Long-toed salamanders (Ambystoma macrodactylum) are common amphibians throughout the Pacific Northwest, making them an ideal species to use for restoration assessment. A successful wetland restoration project would be expected to have a healthy long-toed salamander population, but it was not previously known whether long-toed salamanders were present in the Union Bay Natural Area (UBNA), a Seattle park with many ongoing restoration efforts. The Amphibian Corridor restoration project in UBNA, installed in 2015, aimed to provide amphibian migration habitat. To assess its efficacy, I examine long-toed salamander movements and woody debris microhabitat preferences within and around the corridor. The study also acts as an inventory of amphibians present in the corridor and throughout UBNA. The corridor and surrounding areas are being monitored throughout the 2021 breeding season (January through May) for adults, larvae, and egg masses. To find larvae and egg masses, dip net aquatic surveys are conducted once per week, alternating between day or night surveys. To find adults, visual encounter terrestrial surveys are conducted once per week, alternating between day or night surveys. Individual salamanders are photographed and identified by their unique spot patterns so that individual movement can be tracked using a mark-recapture method. The location and microhabitat of all amphibians are recorded . My surveys have shown that long-toed salamanders use the corridor. To date, 18 individuals have been recorded in the corridor with up to 10 recaptures per individual, and 11 individuals have been recorded in other parts of UBNA with up to 8 recaptures per individual. Three egg masses have also been found in UBNA. UBNA management and future restoration could be informed by this study’s insights about long-toed salamander microhabitat preference and movement strategies. Given this information, similar restoration projects to the Amphibian Corridor in other urban parks may be effective for amphibian conservation.

Microglia, the resident immune cells of the brain, become activated and mediate neuroinflammatory responses in response to traumatic brain injury (TBI). This neuroinflammation can be detrimental to the health of the brain; thus, inhibition of this natural response can benefit TBI patients. Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a technological advancement that allow manipulation of specific cellular signaling pathways. The hM4D DREADD gene was inserted in a viral construct downstream of the CD68 promoter, which is markedly upregulated in activated microglia following TBI. This construct is activated with clozapine n-oxide (CNO) to downregulate secondary messengers in activated microglia and thus attenuates the inflammatory response by reducing microglial activation and proliferation after injury. After viral transfection to deliver the construct to cells, rats underwent a controlled cortical impact (CCI) - an experimental model of TBI - and were treated with a CNO injection at varied intervals after injury. After the rat brains were dissected and sectioned, we used immunohistochemistry techniques to label microglia with an anti-Iba1 antibody, proliferating cells with an anti-BrdU antibody, and cell nuclei with DAPI. This allowed for the visualization of microglia using fluorescence microscopy and microglia were quantified using stereological principles. The aim of this research project is to use pharmacological DREADD receptor-mediated inhibition of microglia at different intervals post-injury to quantify the proliferation of microglia following CCI. We hypothesize that increased duration between CCI and CNO injection leads to more pronounced microglial activation represented by increased numbers and morphological changes. We also hypothesize that microglial activation can be observed as a gradient with the greatest proliferation closest to the CCI epicenter.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a brief ischemic event confers neuronal and axonal protection against subsequent ischemic exposure. The cell types responsible for IPC in the brain are unknown. In a novel model of white matter (WM) IPC and ischemic injury, we identified specific innate immune signaling pathways in microglia as required for IPC-mediated axonal protection, leading us to suspect that microglia are required for IPC. The model of WM IPC involves exposing the mouse optic nerve (MON) to a brief ischemic event 72 hours before the MONs are isolated and exposed to oxygen-glucose deprivation. Animals were treated with PLX5622 - a colony stimulating factor-1 receptor (CSF1R) pharmacologic antagonist – to deplete microglia in the central nervous system, including the MON, to test our hypothesis. By recording axonal function, we determined that microglial depletion eliminated IPC-mediated axonal protection in the WM. This study examines the impacts of IPC and ischemia on the nodes of Ranvier, which we hypothesize are protected by preconditioned microglia. The length of the nodes of Ranvier affects conductance and action potential propagation through an axon, with recent publications suggesting elongation of the notes in disease or injury states. We hypothesize the nodes of Ranvier will be shorter in preconditioned MONs than non-preconditioned MONs, which would support IPC-mediated protection. Additionally, we expect the nodes of Ranvier in PLX5622-treated MONS will be of similar length as non-preconditioned control MONs, indicating loss of IPC-mediated protection in animals without microglia. We will use immunofluorescence and confocal microscopy to measure the nodes of Ranvier and correlate these anatomical findings to prior electrophysiology experiments. The results of this research would contextualize a novel understanding of how microglia and ischemia affect the WM in specific regions of the axon, which is vital for advancing the development of neurotherapeutics for stroke.

Periodontal disease stands to be the leading cause of tooth loss in older adults and is characterized by inflammation of tissues supporting the teeth and periodontal bone loss. This oral disease also occurs during normative aging in mice models. Thus, targeting the biological aging process may provide an innovative approach to reduce the impact of this disease in the elderly population. Rapamycin is a specific mTOR inhibitor shown to delay age-related decline and significantly extend the lifespan of mice models. Our lab has demonstrated that an 8 week treatment with rapamycin in aged animals reversed age-related periodontal disease. However, whether this improvement persists even after the treatment ends is still unknown. To determine whether reversal of periodontal disease persists even after stopping rapamycin treatment, cohorts of aged mice were either started with 8 week treatment with control (eudragit) food or rapamycin (42ppm) food, and then switched to either rapamycin (42ppm) food or control (eudragit) food for another 8 weeks, respectively. High resolution microCT imaging was completed to measure the amount of periodontal bone, and western blot was performed on total alveolar bone extracts and probed for the osteoclast marker RANKL. Our microCT analysis showed there was less periodontal bone loss in aged mice treated with rapamycin, and this result persisted even after 8 weeks on the control diet. Additionally, the age-related increase in RANKL expression was decreased in both treatment groups. In conclusion, the regain of lost periodontal bone in aged mice persisted even after cessation of rapamycin treatment, and the age-related increase in the osteoclast marker RANKL was decreased after rapamycin treatment and such decrease persisted. Overall, our studies indicate that short term treatment with rapamycin is sufficient to rejuvenate oral health and the benefits are maintained even after stopping the treatment.

Antiretroviral therapy (ART) and pre-exposure prophylaxis (PrEP) can treat and prevent Human Immunodeficiency Virus (HIV), respectively. However, good medication adherence (≥4 doses/week) is crucial for these treatments to work effectively. Our research group recently developed the REverse TranscrIptase Chain Termination (RESTRICT) assay, a rapid enzymatic assay that measures the amount of tenofovir diphosphate (TFV-DP) present in blood, a drug that is a good indicator of long-term (1-3 month) ART/PrEP adherence. The goal of this project is to modify RESTRICT to additionally measure the amount of emtricitabine triphosphate (FTC-TP) in blood, which serves as a good indicator of short-term (1 week) adherence. RESTRICT provides the HIV reverse transcriptase (HIV RT) enzyme all the required reagents for synthesizing double-stranded DNA (dsDNA) and measures the concentration of TFV-DP and FTC-TP based on their inhibition of HIV RT activity. We designed custom DNA templates that bind preferentially to either TFV-DP or FTC-TP based on the nucleotides that they mimic. We also designed molecular beacon probes with different fluorescence dyes that bind to each DNA template and can provide fluorescence output corresponding to the amount of complementary DNA (cDNA) synthesized by HIV RT. High drug levels will lead to less cDNA synthesis and lower fluorescence intensities, indicating good adherence to treatment. Preliminary results indicate that molecular beacon probes can distinguish between the cDNA associated with each drug, which would allow for the simultaneous detection of TFV-DP and FTC-TP in a single reaction tube through the fluorescence measurements of the two types of dyes. Ongoing work is focused on optimizing RESTRICT reactions to measure clinically relevant concentrations of TFV-DP and FTC-TP. Gaining information about long- and short-term adherence to treatment through the detection of TFV-DP and FTC-TP respectively, would allow for more informed interventions by healthcare professionals to help patients improve adherence, leading to better health outcomes.

The influence of traditional economic thinking and theory on the development of international commerce and trade policy informs many of the contemporary issues and challenges impacting the global economy today. My research paper addresses the economic and social ramifications of global free trade practices and analyzes an alternative paradigm designed to affect free trade market behavior known as fair trade. Whereas free trade is characterized by the importing and exporting of goods between countries without tariffs or restrictions, fair trade is identifiable based on practices and principles that promote empowerment and equity. My analysis posits fair trade as a policy prescription for some of the deleterious effects of free trade. To compare traditional market-driven approaches to global trade with the contemporary movement of fair trade, my analysis is guided by a series of prompts: What is fair trade’s role in the international political economy sphere and how does it differ from (and complement) free trade? How can the precepts and practices of fair trade counterbalance the inequities of free trade policies? Scholarly resources, including peer-reviewed academic journals, were utilized to develop perspectives and answer these questions. Fair trade practices offer a sustainable improvement for the human condition since they are driven by more meaningful indicators of welfare than just Gross Domestic Product (GDP). Fair trade policies identify and value the needs and well-being of growers, workers, and communities along with the priorities of the developing nation trading partner; free trade focuses mainly on aggregate components that promote efficiency and create market value for the global economy. Social and environmental improvements from fair trade may not be adequately quantified in GDP since they can occur outside of traditional, measurable market-based activity. Therefore, this deficiency could motivate the development of an alternative to GDP to more accurately measure the non-pecuniary welfare effects from global trade.