The dystrophin protein protects cardiac and skeletal muscle from damage during normal contraction and relaxation by acting as a shock absorber in the cell. Mutations in the dystrophin gene lead to Duchenne muscular dystrophy (DMD), an X-linked recessive disease. Boys suffering from the disease become ventilator dependent at a young age and usually succumb to cardiac failure in their thirties. Other symptoms of DMD include muscle wasting, cardiomyopathy and respiratory failure. Currently there is no cure for DMD and while gene therapy has shown great promise, it still needs to be complemented with additional therapeutic interventions in order to fully address the symptoms of DMD. Previous work by our lab identified several drugs that blocked a certain type of calcium channel in cardiac muscle and protected the cells from damage following injury by correcting calcium movement into and out of the cell during muscle contraction. In this study, the leading drugs will be tested further using a cross-platform approach. The drugs will initially be tested in cardiac and skeletal muscle differentiated from healthy and DMD patient-derived induced pluripotent stem cells (iPSCs). The top three drugs that restore normal contraction and relaxation kinetics in vitro will then be tested in the DMDmdx rat. This novel, small animal model has a similar progression of DMD symptoms to human patients. The rats will be fed the drugs in their chow. Skeletal and cardiac muscle performance will be evaluated to determine whether correcting muscle contraction kinetics ameliorates the symptoms of DMD. Furthermore, we will show whether the same drug is equally effective in treating both cardiac and skeletal muscle. The cross-platform approach may help to better predict drug efficacy, leading to a reduced rate of failure in clinical trials. Moreover, this approach may serve as a benchmark for drug discovery in other neuromuscular diseases.

RNA-based therapeutics have attracted significant interest as a promising alternative to traditional cancer therapy methods. Coupled with other technologies such as the application of nanocarriers, RNA-based treatments have shown the potential to regulate gene expression in tumor cells with high efficacy and reduced safety risks. Despite these advantages, delivering sufficient amounts of therapeutic cargo while escaping from the proteolytic environment of the endosome has remained a long-standing challenge. This project focuses on addressing this issue by evolving the I53-50-v4 (V4), a self-assembling synthetic protein nanoparticle engineered to encapsulate its own mRNA, to increase RNA packaging and prevent degradation upon endocytosis. With previous data suggesting a decrease in mRNA encapsulation with an increase in mRNA length, additional designs were generated to further validate this finding. Furthermore, different variations of de novo pH-responsive trimers for endosomal escape were fused to the exterior of the V4 and tested for RNA packaging. Using computational tools, algorithms were also developed to model the porosity of the nanoparticle and to label residues for cationic mutations. Protein nanoparticles were expressed and purified via Immobilized Metal Affinity Chromatography (IMAC) and SEC (Size Exclusion Chromatography). Constructs were then analyzed in their mRNA encapsulation levels through RT-qPCR. Success of this project would demonstrate an increase in encapsulation levels with a decrease in mRNA length. As the phenotype of each nanoparticle design is spatially linked to its genotype, the top performing designs can also be selected and further evolved using both experimental and computational approaches. This will allow the development of a novel assay for screening desired proteins and narrow the gap towards achieving an efficacious RNA-delivery system.

Prescribed opioids are the most common analgesic used for alleviating acute and chronic pain. Despite this positive attribute, opioids are also highly abused drugs that can lead to tolerance and dependence. This abuse has caused a dramatic increase in opioid overdose-related deaths over the past couple of decades, deeming it a crisis. However, cessation of opioid use in tolerant individuals who wish to detoxify can precipitate severe withdrawal symptoms, often leading to relapse in order to avoid experiencing these negative symptoms. In recent studies, modulation of neuropathic and neuroinflammatory responses have been linked to withdrawal symptoms. As a result, we hypothesized that microglia, the resident immune cell of the central nervous system, serve as a potential target for withdrawal treatment. In order to test this, we reduced microglial activity by inhibiting the purinergic signaling pathway. This was achieved by first exposing mice to escalating doses of fentanyl over the course of a few days to create tolerance. Then, we administered clopidogrel, a selective antagonist of the P2Y12 receptors which are expressed in microglia, before inducing withdrawal using naloxone. Subsequently, in order to quantify whether inhibition of microglial P2Y12 receptors mitigated naloxone-precipitated withdrawal in fentanyl-tolerant mice, we measured avoidance of the withdrawal context with the conditioned place aversion (CPA) test, and evaluated somatic signs of withdrawal with EthoVision video analysis. Avoidance of the negative emotional and physical symptoms of withdrawal is a key driver of relapse, therefore the results from this experiment can provide prospective molecular pathways to target for future studies in treating opioid withdrawal symptoms. Reducing the severity of withdrawal would thus allow ease in discontinuing opioid use and diminish relapse.

Infection is the most common cause of mortality among patients with multiple myeloma undergoing autologous stem-cell transplant. Understanding the risk factors that lead to higher probability of infection is instrumental in their prevention. Although some risk factors are known, there are no robust predictive models for infection among myeloma patients receiving a hematopoietic stem-cell transplant. In this research study, we compared a variety of machine learning algorithms to reveal clinical features pre-transplant that are most predictive of infection post-transplant. Using features extracted from the electronic health record by means of natural language processing and manual abstraction, we trained our model using patients diagnosed with multiple myeloma who received an autologous stem-cell transplant at the Seattle Cancer Care Alliance. Along with being robust to missing data, the model is capable of processing diverse, heterogeneous data and identifying a class of predictive factors that can guide treatment decisions. Preliminary results point to lab tests such as lymphocyte, neutrophil, and platelet counts as the strongest predictive features for infection, and further exploration is underway to understand the full impact of these and other features.

The goal of this research is to bridge the gap of stereotypes between homeless adults and other community members. In order to do this, we propose a pen pal application where homeless adults can write and exchange letters with adults who are not homeless. The application provides unique card templates and a pen pal request system that matches two users together based on common interests. By building relationships through customizable letters, our goal is to create understanding and reduce stereotypes between these groups of people. The method we are using to create this application is Value Sensitive Design. Specifically, we are seeking to address the values of storytelling and community, while supporting the interests of the key stakeholders, namely homeless adults, other community members, and Operation Nightwatch. Because we are partnering with Operation Nightwatch, a nonprofit organization that assists the homeless, we are constantly in contact with them, seeking to develop the pen pal application for their organization. However, the main focus are the users of the application so we are making sure to include them in the design process allowing them to share their stories and build relationships through this application. We have interviewed the stakeholders with semi-structured interviews, completed data analysis, analyzed potential stakeholder value tensions and the challenges that may come up, designed wireframes, created a high fidelity prototype, and conducted formative user testing. The main features of the app are: (1) Getting matched to a pen pal based on a profile; (2) Choosing a letter template and writing a letter; and (3) Receiving mail in the inbox. We have kept this application very simple: The focus is on sharing stories and catalyzing new understandings. We are designing and coding the application of the working prototype for the research symposium.

Computational protein design is an emerging field that takes advantage of first principles derived from biological protein-protein interactions and explores the protein space that nature has yet to evolve. The Baker Lab developed a software called Rosetta that enables researchers to explore this space and create brand new proteins more stable than those produced in biological systems via evolution. This software has been adapted to take advantage of a concept that exists everywhere in nature- symmetry. Using this concept, I am building higher-order protein assemblies including protein nanocages and three-dimensional protein crystals. Researchers at the Baker Lab have developed a hierarchical approach to engineer these highly symmetric, complex structures. This hierarchical approach involves combining protein building blocks with different symmetric topologies multiple times to facilitate higher-order symmetric assembly of a three-dimensional protein crystal. By breaking up crystal symmetries into their constituent building blocks, we can design these higher-order symmetries with greater accuracy and troubleshoot experimental difficulties by pin-pointing structural deviations along the way. Here, I will describe my experience using this approach to create a protein crystal from symmetric building blocks.

Gene therapy may be an effective way to treat vascular disease. However, current approaches to achieve efficient gene delivery to the blood vessel wall require invasive surgery. Others have reported that high-concentration lentiviral vectors injected into the jugular vein preferentially transduce cells in the vascular wall of mice. If reproducible, this approach would be a major advance in vascular gene therapy. To test reproducibility, we injected high-concentration lentivirus into mouse jugular veins and measured transduction 4 days later. Eight mice received 10e8 particles of a green-fluorescent protein-expressing lentivirus; six mice received vehicle only. Mice were euthanized and their aortas, hearts, lungs, livers, kidneys, spleens, quadriceps and gastrocnemius muscles, carotid and femoral arteries were excised. These tissues were prepared for DNA analysis (to measure transduction) and histology (to detect GFP expression). Tissues used for DNA analysis were frozen in liquid nitrogen and DNA was extracted. We used quantitative PCR targeting the GFP gene to measure integrated proviral DNA, which we quantified as proviral copies per cell. We found a mean of 3 proviral copies (range 1-6) per cell in the liver samples and 13 proviral copies (range 4-28) per cell in the spleen samples. Essentially no proviral DNA was detected in the liver or spleen of vehicle-injected mice (p<0.003 for both; limit of detection 0.3 copies per cell). Proviral DNA was not reproducibly detected in the other organs/tissues listed above. Tissue for histology was fixed and embedded in frozen blocks for immunostaining. We anticipate that immunostaining will enable detection or exclusion of lower levels of transduction. Intravenously injected lentiviral vectors seem to efficiently transduce the spleen and liver and do not efficiently transduce cells in the blood vessel wall. Our data suggest that better vector targeting is needed to deliver gene therapy to the vessel wall by IV injection.

Sarcopenia, the age-related of loss of muscle mass and function, is associated with a decline in quality of life in the elderly and has few effective treatment options. Sarcopenia is linked to mitochondrial dysfunction and elevated mitochondrial oxidant production. We are investigating the role of elevated 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 in vivo. 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 hypothesize that improving mitochondrial function with SS-31 treatment will delay the decline in muscle function in the Sod1KO mice. To test this, we administered SS-31 to SOD1KO mice through surgically-inserted osmotic pumps for 8 weeks between 3 and 4 months of age, the published timeframe for the onset of skeletal muscle decline in SOD1KO mice. Muscle force generation and fatigue resistance was tested in vivo in the gastrocnemius before pump insertion and monthly after pump insertion for 4 months. At the end of the treatment we used histological and biochemical analyses of mouse tissue samples to determine skeletal muscle fiber type, metabolite and protein concentrations, and muscle fiber respiration and oxidant production. We expected 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.

Atherosclerosis, a cardiovascular disease that results from fat (cholesterol) accumulation in the walls of large arteries, is the major cause of heart attacks and strokes. Gene therapy, delivered directly to the artery wall, has the potential to prevent and reverse atherosclerosis by removing cholesterol from the artery wall. Our group has used gene therapy to prevent and reverse atherosclerosis in a rabbit model; however, the effects are partial. Reasons for partial success likely include expression of inadequate levels of the therapeutic transgene product (apolipoprotein AI; a protein that removes cellular cholesterol and transports it to the liver for excretion). To address this limitation and achieve more-effective gene therapy, we hypothesized that incorporation within our gene-transfer vectors of DNA sequences that activate transcription from nearby gene promoters (sequences known as “cis-regulatory modules or “CRMs”) would increase the amount of apolipoprotein AI mRNA transcribed from our vectors. We used a bioinformatics approach and in vitro measurements of transcript levels in cultured vascular cells, to identify 11 potential CRMs within the sequences of 4 highly expressed vascular cell genes. Some of these CRMs increased apolipoprotein A1 mRNA levels, but not above levels we had achieved using other strategies. We are now repeating this approach, using newly available in vivo data generated with single-cell RNA sequencing of vascular tissue and with a separate technique that identifies highly expressed vascular cell-specific genes. We will use the sequences of highly expressed genes that we identify, along with bioinformatics tools, to identify new CRMs that are more likely—than the CRMs identified previously, using in vitro data—to increase apolipoprotein AI transgene expression above previously attained levels. We anticipate that incorporation of these CRMs into our gene-therapy vectors will increase the efficacy of our vectors in preventing and reversing atherosclerosis.

Latinx individuals feel that mainstream mental health services cannot adequately address their specific needs and that non-Latinx therapists may be insensitive to their psychosocial needs. The Latinx population is a rapidly growing population. In 33 states they account for 58% of the population; in the remaining they account for 7%. As a result of increase in population in the United States, there is an urgent need to understand the Latinx culture and create interventions that are effective for Latinx individuals. Studies reveal barriers around therapists lacking empathy towards Latinx clients, and lack of language metaphors in order to translate mental health diagnosis. To address these gaps, the study aims to discover what are the barriers and needed supports to promote culturally competent care by mental health practitioners serving Latinx communities. This study uses primary data obtained through a quantitative survey from mental health practitioners who are serving or have served Latinx individuals. Chi-square and independent samples t-tests were run to examine bivariate relationships between whether trainings related to cultural competence, humility, and diversity were provided and participant perceptions of cultural competence and understanding of the Latinx culture. A preliminary analysis of 18 participants found no significant differences at the p < .05 level. However, trends suggest practitioners who are provided with training across categories of competence, humility and diversity were likely to report feeling culturally competent. Additionally, no meaningful differences were seen in average ratings of knowledge of the Latinx culture. Preliminary findings suggest trainings are beneficial for practitioners working in mental health within the Latinx community, particularly for facilitating cultural competence but not necessarily for increasing knowledge around the Latinx culture.

Historically, the dominant culture in fraternities and sororities has been heteronormative. Prior studies have found members of the Greek system have higher levels of intolerance towards those in the LGBTQ+ community than those outside of the Greek system. There is little data about the effectiveness of educational initiatives on LGBTQ+ literacy, awareness and acceptance in the Greek system. Thus, we set out to determine the effectiveness of an educational intervention to promote LGBTQ+ literacy and attitudes among new fraternity and sorority members at the University of Washington. To do this, we administered surveys to incoming fraternity (n=383) and sorority (n=208) members to assess literacy, awareness and acceptance before and after a standardized presentation on LGBTQ+ issues developed with content experts from Seattle Children’s Hospital. Post-intervention, significantly more participants reporting knowing that there are more than two genders compared to thinking there are only two genders or feeling unsure. More participants correctly defined the acronym LGBTQ after the intervention than before, and significantly more understood the distinction between gender and sex. More participants understood the definition of heteronormative, pansexual and had an improved understanding of the difference between transgender and cisgender post-intervention. Participants’ level of anxiety about their own sexuality did not change from pre- to post-intervention, but after the intervention, participants rated the Greek System as more inclusive of LGBTQ+. After the intervention, participants reported feeling significantly more prepared if a friend approached them to reveal that they are LGBTQ+. In conclusion, an educational intervention resulted in an increase in LGBTQ+ literacy and self-reports of awareness and acceptance of LGBTQ peers among incoming Greek system at a large public university. Further research can determine if this intervention would be similarly effective in different geographic regions, private university settings and potentially help with academic success and wellbeing.

Gender violence is an extreme manifestation of bias. More and more, research suggests that attitudes surrounding such manifestations are likely the product of a combination of objective psychological mechanisms and more obvious reactions to socialization. This same body of work shows that understanding the combination is key to any attempt to effectively target biased behaviors. When it comes to gender violence and societal attitudes surrounding specific instances, psychological research barely scratches the surface of this interplay. The nearest researchers have come is in studies pertaining to power dynamics and reactions to stereotypes, with a few related tangents in courtroom-based studies and analyses of masculine identity. Database and library searches for research in the fields of group theory, gender identity and stereotype formation and maintenance confirmed the paucity of study and revealed an as-yet unexplored intersection between these fields. This literature review suggests exploring that intersection beginning with research that views gender as a group. Studies of gender salience in children show that where an expectation of gender duality is imposed, group-like behaviors emerge. Similar research into the behavior of minority students when a fellow minority student displays a negative trait demonstrates that when a group member is forcibly reminded of group membership, one of several in-group behaviors emerges. This appears to remain true in the case of a dominant group identity, though this area has yet to be thoroughly explored. This and related research suggests that the stark reminder of gender duality that is gendered violence may also bring psychological behaviors related to group membership into play. This makes the various theoretical frameworks posited for viewing group identity a potentially fruitful place to start identifying the social-psychological interplay at work in this area, providing the basis for deeper work on topics across the spectrum of gender.

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 Spanish 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 Spanish 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 varies 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.