Designing conducting biomaterials capable of long-range electron transfer for the development of bioelectronics and biosensor remains a challenging and unsolved problem in protein design. The overarching goal of this project is to design protein fibers and template redox-active cofactors for long-range electron transfer. Recently our lab developed a method to design proteins that assemble into long fibers with a tunable diameter. We are using these fiber materials to assemble redox-active cofactors within 10-15 of each other. We are using a helical segment of a natural cytochrome-C protein as the electron transfer conduit. First, We docked this helical fragment inside the fiber molecules previously designed in our lab. Promising docks compatible with distance constraints for effective electron transfer (generally within 10-15Á of each other) were chosen for further design calculations to accommodate the cytochrome-C molecule. We ordered 11 proteins from the first round design calculations to characterize experimentally. Proteins were expressed using the usual E. coli. expression system and purified using Ni-NTA affinity chromatography. 9 out of 11 designs expressed and were soluble. For the helical cytochrome-C fragment, we expressed the native protein in E. coli. and obtained the heme-containing helical fragment by trypsin digestion. To ensure that the heme cofactors are incorporated in the intended positions of the fibers, high-resolution cryo-EM will be performed after cofactor incorporation. The redox potential of these fibers will then be measured using cyclic voltammetry (CV). If successful, this will lay the foundation for the development of a wide range of redox-active biomaterials capable of long-range electron transfer.

Self-assembling synthetic nucleocapsids have been at the center of increasing development as a medium for targeted drug delivery. By identifying certain cell markers of tumor cells, researchers at the Baker Lab have been evolving desired characteristics to this protein assembly by attaching different functional domains to specifically target cells. This screening of functional domains is possible because of the nucleocapsid’s ability to encapsulate its mRNA sequence, thereby linking genotype to phenotype. Moreover, this enables us to evolve desirable features in these functional domains through library selection. However, subsequent attachment of functional units to its exterior surface proved to decrease its mRNA packaging efficiency, thus limiting applications like mRNA delivery and evolution of functional units. This project aims to enhance the amount of encapsulated mRNA by mutating certain interior amino acids. Methods include cloning our I53-50-v4 protein assembly gene into Escherichia coli (E.coli) strains and over-expressing the proteins through IPTG induction. After the cells were harvested and lysed, proteins were purified through Immobilized Metal Affinity Chromatography (IMAC). RNA encapsulation levels were measured through Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) to compare between the original and mutated nucleocapsid. Promising results would demonstrate an increase in RNA concentration per nucleocapsid when compared to the original assembly across different constructs. The success of this project would allow for attaching multiple domains for evolution without compromising the nucleocapsid’s capacity to retain its biomolecular cargo. This research would greatly expand the utility of these nucleocapsids for evolving displayed proteins for desirable characteristics as well as enhance their efficacy for drug delivery applications.

Despite the yearly development of a vaccine, influenza (flu) still causes annual epidemics and is responsible for tens of thousands of deaths in the United States. Current flu vaccines are not fully effective in preventing viral infection because they protect people against a finite number of flu strains, which people may or may not be exposed to during a particular flu season. To address this challenge, we are engineering new protein therapeutics to increase the efficacy of flu vaccines. The two major proteins that flu viruses use to infect host cells, hemagglutinin (HA) and neuraminidase (NA), structurally parallel the two proteins that constitute a de novo designed nanocage. De novo protein design allows researchers to create proteins that are more stable than those produced in biological systems via evolution. The focus of this project is to use this designed nanocage to scaffold a more potent flu vaccine through a process called pseudo-symmetrization, which will allow the cage to display many different strains of HA and NA. To pseudo-symmetrize proteins, we used a modelling software to determine which protein components to modify to build a pseudo-symmetric protein. Then, we use molecular biology techniques to make modifications to regions of the protein that interact with other proteins when it self-assembles. As a result, the pseudo-symmetric protein is composed of building blocks that are all slightly different from one another, but can still assemble to create a nanocage with the same structure. So far, we have identified several variants of the modified cage component that can self-assemble, and we are continuing to test more interaction sites that make pseudo-symmetric proteins. These results, and continued experiments, will help us create new vaccines capable of displaying more flu virus antigens, thus protecting people from the flu using a single universal flu vaccine.

Transportation infrastructure like freeways provides an excellent lens through which to look at the issue of environmental justice. There is legislation in place that should prevent or at least draw significant attention to environmental justice, yet new freeways are still being proposed which continue to worsen environmental justices as decades past. With grassroots opposition as a primary form of resistance, this paper investigates the tools available to activists, as well as the ones most effective in ensuring the success of the movement. This paper is based upon three case studies of recent freeway proposals across the United States – the successful resistance to the Harbor Bridge Project in Corpus Christi, Texas; the unsuccessful resistance to the Central 70 Project in Denver, Colorado; and the unsuccessful resistance to the South Mountain Freeway Project in Phoenix, Arizona – as well as ten open-ended interviews with community organizers, government officials, and lawyers involved with the projects’ resistance movements in some way. Through this process, I found that two main factors determine the success of a resistance movement: organization (the timing and coordination of the resistance) and opportunity (the local and federal context and framing of the project and resistance). Through the interviews and in-depth analysis of formal government documents and news articles, I conclude that while aspects of a group’s organizational capacity were important to the outcome, the political opportunity available to them – specifically the attitudes and priorities of the current federal administration – was most critical to the success of a movement. While this leaves several questions for the future of environmental justice resistance, I end with a series of suggestions for how government officials and grassroots organizers might approach the issue with the current federal administration.

Current methods of drug delivery include the use of antibody drug conjugates (ADCs), lipid and polymeric nanoparticles, and top-down modified viruses. While successful in certain applications, these delivery platforms involve laborious production methods and have limited engineering opportunities. Synthetically designed proteins are viable drug delivery candidates that can be precisely modified to overcome the engineering challenges associated with other delivery platforms. Specifically, synthetic nucleocapsids (synNCs) are computationally designed icosahedral protein assemblies evolved to have virus-like genome packaging. The linkage between the encapsulated mRNA (genotype) and exterior proteins (phenotype) allows for directed evolution to be achieved through library selection. The synNC was evolved to increase the in vivo circulation half-life from less than 5 minutes to over 4.5 hours. The next step is to optimize the targeting ability of synNCs toward specific organs. We performed site-directed mutagenesis to introduce several mutations on the exterior of the synNC, performed two rounds of in vivo library selection, and identified several mutations that favored specific organs. Our current work involves producing five promising synNC mutants, biochemically characterizing the mutants, and examining their biodistribution in mice. The ability to target specific organs will be a successful step towards utilizing nucleocapsids in drug delivery and other biomedical applications.

The northern circumpolar landscape holds nearly twice as much carbon as the atmosphere, mostly as organic matter in perennially frozen (permafrost) circumpolar soils. Climatic warming may cause increased greenhouse gas emissions from arctic landscapes linked to thawing and mobilization of stored permafrost carbon. The role of circumpolar lakes in such climate-carbon feedbacks may be important, since lakes cover a disproportionately large fraction of the northern landscape, and emit carbon dioxide (CO2) and methane (CH4) to the atmosphere due to inputs of land-derived organic material. Yet northern lake carbon emissions are poorly characterized due to factors including limited sampling access and restricted sampling during prolonged inclement weather (winter and shoulder seasons). Here, we set out to define the environmental features most related to greenhouse gas build up in thirteen interior Alaskan lakes during winter ice cover, thereby providing information to better model lake emissions and identify hotspots across the regional landscape. We found that lakes with elevated CO2 tended to have elevated CH4 (with one exception), and that these patterns were predicted throughout the study region by variables that characterized the surrounding landscape, lake morphometry, and chemical properties of the lake. Shallow lakes at low elevation had the greatest concentrations of both gases, and also had the greatest quantities of organic matter readily available to fuel greenhouse gas production. Methane was mostly restricted to hypoxic conditions; whereas CO2 was found in both oxic and hypoxic conditions inversely proportional to oxygen. Given the limited information available for northern lakes during winter, this survey provides key information to advance our understanding of the patterns and factors related to winter greenhouse gas buildup in lakes, currently a major unknown in arctic carbon research.

In the last 30 years temperature has risen 0.6°C per decade in high latitude regions, twice as fast as the global average. This extreme warming is causing perennially frozen ground (permafrost) to thaw, thereby changing subsurface hydrology and exposing previously stored, deep millenial-aged soils to microbial activity. These changes are stimulating greater organic matter mineralization and emissions of potent greenhouse gases (GHG), carbon dioxide and methane (CO₂ and CH₄). The magnitude of soil carbon mobilization is poorly contained, in part because it is unclear what fraction of GHGs are emitted to the atmosphere directly, versus released to above ground aquatic networks. To better define the role of streams in the changing arctic carbon cycle, we explored headwater stream carbon chemistry in 10 individual catchments situated in a remote and understudied subarctic landscape of interior Alaska. We found an unexpected, positive relationship between CO₂ and CH₄ across streams, with concentrations peaking in the summer for CO₂, and fall for CH₄, suggesting stream emissions peaked when soil active layers were deepest and permafrost carbon layers were most hydrologically engaged. The positive relationship between surface water temperatures and the concentration of each gas reflected these strong seasonal shifts in stream GHG content. Organic carbon content in stream water was also linked to CO₂ but not CH₄, indicating potential differences in sources and sinks of each GHG that are currently being explored with ongoing stable isotope analyses. Taken together, our findings show that closer-than-expected coupling of CO₂ and CH₄ may make some streams much greater emissions hot-spots than others, and that accounting for seasonality is critical for understanding the greenhouse gas budget of individual streams. 

Since 1982, with the introduction of insulin as the first recombinant protein therapeutic, peptide and protein drugs have grown to encompass 10% of the pharmaceutical market and are the fastest expanding class of drugs. Advantages of using peptides over small-molecule drugs include high potency, selectivity, and capability to be engineered for a diverse range of targets, most commonly to disrupt or facilitate key protein-protein interactions (PPIs) in the human body for a therapeutic effect. To search for strong binders for a therapeutic target, combinatorial peptide libraries of up to billions of different sequences are synthesized and screened against promising targets. Due to the enormous library size, screening for high affinity binders often requires multiple rounds of enrichment in order to isolate the most potent molecules, a laborious and potentially bottlenecking step in developing protein drugs. Current approaches that have strategies for enrichment, such as phage display and yeast surface display, are limited to screening a library of binders against one target instead of multiple targets (library-on-library). This proposal describes the development of a peptide binder screen utilizing a simple workflow of repeated mating and sporulation of genetically engineered S. cerevisiae, or baker’s yeast. This technology improves the throughput of established screening methods through a library-on-library format that efficiently isolates high-affinity peptide binding interactions.

Wildfires play a major role in the structure and composition of landscapes and the general ecology of the Pacific Northwest. The Klamath-Siskiyou eco-region in northern California and southern Oregon has been experiencing an increase in the frequency, scale, and intensity of wildfires in recent years. Understanding the effects of wildfires on small mammal communities is an important, yet understudied, aspect of the response of wildlife to wildfires. In 2014, two wildfires burned areas on and adjacent to a long-term study area of wildlife. We will investigate the effects of these wildfires on the occupancy of small mammals such as Neotoma fuscipes, Glaucomys sabrinus, and Tamiasciuris douglasii, using occupancy analyses of data collected during fall field seasons using track plate stations. The longitudinal data set that we have includes years of data before the fires occurred, allowing us to disentangle any effects of the wildfires from any naturally occurring variation. This research is important in determining what lasting effects these increases in wildfire frequency are going to have on predator-prey dynamics.

Despite possessing only one photopigment in its retina, a feature which should render an animal colorblind, the octopus, like other cephalopods, possesses an unrivaled capacity to camouflage itself with its surroundings. A recent theory suggests that cephalopods discriminate color in their environment through exaggerated chromatic aberrations, or the focusing of different wavelenths of light at different points behind a lens, caused by morphological changes in the eye. By monitoring the blurring of different wavelengths of light upon the retina from the visual field in response to the shape of the pupil, a cephalopod can still perceive color with only one photopigment. We test this theory using conditioning to a two-toned visual stimulus and its two differently-colored halves, to look at whether strobing between the two differently colored stimuli evokes the same response as that paired to the combined stimulus or one of its halves. Evoking the response of the combined stimulus would suggest that they can see the two differently colored halves simultaneously, and that a morphological change is not needed to see two different wavelengths of light. This would present a model for color perception with one photopigment that could be explored in various other vertebrates and invertebrates previously thought to be colorblind.

The majority of research concerning the effect of intranasal oxytocin (inOT) on social cognition have used tasks that explicitly ask participants to focus on social information, such as identifying the emotion of face stimuli or interacting with other participants. As such, the variety of contexts from which data are derived are limited and have not examined the impact of inOT during tasks in which social information is available but not explicitly assessed by the participant. Here, we measured inOT’s effects on face processing with a novel task that does not require participants to explicitly assess social information, but instead relies on the uncanny valley effect. The uncanny valley describes how people tend to rate objects more favorably as they become more human-like until an object is nearly, but not completely human, at which point eeriness or repulsion is evoked. Participants viewed images of faces with varying degrees of human likeness and provided eeriness ratings while reaction time and eye-tracking data were collected. The results revealed that while inOT affected neither the magnitude of eeriness ratings nor eye movement patterns, inOT did decrease reaction times for providing eeriness ratings with the largest effect being for stimuli rated as being intermediately eerie. Thus, our results indicate that oxytocin can modulate the processing of social information in contexts other than explicit emotion recognition or social interaction tasks.

In recent decades, increased nitrogen (N) pollution in coastal aquatic ecosystems caused by increasing agricultural and urban activities has led to extensive habitat degradation and loss, change in the structure of aquatic food webs, and higher frequency of hypoxia. Nitrogen is ubiquitous in the biosphere, entering coastal environments through multiple pathways including ground- and surface waters, plus atmospheric deposition. The fate of much of the N entering coastal environments is not well established. In particular, the magnitude of N consumed at coastal margins, versus exported to downstream estuaries is poorly constrained. It is widely recognized that inland wetlands are important N sinks, as they are sites of fixation of reduced N to inert dinitrogen gas, however the role of coastal wetlands as N sinks is more difficult to establish due to the dynamic nature of these tidal environments. Here, to better understand the role of coastal wetlands in the global N cycle, we established a high-resolution budget of nitrate (the most abundant form of reduced N) from 9/12/2017 to 10/12/2017 at First Mallard Slough within the Suisun Marsh complex, a brackish tidal marsh in the San Francisco Bay Estuary. We modelled nitrate concentrations at 15-minute intervals using a submersible ultraviolet nitrate analyzer (SUNA), and matched these estimates with simultaneous hydrodynamic measurements of water flux. Data were synthesized in Python to establish 15-minute resolution estimates of nitrate mass exchange, showing that the wetland exported a net total of 2.54 Mg of N as NO₃^2- over the complete measurement period, or 84.6 kg N per day. Contrary to other studies showing wetlands are important nitrate sinks, our result revealed that the Suisun Wetland complex was an important N exporter to the San Francisco Bay Estuary, at least over the period measured here. Longer-term observations are needed to confirm this pattern at a complete annual scale.

Despite their sophisticated visual system, convergent and comparable in complexity to that of vertebrates, Octopus rubescens is largely nocturnal and forages mostly at night. Without visual information, their primary means of gathering information from the environment is through the sophisticated chemotactile sensory system within their arms. Octopuses blinded from lesions to their optic nerves have been observed relying on chemotactile perception of their environment with their arms fully extended to maximize their sensory range. Such behavioral profiles optimizing the acquisition of one sensory modality in the absence of another would be critical for navigating and monitoring changes within their environment. Our intention is to characterize how Octopus rubescens modifies its chemotactile range after an acclimation period of either light or darkness, simulating a natural 24 hour light cycle, and then a rapid change to the opposite lighting condition. Using 3d tracking cameras we will be able to quantify the change in the range of arm extension and overall posture that accompanies locomotion during light and dark conditions.

The transfer of carbon in and out of ecosystems is a complex process that is affected by many factors. The largest factor in carbon transfers is the photosynthesis and respiration rates of plants, which sequester and release carbon dioxide. Additionally, processes like soil leaching, sediment burial in lakes, downstream transport and even forest fires and animal migrations have an effect on the movement of carbon throughout ecosystems. In this study we ask the question: “How do physical and climatic conditions influence the concentration of carbon in small streams in the Pacific Northwest?” Our study site includes sixteen watersheds with areas that range from 500-2,500 acres in the northeastern portion of the Olympic National Forest. Using a GIS framework, we compiled satellite and LiDAR datasets of soil type, rainfall, slope, tree age and aspect and summarized these data for each of the sixteen watersheds. Our goal is to better understand which physical factors have the most influence on carbon transport in streams. By comparing our watershed-specific data to measurements of dissolved organic carbon and water quality characteristics in the streams, we identify correlations that can inform on the potential controls on carbon export in the rain-dominated catchments on the Olympic Peninsula.

Sarcopenia, or age-related of loss of muscle mass and function, is associated with a decline in quality of life for elderly populations and few effective treatment options. Sarcopenia is linked to mitochondrial dysfunction and elevated mitochondrial oxidant production. We are investigating the role of mitochondrial oxidative stress in sarcopenia using a mitochondrial targeted therapeutic and a mouse model of accelerated sarcopenia. SS-31 is a mitochondrial targeted peptide that associates with cardiolipin, decreases oxidant production, and increases ATP production. Superoxide dismutase 1 knockout (SOD1KO) mice lack superoxide dismutase 1 (an enzyme that converts the oxidant superoxide into hydrogen peroxide and molecular oxygen) resulting in an accelerated sarcopenia phenotype. We are testing whether treatment with SS-31 preserves muscle function in the SOD1KO mice. We hypothesize that improving mitochondrial function with SS-31 treatment will delay the decline in muscle function in the SOD1KO mice. To test this, we are administering SS-31 to SOD1KO mice through surgically-inserted osmotic pumps for 8 weeks between 3 and 5 months of age (the published timeframe for the onset of skeletal muscle decline in SOD1KO mice) and performing in vivo muscle function measurements of the gastrocnemius before pump insertion and monthly after pump insertion for 3 months. We compare muscle functional measurements with histological and biochemical analyses of mouse tissue samples upon euthanasia and determine skeletal muscle fiber type, metabolite and protein concentrations, and muscle fiber respiration and oxidant production. We expect SOD1KO mice with SS-31 to have a lower rate of decline in muscle force production and increased fatigue resistance over time, higher max ATP production, and decreased oxidative stress. The effect of SS-31 on muscle function, mitochondrial quality, and redox homeostasis has exciting potential as a translational therapeutic treatment for human sarcopenia.

Gut Microbiome is increasingly recognized as a pivotal player in toxicological responses, thus dysbiosis or microbial imbalance may worsen chemical-induced adverse outcomes such as inflammation, metabolic syndrome, and cancer. Early life exposure to environmental contaminants may produce long term toxicities in adulthood, and little is known to what extent early life exposure to environmental contaminants modulate the gut microbiome beyond adulthood. Therefore, this study tested the effects of perinatal exposure to 3 human health relevant environmental contaminants (BDE-47, TBBPA, and BPS), on the composition and functions of the gut microbiome of perinatally exposed adult male mice. CD-1 mouse dams were orally exposed to vehicle (corn-oil, 10ml/kg), BDE-47 (0.2mg/kg), TBBPA (0.2mg/kg), and BPS (0.2mg/kg) once daily from gestational day 8 to the end of lactation (postnatal day 21). Feces from male pups were collected at 12-weeks of age (n=14-23/group). Microbial DNA was isolated, subjected to 16rDNA sequencing, and analyzed using QIIME. Microbial biomarkers for each chemical exposure were predicted using LefSe. Microbial functions and key taxa that drive functional changes were predicted using PICRUSt and FishTaco, respectively. None of the 3 chemicals markedly altered the overall richness of the gut microbiome in adult male pups. However, principle coordinate analysis showed a distinct separation among different exposure groups, and especially between BPS and vehicle exposure groups. A total of 73 taxa were persistently altered by at least 1 chemical exposure, among which 12 taxa were commonly regulated by all 3 chemicals. The most representative microbial biomarkers for each exposure condition were Clostridiales for vehicle, S24-7 for BDE-47, Rikenellaceae for TBBPA, and Lactobacillus for BPS. Together, these observations suggest early life exposure to these human health relevant environmental contaminants produce persistent gut dysbiosis in adult male offspring, leading to functional shifts that may play important roles in regulating certain diseases of the host.

Increasing energy demand coupled with over-reliance on fossil fuels and other non-renewable energy sources has created a need for alternative renewable energy sources. The sun is one of the most promising sources and photovoltaic cells are one way to capture solar energy. Halide perovskite thin-films have recently emerged as ideal materials for solar cells due to low fabrication costs, bandgap tunability, high extinction coefficients, and high carrier mobility. Moreover, they have demonstrated rapid gains in power conversion efficiencies from 3.8% to 23.7% in nine years. Halide perovskites have the molecular formula ABX3, where A and B are cations while X is a halide. Past research has shown that methylammonium(MA) lead triiodide, a commonly used perovskite, can be changed into formamidinium(FA) lead triiodide by exchanging the A-site cation in a formamidinium iodide solution. This highlights high ion mobility and interchangeability in perovskites. However, in perovskites with mixed-ion composition, high ion diffusion adversely affects the device performance due to ion segregation, but little is known about inter-diffusion of different ions in perovskites. Here, we investigate the inter-diffusion of A-site and X-site ions in halide perovskite films by creating a lateral heterojunction of the ions. We confirm the creation of the gradient using UV-Vis and steady-state photoluminescence (PL) measurements. We further confirm that there is no change in the film morphology and crystallinity as evidenced by SEM and XRD, respectively. With PL line scans across the lateral gradient, we image the inter-diffusion of the ions as a function of position and time. Using Fick’s Diffusion equations to fit the PL line scans, we determine the ion inter-diffusion coefficient and extract the activation energy using temperature dependent measurements. This study demonstrates a facile quantitative method of probing the ion inter-diffusion in halide perovskites and furthers understanding of mixed-ion perovskite compositions.

During the early stages of vertebrate embryonic development, blocks of muscle tissue called somites form progressively along the anterior-posterior (head to tail) body axis. As the embryo grows in length, new somites are continuously added at the posterior end until the tail reaches its final length. Our work focuses on a subset of genes called the hox genes. These genes encode transcriptional regulatory proteins that are involved in controlling the formation of the body plan along the anterior-posterior (AP) axis. In vertebrates, these genes are present in four major clusters (A, B, C and D) and within a cluster they are expressed temporally from 3’ to 5’ of DNA, with hox13 being at the very 5’ end and thus the most posteriorly expressed hox genes. In this study we use zebrafish as a model organism. Zebrafish embryos are excellent for investigation because: 1) the genome has been fully sequenced to a very high quality allowing the use of CRISPR mutagenesis; 2) the zebrafish embryos are transparent and so very easy to study using live microscope imaging; 3) much of the early development is similar among all vertebrates including humans. The role of the hox genes during the somite-forming states has almost entirely been characterized based on the overexpression of individual hox genes in previous research. We developed a zebrafish loss-of-function hoxa13 CRISPR mutant and are investigating the roles of this gene on the formation of the body plan. My research focuses on understanding how a loss of hoxa13 gene affects cell movement as the AP axis forms using spinning desk confocal microscopy to capture cells and Imaris software to track them, and I will present the results of this analysis. We expect to observe abnormal or reversal of cell movement in the prognitor area of the tailbud.

Studying zebrafish embryos allows us to understand features of vertebrate embryonic development. Neuro-mesodermal progenitor cells at the very posterior end, or tailbud, of an embryo are bipotential. This is because the presence or absence of Wnt signaling commits them to either neural or mesodermal fate. Directed by environmental cues, mesodermal cells exit the tailbud, migrate anteriorward, and become somites, structural segments from which muscles differentiate. The Kimelman lab has found that Tbx16/Spadetail, a major driver of mesodermal morphogenesis, downregulates Arhgap29 and Arhgap35, members of Rho family GTPase activating proteins. This suggests Arhgap29 and Arhgap35 may be involved in mesodermal cell movement. My work in the lab is focused on finding out what roles these two genes play. I used heat shock promoter hsp70 to overexpress Arhgap29 and Arhgap35 in transgenic fish lines. Previously, our lab showed that sustained Arhgap35 affected somite morphology, and that sustained Arhgap29 also decreased the number of somites. In my experiments, I carried out in-situ hybridization in wild-type, Arhgap29- and Arhgap35-expressing embryos to examine genes regulating specification/differentiation of muscle cells and genes involved in transmembrane cell adhesion. I will present data on cell tracking and cell protrusions collected from Arhgap29- and Arhgap35-expressing embryos. These results will help me compare cell migration between Arhgap-expressing and wild-type embryos. The purpose of these analyses is to understand how these two proteins control cell movement in the embryo. In the future, I will continue to investigate cellular mechanisms underlying vertebrate posterior elongation.

Atherosclerosis, the underlying cause of most heart attacks and strokes, results from lipid accumulation in cells of the artery wall. Gene therapy, delivered directly to the artery wall, has the potential to prevent and reverse atherosclerosis. However, lipid accumulates primarily in cells below the endothelium, which are difficult to reach with gene therapy vectors. Our goal is to remove lipid from these cells by delivering therapeutic microRNA (miR) that increases cholesterol export from the cells. We hypothesized that if we introduced a therapeutic gene expressing the miR to endothelial cells (cells along the artery lumen), the endothelial cells would release this miR (anti-miR-33a-5p) via extracellular vesicles (exosomes) that transport miR between neighboring cells. We also hypothesized that smooth muscle cells (SMC) and macrophages would take up the therapeutic miR-containing exosomes, leading to higher expression of a critical cholesterol export protein (ABCA1) and increased cholesterol export. To test this in vitro, we introduced a therapeutic gene encoding the miR into endothelial cells and used RT-qPCR to test if the miR was released into the endothelial cell culture medium (CM) via exosomes. After confirming the presence of the therapeutic miR in exosomes purified from CM, we treated SMC and macrophages by incubating the cells with the exosome-containing CM. After incubation, we measured ABCA1 protein expression and cholesterol export. Expression of ABCA1 protein increased by 1.6- and 2.2-fold in SMC and macrophages, respectively, while cholesterol export increased by 1.4- and 1.6-fold. We conclude that gene therapy delivered to endothelial cells can produce therapeutic miR that is transferred to neighboring artery wall cells via exosomes, and increases cholesterol export in these target cells. If also effective in vivo, our approach has potential for reducing the severity of atherosclerosis by delivering therapeutic miR to cells that are difficult to reach with gene therapy.

Have you ever had to motivate the team? What was the result? The English language Wikipedia is notable for its large number of articles. However, 288 other active language editions of Wikipedia have also developed through the intricate interactions of contributing editors. While the editor interactions in the English Wikipedia have been researched extensively, these other language editions remain understudied. To understand how editors currently come to consensus in article building in the French language, a team of researchers has leveraged an existing English framework that depicts how power and policies play a role in mass collaboration. Using this English language framework, we are using qualitative coding methods to build a unique model of the editor interactions on the French language Wikipedia. The results of this study will help contribute to a deeper understanding of how a framework in a different language edition of Wikipedia differs from the English. Our preliminary results show that policy plays a large role in justifying editor decisions for the edits they make on various articles. Furthermore, our research findings have expanded our knowledge of the issues surrounding replication of an English framework in a different language platform.

Currently at the UW Molecular Hematopathology lab, peripheral blood and bone marrow samples stored at room temperature and at 4°C are used to perform molecular testing for clinical diagnoses. The current maximum storage length for both specimen types is established at 4 days, and samples received that are older than 4 days must have degradation and DNA quality assessed, a specimen redraw is requested, or the specimen is even rejected. To test the possibility of extending sample storage life by analyzing DNA quantity and quality of samples older than 4 days, I collected peripheral blood and bone marrow specimens and I stored them respectively at either room temperature or storage temperature conditions. I then extracted DNA from the samples on set days throughout a 14-day period, where I then quantified the DNA by spectrophotometry. Other technologists within the lab then qualitatively assessed the extracted DNA samples by fragment analysis. DNA concentrations appear stable through Day 14 and Day 11 for PB and BM, respectively, well beyond the current 4 day age limit for samples. Temperature did not seem to contribute to decline in DNA concentrations, though did seem to affect DNA quality. It would be beneficial to extend storage beyond 14 days to see when quantity and quality eventually do decline. There is good reason to believe sample holds and quality assessment can now be reliably deferred for samples older than 4 days, at least until the 14 day age mark for peripheral blood specimens and the 11 day age mark for bone marrow specimens. The quantity and quality of PB and BM samples did not decline significantly when held for longer than 4 days, nor did temperature or storage time up to 14 days significantly affect sample viability.

The English language Wikipedia is notable for its large number of articles. The development of this online encyclopedia would not be possible without the intricate interactions of editors that help sustain the virtual collaborative platform. Editors have a role in creating and reshaping articles and therefore have a role in how Wikipedia evolves over time. Our study replicates a qualitative coding scheme created over 12 years ago that demonstrates that policy and power play a role in mass collaboration. The prior work shows that user interactions including collaboration, conflict, coercion, and consensus have influenced changes on Wikipedia articles. A team of researchers are currently working to replicate this qualitative coding scheme on English Wikipedia talk pages to understand how these different types of user interactions occur in current editor discussions around article construction. Our findings will help contribute to a deeper understanding of how power plays between users have changed since the initial study. Our preliminary results, show that editor debates often lead to questions around article scope and legitimacy of sources. Furthermore, while policies play a large role in article building, editors own opinions influence how editing occurs.