Disruption of ion channel function is increasingly associated with neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD). ASD has complex etiology and symptoms, but recent studies suggest that individuals with ASD have disrupted mesostriatal network activity associated with social interaction deficits. Although mutated ion channels play a key role in NDDs, the degree to which they disrupt mesostriatal network activity is not well understood. The Kv7 family of voltage-gated potassium channels encoded by KCNQ1-5 genes are increasingly linked to NDDs including ASD. These channels are broadly expressed, including within the ventral tegmental area (VTA), a nucleus of the mesostriatal pathway that plays an important role in social behavior. Here we assessed the impact of the ASD-associated variant KCNQ3(R2C) on VTA dopamine neuron excitability and social behavior in mice. We used a viral-based strategy to conditionally inactivate endogenous Kv7.3 with CRISPR/SaCas9 and re-express human wildtype (hKv7.3/WT) or mutant (hKv7.3/R2C) KCNQ3. Whole-cell patch clamp electrophysiology in brain slices revealed that hKv7.3/R2C significantly decreased excitability of dopamine neurons. I also observed a loss of social novelty preference using the three-chamber social interaction task in mice expressing hKv7.3/R2C but not in controls expressing hKv7.3/WT. These results suggest that hKv7.3/R2C and other similar Kv7 mutant variants may disrupt mesostriatal network activity and social interaction by decreasing dopamine neuron excitability.

Late Onset Alzheimer’s Disease (LOAD) is a common neurodegenerative disorder that affects ~5 million Americans. A primary risk factor in sporadic LOAD is the apolipoprotein E (APOE) gene, with carriers of the É›4 allele (É›3/É›4 heterozygotes or É›4/É›4 homozygotes) being at highest risk compared to É›3/É›3 homozygotes. LOAD is strongly sex-biased, with increased frequency in women (XX), but also increased severity in men (XY). The impact of genetic sex differences combined with APOE genotypes has not been studied. Our goal is to build models with various sex chromosome complements and APOE genotypes to understand interactions of these genetic factors in sex differences in LOAD pathology. The Disteche lab has derived isogenic pairs of human induced pluripotent stem cell (iPSC) lines with different numbers of sex chromosomes, e.g. XY/XXY, X0/XX, or X0/XXX. These pairs are genetically identical, save for their sex chromosomes. My project is to use CRISPR/Cas9 editing to generate different APOE genotypes in these paired lines. Starting from an XXY/XY isogenic pair from a heterozygote É›4/É›3, I am generating É›3/É›3 clones with either the XXY or XY genotype. To accomplish this, I transfect iPSCs with Cas9 and APOE-specific guide RNAs in the presence of a single-stranded DNA donor that contains the É›3 allele. After DNA cleavage and replacement, edited É›3/É›3 single-cell clones and control É›4/É›3 clones are identified using PCR and DNA sequencing, followed by karyotyping and verifying the absence of off-target effects and epigenetic instability. The new isogenic iPSC lines with different APOE genotypes and sex chromosome complements will be differentiated to LOAD-relevant cell types, including neurons, microglia, and cortical organoids, for transcriptomic and functional analyses to better understand how APOE genotypes and genetic sex interact to modulate risk in LOAD pathology.

Ewing sarcoma (ES) is a bone and soft tissue tumor that primarily occurs in children and young adults. The tumor is driven by an oncogenic fusion gene that fuses the EWSR1 gene located on chromosome 22 to FLI1, an ETS family transcription factor located on chromosome 11. EWS-FLI1 promotes tumorigenesis through transcriptional and epigenetic dysregulation. Despite maximally intensive cytotoxic therapy, the outcome for patients with metastatic ES remains poor, thus the need to identify new therapeutic agents. Given ES’s epigenetic dependencies, there is strong rationale to investigate epigenetic modifying drugs. Bromodomain and extra terminal domain (BET) proteins function as epigenetic readers that facilitate transcription and are upregulated in many tumors. I have shown that BET inhibition slows the growth of ES cells in vitro and in mice. However, BET inhibitors (BETi) will not be successful as a single agent. I hypothesize that combining BETi with other biologically targeted agents will lead to synergistic effects and tumor regression. Based on established literature, RNA-seq data of BETi-treated ES cells, and in silico drug screen, we selected Copanlisib (PI3K inhibitor), GSK-126 (EZH2 inhibitor), PP121 (Src, mTOR, VEGFR2, PI3K inhibitor), Danusertib (Aurora Kinase inhibitor) and Infigratinib (FGFR inhibitor) to test in combination with BETi (BMS-986158). Using standard in vitro cytotoxic assays and calculating synergy using the Chou-Talalay method, my preliminary results showed strong synergy between Copanlisib and BMS-986158. I detected no synergy between BMS-986158 and GSK-126. Our initial results from our in silico drug screen predict that Danusertib, Infigratinib and PP121 may be synergistic with BETi. Ongoing in vitro studies are testing this. Promising combinations will be tested in vivo xenograft models. It is our goal to identify drug combinations from the drug screen that will enhance the cytotoxic effects of BETi in vitro and lead to tumor regression in vivo.

Despite the significance of North Korea’s state ideology of Juche (translated as "self-reliance") on all aspects of life and policy in the country, scholars have generally neglected to cite ideology as a cause for nuclear weapons development. In this research, I explore how Juche affects nuclear weapons development through a text analysis of close to 10,000 pages of primary documents produced by North Korean leaders from 1980 to 2007, utilizing keywords related to Juche and nuclear weapons to identify relevant texts. In this study, I find that the Juche ideology is the primary way in which North Korea justifies its nuclear weapons development up to 2007. In particular, North Korea justifies its nuclear weapons through Juche in four ways through the Juche core pillars of political independence (jaju), economic self-reliance (jarip), loyalty to the leader, and self-reliance in defense (jawi). I also find that the relationship between Juche and nuclear development has evolved over the years (from theoretical foundations in the 1980s to justification of nuclear weaponry in the 2000s). This study has important practical implications as it shows that an important reason North Korea elected to pursue nuclear weapons development is because it feels that doing so is the only way to uphold the core values of the Juche ideology and preserve its independence. It also shows that a central mission of Juche and North Korean nuclear weapons is ensuring that the Kim dynasty continues to rule and to maintain their hereditary succession. This means that the North Korean nuclear logic is significantly different from other countries like China, Israel, Pakistan, and India where the operative factor is primarily the “national interest.” As previous denuclearization negotiations have mostly neglected this aspect, future negotiations may benefit from keeping this unique characteristic of North Korean nuclear weapons development in mind.

Body shape varies drastically across vertebrates, making it an effective trait to study when trying to understand macroevolutionary patterns of phenotypic variation. Body shape has been quantified in many ectothermic clades, but rarely in mammals. The goal of our research is to quantify body elongation in the family Sciuridae, as this area has been understudied thus far. Squirrels (Sciuridae) can be sorted into three distinct ecotypes based on life history and locomotion: ground, tree, and gliding. This leads to questions regarding differences in body shape between ecotypes in their respective environmental niches, and how differences in elongation correlate to different types of locomotion. We hypothesize that tree squirrels will be the most elongate, followed by ground squirrels, then gliding squirrels due to ecological and functional adaptations. To determine the potential differences in elongation between ecotypes, we will calculate the head-body elongation ratio (hbER) from skeletons held at natural history museums. We will use phylogenetic comparative methods to compare hbER between the three ecotypes. Thus far, our preliminary data shows both gliding and ground squirrels to have a statistically significant difference in hbER from tree squirrels. Tree squirrels are the most elongate, followed by ground, then gliding squirrels. We hope to further test differences between the hbER of ground and gliding squirrels with an increased sample size. Research on correlations between robustness and bone density in this clade is already underway, which will complement our results on elongation ratios between ecotypes.

Bats are the only mammals capable of powered flight and have correspondingly specialized body plans, apparent in the limbs. These specialized morphologies are thought to be the result of adaptations for the demands of flight; the skeletal elements of the bat forelimbs are elongated in order to support flight membranes and increase aerodynamic efficiency, whereas bat hind limbs are relatively short and specialized for hanging and catching prey in flight. Due to a deficient fossil record, the evolution of bat flight is still not fully understood but is hypothesized to be the result of an ancestral transition from gliding to flying. This hypothesis is plausible considering the morphological similarities between bat and glider forelimbs (both elongated) and the contrast between bat and glider hind limbs (shorter versus elongated). In this study, I collected linear measurements of the forelimb and hind limb skeletal elements of bats to add to a dataset of gliding, arboreal, and terrestrial mammals. I then fit evolutionary models to the data to test the hypothesis that A) selective pressures for flight drove the evolution of bat forelimb skeletal elements from glider-like forelimbs and that B) bat hind limbs evolved to become morphologically distinct from those of other mammals. Based on this hypothesis, I predict that A) bat and glider forelimb trait optima will fall progressively farther from arborealist optima and B) bat hind limb trait optima will be located in a unique region of morphospace. Preliminary results show that forelimb long bone lengths have evolved to be progressively longer from arborealists to gliders to flyers, supporting my hypothesis. This research helps address the longstanding question of how bats may have evolved flight from ancestral gliding mammals.

In vertebrates, differences in limb morphology are often the result of adaptions to locomotion. While previous researchers have examined the external shape of skeletal elements, there have been relatively fewer studies examining internal bone structure despite its potential significance to locomotor biomechanics. This study aims to help fill this gap by quantifying internal differences in forelimb skeletal morphology of squirrels (Sciuridae) across three locomotor ecologies: ground, tree, and gliding. We test the hypothesis that forelimb internal bone structure reflects adaptations to these ecotypes. To test our hypothesis, we micro-CT scanned the humeri of 61 species of squirrels and conducted bone structure analyses in the open-source software 3D Slicer. We assessed cortical bone composition by measuring material properties including global compactness (bone density), diaphysis (shaft) elongation, and second moment of area (bending ability). Based on biomechanical demands,we predict that A) gliders will have relatively less compact long bones with more elongated diaphyses due to the gravitational/aerodynamic constraints of gliding and B) ground squirrels will exhibit highly compact long bones with more robust diaphyses to gain more force while digging burrows​​. Preliminary results support our prediction that larger ground squirrels exhibit relatively more compact, robust, and bend-resistant humeri in accordance with their digging locomotion. This research furthers the understanding of diversity in forelimb morphology across mammals and the connection between forelimb morphology and locomotion. This study also lays the groundwork for future biomechanical and behavioral work to examine the evolutionary ties between form and function.

In mammals, males have sex chromosomes XY while females have XX. To balance out the extra X sex chromosome, females undergo X-chromosome inactivation (XCI) which silences most genes on one of the two X chromosomes. However, some genes escape XCI and continue to be expressed on the inactive X chromosome causing a high expression level of these genes in females compared to XY males, leading to potential sex differences in health and disease. Shroom4, an X-linked gene that encodes an essential protein for cytoskeletal architecture, is an example of a gene that escapes XCI in mice. How Shroom4 escapes XCI is unclear. It has been proposed that CTCF, a master chromatin regulator that controls gene transcription through histone or chromatin modifications, could play a role in insulating escape genes from the silencing environment on the inactive X chromosome. Indeed, we found there is a strong CTCF peak between Shroom4 and the neighbor silenced gene Bmp15. To functionally test this insulation model, I am using CRISPR/Cas9 to edit the CTCF binding site and examining the effects of deletion and inversion of the site on Shroom4 allelic expression levels. This analysis will show whether the CTCF binding site and its correct orientation are necessary for Shroom4 escape from X inactivation. Through this project we are able to improve our understanding of the complex nature of XCI.

Sexual dimorphism is the presence of morphologically distinct characteristics between males and females of the same species. Our project investigates the evolution of sexual dimorphism in the mandible across the family Canidae. We have created a database of 3D scans of specimens from the Burke Museum's mammalogy collection. We used a macroevolutionary approach to understand why sex-based differences exist today. Rensch's rule states that in male-biased species, sexual dimorphism is more evident in larger species. In female-biased species, the opposite effect is observed with smaller species exhibiting a greater degree of sexual dimorphism. Our project tests Renschs rule across a variety of Canids to determine if the degree of sexual dimorphism is greater in larger taxa. Using 3D models and geometric morphometrics, we placed landmarks on digitized mandibles to quantify sexual dimorphism in mandibular traits between males and females across various Canid species. Landmarked differences quantify sexually dimorphic features in the mandible, which we incorporated into evolutionary models to hypothesize how sexual dimorphism arose through evolution. Our project investigates whether Rensch's rule is applicable in Canids of various body sizes. We hypothesized that Canids will conform to Rensch's rule and that in larger Canid species, there will be a larger degree of sexual shape dimorphism for male-biased species. Rensch's rule is often tested with respect to size dimorphism, but limited studies use a multivariate approach to look at interspecific differences in sexual shape dimorphism. Our study will help answer ecological questions such as whether size and shape dimorphism is more significant in larger Canid species and whether sexual selection or niche divergence may be attributed to their evolution.

Joubert syndrome (JS) is a neurodevelopmental disorder diagnosed by the appearance of a “molar tooth sign” on an axial brain magnetic resonance imaging (MRI). JS patients present with hypotonia (low muscle tone), abnormal eye movements, and ataxia (uncoordinated movements). Subsets of patients develop progressive medical complications. Autosomal recessive, X-linked and rare dominant causes of JS in >40 different genes can be identified by DNA sequencing in ~75% of families. Cryptic DNA variants and alternative inheritance mechanisms are thought to account for the other 25% of families. The goal of this project is to evaluate the role of non-canonical splice variants in the pathogenesis of JS. We identified candidate splice variants in MKS1 from whole genome and targeted sequencing data and prioritized using SpliceAI annotation. A synonymous variant in patient 1 and a 30 base pair intronic deletion in patient 2 were identified. For each variant, we designed two sets of primers to flank the affected splice junction. Next, we extracted RNA from patient fibroblasts. We converted RNA into complementary DNA (cDNA) and amplified using polymerase chain reaction (PCR). Using gel electrophoresis and Sanger sequencing, we compared PCR products from patients versus controls.  We identified differences in DNA band sizes between unaffected control and patient samples. Based on Sanger sequencing, we determined that exon 4 is skipped in patient 1, but we were unable to determine the specific aberrant splicing event in patient 2. We confirmed the pathogenicity of candidate splice variants, identifying the precise genetic cause. An accurate genetic diagnosis informs prognosis, avoids unnecessary work-up, and guides monitoring for associated complications. Defining all genetic causes of JS expands our knowledge of the genetic mechanisms underlying recessive Mendelian conditions, confirming a substantial role for non-canonical splice variants.

Individuals experiencing homelessness and housing instability in King County have inadequate access to water, sanitation, and hygiene (WASH) services. This urgent matter of community health has only been exacerbated by the coronavirus disease (COVID-19) pandemic in recent years and can necessitate open defecation or unsafe disposal of wastewater. Particularly among those residing in recreational vehicles (RVs) and use their bathroom facilities, unsafe sanitation practices increase the risks of intestinal pathogen transmission and infection in densely populated communities. In a continuation of efforts to address this, I have developed a study that aims to identify an optimal method of RV wastewater concentration for downstream detection of pathogens. With informed consent from RV residents and sampling assistance from Seattle Public Utilities, I have began collecting a representative collection of samples of RV wastewater from multiple neighborhoods across Seattle. I have utilized a split-and-seed approach in which half the samples were seeded with known amounts of target bacterial, viral, and spore-forming organisms, and the other half left unseeded as a control. These samples will be used to compare two methods of sample concentration, skimmed milk flocculation and membrane filtration, using a weighted rubric that evaluates biosafety, seeded organism recovery efficiency, personnel time, and cost. Upon observation in the lab, I have found that skimmed milk flocculation led to inhibition during detection via molecular assays. Additionally, high turbidity of the samples yielded significant logistical challenges with processing skimmed milk samples, leading my team and I to instead favor the membrane filtration technique. As we move forward, I anticipate detecting elevated levels of pathogens in wastewater from RVs when compared to samples from control locations chosen to represent populations living in sewered and unsewered environments representing populations. Upon development of the optimized protocol, my work will be prepared for publication in a peer-reviewed journal and shared with the City of Seattle.