Hair cells are mechanosensory receptors in the inner ear used for hearing and balance. When hair cells are damaged or die, hearing is lost or impaired. Many studies have also shown that genetic mutations affecting mitochondrial function are another cause of hearing loss. However, it is unknown which cells in the auditory system are affected. In this study, we sought to determine whether or not these deafness-causing mutations impact hair cells. To do so, we took advantage of zebrafish as a model system and studied hair cells of the lateral line. To generate mutant fish, we used the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas system, a genome editing tool that allows for gene-specific changes to be made to DNA. If these mutations do affect hair cells, we predict to see changes in mitochondrial and hair cell function. Characterizing these mutations in zebrafish will inform our understanding of the cellular mechanisms of hearing loss.

Calcium is an ubiquitous and critical signaling molecule that mediates a wide range of cellular processes. Cytosolic calcium is maintained at concentrations that are orders of magnitude below both the extracellular space and intracellular organelles. Even small, sustained increases in cytosolic calcium can have harmful effects on the cell. As such, calcium homeostasis mediated in large part by the endoplasmic reticulum (ER) and mitochondria that serve to sequester calcium enable normal functioning of the cell. To examine cellular mechanisms that mediate calcium homeostasis, we are using mechanosensory hair cells of the zebrafish lateral line. Mechanosensory hair cells can be stimulated via a mechanical stimulus or drug-mediated calcium release from intracellular stores. The hair cells' abilities to easily uptake drugs and respond to physical stimuli make them an ideal tool to examine calcium homeostasis. Preliminary studies using transgenic zebrafish in which the calcium indicator GCaMP3 is targeted to the cytosol vs. the mitochondrial matrix demonstrates that stimulation of hair cells causes an immediate increase in cytosolic calcium in contrast to a gradual increase in mitochondria calcium. The question is whether the calcium in the ER is changing in response to the changes in mitochondrial and cytosolic calcium. As a first step to investigating the role of ER in calcium homeostasis, we will use gateway cloning to generate transgenic zebrafish lines in which calcium indicators are targeted to the ER. We predict that stimulation of mechanosensory hair cells will cause changes in ER calcium levels. These studies may provide valuable insight in how cells maintain health by buffering intracellular calcium levels in response to activity. 

Historically, only clinicians and researchers have been involved with decisions about what research topics to pursue, and patients have been left out of the process. This creates a gap between clinical application of research and patients’ experience. By expanding the scope of how researchers and clinicians obtain patient data to include research topic priorities, we explore how to incorporate patient perspectives better. Although there are many studies that aim to analyze patient registries, there are limited studies that attempt to elucidate the degree of the participants’ interest, or willingness to get involved in providing input on research activities. Through a study funded by the Patient-Centered Outcomes Research Institute (PCORI), we developed a two-phase approach to prioritize topics for low back pain research. In phase one, we invited participants from a patient registry of older adults with back pain , and in phase two, invited participants using Amazon Mechanical Turk (MTurk), an online crowd-sourcing platform. Participants completed an initial screening survey comprised of the Roland Morris Disability Questionnaire, a standard tool to identify individuals experiencing back pain. Those scoring 7 or higher on the RMDQ were sent a research topic prioritization survey. 1966 people completed the screening survey, and 480 qualified, of which 353 completed the prioritization survey (completion rate: 74%). Using both recruitment strategies, we had a high response rate, and participants are diverse in terms of pain, gender, and age. Responses show participants’ effort to fully answer the survey and voice their opinion, including providing additional topics that had not been identified previously.This work highlights that recruiting from a patient registry or using MTurk are both effective tools to obtain patient input, and increases the flow of information from the public/patient to the researcher, and inevitably the clinician; we expect incorporating these methods will increase patient voice in research.

Existing work argues that intergenerationality and interraciality in LGBTQ communities is in need. In one report, “many of the issues facing both younger and older LGBT people are related to weak intergenerational ties, particularly with LGBT people of a different generation.” Additionally, the Group of the Advancement of Psychiatry argues, “often, ‘the gay community’ being studied consists of gay, white men. One reason for a paucity of literature on gay people of color, for example, is researchers' lack of recognition of diversity within the gay community.” As a young queer person of color, I see and feel the imperativeness of intergenerationality and interraciality in the LGBTQ community. Many young LGBTQ people don’t have many intergenerational relationships with other LGBTQ people, resulting in a lack of much needed mentorship. Likewise, many spaces that I and other LGBTQ people of color exist in are not representative of racial diversity or our own races. Following the work of the “Dragon Fruit Project,” a LGBTQ intergenerational storytelling organization within the Asian Pacific Islander community, I conducted interviews with people, diverse in age and race, in San Francisco, Seattle, and New York about their experiences in interracial or intergenerational communities. I used academic sources regarding LGBTQ intergenerationality and interraciality, and connected the key academic findings to the themes brought out of the interviews to create a prototype of an intergenerational and interracial LGBTQ community. As a result, I connected LGBTQ people in the Seattle community of all races and ages. With the LGBT elders of color population “projected to increase by 217 by 2030” and the rise of LGBT people being out in the future, the need to redesign and incorporate interraciality and intergenerationality in the LGBTQ community is imminent to accommodate the future of what the LGBTQ community will be.

Himalayan adventure travel is a burgeoning industry in some mountainous regions of Nepal, India, and the Tibet Autonomous Region of the People‘s Republic of China. The development of a trekking and expedition mountaineering infrastructure has created employment opportunities in remote areas and allowed visitors from around the world to embark on life-changing explorations to some of the planet‘s most unique destinations. However, with the rapid, uneven, and largely unregulated growth of this industry have come environmental and resource challenges, the creation of new economic and social arrangements, and renewed questions of equity and safety. Despite—or perhaps because of—the prominence of Himalayan adventure travel in the popular consciousness, the historical, social, political, and economic drivers responsible for maintaining and reproducing the current paradigm remain poorly understood. The industry‘s growing pains are highlighted by the mounting human toll of disasters in the mountains, most recently the earthquake that destroyed Everest Base Camp in April 2015. Although the proximate cause of these disasters were natural events, growing unrest suggests that their ultimate causes are in fact chronic and unnatural. My research is based on eight months of fieldwork in the Himalaya and combines analysis of theoretical, historical, and contemporary sources with personal experience as a Himalayan expedition leader. I argue that the current Himalayan adventure travel paradigm is unsustainable, and that only a profound reimagining of sociopolitical relationships—those between the state, local and global civil society actors, and adventure travel practitioners and participants—will allow it to continue. My findings demonstrate that recent developments reflect longstanding systemic problems and that current events may serve as a catalyst for previously underrepresented stakeholders in adventure travel to translate their growing assertiveness into meaningful and lasting solutions.

Interactions between single nucleotide polymorphisms (SNPs) and complex diseases have been an important topic throughout epidemiological studies. Previous genome-wide-association studies have mostly focused on gene variables at a single locus, but did not obtain strong evidence for SNPs with complex diseases such as cancer. In our project, we performed a focused candidate gene study to test the interaction of multiple SNPs with the risk of different types of cancer. Using the R package algstat, developed by Kahle, Garcia-Puente, and Yoshida, we developed an algorithm which can test for independence between several variables and the disease. We applied our methods to the study of gene-gene interaction on cancer data obtained from the European case-control study Gen-Air. Previously pairs of SNPs were tested for independence versus certain cancers in this study, but it was not computationally feasible to test triplets of SNPs with the methods available. The algorithm developed was significantly faster so we were able to test for independence of SNP triplets and found strong evidence to reject independence of many triplet combinations of SNPs with the disease, suggesting correlation between having a certain combination of SNPs and risk of certain cancers. These results can be used for further testing of associations of these SNP combinations and certain cancers. Outside of the study of SNP-cancer association, this algorithm can be easily adjusted to perform other studies using arbitrary log-linear statistical models.

The Redbluff landslide, situated at Washington-Oregon state border, is part of the larger Bonneville landslide complex. In 1450 A.D., the Bonneville landslide dammed the Columbia River, but the river eventually breached it. In the present day, only one part of the landslide complex, namely the Redbluff landslide, is still slowly creeping into the Columbia River. It also poses a risk of damaging nearby public infrastructure. Interferometric synthetic aperture radar (InSAR) is a remote sensing technique used to measure millimeter-scale surface deformation. These satellite data are complemented by Light Detection Ranging (LiDAR), which is used to produce high-resolution topography images. Using these observations, I seek to understand how the movement of the Redbluff landslide relates to the surrounding geology and morphology. The downslope landslide movement is computed by back-projecting the line-of-sight InSAR observations onto a downslope vector, as done by previous InSAR glacier studies. ArcGIS software will be used to produce a higher-resolution model of the landslide movement. Furthermore, surface roughness will be computed as a geomorphic proxy of active movement on the slope. I expect that the InSAR surface displacement will show active movement in the regions with highest surface roughness. The InSAR and LiDAR data sets are highly complementary data sets and both help to better characterize landslide hazards along the Columbia River.

Chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is the gold standard for assaying the binding sites of chromatin-binding proteins and their modifications, such as histone tail modifications. One major limititation for this method is that it requires large amounts of starting material, therefore limiting applications in those cases where only a limited number of cells can be recovered, e.g. rare cell populations obtained directly from patients. In the last few years, great efforts have been made to develop new ChIP-seq protocols to overcome this limitation. In my lab, they have developed a method called carrier ChIP-seq (cChIP-seq). The method is based on the use of a DNA-free carrier to enhance recovery of DNA after IP. Using cChIP-seq they have been able to perform ChIP-seq on 10,000 cells for several histone tail modifications. I tested a series of modifications on the cChIP-seq protocol. The goal of my project was to develop a protocol that can be performed in one day. Currently the cChIP-seq protocol takes 3 days to be complete. This is mainly due to two overnight incubations. The first one is the immunoprecipitation, so I performed multiple trials to identity the minimum time sufficient for immunoprecitating the chromatin. The second overnight incubation is to reverse crosslink immuneprecipitated chromatin. I tested a version of the cChIP-seq protocol for native chromatin, meaning the DNA is not crosslinked. By optimizing and combining these two modifications I was able to shorten the cChIP-seq to a single day. Through my project I contributed to a larger project with the goal of scaling down the number of cells used for ChIP-seq.

Gastrulation is a stage in embryogenesis in which most progenitor cells commit to a muscle or neural fate. However, a population of bipotential progentitor cels does not differentiate during gastrulation and allows the embryos to dynamically allocate neural and muscle tissue in the correct proportion as the body axis is formed. The regulation of this process is crucial for proper neuromuscular development. The maintenance of these bipotential progenitor cells requires the repression of tbx16, a transcription factor that progenitors express when they commit to a muscle fate. Here, we examine two genes of interest termed eve1 and vent, and investigate their roles in the regulation of tbx16. While previous studies relied on Morpholino anti-sense oloigonucleotides (MOs), the credibility of MOs has recently become seriously questioned because they can cause off-target effects, generating misleading effects that don't reflect genetic mutants. Revolutionary new gene-editing techonology known as CRISPR, is hailed as the new standard for gene analysis because it allows the production of true genetic mutants in specific genes. CRISPR accurately and efficiently creates small deletions in target genes, which can result in complete loss of function. Using the CRISPR method, we will generate F1 lines of zebrafish with eve1 and vent mutations. Through high-resolution melt anaylsis (HRM) and DNA sequencing, we have thus far identified two eve1 F0 founder fish. We will then test how the absence of eve1 and vent affects the expression of tbx16 during zebrafish embryogenesis using in-situ hybridizations. By analyzing how embryogenesis in normal zebrafish embryos differs from that of eve1 or vent mutant embryos, we can identify the role that these genes play during development. Ultimately, our goal is to gain a better understanding of how serious birth defects and genetic disorders arise from complications in development. 

Honeybees (Apis mellifera) use a caste system to organize individuals into different roles. The queen is the only bee in the hive to ingest royal jelly and the only fertile female. Many sterile workers perform different jobs in their short lives, moving from larvae-nurse to keeping the hive clean to foraging for food. At each stage the worker bees learn a new task. Such different phenotypes and behavior would indicate a difference in gene expression in the brain. In a population of organisms that are genetically similar, these changes in gene expression must be explained in chemical modifications of the genome, or the epigenome. Thus far, much attention has been given to DNA methylation as an epigenetic feature known to be associated with variation in gene expression. Royal jelly, however, contains butyrate, a histone deacetylase inhibitor, which suggests that histone tail modifications could be a major mechanism for regulating gene expression in queen bees. To further investigate the role of histone modification in determining honeybee caste division, we are performing chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-seq) to profile key chromatin modifications: H3K27ac, and H3K27me3. H3K27ac and H3K27me3 are histone modifications that have opposing effects; where H3K27ac marks active regions of the genome, and H3K27me3 is indicative of repressive chromatin. We anticipate when using ChIP-seq to map chromatin modification in the brains of queen and worker bees, we will observe key differences in genome regulation and be able to identify genes important to honeybee behavior. As an initial step toward achieving our goal, we describe the chromatin state of brains from forager honeybees.

Seattle’s young homeless population has long been underrepresented and misunderstood—their experiences are largely absent from public conscious. In the University District, young people often face discrimination, stereotypes, and violence rooted in negative perceptions from University of Washington students. These two groups share a history of mutual distrust and even fear. Exposure is creating a 3D interactive narrative to challenge stereotypes toward homeless youth and allow an audience of University of Washington students to step into the shoes of a young person experiencing homelessness as a way to confront misperceptions. We took a community-grounded approach to researching this marginalized group. Through volunteering with service organizations and meeting with community leaders, we have curated stories and experiences by which to design this dramatic narrative. We also draw upon existing literature on daily youth community practices as well as game design for addressing social issues. We have then developed requirements for key scenes and interactions for this narrative. For example, cell phones, which play a large role in managing young people's’ lives, will be represented in the narrative. The diversity of the community across race and gender identity will manifest as playable characters with narrative outcomes distinct to those identities. As of today, Exposure is completing a working prototype for this narrative which will then become a playable demo. Key elements of our narrative have been written and storyboarded. By exposing our audience to the youth community through this fictionalized interactive experience, we hope students will confront prevailing misperceptions of young people, develop empathy for this underrepresented community, and engage with movements addressing structural causes of homelessness. In the same way film and literature are used to communicate our lived experiences, we hope that the use of an interactive medium will be a novel and impactful approach to represent a community long neglected.

X-Link Myotubular Myopathy (XLMTM) is a progressive congenital pediatric disease that affects one in every 50,000 male births. XLMTM is caused by a mutation of the MTM1 gene. MTM1 codes for myotubularin, an enzyme that is involved in the excitation-contraction coupling of muscle cells. XLMTM is characterized by a severe phenotype marked with muscle weakness and hypotonia that can result in many life-threatening complications, including diaphragmatic weakness leading to respiratory failure. Adeno-associated virus (AAV)-mediated gene therapy has reversed muscle pathology in murine and canine models. Treatment of the canine models with an AAV8-MTM1 vector indicated improved muscle strength as well as prolonged life. The field of muscle disease research would benefit greatly if an in vitro patient-specific induced pluripotent stem cell (iPSC) model were utilized to generate XLMTM-null muscle myotubes. It is necessary to develop an in vitro model of the disease using specific patient-derived cells because the patient-specific disease model has the predictive utility to determine if there are aspects of the disease not mitigated by gene therapy. This will help pinpoint the areas the disease affects in order to come up with a cure. To do this, MTM1-mutant iPSCs were differentiated stepwise to myogenic precursors and then to multinucleated myotubes using a small molecule approach that has been shown to mimic embryonic muscle specification. Extent of myotube differentiation was qualified with immunochemistry, using muscle-specific markers such as Myogenin, MyoD, and MF20. Experiments are currently underway to identify the disease phenotype in vitro and correlate with known pathology observed in the patients with XLMTM.

Patient specific individualized pluripotent (iPS) cells can be differentiated into cardiomyocytes that beat spontaneously, express sarcomeric proteins, and contain ion channels. However, these differentiated cardiomyocytes express a “fetal” like phenotype in that they are smaller, irregularly shaped, have a shortened sarcomere length, and lowered excitation-contraction rates that prevents their clinical applicability. In addition to executing their intrinsic genetic program, functional cardiomyocytes also receive inductive cues from their microenvironment. In the intact heart, those cues are supplied by the extra cellular matrix (ECM). The marriage of iPSC-derived cardiomyocyte technology with ECM differentiation cues will result in more mature cardiomyocytes that better represent natural human cardiomyocytes. Therefore, we are using a “native” cardiac matrix derived from a decellularized pig heart to try and promote maturation in cardiomyocytes using no mechanical inductive cues. We are particularly interested in the fibronectin to laminin ratio because the work of Wang, Lei, and Weighton (2015) suggests a proper ratio of these ECM components promotes maturation. Additionally, we are interested in the stiffness of the ECM coating as the cardiac environment has an optimal stiffness level. The iPSC will be differentiated into cardiomyocytes by using chemicals that alter the WNT signaling pathway. Some cells were exposed to the ECM during differentiation to measure the extent of cardiomyocyte maturation in the presence of ECM. Other cells were transferred to the ECM after the differentiation process. The extent of maturation will be measured by gene expression profiling and RNA sequencing. We hypothesize that the presence of cECM will enhance maturation based on the work of Oberwallner et al. (2014). If successful, this work will lead to improved drug screening and allow for tissue engineering for cell replacement therapies.

The diffusion of ions into a nano-scale ZnO lattice has gained attention in the past decade as a possible solution to the rapid fracturing of ZnO electrodes in bulk batteries and as an alternative to conventional LiCoO₂ and LiFePO₄ materials which are expensive to manufacture and environmentally hazardous. Experiments have shown that utilizing bulk (ie. not nano-structured) ZnO electrodes fracture in discrete segments (a leapfrog effect) after only a few charge/discharge cycles, diminishing the effective charging/discharging capacity. Current efforts are aimed at using nano-scale lattices which fall within the size range of the cracking separations and thus are not susceptible to leapfrog fracturing. Using an optimized quantum dot structure and conventional ab initio electron structure theory methods, the effects of lithium ion intercalation into a Zn₃₃O₃₃ quantum dot lattice were investigated. The main research questions were: (1) where are the potential energy valleys in which lithium ions can settle; (2) What effect does lattice expansion have on the energy of a lithium-ion charged quantum dot; and (3) how does doping the dot with iron affect the energy? Using a rigid and a relaxed scan of a lithium entering and exiting through the C3v axis of the quantum dot, the potential energy landscape was mapped, illustrating the presence of multiple potential valleys in which ions could settle. We used single point calculations to determine the optimal expansion factor after observing expansion in previous molecular dynamics simulations. Currently preliminary results indicate a possible trend regarding iron positioning within the lattice and we hope to improve our data by introducing a larger Zn₈₄O₈₄ dot. Hopefully our findings will demonstrate that nano-scale iron doped ZnO lattices are not subject to leapfrog fracturing and may therefore be cheap and viable options as battery electrodes.

There are many different Danio species that each have their own very unique pigment pattern. Even though each pattern is different, they are all made up of the same pigment cell types. Different interactions between black melanophores, yellow/orange xanthophores, and iridescent iridophores establish and maintain the Danio pigment patterns. Changes in these interactions allowed the species to diverge and created their own unique phenotype. Our project is focused on determining the differences that allow for the development of different pigment patterns within Danio species, such as Danio rerio, D. margaritatus, D. erythromicron, D. choprae, D. tinwini, D. alboliniatus, D. aesculapii, and D. nigrofasciatus. One of the more obvious differences in the development of these patterns is the order in which the adult pigment cells differentiate. In some species melanophores differentiate first, while in others xanthophores differentiate first, and in some species multiple pigment cell types differentiate at the same time. Previous studies have shown that xanthophores are very important in determining the final pigment pattern in D. rerio. In order to test what roles the xanthophores have in determining the final pattern for other species we generated colony stimulating factor 1 receptor (csf1r) mutants using CRISPR/cas9 mutagenesis. Without csf1r there cannot be any csf1 signaling, which is required for xanthophore development. By knocking out csf1r we can knock out xanthophores and the cell-to-cell signaling involving xanthophores that was necessary to form the normal pattern in Danios. So far we have created mutants for csf1r in D. rerio, D. alboliniatus, D. aesculapii, and D. choprae. We have also started to quantify the phenotypes for these mutants to determine the overall effect of knocking out xanthophores. Understanding the cellular mechanisms that allow for all the different patterns in Danios will help our understanding of the evolution of different Danio species.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by mutations in the gene that encodes the protein dystrophin. This disease is incurable and causes severe muscle weakness, respiratory failure, cardiomyopathy, and early death. Understanding the causes of DMD and developing new treatment strategies relies on in vitro models. However, current animal and cell based disease models of the cardiomyopathy experienced by patients with DMD do not accurately recapitulate the human presentation of the disease. Stem cell derived cardiomyocytes provide a platform for improved and personalized disease modeling, however the cells are functionally immature, exist in two dimensional cultures, and do not experience the mechanical stresses of the human heart. There is a need, therefore, for a way to generate three-dimensional constructs of cardiomyocytes and subject them to physiologically relevant mechanical loads for the purpose of promoting maturation and disease phenotype testing. Induced pluripotent stem cells (iPSC) derived from the urine of healthy and diseased patients were used to generate cardiomyocytes in vitro (iPSC-cardiomyocytes). Differentiations of stem cells were enriched using metabolic methods, allowing us to generate cardiomyocyte cultures with greater than 90% purity. Three-dimensional cardiomyocyte constructs were generated in polydimethylsiloxane (PDMS) molds using a cardiac-stromal co-culture method with a combination of collagen and geltrex. In order to further replicate the human heart, a device was designed and fabricated that was capable of applying controllable, uniaxial mechanical loads to the three-dimensional cardiomyocytes and was used to measure the force generation of DMD or healthy iPSC-cardiomyocytes. Future studies involving the device will increase our understanding of the pathology of DMD and allow for novel, personalized treatments to be developed.

Autism Spectrum Disorder (ASD) describes a set of neurodevelopmental conditions characterized by behavioral and cognitive phenotypes with multiple gene involvement. There is a strong association between bowel disorders and ASD, making the enteric nervous system (ENS) a potential target for drug treatment and therapy. The ENS innervates the gastrointestinal system and regulates its function. Current studies of the ENS are limited to the invasive procedure of removal of gut tissue, or animal models that do a poor job of recapitulating the human disease. The work presented here uses induced pluripotent stem cells (iPSCs) from ASD patients that have been differentiated into enteric neurons to model the ENS for study. Fully differentiated enteric neurons pass through an intermediate cell type called neural crest (NC). Here, we report the successful differentiation of iPSCs to neural crest progenitor cells (NCPCs). Treatment of iPSCs with two inductive factors, SB431542 (a TGF-B Inhibitor) and BIO (a GSK-3 inhibitor) generated a high yield of NCPCs. The expression of NCPC markers by TaqMan® PCR gene expression assays, was used to quantitate the extent of NCPC differentiation. To demonstrate the specificity of the differentiation protocol, immunocytochemistry results between NCPCs and forebrain neurons were compared. Together, these findings support the conclusion that iPSCs can be efficiently differentiated to neural crest progenitors as an intermediate in the generation of an “ENS in a dish” to study the chronic constipation suffered by patients with ASD.

The loss of mechanosensory hair cells in the inner ear gives rise to many hearing and balance-related disorders in humans. The permanent loss of these cochlear hair cells can be caused by many factors, including age and noise-related hearing loss, and ototoxic medications. A certain class of antibiotics known as aminoglycosides as well as a number of chemotherapy drugs such as cisplatin have been identified as ototoxic (being toxic to inner ear structures responsible for hearing and balance), with usage resulting in varying levels of temporary or permanent hearing loss and vertigo. Because of the inaccessibility and complexity of the mammalian ear, the zebrafish has emerged as an effective animal model for studying mechanosensory hair cells due to comparable external mechanosensory hair cells clustered along the lateral line of fish. The external location of the hair cells allows for ease of access and imaging of hair cells for large drug library screens for compounds which protect against ototoxic effects. I followed up on a recent drug library screen of bioactive small molecules which produced three possible protectants against neomycin-induced hair cell loss. To characterize their protective qualities, I exposed zebrafish to different dosages of the protectants and neomycin and measured the amount of hair cell loss to create dose-response profiles for each protectant. Higher protectant doses will increasingly attenuate neomycin-induced hair cell loss. The six tested compounds’ efficacy will be compared to a known protectant compound to see whether they can be further investigated to as a potential ototoxicity-blocking compound (blocking the effects of ototoxicity). The results of this study will identify and describe the efficacy of these putative hair cell protectants and will lead to a better understanding of the role these compounds may play in reducing the effects of ototoxins.

The goal of this project is to better understand the functions of symbiotic bacteria that live in earthworm kidneys. This project will be focusing on the composting earthworm, Eisenia fetida. Earthworms play an important role in soil ecosystems, breaking down organic matter, aerating the soil, and enriching soil with nutrients beneficial for plants. Many earthworms harbor bacterial symbionts in their osmoregulatory excretory organs, the nephridia, which function like kidneys to manage salt balance and process waste. Two nephridia occur in each segment of the worm (~ 200 nephridia total) with each containing symbiotic bacteria. Eisenia fetida has three species of bacteria that live within their nephridia: Verminephrobacter eiseniae, a Cytophagales member, and an Actinobacteria. These bacteria are passed into egg capsules from the mother to the next generation. The function of these bacteria is unknown. This project is testing the hypothesis that these bacteria are important for supplementing the diet of the worm. To test this, worms are being cured of their symbionts by treating them with antibiotics during early development to prevent colonization. A colony of cured worms has been established and control and erythromycin-treated stocks will be fed various diets to test their ability to handle a lack of nutrients. Their mortality and fertility will be observed to determine the contribution of the nephridial bacteria to the success of E. fetida. We expect that treated worms will have higher mortality than the control group. 

Marine ammonia-oxidizing archaea (AOA) gain energy through the conversion of ammonia (NH₄⁺) into nitrite (NO₂^-) and are among the most abundant microorganisms in the oceans.  AOA are widely accepted as the main drivers of marine nitrification processes, and therefore play a major role in controlling the amount of fixed nitrogen available for primary production.  In order to gain a better understanding of the regulation of the marine nitrogen cycle, the factors influencing AOA abundance and activity in the environment must be studied.  Several Nitrosopumilus maritimus strains isolated from Washington's costal waters were evaluated for their physiological response to key environmental variables, including salinity, temperature, ammonia concentration, and pH.  The AOA isolates display wide ecotype variation and show ranges of growth responses for each environmental variable.  The growth of some of the costal AOA isolates requires a supplement of organic acids, such as pyruvate, oxaloacetate, and alpha-ketoglutarate.  This study provides further insight into the ecological and trophic role of N. maritimus in the environment, and potential responses to changing environmental conditions such as ocean acidification.