Stress-related disorders impact over 18% of the United States population. Further definition of the mechanisms behind stress responses in the brain is necessary for understanding and treating stress related disorders, such as anxiety and depression. Specifically, the lateral habenula (LHb) is a brain region known to modulate stress and anxiety responses, but the ways in which it modulates these responses is relatively unknown. In order to investigate potential mechanisms for this modulation, we examined differential gene expression in the pathways from the LHb to three brain regions: the ventral tegmental area (VTA), the dorsal raphe nucleus (DRN) and rostromedial tegmental nucleus (RMTg). Rats were stressed using a forced swim protocol and tissue from the LHb was homogenized in order to extract RNA using RiboTag. Using a combination of transgenic mice and a RiboTag viral vector we were able to extract highly specific RNA from complext brain tissue. We used a portion of our collected RNA to perform RNAseq, a molecular tool used to identify differential gene expression in certain pathways. Using the results from our RNAseq analysis, we used extracted RNA to perform quantitative PCR to verify the pathway specific genes found in the LHb. By determining the pathway specific patterns of differential gene expression, we propose a molecular mechanism for neuronal response to stress in the LHb.

Methane reservoirs are commonly found throughout the world’s oceans and the release of methane from seafloor reservoirs is thought to make up 5 to 10% of the global atmospheric methane. In fact, the greatest deep-sea mass extinction in the last 97 Myr during the Paleocene-Eocene Thermal Maximum (PETM) may have been caused by methane release from seep sites along the upper continental slope margin. Recently, methane reservoirs along this margin have been gaining attention due to their potential to accelerate current global warming. Changes in seafloor pressure and temperature could destabilize these seafloor deposits and cause methane bubble plume release into the ocean. At SHR, an extensively studied active seep site located ~ 90 km offshore Oregon, discontinuity in methane plume release was observed, but still not well understood. Hence, using Acoustic Doppler Current Profiler (ADCP) and pressure data archived by the Ocean Observatories Initiative (OOI) Cabled Array, we are investigating the potential correlation between tides and the presence of methane plume at SHR. Our study detects methane plume structures based on the proxies of echo contrast caused by acoustic-bubble interaction. By analyzing the derived plume structures and their correlation with 226 tidal cycles, we expect a trend of plume release triggered by low tides. Our study provides the first high-temporal-resolution analysis on the methane plume release at SHR using OOI acoustic data. 

Rats produce ultrasonic vocalizations (USVs) in a range from 20-kHz to 95-kHz that vary in frequency and shape across social and motivational contexts and can correspond to the affective state of the animal. To assess these USVs accurately and efficiently, our lab created DeepSqueak, a novel machine learning software package that expedites the detection and analysis of rat USVs by using neural networks to differentiate them from noise. DeepSqueak also allows for automatic and unbiased classification of USVs into discrete categories using call parameters such as shape, frequency and duration. Prior to this unbiased categorization method, identified 14 subjective categories in 50-kHz rat vocalizations that could be manually identified by a trained experimenter. These categories have received some limited study, but the excessive labor and time needed for manual classification restricted broad adoption. We aim to use neural networks to quickly and automatically classify USVs into these categories to promote broad adoption and better our understanding of the relationship between USVs and behavior. The process of training our neural network to differentiate between vocalizations was approached in two ways. Audio files were converted to sonograms through DeepSqueak and manually labeled. Thousands of these labeled calls were then inputted as training data for the neural network. This method allowed the network to learn using a large set of labeled vocalization data. The second method is based around the manual selection of an optimal call for each subtype using DeepSqueak's "call clusters" function; the neural network was then trained around how closely vocalizations matched the optimal calls. We now plan to compare DeepSqueak's automated calls and clustering to manual scoring in order to develop the best possible system that reliably categorizes USVs, thus allowing for more specific analyses of USV categories and behavior.

During the Last Glacial Maximum (LGM), the Cordilleran Ice Sheet advanced into the northwestern United States and dammed the Clark Fork river in western Montana, forming Glacial Lake Missoula. The ice dam in the valley repeatedly broke, sending at least 100 massive outburst floods known as the Missoula Floods into eastern Washington where they incised deep canyons forming the Channeled Scabland. During the period of flooding, the Okanogan Lobe of the Cordilleran Ice Sheet crossed the Columbia Gorge onto the Waterville Plateau, diverting floodwaters away from the Columbia River into Grand Coulee, before blocking Grand Coulee itself. My research project examines the timing of glaciation of the Waterville Plateau and how it influenced the paths of the successive floods. To determine the timing of glaciation, my advisers and I studied glacially transported boulders that began to accumulate the cosmic ray produced isotope beryllium-10 (10Be) after they were exposed by ice sheet retreat. In the lab, we separated and dissolved quartz from our samples, purified beryllium, and then measured 10Be/⁹Be ratios using Accelerator Mass Spectrometry (AMS). From AMS measurements, we calculated the exposure ages of our samples. We expect the ages to fit within the established sequence of ice advance across the Columbia River, redirection of floodwaters down Grand Coulee, and eventual ice retreat to north of the Columbia River. Following this sequence, we expect our samples from the northern edge of the Waterville Plateau to be younger than the last floods down Grand Coulee (15,300 years) but older than samples from the Omak Plateau north of the Columbia Gorge (13,900 years). Along with dates from previous studies, the new exposure dates from this project explain how the Okanogan Lobe guided Missoula floodwaters and influenced landscape evolution in eastern Washington.

Mitosis is an essential cellular process in which a cell divides to produce two genetically identical daughter cells. When this process becomes dysregulated cells divide uncontrollably leading to diseases such as cancer. Polo-like kinase 1 (Plk1) is a key enzyme that is necessary for coordinating numerous events during mitosis. When Plk1 becomes dysregulated or mislocalized, mitotic spindle assembly, protein organization, and mitotic timing impairments may occur. One of the many subcellular locations where Plk1 carries out essential mitotic functions is the kinetochore. The kinetochore is the interface between the chromosomes and the mitotic spindle and is critical for ensuring proper DNA to microtubule attachments early on in mitosis. Historically, the small-molecule inhibitor drug BI2536 has been used to inhibit the activity of Plk1 in order to study its role in regulating various mitotic processes. However, traditional inhibitor drugs turn off entire protein kinase populations, inhibiting the activity of Plk1 all throughout the cell, not just at the kinetochore. This can lead to unwanted side effects and limits our understanding of Plk1’s role at specific subcellular locations. To improve the specificity of BI2536 drug delivery, we utilized SNAP-tag, a tool in which a self-labeling enzyme can irreversibly react with substrates linked to a chloropyrimidine (CLP) functional group. By genetically manipulating human bone cancer (U2OS) cells, we expressed a kinetochore localizing SNAP. We also generated a BI2536 conjugated to a CLP group. By treating our genetically modified cells with CLP-BI2536, we can target Plk1 inhibiting drug to the kinetochore to study Plk1’s role at this specific location. Using super-resolution structured illumination microscopy (SIM), we demonstrate that we can effectively target fluorescently labeled CLP substrates to kinetochores in our cell line. In future work, we will target our CLP-BI2536 drugs to the kinetochore and investigate how local Plk1 inhibition affects mitotic timing.

Field research is an important way to understand complex ecosystems and the roles of individual wildlife species within these systems. As climate change and other human factors continue to affect our global patterns, studies must be conducted to assess the health of current populations and predict future trends. Many studies involve direct or indirect contact with the focal animals, which may impact the fitness of study animals despite efforts of researchers to minimize cost. Long-tailed Jaegers (Stercorarius longicaudus) in Denali National Park, Alaska were captured and fitted with satellite-linked GPS trackers to study migratory routes. The birds were tagged by other researchers while on their summer nesting grounds caring for eggs or recently hatched chicks. This disturbance has potential to affect the nesting success of the jaeger which is thought to already be in decline in the area. A long-term study of the nesting success of jaegers and other shore birds in DNP is being conducted to assess the suggested decline in these species. What impact did radio tagging nesting jaegers have on nesting behavior and success? I collected behavioral data in the field during a four-week period in July of 2018. With a UW grad student, I directly observed 2 breeding pairs of jaegers that had been tagged and 3 that had not. We observed the birds from over 150 meters away, recording parental feedings, chick protection, and vigilance. Preliminary analysis suggests that the two subsets of jaeger behaved differently during chick fledging. This could have impacted the way chicks were raised and the nesting success rate of tagged parents. Further analysis into the feeding rates and protective behavior is underway to determine the complete impact of radio tags on the population.

As of now, mRNA vaccines have been deemed as a potent replacement for current vaccine models against infectious diseases for their improvements in B-cell and T-cell immune responses. Usually, when soluble, subunit antigens are delivered, they are scattered and randomly bind to B-cell receptors, often loosely. However, with a nanoparticle carrier for antigens, there would be more effective crosslinking with B-cell surface immunoglobins as there is a higher density of structurally ordered antigen arrays presented by the nanoparticle. As a result, the B-cell creates a stronger immune response. Additionally, the multivalent particles also favors the creation of long-lasting immunity against a given virus. My team and I are currently developing a self-assembling protein platform using dn5A and dn5B protein components as a carrier for an mRNA vaccine against the flu. My project mainly focused on optimizing the co-secretion of the two particles by exploring different models and combinations of both. This is important as the translated cage not only has to be able to self-assemble but also be capable of doing so without producing excess protein in order achieve its purpose. To do so, I investigated 12 different constructs of dn5A and dn5B through transfections and analysis with western blots and electron microscopy. We used the data collected to improve the dn5A/dn5b protein platform utilized alongside flu mRNA vaccines, helping them better achieve potency. Overall, if effective, the new vaccination model can be utilized for other infectious diseases, including HIV and meningococcus.

Theiler's murine encephalomyelitis virus (TMEV) is a positive-sense RNA Picornavirus used to model epilepsy, poliomyelitis and multiple sclerosis. Previous research has demonstrated a dependence of some Picornaviruses on host glutathione (GSH) in order to produce viable progeny. GSH depletion in host cells has resulted in unstable viral capsids, reducing viral fitness due to a lack of oxidative homeostasis. The objective of this study was to investigate the dependence of TMEV on GSH and determine the consequences of oxidative stress in the host cell due to the absence of GSH. Incubation of TMEV at denaturing temperatures with increasing doses of reduced GSH demonstrated that GSH stabilizes viral particles and 54% viral infectivity was retained with doses as low as 25mM GSH. Using L-Buthionine-sulfoximine (BSO) to deplete GSH in host cells, a 3-fold decrease in viral production was observed when compared with untreated cells. Interestingly, when GSH was restored in BSO treated cells using cell permeable GSH ethyl ester, TMEV viral production was partially restored and viral plaque formation was comparable in yield to an untreated viral infection. Virus production and viability were quantified using plaque assays on host cell monolayers. Next steps include monitoring viral genome production via RT-qPCR and quantifying GSH levels using fluorescence plate assays. RT-qPCR will be further utilized to examine other host stress pathways. This project will use TMEV to look at a host-virus relationship as it relates to an important host antioxidant system interacting with the capsid, assembly, and genome replication of the virus life cycle.

For many years, concerted efforts to combat malaria through the use of antimalarial drugs, bed nets, and other public health measures led to marked reductions in morbidity and mortality. Unfortunately, progress has stalled. Reductions in malaria have leveled off and even reversed in certain areas (WHO, 2017). As of 2016, there were 216 million cases and 445,000 deaths annually due to Plasmodium infections (WHO, 2017). To regain momentum and accelerate malaria eradication efforts, an effective and durable vaccine is needed. The Murphy Laboratory focuses on developing novel pre-erythrocytic (PE) malaria vaccines that can effectively stop the Plasmodium sporozoite (spz) before the clinically symptomatic blood stage begins. Identification and inclusion of multiple different protective Plasmodium antigens is thought to be crucial to developing a broad immune response and durable protection against this intracellular parasite. To test and define protective antigens, the Laboratory developed an “Acute Challenge” (AC) model in order to sensitively measure T-cell responses that are completely or partially protective. In this model, DNA vaccines encoding Plasmodium yoelii proteins are delivered by gene gun to induce CD8+ T-cell responses in BALB/c mice. At the peak of the immune response, we challenge the mice with luciferase-expressing P. yoelli sporozoites and measure the parasite burden and protection using IVIS imaging. A known protective epitope derived from P. yoelii circumsporozoite protein (CSP) induces a potent and protective response in this system. My project is to utilize the AC model to assess P. yoelli candidate antigens, of unknown protective potential, that are putatively exported or secreted from the parasite-containing vacuole into the host cell cytoplasm. Confirmed protective antigens will then be assessed for their localization and defined T-cell epitopes. The results will be used to create vaccines designed to maximize such responses and target the responding T-cells to the liver.

Periodic orbits provide an underlying structure to chaotic attractors, which provides order and low-dimensionality to complex, chaotic systems. The trajectory of chaotic systems proceeds to infinity for an arbitrary initial condition; for this reason, periodic orbits are useful as a finite set of finite mathematical objects to characterize the infinite, complex behavior of a chaotic system. In particular, the mathematical framework of chaos, chaotic attractors, and unstable periodic orbits allow for a new understanding of self-organization in complex physical systems like turbulence. In this study periodic orbits as an organizing principle will be examined in the Lorenz system and the double pendulum system. This study will find the unstable, periodic orbits of the Lorenz system and the double pendulum system using Newton’s method. The expected results of the study are that periodic orbits of both systems will both be found. This study presents an opportunity for an understanding of chaotic behavior which will lead to engagement with the current research on turbulence.

Neurodegenerative diseases, such as Alzheimer’s (AD), Parkinson's, and Huntington’s, affect millions of people. In AD, prior studies indicate the formation and accumulation of amyloid-beta proteins may play a crucial role in the pathology of the disease. The Herpes Simplex Virus (HSV-1) encodes an alkaline nuclease (UL12.5) known to cause degradation of the mitochondrial genome. HSV-1 infection has been previously associated with AD brain pathology. We hypothesize that UL12.5 activity in the brain may predispose an individual to amyloid-beta aggregation and AD neuropathology. Here, we controlledl the amyloid-beta protein aggregation using a degron attached UL12.5, which is induced by the plant hormone auxin through a molecular signaling pathway known as auxin-inducible degron. We have engineered an auxin UL12.5-degron construct in order to precisely control the temporal and cell type expression of UL12.5 in Caenorhabditis elegans (C.elegans). This construct was microinjected into the worms and by using auxin, we controleld the expression of UL12.5 and tested its effects on amyloid-beta and Huntington protein aggregation. Here, we have elucidated the relationship between HSV-1 infection, UL12.5 expression, and neurodegenerative disease which may form the basis of novel treatments.

Thermoregulation, the maintenance of core body temperature in a constantly changing enviroment, is a critical aspect of homeostasis. Despite its importance, the neural mechanism by which thermoregulatory processes occur is not very well understood at the circuit level. Afferent skin temperature information travels through the spinal cord to the parabrachial nucleus (PBN), where it passes on to the preoptic area of the hypothalamus (POA). A subset of prodynorphin (Pdyn)-expressing neurons in the PBN (PdynPBN neurons) are activated when mice are exposed to warm environments, and 80% of these neurons project to the POA. The exact role of PdynPBN neurons has not been characterized, however, and their full projection profile is not established. Using genetic and viral techniques, we inserted a Cre-dependent designer receptor exclusively activated by designer drugs (DREADD) into mouse PdynPBN neurons and labeled their synaptic projections with GFP-bound synaptophysin, an abundant synaptic vesicle protein used for neurotransmitter trafficking. The use of Cre-dependent DREADD and synaptophysin-GFP allowed us to specifically label and activate PdynPBN neurons. We found that activation of these cells increases tail-skin temperature with a concurrent drop in core-body temperature. These data suggest that PdynPBN neurons may convey environmental temperature information that is sufficient to activate heat-defense responses. Establishing the genetic identity of neurons in a circuit that helps to maintain constant core body temperature will allow for the elucidation of downstream nodes in this circuit.

Hair cells are sensory receptors responsible for hearing and balance in the auditory and vestibular systems. These cells are surrounded by supporting cells, which are non-sensory cells similar to glia. In non-mammals, hair cells get replaced after damage through conversion of supporting cells. Recent studies have shown supporting cells can also transdifferentiate into type ll hair cells in the vestibular system of adult mice. The signals that control hair cell regeneration in mammals are poorly understood. It is known that the transcription factor Sox2 is utilized for specification of hair cells in the neurosensory domain of the otocyst. This project tests the premise that Sox2 is required in adult supporting cells for their transdifferentiation into type II hair cells after damage. We used time specific gene deletion technology, CreER-LoxP, to delete the gene encoding Sox2 in supporting cells prior to damage. The mice used include the following alleles (genetic segments): 1) the floxed Sox2, or Sox2loxP/loxP allele, which enables Sox2 deletion when Cre is active; 2) the Sox9CreERT2 allele, which targets Cre expression to supporting cells; Sox9CreERT2 is tamoxifen-dependent, so the timing of Sox2 deletion is controlled by tamoxifen injection; and 3) the Pou4f3DTR allele , which enables hair cell killing upon injection of diphtheria toxin (DT). Hair cells were identified and “typed” using several markers, including antibodies to myosin VIIa, neurofilament, and ßIII tubulin. I compared the number of regenerated hair cells of Sox2 expressing supporting cells and Sox2 negative supporting cells. Sox2 deletion in supporting cells resulted in a significantly reduced number of type ll regenerated hair cells. This result provides evidence that Sox2 is required for transdifferentiation of supporting cells. Understanding the function of Sox2 will give insight into potential downstream molecules that can become prospective targets for future research in understanding cellular regeneration in adult mice.

There has been growing concern regarding rising carbon dioxide concentration in the atmosphere because CO₂ traps excessive heat and warms the planet through the greenhouse effect. One strategy to mitigate this problem is through the capture and conversion of CO₂ via hydrogenation to fuels and chemical feedstocks that are currently produced from fossil fuels. Given the complexity of these multi-electron, multi-proton transformations and the multitude of products that can result, catalysts are required to lower the energy barrier and direct the selectivity of CO₂ conversion reactions. One catalyst currently under development is a Ru(II) bis-(protic N-heterocyclic carbene) phosphine catalyst, which incorporates protic N-H wingtips adjacent to the metal center. The N-H wingtips are an interesting feature due to their ability to activate CO₂ through metal-ligand cooperation, their accessibility as a proton source near a metal active center, and the likelihood that they aid in splitting H₂ between the metal center and the nitrogen. Preliminary results have shown moderate turnover numbers (TONs) for both formate (130) and methanol (7), the latter of which is a rare transformation in a single catalyst system. In order to improve catalytic TONs and understand the role of protic N-H wingtips, a library of catalysts with varying ancillary ligands, including 2,2’-bipyridine, 4,4’-dimethoxy-2,2’-bipyridine (electron donating), 4,4’-dibromo-2,2’-bipyridine (electron withdrawing), and 1,2 bis(diphenylphosphino)ethane (sterics), is synthesized and screened under high pressure and temperature conditions using THF solvent and varying additives (e.g. Li₃PO, K₃PO₄, KPF₆) and additive concentration. It is expected that the ancillary ligands, bound trans to the bis-carbenes, will influence the proton donor ability of the N-H wingtips and catalytic turnover.