Ischemic preconditioning (IPC) is a phenomenon in which a brief ischemic event confers protection against subsequent prolonged ischemia, such as stroke. Understanding the mechanisms underlying this endogenous neuroprotective response could lead to advances in stroke therapeutics. Most experimental rodent models of stroke focus on injury to grey matter. However, injury to the white matter (WM) is a significant component of ischemic stroke in human patients and is poorly understood. Our laboratory has shown that microglial innate immune signaling is required for WM IPC-induced protection. The nodes of Ranvier (NoR) are critical WM structures that control conductance and action potential propagation along axons. Recent studies have shown elongation of the NoR in the context of WM injury and disease. The mouse optic nerve (MON) is a fully myelinated central nervous system WM tract. We expose MONs to a brief in vivo ischemic event to induce IPC. Seventy-two hours later, we isolate MONs and expose them to ex vivo oxygen-glucose deprivation (OGD) to mimic stroke. Using MONs from mice that underwent pharmacologic depletion of microglia followed by in vivo IPC and then ex vivo OGD, I performed immunohistochemistry with Nav1.6 and Caspr antibodies to label the NoR and image them with a confocal microscope. NoR size, diameter, and length are quantified with ImageJ in conjunction with an in-house MATLAB script. I hypothesize that IPC will protect and conserve structural parameters of the NoR from OGD-induced injury and that this protection will be eliminated by microglial depletion. Our preliminary data show feasibility of this project (i.e., all the methods to carry out this work are in place) and we can generate precise and reproducible measurements of the NoR in MONs. Findings from this project will provide a better understanding of the mechanisms by which microglia mediate IPC-induced axonal protection in WM.
 

Platelets aggregate at the site of injury to stop bleeding, but disruptions to hemostasis can cause bleeding or thrombosis. Studying platelet-plug area and contractile force can predict whether bleeding or thrombosis is likely to occur. Microfluidic devices, composed of polydimethylsiloxane (PDMS), are used to study these biomechanics by inducing aggregation through shear flow. As blood flows through the device, the platelets pass over a rigid block in the channel which causes platelets to activate, deflecting a flexible post within the channel. This deflection is used to calculate the platelet forces based on the material properties of the PDMS. These microfluidic devices are single use and require a fabrication process that spans multiple days. Additionally, creating duplicate silicon master molds is a laborious and expensive process that necessitates cleanroom training. I am engineering and implementing a more efficient process for the fabrication of these devices, while limiting the use of the master mold that undergoes long-term wear from repeated uses. My focus is on improving the efficiency of the initial negative mold creation process by using a different material, urethane resin, to replicate the master, which allows us to make more negatives simultaneously without needing to fabricate another silicon master. To compare the devices produced using the onyx master with the silicon master, I am running three blood experiments, each with varying levels of antibodies, on two devices fabricated by the onyx and silicon masters. The aggregation sizes and forces are being observed between each of the experiments. I expect the results to be similar within a degree of certainty, proving the onyx master is equivalent to the silicon master and can be used to increase microfluidic device production and increase the availability of platelet biomechanics studies.

The world’s population is rapidly aging, and we now increasingly face challenges related to the prevalence of age-related diseases. Studies from Geroscience have identified hallmarks of aging, such as the cellular senescence and mechanistic target of rapamycin (mTOR) pathways, that can be disrupted to improve health and lifespan in model organisms. Previous studies have shown that these pathways can be targeted pharmacologically by the mTOR inhibitor rapamycin or the senolytic drug combination Dasatinib + Quercetin (DQ). Our laboratory’s published data shows that rapamycin restored oral health in aged mice and reversed periodontal disease. While attempting to uncover the mechanism behind this reversal, our laboratory discovered that the age-related increase in cellular senescence was attenuated by rapamycin treatment. Thus, we hypothesized that targeting cellular senescence may provide an alternative therapeutic strategy to phenocopy the impact of rapamycin on aging oral tissues. I compared the effects of control (n=5), rapamycin (n=5), and the senolytic drug combination, DQ (n=5), in aged male mice (20-21 months old). Western Blot was performed on the mandible and salivary gland tissues to analyze the senescence marker p16INK4a. Histological assessment of H&E and lipofuscin staining was completed on the salivary glands to compare the effects of age, rapamycin, and DQ treatment. In male salivary glands, I discovered the age-related increase in p16 expression was decreased after rapamycin treatment, but not with DQ treatment. There were no significant changes in levels of p16 in the mandible with age or with the administration of rapamycin or DQ. Rapamycin treatment attenuated cellular senescence in male salivary glands, while the senolytic cocktail DQ had no impact on the aging male salivary glands. Future studies could be performed on female mice, with the addition of alternative senolytics which may target other markers of cellular senescence beyond p16. 

Mount St. Helens shook local communities and spewed volcanic ash into the sky for two months before it finally had its major eruption. In those two months newspapers eagerly tracked the activity, crafted a story, and relayed it to their audiences. When the mountain erupted on May 18, 1980, the world caught a glimpse of nature’s power and found a dramatic climax to their two-month story. In the immediate aftermath, accounts of what had happened took various forms, ranging from personal hymns to films, with each of them showing a different response to the eruption. These responses showcase a population reconsidering what it means to live alongside nature. Today, forty years later, Mount St. Helens’ story is still being told through a wide array of sources. In my research, I analyze works approaching the eruption, reacting in the immediate aftermath, and those which have come out in memory. I note the content of these sources as well as their framing to construct an analysis of how changing treatment of the Mount St. Helens story reveals society’s approach to nature before the eruption and how that approach changed in response to the events of May 18. I specifically focus on sources consumed and produced by the broader public, such as films and songs, using private correspondence or scientific conferences only as a source of what does not make widespread narratives. This approach encapsulates how people of various communities make sense of living alongside the natural world, and especially how they conceptualize sudden change events like volcanic eruptions. My research uncovers both flaws in the population’s conceptual relationship to nature as well as their tendencies to remember natural events, specifically Mount St. Helens, in a way that maintains or minimally changes the way the see themselves in the world.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that have been engineered into three-dimensional heart tissues (EHTs) are valuable research tools for investigating debilitating genetic diseases that afflict the heart, such as Duchenne muscular dystrophy (DMD). Ensuring iPSC-CMs can be sufficiently matured to model such diseases remains a hurdle in current research, and maturational analysis techniques for iPSC-CMs are either qualitative, manual, or primarily based in two dimensions, leaving much to be desired. This poster details the creation of a suite of MATLAB image-processing scripts that can quantify the effect of three-dimensional culture and disease-causing DMD mutations on cardiomyocyte structure and maturation state. The iPSC-CMs were differentiated from stem cells, cast into EHTs, stained using immunofluorescence, and imaged using confocal microscopy. Using the scripts to analyze these 3D images of iPSC-CM stains, key maturational features of the cells can be quantified such as nuclei count; cardiomyocyte area; and sarcomere length, orientation, and z-disk width. Analyzing cardiomyocyte area can give key information on cardiomyocyte hypertrophy while examining sarcomere length, orientation, and Z-disk width can provide information on myofibril structure and organization. The suite allows analysis of these maturational features in both 2D and 3D cultures and offers a method for quantitatively assessing maturation in an automated manner. Measuring iPSC-CM maturation will also allow better comparison of existing maturational methods, such as mechanical loading, electrical stimulation, and small molecule treatment. The suite can also create graphical outputs to elegantly display data. Recent progress also includes a script that can count cell nuclei and quantify cell area. Overall, the suite will help improve maturational analysis of EHTs, and hopefully contribute to the discovery of new treatments for diseases that affect the heart. 

Hepatitis C (HCV) is a liver disease caused by the bloodborne HCV virus. When left untreated, HCV can lead to cirrhosis and liver failure. Recent developments in therapeutics present a cure for HCV; however, treatment must be received soon after infection to be effective. Thus, limited availability of HCV testing creates a barrier to treatment distribution as chronic HCV is identified through a detectable viral load. Current HCV testing involves polymerase chain reaction (PCR) testing of blood samples, requiring a central laboratory and technicians to run them. The delay between appointments, sample transportation, running PCR, and receiving results can lead to lost contact with patients, making it difficult to connect them with timely treatment. The goal of the project is to develop a rapid point-of-care assay for HCV nucleic acid testing that allows healthcare providers to diagnose chronic HCV in 30 minutes and immediately prescribe treatments. We designed and validated an isothermal nucleic acid amplification assay for detecting HCV RNA: a two-step process involving reverse transcription of HCV RNA into complementary DNA (cDNA) which is detected by recombinase polymerase amplification (RPA). RPA is an isothermal process held at 40℃ with a runtime of 15 minutes, where a fluorometer collects data from the reaction. We compared the results of our RPA detection assay to the PCR-HCV assay used by the UW Clinical Virology Lab. We tested RNA from all six major genotypes using serum samples from Harborview Liver Clinic, where we had a limit-of-detection of 25 copies per reaction. We were able to match the results of the RPA and PCR assays with 100% agreement. By developing a streamlined detection assay for HCV, we will contribute to HCV testing without the need for expensive machinery or trained technicians, increasing the testing availability to increase HCV treatment rate and decrease HCV prevalence.

Melusin, a chaperone protein expressed in cardiac tissue, is known to induce a protective hypertrophic response in response to chronic mechanical stress. This protective hypertrophic response prevents the progression of cardiomyopathy into heart failure. In previous work done in wild-type (WT) and melusin knockout (melKO) mice, the absence of melusin was correlated with a hypertrophic response indicative of heart failure. I plan to further investigate the biomechanical role of melusin in humans using human engineered heart tissues (EHTs) created from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that lack melusin and their isogenic controls. EHTs are more representative of the human heart, making them an ideal model for studying the role of melusin in humans. I hypothesize that WT EHTs subjected to different mechanical stress conditions, i.e., high afterload, will outperform the melKO EHTs. In order to measure this, I increased the stiffness of the EHT posts and measured contractile force. I have been successful in differentiating high purity WT cardiomyocytes from iPSCs, essential for creating healthy EHTs. I also differentiated the melKO iPSCs and cast both WT and melKO tissues. The EHTs were planted on a bed of silicone EHT posts that can then be stiffened to induce mechanical stress on the cells. I compared the contractile force between the WT and melKO tissues. Improving our understanding of the role of melusin in humans can lead to further research into therapies and treatments for heart failure.