua (spectra without absorption) and removing metal (predominantly carbon-4) contamination from the Lyman-alpha forest. I used Principal Component Analysis to predict the continuum spectrum but found that a fit by eye was more effective. Through analysis of absorption probabilities outside the Lyman-alpha forest, I statistically removed carbon-4 from the data inside the Lyman-alpha forest. Ensuring the most precise flux PDF measurements are used is critical to the comparison of observations to simulations and to the constraints of parameters governing the universe.

Through analyzing the properties of the host galaxies to Supermassive Black Hole (SMBH) mergers, we will be able to determine what phase of galaxy evolution these mergers track. We do this by analyzing ROMULUS25, a large cosmological simulation of the universe, from the Big Bang to present day. The data from ROMULUS25 includes the evolution of thousands of galaxies and is able to resolve the internal structure of galaxies. Most galaxies have SMBHs in their core, and when galaxies merge, it is believed that their SMBHs merge as well. Project LISA, as well as other future projects, will be able to detect gravitational waves coming from these mergers. With simulations like ROMULUS25, we will be able to better predict which galaxies are likely to host these SMBH mergers, which will give context to future gravitational wave studies with respect to galaxy evolution theory.

Despite their inability to express proteins, long intervening noncoding RNAs (lincRNAs) have demonstrated significant biological function; they regulate both cancer and physiological epidermal development and target chromatin-remodeling genes. Their repertoire of activity is constantly growing with the discovery of each new lincRNA. The lincRNA OIP5-AS1 (Cyrano) is a 4.5kb transcript required for proper embryogenesis in zebrafish, and unlike most lincRNAs, possesses multiple stretches of conservation found in at least 52 vertebrates. While the mechanism for how Cyrano regulates embryogenesis is unknown, it is believed that Cyrano functions primarily through a 26-nucleotide region that is perfectly conserved in every tetrapod; perturbation of this region resulted in a lack of proper development of zebrafish embryos. The secondary structure of the 26-nucleotide conserved region was revealed to contain an RNA binding domain, 5’–CCAU-3’, for RBMX, an RNA binding protein that is highly conserved in mammalian species. I hypothesize that Cyrano interacts with RBMX to aid in the process of embryogenesis, not only in zebrafish, but in other species as well. By using in vitro gel shift assays and in cellulo pull-down experiments, I aim to determine if the lincRNA Cyrano binds to the RBMX protein. Understanding this protein-lincRNA interaction will give more insight to the process of embryogenesis, and how lincRNAs function overall.

Little is understood about how supermassive black holes affect their host galaxies in terms of growth and shape. Using a large simulation called Romulus25, we are able to study how galaxy properties correlate with black hole properties throughout time. From this simulation we draw a sample of 40 massive galaxies and analyze their properties to understand how they are affected by the growth of supermassive black holes. We examine galaxies with a variety of star formation histories and investigate how the black hole is involved in determining these histories. We seperate the galaxies into three categories: 1) Galaxies with on going star formation 2) Galaxies that stop forming stars 3.) Galaxies that stop and then restart star formation. We show how the black hole activity changes between categories. These results may lead to solving the mystery: what causes the star formation in the galaxies to quench, (and in some cases) un-quench?