DREADD receptors (Designer Receptor Exclusively Activated by Designer Drugs) are engineered G-protein receptors which are exclusively activated by the inert-molecule CNO (clozapine-N-oxide) and to a lesser degree clozapine. DREADD receptors are often used, to identify the circuitry and cellular signals that specify behavior, perceptions, emotions, motivation, and motor functions in species ranging from flies to nonhuman primates. Heterologous overexpression of non-native genes in biological systems, is often confounded by varying degrees of overexpression and result in non-specific effects. Thus, our purpose is to determine effects on cell signaling pathways following varying levels of DREADD receptor expression. Our hypothesis is that, increased levels of DREADD receptor expression increase non-canonical cellular signaling pathway activation. We used human embryonic kidney cells (HEK cells), as our model system, then expressed various levels of DREADD receptors using Lipofectamine transfection and DNA-plasmids expressing Gs- and Gi-coupled DREADD receptors (Canonically, Gs-increases cAMP and Gi-decreases cAMP). After transfection, we treated transfected cells with 1µM of CNO or VEH (a control) for 5-30min and then lysed the cells with RIPA Buffer, and prepared samples for western blotting to detect activation of proteins of interest in the GPCR non-canonical signaling pathways (example: kinases and transcription factors). We used anti-HA antibodies to ensure dosage of receptor expression, and used a panel of antibodies to detect activated proteins in well-established non-canonical GPCR signaling pathways. Western blot results were measured using Odyssey scanner and the Image studio software suite. We expect to see an increase in non-canonical signaling with increased expression of both Gi- and Gs- coupled, specifically in the MEK/ERK and CREB signaling pathways. In summary, our findings will highlight additional considerations regarding the level of receptor expression when using DREADD receptors in vitro and in vivo experiments, and specifically measure the off-target “non-canonical” signaling pathways induced by DREADD receptor overexpression.

Understanding the population structure and biogeography of avian fauna is an important tool for gaining insight into the historical and evolutionary processes leading to the diversification of North American birds. Research into the extensive adaptive radiation by the New World Setophaga warblers of the Parulidae family has been especially useful in illustrating these processes. Through phylogenetic analyses, we investigated the population structure of black-throated gray warblers (Setophaga nigrescens) in the Western United States. Previous analysis describes two distinct groups; a northwestern clade, known as S. n. nigrescens, and a southwestern clade, known as S. n. halseii. To elaborate on this study, we analyzed a larger number of Setophaga nigrescens samples that encompass a wider distribution across the Western US. Using mitochondrial sequences of the ND2 gene, we constructed several phylogenies using 48 S. nigrescens samples and 1 S. graciae sample as the outgroup. Our results support subpopulation structure as indicated by two haplotypes. One haplotype is regularly found in populations west of the Cascades and Sierra Nevada, while the second haplotype is common in the Southwestern US. Admixture appears to be occurring over a wide geographic range extending throughout Northern Arizona, Utah and Nevada, and possibly spanning farther north based on both haplotypes occurring in Washington State East of the Cascades. With a sequence divergence of about 0.8%, a Pleistocene origin seems likely; numerous glaciation cycles during this period may have facilitated this genetic divergence. However, additional samples will be needed to better illustrate the extent of their structure. This will have important implications for the diverse relationships found within the Setophaga genus of the Parulidae family and may benefit future studies involving the evolutionary processes that have led to the diversification of this group and North American birds in general.

Stress is highly correlated with negative physical health impacts and an estimated 75 to 90% of primary care visits are attributed to stress related conditions. Currently available treatments for anxiety are not always effective and very limited, therefore identifying new targets for the development of medication is important. Recently, neuropeptides have drawn interest as potential targets for drug therapies. Pituitary adenylate cyclase-activating polypeptide, or PACAP, is a neuropeptide that has been linked to stress responses in a variety of brain regions. For example, the amygdala is a crucial brain region for mediating chronic stress, acute stress and fear and contains numerous PACAP-expressing neurons. My research focuses on the amygdala and the expression of PACAP following an acute stress. In my experiment, I quanitfied PACAP levels both with and without acute stress. After stressing rats using a forced swim protocol, I measured PACAP and mRNA expression in the amygdala using RNAscope in situ hybridization. Brain slices were imaged using fluorescence microscopy to quantify the RNA scope signal. To analyze these images quantitatively, I have developed a CellProfiler code to accurately and rapidly analyze the proportion of neurons expressing PACAP mRNA as well as the intensity of expression within the neurons. I hypothesize that PACAP mRNA expression will be increased in the rats subjected to the acute stressor, indicating that PACAP is an important mediator for the neural stress response. By determining this relationship between neuropeptide expression and stress response, PACAP antagonists could be potentially used in drug therapies to help patients experiencing stress related health conditions.

One of the most inflammatory topics in animal welfare and wildlife conservation is the issue of feral domestic cats. Wild bird advocates favor lethal eradication programs to protect wildlife from cats, and feral cat advocates support reducing their populations non-lethally through trap-neuter-return programs. Little research has been done regarding the motivations and beliefs of the humans behind this conflict, a gap that this study has sought to remedy. Feral cat and wild bird advocates are as worthy of investigation for potential commonalities as they are for their history of strife. Both groups express a connection to animals which they cannot share much, if any, contact. Most human-animal bond research focuses on traditional pets, and questionnaires routinely use physical closeness as a metric of one's bond to an animal. An online survey was conducted of 3859 adult Americans who identify as advocates for either feral cats or wild birds. Questions included demographics, motivations, political engagement, how the animals make them feel, and what they enjoy or admire about feral cats or wild birds. The groups had statistically significant differences in more areas than they had commonalities. Areas of difference include number of pets, level of emotional attachment, and why they like their focal species. The groups did not have significant differences in subjects such as household size, enjoying feral cat/wild bird activities because they are challenging, and reporting that being a "feral cat person" or "wild bird person" is an important part of their identity. This is the largest study that has been conducted on a human-animal bond topic, as well as the most comprehensive study examining the people behind an important area of policy debate. These results could be useful in deescalating future policy battles and trying to pave a path forward to compromises that both stakeholder groups could accept.

The ability to quantify rat and mice ultrasonic vocalizations (USVs) can provide ethological validity to animal models of affective disorders. Vocalization frequencies are key indicators of a rodent’s subjective state and are closely tied to their behavior. For example, 50-kHz calls in rats are emitted during positive events such as sucrose drinking, and are attributed to a positive affective state. In contrast, 22-kHz calls are ascribed to a negative affective state, and are emitted during negative events such as shock. Current methods for manually scoring vocalizations are slow and labor intensive, while automated programs lack the accuracy to produce satisfactory scoring under non-ideal recording conditions. In order to create a more precise program for detecting these USVs, we applied deep learning, which has revolutionized the field of bioacoustics through image detection and recognition of audio files. By converting soundwaves produced by mice and rats into spectrograms, we can train convolutional neural networks to distinguish between vocalizations and noise. Using these methods, we created the software DeepSqueak©, a Deep Learning Based System for Quantifying USVs and Extended Analysis Kit, which outperforms current state-of-the-art, commercially available software. Hundreds of rat calls from the 18-75kHz were manually isolated from spectrograms and used as training data for our neural network, allowing it to differentiate calls from noise. As a result, USV detections using DeepSqueak are far more rapid and accurate in comparison to existing software. The detected calls produced from the software were then reviewed and re-entered into the neural network as another training set to consolidate its precision and efficiency. With the speed and reliability of DeepSqueak, quantification of USVs can become more widespread and lead to a wealth of new knowledge relating to affective disorders.

Some of the most widely used medications have unknown side effects on wildlife after disposal. One of these compounds is 17α-ethynylestradiol (EE2), a potent synthetic estrogen and endocrine disrupting compound. EE2 is an active ingredient in birth control pills and has been found in environmentally relevant concentrations in waterways due to the inability of wastewater treatment systems to completely remove it. Even at parts per-trillion, EE2 can cause male fish to develop female sex organs in a process known as intersex. EE2 mimics estradiol, the natural female sex hormone that causes feminization, and affects reproduction and development. Another role of estradiol is it binds to estrogen receptors on cells to modulate immune responses. Our group has observed increased mortality rates in fish exposed to EE2 prior to pathogen challenge. Additionally, we have shown that EE2 down regulates genes and pathways involved in innate immunity in fish, some of which are tied to phagocytosis, a process essential for ridding the body of pathogens and cell debris. Therefore, altered phagocytosis rates may be contributing to the increased mortality of fish when they are exposed to EE2 and challenged with pathogens. My hypothesis is that EE2 alters phagocytosis. To test this hypothesis, I’ve developed an in vitro phagocytosis assay using a rainbow trout macrophage cell line (RTS11). In this assay, RTS11 cells are cultured with fluorescently tagged bacteria that only fluoresce when eaten by phagocytic cells. Cells can be cultured in the presence or absence of endocrine disrupting compounds with potential changes in phagocytosis being measured using a flow cytometer and associated statistics to assess if a compound alters phagocytosis—a key component of innate immunity. This study will provide insight into the biological mechanisms that contribute to higher mortality rates in fish exposed to EE2 prior to being infected with pathogens.

Current studies have shown that changing climate is responsible for methane releases from the seafloor, making it important to understand how methane plumes can impact the ocean. Methane plumes impact the stratification and circulation within the water column which can influence primary productivity and seawater chemistry and potentially atmospheric greenhouse gas levels if the methane from the plume undergoes exchange with the atmosphere. Along the Washington margin there has been over 1772 individual bubble plumes located and identified in depths ranging from 40 meters to 1988 meters water depth. Compilation of archived CTD (conductivity, temperature, depth) data at active methane plume sites including Southern Hydrate Ridge, Grays Harbor, and Vancouver Island, can provide estimates of the plume fluids temperature and salinity. Using these estimates, the point where buoyancy reaches zero determines the possible depth of horizontal plume fluid intrusion into the water column and the entrainment coefficient to quantify the fluid that’s entrained by the plume can be calculated., I can determine the plumes’ ability to penetrate through sea water density interfaces such as the thermocline by calculating the Richardson number for the plumes. Using pre-established bubble models, I will also quantify sea water entrainment by the plumes. Through these methods, I will be able to determine how methane plumes impact water column stratification on the Washington Margin.

Glioblastoma (GBM WHO grade IV) is the most dangerous brain tumor in adults and comes in two types, primary and secondary. Primary GBMs form de novo and are more common and more aggressive than secondary GBMs, which develop from grade 2 and grade 3 gliomas. Despite primary and secondary GBMs displaying identical tissue, there are significant differences in the genes expressed that affect the GBMs responsiveness to alkylating treatment. Our research focuses on secondary GBMs and why they have an association with higher progression free survival (PFS) than primary GBMs, in hopes to aid the personalization of treatment plans. Our hypothesis that O6-methylguanine-DNA methyltransferase (MGMT) activity is inversely related to PFS. Our method of experimentation includes taking the MGMT content of the extract, which is supernatant liquid that lies above a precipitate of whole tissue lysates. We then use the radioactivity transfer from a DNA substrate containing O6-methylguanine-DNA to protein as a quantitative measure. In secondary GBMs, there are sometimes mutations in the Isocitrate Dehydrogenase 1 (IDH1) gene. These mutations reduce substrate affinity, decreasing the enzymatic activity. Furthermore, the enzyme develops the ability to produce an oncometabolite, D-2-hydroxyglutarate (2-HG). 2-HG synthesis leads to genome wide hypermethylation of CpG islands potentially causing the MGMT gene to be silenced. The MGMT protein repairs DNA and prevents it from breaking. By silencing the MGMT protein activity, DNA gets repaired less and usually results in double strand breaks. The higher PFS associated with secondary GBMs could be affected by the IDH1 mutation, which can reduce MGMT protein activity and lead the tumor DNA to break, making treatment more effective. Establishing this association could lead to personalized treatment because patients with IDH1 mutations respond better to alkylating agents, such as TMZ.

Inferior olive (IO) is part of the brain stem that projects their climbing fibers in the cerebellum's Purkinje cells and receives input from the deep cerebellar nuclei DCN to regulate the strength of electrical coupling in the IO. The structure of the IO contains a high density of gap junctions that are responsible for weak spontaneous subthreshold oscillation (STO). The weak electric potential STO can depolarize the neuron and open the N-methyl-D-aspartate receptor (NMDA) channel that is located close to the gap junction by removing its Mg ion. The activation of the NMDA receptor can play a role in strengthening the STO, increasing its synchrony, and inducing a new STO. Increase the synchrony in IO may contribute to the precision learning of the motor skills. Therefore, our study was done on mice in vivo, and extracellular recording technique was used to record from IO before and after the addition of NMDA. It was found that adding NMDA changed the firing of the neuron relative to itself. Second, NMDA decreased the power spectra density's raw power in both groups 2 Hz and 3-6 Hz, however, there was some increasing in the PSD at 5 Hz in the group of neurons that fire with 2 Hz. Third, the coherence value between the neuron and the local field potential (LFP) decreased after the addition of NMDA. There was an exaption in which the coherence value increased at specific frequences. Finally, we found that a neuron showed phase locking its firing activity with the LFP, which was in the negative firing phase. In conclusion, NMDA receptor plays a role in increasing the synchrony of neuronal electrical synapses in IO, involves in the IO rhythmogenesis, and may add temporal precision to coordinate the muscle activity.

Religious rhetoric has been a common tool in politics for the majority of American history, from Thomas Jefferson being “unfit to lead a Christian Nation” to Ronald Reagan’s “City Upon a Hill”. Although the Constitution declares the government to be secular, members of Congress, to varying degrees, regularly invoke religious imagery and rhetoric in the public communications. This study seeks to understand why. I examine congressional religious rhetoric using a unique data source of congressional webpages from the massive .GOV collection of the Internet Archive Project. I hypothesize that members' personal beliefs and electoral strategies help to explain differences in website content. I measure the personal religiosity of members using a carefully constructed scale of involvement in their religious communities. I measure district religiosity using information about district-level church attendance and prayer frequency from the Cooperative Congressional Election Survey. Finally, I draw from established religion lexicons to measure religious emphasis in congressional website content. I find positive, statistically significant support for both hypotheses. This work sheds light on politicians in particular members of congress are actively seeking to brand themselves through the internet for political purposes.

In 2015, Seattle voters overwhelmingly voted in favor of Honest Elections Seattle. A citizen-led initiative that significantly changed the way Seattle City elections were financed. By providing every resident of Seattle with Democracy Vouchers, four $25 credits that can be given to participating candidates, these reforms have provided a new opportunity for citizens to become political donors and participate in a process that has been dominated by wealthier and more educated elites. My research analyzes every political contribution made in the 2017 and 2015 electoral cycles for two city council races that are city wide. I break down the city of Seattle into ZIP codes and compare political contributions from each ZIP code in 2015 and 2017. I then use the American Community Survey data to compare each ZIP code by mean income, percent of residents with a bachelor’s degree or higher, and percentage of white residents. I predict that Seattle’s Democracy voucher program has significantly changed the profile of the average political donor in Seattle and made them more representative of the city as a whole. I hope to find that through publicly financing elections, we can create an electoral system where everyone can participate, not just the wealthy elite. This work is critical for evaluating the success of a multi-million dollar public program and whether it ought to be adopted in other cities throughout the United States.

The North Korean nuclear crisis is severe, with North Korea having developed missiles potentially armed with nuclear weapons that can reach the continental US. However, the international response has mainly been ineffective in stopping North Korea. Additionally, South Korea’s new attitude going towards negotiation with North Korea is an important game-changer. Through this research, I plan to explore the questions of why the international sanctions have been ineffective, how South Korea’s changing attitude can influence this issue, and what can be done to solve this issue completely. For this research, I have collected information from many sources both from the US and in Korea, including books, journals, and newspapers. Some of my findings are that there are loopholes to the sanctions, especially China not enforcing the sanctions and trading key goods with North Korea, especially oil. Additionally, the Moon Jae-In administration’s new strategy towards the issue, going more towards negotiation, may be catastrophic and must stop immediately. To solve this issue, South Korea must stop doing peace talks with North Korea, as such talks were catastrophic during the past, and stay on the same page with the US to motivate China from not trading with North Korea, especially oil which is crucial to its survival, as North Korea cannot produce its own oil. This may be enough to force North Korea to denuclearize. A better solution is to assassinate the dictator of North Korea, Kim Jong Un, which will lead to disorder in North Korea as Kim Jong Un does not have a successor currently, and South Korea and the US can use this opportunity to destroy the weapons of mass destruction in North Korea. This research is crucial for maintaining the peace and security not only in the East Asia region, but also for the entire world.

On top of being iconic symbols of climate change, glaciers integrate year-to-year precipitation and temperature variability, which can mask their response to a climatic trend. For this reason, it is vital to identify the variability in their records to better detect regional climate change. With time-varying sea-level pressure (SLP) and sea-surface temperature (SST) fields, we examine the influence of natural climate variability on 14 glaciers in the North Atlantic region by using a statistical method called dynamical adjustment. The SLP and SST fields each explain 50-60% of variance in the winter mass-balances and 30-40% in the summer mass-balances of the maritime glaciers. The continental glaciers are less closely linked to oceanic variability, with SST explaining roughly 30% of variance in winter and summer mass-balances. The glaciers located further south fluctuate with the North Atlantic Oscillation (NAO) in the winter, while glaciers near the Arctic show influence from the Atlantic Multidecadal Oscillation (AMO) in the winter. To investigate how this variability affects glacier trends, adjusted mass-balances are compared to observational mass-balances with no signs of variability forcing trends. The observed and adjusted summer mass-balance trends of the southern glaciers are significantly negative, while the observed and adjusted winter mass-balances of the northern glaciers are significantly negative. This indicates that winter trends are more likely to emerge in glaciers near the Arctic, while summer trends are more likely to emerge in glaciers further south which is consistent with surface temperature time of emergence for global warming.

Mitosis is a process important for the regulated division of cells. In cancer cells mitosis is often dysregulated, which can cause cells to split uncontrollably. One important protein that plays a key role during mitosis is the protein kinase Polo-like Kinase 1 (plk1). The function of Plk1 is to regulate centrosome maturation, bipolar spindle assembly, and mitotic entry, making it a very important protein throughout mitosis. To understand how it functions during mitosis we can use drugs that turn off the activity of Plk1 to mimic what occurs when the protein is malfunctioning. Drugs like BI2536, a small-molecule inhibitor that turns off Plk1 activity in mitotic cells, have provided insight into how Plk1 functions; however, these drugs are not selective enough to inhibit protein activity at distinct locations in the cell. To address this problem, we use a technique called SNAP-tag to localize BI2536 to specific organelles within cells. Using genetic engineering approaches like cloning, virus generation and stable cell production, I helped make cells that express a SNAP protein linked to a PACT domain. PACT localizes to the centrosomes in dividing cells and allows us to bring SNAP protein only to that location. Our modified drug enters cells, binds to SNAP-PACT, and selectively inhibits Plk1 activity at the centrosome of cells. Through experiments I have run, we have shown that we are able to inhibit Plk1 activity in vitro and in vivo. Using immunofluorescences and super-resolution microscopy, I have shown that local targeting of CLP-BI2536 to the centrosomes causes a greater reduction in active Plk1 (p-plk1) than traditional global drug delivery. In the future, I hope to use this approach to investigate the activity of another mitotic protein, Aurora A, in order to further explore the role of mitotic kinases during the cell cycle. 

Past research shows that white and wealthy men are the most prevalent donors to local and state political campaigns, maintaining political influence through ineffectively regulated systems thereby threatening the quality of American democracy. As regulation-based campaign finance reform has been unable to mitigate the influence of large donors in local campaigns, city councils and grassroots organizations have started to present incentive-based policies to provide average Americans with the means to donate, though few have actually implemented them. Budding reforms like that of the Democracy Voucher program in Seattle, though showing promise, demand the attention of researchers to analyze their efficacy. Through comparing both the demographics and the histories of political engagement between donors, program participants, and other voters, this study seeks to determine the initial success of the Democracy Voucher program in Seattle. It concludes that one can observe a minimal increase in donors and more diverse donors, suggesting that the Voucher Program was marginally effective in achieving its goals of diversifying the donor pool. This research contributes to the literature available to policy makers and other researchers interested in developing public campaign financing reforms at the municipal and, potentially, state level.

In Washington State, the goal of the Washington Fish and Wildlife Department’s gray wolf (Canis lupus) conservation and management plan is to not only help wolves naturally re-establish a sustainable population across the state, but also to reduce the conflicts with humans, livestock and other wildlife. A successful implementation of this requires us to understand how the public values wolf management and what factors could shape this kind of value. The purpose of this study is to examine Washington State residences’ willingness to pay for wolf-livestock coexistence projects from a statewide survey including an attribute-based choice-experiment. Willingness to pay shows the maximum amount of money an individual wants to sacrifice to procure a good or service, it can be influenced by personal interests, preferences, incomes and demographic background. In this study, I explore the survey data using random utility maximization (RUM) models to identify both heterogeneity and homogeneity factors that may affect Washington residents’ willingness to pay for wolf-livestock coexistence. I also apply Machine Learning to select covariates that help to generate highly accurate prediction models. An individual’s willingness to pay for wolf-livestock coexistence projects is expected to increase as their income, knowledge about wolf conservation status, and attitudes to wolf conservation increase. The aims of this study are to have a comprehensive understanding of people’s willingness to pay for projects designed to promote human-wolf coexistence and to provide implications for conservation groups and organizations to find alternative funding sources in the future.

Connecting patients to the appropriate health or social service intervention is critical for their long-term health and well being. The 911 system is increasingly being utilized for non-emergency medical situations that originate with non-acute health issues and poor health care access. This diverts resources away from the core mission of fire departments which is to respond to emergencies in a timely manner. This study utilized data from South King County’s 911 dispatch center, Valley Communications, and the American Community Survey to identify socioeconomic factors that may correlate with non-emergent 911 responses. Ultimately, this demographic study identified areas with high primary health needs that are ineffectively addressed and characterized their demographic profiles. Of particular interest are high frequency non-emergent callers that rely on the 911 system for chronic and reoccurring health issues. This information was mapped and related to health care facility locations to determine how health access played a role in this behavior. Researchers and policymakers can expand upon the conclusions of this study to create more targeted, effective, and efficient alternatives to the emergency medical system that can serve regions with the greatest health access and health delivery needs.

Scholars continue to be perplexed by the low rates of Asian American political participation despite their relatively high levels of education and income, two factors which have been shown to be strong predictors of political behavior among whites, African Americans, and Latinos. What is different about Asian Americans that make them largely unaffected by these factors? If their low rates of participation remain unmoved, they risk being systematically neglected from the democratic processes of the government. This study hypothesizes that the deeply rooted Asian cultural values of respect for authority and hierarchy dampen defiance and encourage passivity, therefore causing Asian Americans who are closely tied to their Asian culture to be less politically active. This study will measure political participation through self-reported voting and registration, while using a series of variables to determine levels of engagement with Asian culture. The dataset used from National Asian American Survey captures Asian Americans’ political attitudes and behaviors during the Presidential elections of 2008.

Environmentalism has often been considered a moral commitment. However, this paper investigates the hypothesis that environmentalism is not just a moral commitment, but a highly logical economic commitment. I argue that environmentalism is not contrary to economic motivations: instead, states fund their departments of ecology when they stand to gain economically from doing so. This paper compares the percentage of each state’s budget that went towards funding their ecology department to the tax revenue generated by two types of state resources: the outdoor recreation economy in each state and each state’s proven crude oil, shale gas, and natural gas reserves. When states receive more tax revenue from the outdoor recreation economy, they allocate a larger percentage of their budget to ecology departments. On the other hand, the presence of proven crude oil, shale gas, or natural gas reserves will decrease the amount of funding for state ecology departments because states generally gain more economically from the exploitation of those resources than from the outdoor recreation economy. Broader implications of this paper include the conclusion that the best way to ensure that states will be environmentally conscious is to make it economically advantageous to do so as states act primarily on economic incentives. This study uses data sets from Ballotpedia and American FactFinder and is supplemented with data from the National Conference of State Legislatures and the Outdoor Industry Association.
 

Due to urbanization and city development, more people are losing contact with nature that earlier generations were more likely to have had. Children who grow up in urbanized, and poverty impacted spaces are likely to have less experience of fully immersing themselves in nature. All children should receive equal or similar benefits from their environment. Hence, for those children attending preschools in the urban grid, we can think about ways to create opportunities for nature-based play and exploration in order to bring benefits associated with nature to all children for an optimal childhood development. Playspace is a space for an outdoor learning environment, where children learn to socialize, wonder and grow. Naturalized outdoor environments offer developmental benefits—psychological, social, physical, cognitive, health—for the growing young child. Children will grow stronger immune system, growth and physical advantages through interaction with nature. Children can also experience increased concentration, reduced stress, better social-communication skills and academic performance. It is where children can reclaim their freedom of exploration in the natural world. My research analyzes how children, in the context of an existing naturalized outdoor preschool, use the space for play and learning in a number of areas of development. I apply this study of the outdoor preschool to a more urbanized project site, University of Washington Children’s Center at Portage Bay. I conduct interviews with teachers from both centers and observe children to gather information on what is missing and what can be supplied. Using in particular the study by Robin C. Moore, Nature Play and Learning Spaces, I propose a framework of playspace elements that can convert a more traditional childcare center’s outdoor space into a place where children can receive a “daily minimum dose”—as Moore states—of natural experiences and acquire love and respect for the natural world.

Leigh syndrome is a severe pediatric mitochondrial disease for which no effective treatments currently exist. One of the defining features of Leigh syndrome is the appearance of progressive necrotic lesions in the brain, specifically in the brainstem, cerebellum, and olfactory bulb. In humans, the disease is caused by mutations in Ndufs4, a subunit of the mitochondrial electron transport chain complex I. However, lesion development is not directly linked to impaired mitochondrial function. At the Seattle Children’s Research Institute, I am assisting as an undergraduate researcher in a project aimed at uncovering the pathogenesis of these brain lesions. Homozygous loss of Ndufs4 in mice closely resembles the human disease, making mice a valuable model for study. For my project, I have been longitudinally tracking circulating metabolites in knockout and control mice, measuring bulk tissue metabolite levels via biochemical assays and examining brain slices using immunological and histological staining techniques to analyze glycogen (stored glucose) and lipid localization in lesion areas. Using these techniques, we have identified a developmental shift in circulating metabolites at the age where Leigh syndrome mice first develop disease (around post-natal day 35). Starting at this age, glycogen and lipid droplets accumulate at lesion sites, co-localizing in regions where loss of neuronal marker expression is evident. Because astrocytes (neuron supporting cells,) play a large role in neuron metabolism and survival, we also utilized fluorescent markers to assess the localization of accumulated astrocytes at areas of high lipid concentration. Using 3-D microscopy, we found that the smaller accumulated lipid droplets co-localize with astrocyte processes. Our findings support a role for altered circulating and tissue level metabolism in the pathogenesis of Leigh syndrome. This study is not only helpful in understanding the pathogenesis of Leigh syndrome, but also in linked metabolic and neurodegenerative diseases, leading us closer to effective treatments.

In the past few years, tetanus has caused many cases of the disease, according to the World Health Organization, specifically 34019 newborns died in 2015 from Maternal and Neonatal Tetanus (MNT). Many studies have been done on its transmission pathways and its effect on patients. This project explored research in academic journals and other professional publications with keywords such as “maternal and neonatal tetanus” and “transmission.” We explored existing transmission pathways of tetanus, as well as current recommendations for these specific cases, such as proper wound care regarding umbilical cord cutting and vaccination of pregnant mothers. Such recommendations would be to prevent illness and death, as well as diminishing the risk of infant demise. It was found that tetanus is endemic in certain regions. Its contamination can be prevented through proper wound care, vaccination, and proper umbilical cord practices. This review used the disciplinary lenses of microbiology, clinical medicine, epidemiology, and public health. There is overwhelming evidence pointing to the recommendation to vaccinate in the circumstances of a wound or as a form of prevention. This review will suggest implementation in regards to vaccination and prevention options to accompany current healthcare provider efforts to further assist their endeavors in eliminating the specific presentation of MNT in the remaining 15 countries where it remains a public health threat, such as Mali or Pakistan.

Monitoring bacterial indicators and pathogens in environmental waters via filtration is critical in preventing the spread of water-borne diseases. Despite the many filtration methods described in the literature, few have demonstrated applicability to multiple target agents and are adapted for collection of water samples in rural or remote areas. Recently, a novel system for collection and concentration of samples in remote settings was designed and validated. This bag-mediated filtration system (BMFS), originally developed for environmental surveillance of poliovirus, collects water-based samples in a bag then gravity filters the sample through a capsule filter, which can be easily transported back to the lab. The objective of my project is to adapt and validate the BMFS for capture and concentration of bacterial indicators and pathogens found in environmental waters. To evaluate the adaptability of the BMFS method to bacterial targets, I inoculated a series of 10L of tap water, with either E. coli, enterococcus, or an attenuated vaccine strain of S. typhi (Ty21a). I filtered the water through a ViroCapTM filter, then eluted the filter using 0.1% tween-20. Recovery rates were quantified using qPCR, defined substrate MPN methods, and spread plating techniques. Results were expressed as percentage of the bacterial inoculum recovered in the eluates. My next steps will be to filter various environmental water types obtained near the University of Washington, in order to assess the effect of water quality on BMFS performance. The experimental design will mimic the procedure described above, except we will be innoculating environmental water instead of tap water. It is our hope that the results of this study demonstrate the potential for environmental surveillance of multiple bacterial targets in remote settings where site location, climate, and road conditions make sample-site access challenging.

In some locations (e.g., refugee camps or rural areas in countries like Haiti), fecal waste systems are not water-based, but resemble a pit latrine, for which current sampling and processing methods were not designed. The fecal waste is collected and held, often under poor infrastructure. The fecal sludge management process is important to consider for environmental surveillance, as this may be the only source for screening. This surveillance is important for the continued eradication of poliovirus (PV). The goal of my research was to develop a method for PV extraction from sludge in pit latrines for use in locations with concentrated fecal waste. Towards this goal, I modified two existing protocols for extraction of various viruses from different solid media and applied them to evaluate virus recovery from primary sludge. The first (M1) was originally designed to recover hepatitis A from oysters, while the second (M2) was originally developed to extract somatic coliphages from sewage sludge. The modified methods were evaluated using primary sludge collected from a wastewater treatment plant in Seattle and spiked with the vaccine strain of poliovirus type 1 (PV1). Virus recovery was assessed by a plaque assay. M1 involved acid adsorption followed by glycine elution, threonine elution, skimmed milk flocculation, vertrel and threonine extractions. For M2, liquid and solid fractions were initially separated by centrifugation. Processing of the solid fraction involved beef extract elution, sonication, skimmed milk flocculation, and vertrel extraction, while the liquid fraction was concentrated using membrane filtration and beef extract elution. The preliminary data indicate that M1 may have a higher recovery, and therefore be the preferred method for use during environmental surveillance of poliovirus. Future work should include additional replicates and optimization of the preferred method. 

Serotonin (5-hydroxytryptamine, or 5-HT) signaling plays a key role in regulating a range of behaviors related to reward learning, motivation, and addiction. 5-HT6 receptors in particular are notable for their localization to primary cilia in neurons, and abundant expression in the striatum, a brain region critical for its role in mediating reward seeking behavior. In addition, 5-HT6 receptors signal through the cilia-specific type 3 adenylyl cyclase (AC3). Previous overexpression studies in my lab show that 5-HT6 receptors in the nucleus accumbens (NAc) modulate cocaine taking in a pathway-specific manner. To further characterize the role of endogenous 5-HT6 receptor signaling in cocaine taking, we are knocking out 5-HT6 receptor or AC3 expression in vivo using a pathway-specific CRISPR-Cas9 approach. We are injecting Cre-dependent AAV CRISPR-Cas9 into the NAc to target specific pathways, and we are validating successful knockout using immunohistochemistry (IHC). We are using an intersectional surgery approach, injecting CRISPR-Cas9 into the NAc, and Cre into either the VTA (direct pathway) or the Ventral Pallidum (indirect pathway) therefore only neurons with projections to both regions will express the CRISPR-Cas 9 enzyme. IHC staining is carried out on brain slices to confirm successful knockout of 5-HT6 or AC3 by staining for Cas9 and either AC3 or 5-HT6. The presence of Cas 9 positive neurons that lack 5-HT6 or AC3 staining will confirm the approach. We effectively stained for AC3 in rat brain slices, allowing us to validate AC3 knockout. Explicit staining of 5-HT6 and AC3 is crucial for continuing the study of 5-HT6 receptors signaling and their effects on cocaine reinforcements. This project aims to provide insight into the endogenous function of 5-HT6 receptor signaling in drug-seeking behavior.