Engineering metabolic pathways in bacteria requires regulation of cellular biology with predictable genetic control devices. Characterizing these controls is key to optimizing biochemical pathways for the sustainable production of high value chemicals. In prokaryotes, small RNA (sRNA) downregulate gene expression by antisense binding to messenger RNA (mRNA) transcripts. The relative ease of re-engineering sRNA targets by replacing its antisense targeting sequence makes sRNA an ideal candidate for implementing post-transcriptional controls in metabolic engineering. In previous work, we incorporated sRNA into metabolite-responsive RNA controllers, but in many cases did not observe the hypothesized trends in mRNA repression. However, we observed the lack of secondary structure within the antisense targeting sequence correlated with higher mRNA repression. To understand these effects and refine the functionality of our constructs, we built a range of new sRNA devices with additional sequence at the 5’ end. We targeted the sRNA to repress fluorescent protein expression, and used functional assays to measure fluorescence across a range of sRNA induction levels. We hypothesized that sRNA carrying a 5’ hairpin will result in greater repression than sRNA lacking structure at the 5’ end, and that base-pairing interactions between the 5’ sequence and the targeting sequence will lead to lower repression. This work aids in the development of rational design principles for using sRNA in RNA-based controls. By understanding the effects of sRNA structure, we will be better able to expand the post-transcriptional controls available to metabolic engineers. Ultimately, this extends our understanding of cellular biochemistry, while providing novel means by which to optimize engineered biochemical pathways.

Current synthesis for thermoresponsive magnetic nanoparticles such as iron oxide nanoparticles coated with poly(N-isopropylacrylamide), pNIPAm, usually requires extensive surface functionalization or high temperature procedure, and the yield is very limited. Here we present a simple and reproducible approach for the synthesis of thermosensitive iron oxide nanoparticles, via coprecipitation of Fe2+ and Fe3+ ions from basic aqueous solutions at room temperature in the presence of block copolymer, poly(N-isopropylacrylamide)-block-poly(acrylic acid) with a dodecyl tail following the acrylic acid block. The polymer facilitates iron oxide nanoparticle synthesis by forming micelles via hydrophobic dodecyl tail assembly and coordinating iron oxide formation via complexation with the poly(acrylic acid) segment. The block copolymers were synthesized through reversible addition-fragmentation chain transfer polymerization. Proton NMR characterization shows incorporation of 11 acrylic acid monomeric units to pNIPAm macro chain transfer reagent with 150 degree of polymerization. The new synthesis approach is rapid, taking one hour, and can be easily purified via size exclusion chromatography. The resulting magnetic nanoparticles’ surfaces display pNIPAm, which enables particle aggregation when the solution temperature is above 34°C. The particle aggregates exhibit high magnetophoretic mobility, which results in rapid and efficient magnetic separation, ≥95% in few minutes. The particle’s hydrodynamic diameter is ca. 30 nm, characterized using dynamic light scattering. This simple method overcomes the problem of using high temperatures while maintaining reproducibility, which could make it useful for larger scale synthesis.

The majority of the population living with HIV resides in African countries where the cost-effectiveness and ease of implementation of the technology is imperative. Our goal is to develop an assay that eliminates the need for expensive nucleic acid sequencing technology, to test and diagnose drug resistance by detecting variation in nucleic acid sequence of the virus. One of the missing pieces that will allow us to reach this overarching goal is to design a method for isolating the viral particles in a manner appropriate for point-of-care application. Our approach, which will contribute to the assay being developed at the same time, is to use protein G magnetic beads, which are affinity molecules, to isolate the HIV virus from plasma sample. The protein G on the magnetic beads interacts with the coat antibodies on the viral particles and facilitate their removal from plasma. We show that the protein G magnetic beads have comparable efficiency at isolating the viral particles compared to the gold standard, Qiagen extraction, without the use of strong chemical reagents and complicated instruments. Yet, further work is needed to demonstrate that the viral RNA can be extracted starting from clinical plasma specimens with a protocol applicable in point-of-care settings defined by limited use of complicated insturments and cost effectiveness of the solution. 

The Burmese political system has entered a period of gradual democratization since the rise of the pro-Western National League for Democracy (NLD) in 1988 against the longstanding military regime. Myanmar/Burma is progressing insofar as democratic ideals are theoretically implemented; however, these provisions realistically function primarily to position the state more favorably in the international arena. This project describes the evolution of foreign diplomacy and the relationship between economic liberalization and the most recent period of democratization. To approach this subject, I focus on the legacy of British colonialism as establishing weak public institutions that promoted political disorder and exacerbated domestic conflict with the politicization of race. I look into the shift of public discourse from this hopeful unification to a regime that adopted an instutional reinforcement of colonialist racial hierarchies through militarization. My research seeks to answer why Myanmar/Burma has oscillated between democracy and authoritarianism historically based on the effects of economic growth and the potential for economic crises related to the globally interconnected economy. I emphasize the contrasting ideas between foreign and domestic actors to explicitly contextualize the ways in which political order is developing with the emergence of democratic ideals. The focus is on building state capacity since independence by using the analytic tools of modern political economy as informed by the growing importance of foreign influence on the integration into a global economy. I use the internationalization of Burma's politics to analyze the impact of foreign political support on acting as a stabilizer of domestic conflict. I conclude that the implications of foreign values taking root in Myanmar/Burma undermine domestic growth by adopting the faulty assumption that democracy is both necessary and sufficient for producing positive social and economic policies.

This project will inform a systems biology platform for identifying adverse responses to environmental compounds on testicular development in mice, a commonly used model system in the field of Toxicology. In order to validate the organotypic culture system, we have investigated the adverse effects of the known testicular toxicant, cadmium. To initiate the culture model, we isolate and plate testicular cells from immature mice on postnatal day (PND) 9 in order to capture a developmental window of susceptibility. Based on a literature search derived developmental timeline, we chose to treat the culture on days in vitro (DIV) 2, 5 and 15 with cadmium and quantify effects 24 hours later. We hypothesized that dosing with cadmium during these time points would correspond to impacts on steroid regulation, proliferation, and spermatogenesis processes. In order to evaluate a dose response relationship in our testicular culture, we treated the testis cells with 2.5, 5.0 and 10 uM concentrations of cadmium. To quantify the effects of cadmium in our cell culture model we evaluated; total protein, testosterone production, cell type specific proteins with western blots, LDH cytotoxicity, 3 color assay for live and dead cells and immunofluorescence to capture morphology for DIV 3,7 and 16, 24 hours post treatment for all three doses. Our initial studies have observed that cadmium treatment for all three doses impacted testosterone production, germ cell morphology and proliferation on DIV 7 and 16.  

Explosive boiling is characterized by an unstable, non-uniform liquid/vapor interface consisting of multiple gaseous lobes. Explosive boiling at microscales is a process of extreme interest in the studies of micromechanical systems and actuation due to the nearly instantaneous pace and large amounts of kinetic energy associated with the process. These characteristics that make the explosive boiling phenomenon highly attractive for certain microscale applications also hinders the study and understanding of the dynamics of the unstable liquid/vapor interface, and thus the prediction of the overall behavior of the process. The objective of my research is to overcome the challenges posed by the small temporal and spatial scales by designing and building an experimental apparatus to observe the explosive boiling phenomenon. This apparatus consists of a test chamber in which the explosive boiling of a droplet of test fluid will be controlled by the host fluid temperature gradient, and a high speed camera used to capture the process in a high level of detail. The experimental results will be compared with theoretical results acquired from direct numerical simulation to elucidate the dynamics of the liquid/vapor interface. The experimental apparatus design and preliminary imaging results are presented.

Myths of the quest for the Golden Fleece and Jason’s relationship with Medea have survived whole or in fragments for almost 3,000 years.  Our analysis of the many variants of the myth used a Google site we developed to facilitate collaboration.  We examined key literary works written over a span of roughly 1500 years, starting with Homer.  Our base text was a summary by “Apollodorus” (1st-2nd Century CE) supplemented by entire works such as Apollonius’ Argonautica, Pindar’s Pythian Ode 4, Euripides’ Medea and by many fragments from lost epics.  We identified many variations in the story as it developed over time observing major differences in the way key characters are depicted.  We also explored artistic representations of the myths that have survived, such as Greek vase paintings illustrating different interpretations of myth unknown in literature.  Our research found that early Greek colonization of territories bordering the Black Sea and later geopolitical factors affected the development of what once may have been a simple folktale.  We noted how authors (e.g. Ovid, Valerius Flaccus) manipulated the narrative to serve artistic and political purposes and hypothesized why.  We completed our survey with two cinematic adaptions of the myth (Jason and the Argonauts 1964, 2000) examining how modern conceptions of narrative have changed the stories.  Our project attempted to answer various questions.  Where did the myths originate?  Homer tells us that Jason’s expedition was well known at the time of his writing but does not mention Medea.  Why was Jason heroic to Homer, then clueless to Apollonius?  Why was Medea first a goddess, then heroine, witch, murderess, then filicide?   Why does Hercules haunt the myths?  How did Roman authors receive and develop the myths from their own cultural perspective? Through this project we observed first-hand how cultures collide but creatively coalesce.

Multiple strains of rats and mice are used to evaluate differences in response after exposures to various toxicants at the molecular or cellular levels. However, neuronal toxicity in different strains of species has received much less attention. Therefore, the objective of our research was to compare and characterize fetal mouse micromass culture system using two different strains of mouse—C57BL/6 and A/J—which may be used to screen and prioritize variety of neurodevelopmental toxicants. Our mouse midbrain micromass culture system, a high-density in vitro cultures of primary midbrain cells, is an alternative method to in vivo neurodevelopmental testing because of the ability of cells within the cultures to differentiate in vitro without additional stimulants. Differentiation is purely driven by high cell density. For our 3D in vitro micromass culture system, we isolated the primary midbrain cells from fetus at gestational day 11 (for C57BL/6 mice) or 12 (for A/J mice). The embryos were surgically removed, and midbrain cells of fetal mice were digested into single neuronal cells. These primary in vitro neuronal cells were plated onto plates and cultured up to 22 days in vitro. When morphology was examined throughout 22 days in culture with a 400x phase microscope, cell proliferation, differentiation, and migration were observed for both strains of mice. Proliferation (PCNA, Nestin) and differentiation (B-tubulin, EN1, MAP-2) related protein expression levels were examined using western blot for both C57BL/6 and A/J mouse strains. Dynamics of protein expression levels in western blot indicated that proliferation may be at its highest point between DIV 4-6 while differentiation is between DIV 8-14. If midbrain cells from multiple mouse strains can be harvested at similar neurodevelopmental stages, differences in response due to genetic variability may be studied using our 3D fetal mouse micromass model.

Sea otters (Enhydra lutris spp.) play a significant role as a keystone species in shaping nearshore marine ecosystems of the northern Pacific coast through their primary diet of bottom-dwelling invertebrates, like sea urchins. Northern sea otters (Enhydra lutris kenyoni) were hunted to extinction from Washington State in 1911 due the fur trade of the 18th and 19th centuries, and remained locally extinct until their reintroduction to Washington State from Alaska in 1969 and 1970. We studied the foraging ecology of this species since this reintroduction, specifically how the diversity of prey species consumed by sea otters has changed from the 1990s to the present day. The prey switching hypothesis suggests that predators preferentially consume the most abundant prey species, then as a given prey item becomes rare, the predator will add a prey item of the next highest rank to their diet, increasing their diet diversity. Based off of this hypothesis, we expected sea otters in the 1990s to consume a lower diversity of prey species compared to recent years. Using sea otter diet data we collected from 2014-2016 as well as data collected by the U.S. Geological Survey from 2010-2011, we compared prey species diversity from 1990s data to present day data with the Shannon-Wiener Diversity Index. Preliminary analyses suggest that sea otter diet in the 1990s consisted primarily of clams, while it consisted primarily of snails in the 2010s.

This project intends to answer the following questions: (1) how knowledgeable are teens and young adults on how they can maintain confidentiality when seeking sensitive health care services while under their parent or guardian’s health insurance, and (2) what is the most effective tool to disseminate this kind of information? Although health care confidentiality is protected by law, there still remains the possibility that health information may be disclosed when documents such as the Explanation of Benefits (EOBs) and invoices are sent home. For adolescents and young adults insured under their parent or guardian’s health insurance plan, the potential breach of confidentiality may deter them from seeking appropriate health care. We hypothesize that teens and young adults have little knowledge about how to protect to their health care confidentiality and that a pamphlet is the most effective tool to educate them. To test this hypothesis, we created a pamphlet that provides options on how to maintain confidentiality when seeking sensitive services while on their parent or guardian’s health insurance. We will survey 20 adolescents, aged 13-25, on the pamphlet information, including the content and lay-out. Additionally, respondents are asked about other tools that would be effective in disseminating this information. Survey responses will be analyzed for themes. Thus far, we have interviewed 9 persons between ages 13 and 25. Each respondent stated that he/she was unaware of some information provided in the pamphlet. For example, one young adult stated, “I didn’t know that insurance or laboratory testing could accidentally make it easier for privacy to be lost in the billing process.” Respondents stated that tools such as a website or videos may be more effective to publicize this information. This project highlights the need to inform adolescents and young adults about their rights to maintain confidentiality when under their parents’ health insurance plans.

Short-term inflammation processes are commonplace following traumatic brain injuries (TBI), as they are a normal defense mechanism the body elicits to heal itself. However, when prolonged or chronic, inflammation can become detrimental. One inflammatory cytokine, Interleukin-6, has been shown to be increased in cerebrospinal fluid and blood post-TBI. In other non-TBI related clinical studies, Interleukin-6 has been associated with pain, fatigue, sleep disturbance, and depression. In this study, we examined whether plasma levels of interleukin-6 were associated with a higher symptom levels of four symptoms (headache, fatigue, sleep disturbance and depression) in the post-acute phase in patients with mild TBIs. To answer this question, secondary data analysis from a study that is currently underway was used. In the parent study, interested participants were enrolled from the Harborview Medical Center ED if they were 21-54 years of age, had sustained a mild TBI within the last 24 hours, and met other inclusion/exclusion criteria which would have impacted the immune response. Endorsement of symptoms and total symptom burden at 3 and 6 months was measured using the Rivermead Post-Concussion Questionnaire. Interleukin-6 levels at 3 and 6 months post-injury were obtained from banked plasma samples using a multiplex assay (Millipore, Minneapolis, MN). The sample (N=84) had a mean age of 34.7 years (SD 9.4). At 3 months post-injury the percent of patients endorsing symptoms as a problem were, 35.9% for headaches, 44.9% for sleep disturbance, 50% for fatigue and 32% for feelings of depression. At 6 months, the percentage reporting the symptom was higher for headaches (38.2%). Data analysis is currently underway using chi-squared tests and regression analysis to assess for the association between Interleukin-6 levels. If such an association is shown, this understanding of disease progression in mild TBIs could bring new treatment modalities to the medical realm concerning chronic inflammation.

Mild traumatic brain injuries (MTBI) affect millions every year. Symptoms are typically transient in nature but recovery time can span weeks to months. The neurobehavioral disturbances resulting from MTBI are associated with lower functional outcomes. Research suggests that well-developed social support networks contributes to improved health outcomes. However, less is understood regarding the existence of a relationship between social support networks and MTBI outcomes specifically. The objective of this study is to establish whether or not there is a relationship between social support availability and perceived symptom burden after MTBI. This study is a secondary analysis of data captured by the AIm:TBI study, which tests whether poorer outcomes from TBI in older adult survivors are associated with an altered post-traumatic immune response. In the parent study, participants were enrolled from the ED if they were 21-54 years of age and had sustained a MTBI within the last 24 hours (N=78). Social support was measured by the MOS-Social Support Scale (range 19-95; higher scores mean more social support). Endorsement of symptoms and total symptom burden at 3 and 6 months was measured using the Rivermead Post-Concussion Questionnaire (range 0-64; higher scores mean more burden). At baseline, the average social support score was 79.7 (SD 14.9). At 3 and 6 months post-injury the mean total symptom burden was 16.2 (SD 14.4) and 15.5 (SD 15.0) respectively. Analysis of the relationship between the two includes the assessment of relative influence of the type of social support received on symptoms. If the results of this study suggest that there is an inverse relationship between the severity of an individual’s symptoms after MTBI and the quality of their social support network, it may justify the development of interventions to increase social support in specific areas in order to facilitate better outcomes for MTBI patients.