Chronic stress induces the release of hormones and neuropeptides including dynorphin, which activates Kappa Opioid Receptors (KOR) to encode the anxiogenic components of the stress response. Previous studies have identified the specific dynorphin-KOR neurochemical signaling responses in the regions of the brain known for regulation of reward systems, and thus may be potential therapeutic targets for stress-related conditions such as depression, anxiety, and substance use disorders. Formally, cells containing dynorphin-KOR have been difficult to study due to technical limitations of the available reagents. However, the generation of new tools including mice expressing promotor-driven cre-recombinase (e.g. KOR-Cre and prodynorphin-Cre) have allowed cell-specific gene expression using Cre-dependent viral expression of fluorophores combined with in-situ staining. In this study, the reliability of a phospho-KOR antibody was tested. The specificity of the immunostaining was verified by administering a KOR agonist, U50,488, in wild-type animals or transgenic animals lacking the G protein-coupled receptor kinase 3 (GRK3), which phosphorylates the KOR. Additionally, wild-type mice with a pre-treatment of norBNI, a KOR antagonist, followed by U50 were also tested. Immunohistochemistry of animal brain tissue showed that the norBNI pre-treated, wild-type animals and GRK3 knock-out animals had lower KORp-IR fluorescence compared to their wild-type counterparts treated with U50,488, verifying that the KORp antibody was reliable. After verification, this KORp antibody was utilized to characterize the dynorphin-KOR circuitry in the mouse brain. To do so, an excitatory opsin was injected into the DR of pDyn-Cre mice with an optic fiber implant. The DR region was then optically stimulated with blue light and dynorphin-releasing projections into the ventral tegmental area were verified by measuring KORp fluorescence. This experiment details one specific dynorphin-KOR pathway in the mouse brain that was not previously studied, and future experiments will utilize this approach to map the extent of dynorphin/KOR localization within the reward circuitry.

High Mass X-ray Binaries (HMXBs) are some of the most physically extreme sources in the universe. They comprise of a compact object such as a black hole or a neutron star, and a high mass companion star. The compact object accretes matter from the companion star and forms an accretion disk of material that has temperatures in excess of ten million degrees Kelvin. This accretion disk then radiates out in the X-ray energy band and allows us to ascertain physical properties for the source. However, the very nature of an HMXB constrains the age of the source to a very high degree--this is because the source needs to be old enough for the compact object to form yet it needs to be young enough for the high mass companion star to still have fusion fuel in its core. In this project, we have created star formation histories at the locations of unknown X-ray emitting objects using optical observations from Hubble Space Telescope. These star formation histories determine if there are stars that have an age consistent with HMXBs present. The age of these unknown sources may be associated with these stars, which means they could be HMXBs. We then looked for star formation histories that have constrained ages consistent with an HMXB to try and give more evidence for an unknown object being an HMXB.

National interest in the fatal shooting of civilians by police is growing, driven in part by several high profile cases that were captured on video. Several news organizations, including the Washington Post and the Seattle Times, have done in-depth reporting on local or national trends. While the data these organizations used has been made public, the technical skills needed to access and analyze the data present a barrier to public use. This project seeks to develop browser-based software that will support simple access to the data, along with tools for Exploratory Data Analysis (EDA), that will make it easier for others to learn from these data. For this project we are working in the programming language R, writing a shiny app to provide the browser-based interface to R’s powerful EDA tools. Shiny apps involve writing two software components: the underlying R code for analyzing the data, and a user interface (UI) that provides a simple point and click tools for running the code. For the R code, we will focus on graphical exploration of the data: time series charts at different levels of spatial aggregation, interactive maps where users can zoom in and view online links for individual cases, choropleth maps to visualize racial disparities, and animated cartograms. For the UI, we will develop a webpage with tabs for each graphical option, and a range of filters and aggregation options on each tab. Users will be able to easily view and interact with each visual and modify it to display what they are interested in. Our codebase will follow the guidelines for reproducible research, and be uploaded to a public GitHub repository. This will support both public use and public development; users interested in contributing to the codebase can clone our repository, and submit suggested edits via pull requests.

For those interested in understanding more about fatal shootings of civilians by police, a natural place to start would be data on the events. Such data is, however, surprisingly hard to access. There are no official sources, but there are several crowd sourced data sets available online. These data sets each contain some common and some unique fields, and they are stored in very different formats. Hence, these data sets require preprocessing before the data can be used for other practical purposes like analysis and visualization. In this research, we focus on cleaning such heterogenous data sets using statistical methods with major focus on data matching. Data matching refers to matching data from different sources and recognizing the matching entities. It is an essential step in data cleaning that helps in resolving the conflicting information provided by the data sets---clerical errors, missing data, differently stored data, etc. Traditional statistical procedures and classification algorithms are based on supervised learning which requires training data. However, it is quite common that the training data is not available at hand and it might not be cost effective to prepare one from scratch. We explore a two step unsupervised learning algorithm that might achieve the same performance as the supervised counterparts. In the first step, it prepares a seed data set containing data points that are relatively extreme matches and non-matches based on discriminating fields. This prepared data set can be used as training data to train classical classification algorithms---k-nearest neighbors, Support Vector machines, Random Forests---which in turn can be used to classify the remaining data points. In the process, we examine missing data, geocode matching, and feature selection methods that increase classification accuracy by selecting the most discriminating fields. Finally, we examine the prospect of generalizing this approach to suit differently aligned data.

Kidneys are responsible for regulating blood glucose levels in the body through the reabsorption of sugars in the proximal tubule of the nephron. Issues with glucose absorption can lead to diabetic nephropathies where too much glucose is excreted. To study kidney glucose absorption, the lab made kidney organoids, which are miniature stem cell-derived in vitro organs that mimic the structure and function of kidneys. We used induced pluripotent stem cells (iPSCs), a type of stem cell that can be generated directly from one’s own adult stem cells. In a previous experiment, the lab found glucose absorption was present through the Sodium Glucose Co-Transporter 2 (SGLT2). The lab wanted to see if this was the only channel responsible for glucose absorption. Using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, we knocked out SGLT1 and SGLT2 separately and also knocked out Glucose Transporters (GLUT1 and GLUT2) separately in the WTC11 kidney stem cell line.  We selected a guide RNA corresponding with each glucose transporter in the cell membrane of kidney organoids.  Then RNA-guided Cas9 created the mutation by breaking the sequence for each glucose transporter in a specific site of the genome.  We verified our mutations were done correctly by sending our results to a DNA sequencing company.  We expect to find mutant lines will have less glucose absorption than our control and hope to learn which transporters are more active.  We are further using this method to create double and quadruple knockout lines with multiple glucose transporters knocked out at once.  We hope to learn which transporters are most responsible for glucose absorption, and in the future, can assess when certain transporters are used over others, which will open the doors for disease models for diabetes not only in kidneys but also in other organs.

Treatment options for kidney disease are limited to dialysis and transplantation, which are of limited efficacy and availability. Human pluripotent stem cells (hPSCs) can be used to generate immunocompatible kidney organoids, which contain the major proximal structures of the nephron, including podocytes, proximal tubules, distal tubules, and endothelial cells. They can be made on-demand to study kidney disease and regeneration. Unfortunately, these organoids lack a collecting duct system, a crucial component of the kidney. To address this need, we have developed a protocol to differentiate hPSCs into ureteric bud (UB) cells, which are the precursors of collecting duct cells. First, we identified markers for the collecting duct lineage in developing kidneys. Immunofluorescence analysis of human kidney tissue revealed co-expression of cytokeratin 8 and Dolichos biflorus agglutinin (DBA) in the collecting ducts. These markers were not expressed in the proximal tubules. Next, we investigated the ability of hPSCs to express these markers. Undifferentiated hPSCs were treated with a concentration gradient of small molecules known to induce kidney lineage differentiation, including CHIR99021 and glial cell line-derived neurotropic factor (GDNF); their effects were jointly and individually examined. Immunofluorescence was used to characterize the resulting cells, which were initially stained with DBA and Lotus tetragonolobus lectin (LTL), a proximal tubule marker. We found that CHIR99021 alone was sufficient to produce DBA positive and LTL negative cells in vitro. Notably, the DBA levels in these cells were lower than observed in kidney tissue, indicating that the structures we obtained were not yet fully mature. These studies provide the first evidence that collecting duct cells can be generated from hPSCs. Optimization of this differentiation protocol will yield kidney organoid structures that include the collecting duct system, which will make them ideal for kidney disease modeling and regenerative medicine approaches to reduce the need for kidney transplants.

Primary cilia are antenna-like structures on the surface of diverse cell types. Cilia are important for signal transduction and processes such as cell division and development, but their precise role in the cell remains poorly understood. Defects in cilia result in a wide spectrum of genetic disorders called ciliopathies, which commonly involve polycystic kidney disease (PKD), the formation of fluid-filled sacs in the kidney which eventually result in kidney failure. To understand the role of cilia in these diseases, we generated human pluripotent stem cells (hPSCs) with defects in cilia formation for the first time. We used the CRISPR-Cas9 genome editing system to introduce mutations in two different genes that are required for cilia formation, kinesin family member 3A (KIF3A) and kinesin family member 3B (KIF3B). We isolated four independent cells lines with indel mutations in exon 3 of the KIF3A gene and two in exon 2 of KIF3B. The introduced mutations led to frameshifts and protein truncations. Immunoblot analysis confirmed that no full-length protein was produced in these mutant cells. The cells were stained with acetylated alpha-tubulin, a marker of cilia. No cilia were detected in the mutant hPSCs, while an average of 48% cilia were detected in controls of identical genetic background. Surprisingly, the absence of cilia did not alter hPSC pluripotency, self-renewal, amniotic cavity formation, or growth. Further, we differentiated these cell lines into kidney organoids. The cilia deficient kidney organoids formed cysts reminiscent of PKD, while the isogenic controls did not. In conclusion, we have generated human cells and organoids lacking an entire organelle, the primary cilium, and used these to conclusively link cilia to PKD. These cilia deficient hPSC lines can be used to study cilia function, to model various ciliopathies, and to screen for drugs that can ameliorate the disease phenotypes associated with them.

Multiple factors interact to determine the lifespan of an organism. The Promislow lab uses the fruit fly Drosophila melanogaster to study the interaction between the metabolome (the profile of all small molecules within an organism), and the lifespan of a fly under stress. In a study of metabolome and lifespan data for many genotypes of Drosophila, our lab found that fly strains with relatively long lifespans when exposed to oxidative stress (peroxide food), had high levels of maltose, a disaccharide of glucose. We hypothesized that maltose was beneficial to flies on peroxide food, and tested this by supplementing the diet with maltose to see if this would extend lifespan. Flies fed supplemental maltose prior to exposure to peroxide food lived longer than flies fed unsupplemented food, supporting our hypothesis. Maltose could extend lifespan by providing energy to the fly via metabolism into glucose, or through another function as a disaccharide. To distinguish between these possibilities, we tested lactose, a disaccharide, to determine if any disaccharide could extend lifespan. Lactose did not extend lifespan, suggesting that disaccharides in general do not extend lifespan under stress. We found that glucose extended lifespan, supporting the hypothesis that maltose extends lifespan via conversion to glucose. Maltose can be stored as glycogen, a polysaccharide, and glucose is derived from glycogen by glycogen phosphorylase, encoded by the gene GlyP. To test the role of glycogen metabolism on lifespan under stress, we manipulated the expression of GlyP. Several transgenes were used to reduce the expression of GlyP by RNA interference (RNAi). RNAi of GlyP decreased lifespan, which supports our hypothesis that glucose derived from glycogen promotes survival. Our work suggests that glucose derived from glycogen or maltose is an important determinant of lifespan under stress, furthering our understanding of links between metabolism and complex phenotypes, like lifespan.

Reactive oxygen species (ROS) are byproducts of photosynthesis that can damage plastid DNA (ptDNA), and 8-oxoguanine (8-oxoG) is the most common DNA lesion. Our lab previously showed that maize ptDNA damage, in general, increases during the development of non-green proplastids to green, photosynthetic chloroplasts. The objective here is to determine the amount of 8-oxoG in ptDNA during plastid development under light and dark growth conditions. DNA damage will be assessed using a semi-long qPCR procedure and the enzyme formamidopyrimidine DNA glycosylase (Fpg) which selectively removes 8-oxoG lesions. Testing of the semi-long qPCR method was performed with control DNA treated with damage-inducing UV light. After UV treatment, PCR amplification decreased, as expected for Taq DNA polymerase inhibition by bulky lesions. PCR amplification of ptDNA also decreased after UV light treatment of light-grown, dark-grown, and dark-to-light transferred seedlings. When ptDNA was extracted from plants grown in these three conditions, treatment with Fpg affected the PCR amplification, indicating removal of 8-oxoG lesions. 8-oxoG will now be assessed along the developmental gradient from proplastid to mature chloroplast. We may find more 8-oxoG lesions in mature chloroplast due to high ROS from photosynthesis than in the undeveloped proplastids, as well as more 8-oxoG in light than dark growth conditions.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder in which fluid-filled cysts form in the kidneys and other organs. Mutations in the PKD1 and PKD2 genes, encoding the polycystin-1 (PC1) and polycystin-2 (PC2) proteins respectively, result in ADPKD. The underlying mechanisms that connect these mutations to ADPKD pathogenesis are poorly understood. Therefore, there is a great need to understand the mechanism of this disease and find an effective treatment. Our laboratory has developed an ADPKD cellular model by differentiating human pluripotent stem cells (hPSC) with mutations in PKD1 or PKD2 into kidney organoids. We are using this model to screen drugs that potentially induce or inhibit cyst formation in a dish. The cyclic AMP signaling pathway is hypothesized to contribute to cystogenesis in PKD. To test this, PKD mutant hPSC lines and isogenic controls were differentiated into kidney organoids and treated with forskolin, a strong cAMP agonist. Images of live organoids in adherent cultures were acquired in intervals and quantified for cyst formation using the cell counter feature of ImageJ. Organoids were subsequently fixed, stained, and imaged with fluorescent microscopy. Forskolin, induced a rapid and dose-dependent swelling in kidney organoids derived from both PKD hPSCs and isogenic controls. After removal of the drug, the tubules returned to its original form. These studies show that chemical modifiers of cystogenesis can be successfully tested using this system. Surprisingly, cyclic AMP does not appear to have effects that are specific to PKD. We are currently testing other drugs and culture conditions for cyst-inducing or –inhibiting effects. Interestingly, PKD kidney organoids form cysts in suspension cultures at significant higher rates than adherent cultures, revealing a role for the microenvironment in cystogenesis. These findings help us gain insight into the disease pathophysiology, which will ultimately guide us to discoveries of better treatments for PKD.

Antibiotic resistance is an enormous public health problem and causes death for 23,000 Americans each year due to ineffective treatments. Many strategies to target resistance have emerged, the majority of which target resistance machinery directly. In Escherichia coli, the TEM-1 gene encodes for the β-lactamase enzyme which can degrade penicillins, a subclass of β-lactam antibiotics. Alleles of this gene can either improve or weaken the ability of β-lactamase to degrade other subclasses of β-lactam drugs. One strategy that can potentially slow the evolution of bacteria and select against antibiotic resistance is exploiting collateral sensitivity – mutations that confer resistance to one drug and confer sensitivity to another. Utilizing a technology called Deep Mutational Scanning, we are screening every amino acid variant of the TEM-1 gene, and examining growth patterns in twelve different β-lactam antibiotics. This will allow us to quantitatively measure the effect of every single protein variant on the fitness of the organism in hopes of identifying patterns of collateral sensitivity. This is done by first optimizing growth parameters and next sequencing the DNA of each amino acid variant before and after exposure to drug. The large data set produced allows us to uncover common evolutionary trends. This foundational knowledge will potentially allow us to predict drug pairings that exploit collateral sensitivity, leading to more successful treatments.

Flowering plants exhibit striking diversity in their floral symmetry due to independent genetic changes throughout their evolutionary history. Diversity is also observed within the flowers of genus Rhododendron, which displays a variety of radially and bilaterally symmetric flowers across its approximately 1000 species. Floral symmetry is governed by an interaction between one TCP transcription factor, CYCLOIDEA (CYC), and two MYB-class genes, RADIALIS (RAD) and DIVARICATA (DIV). This interaction initiates when CYC is expressed in the floral meristem, leading to the activation of RAD in the dorsal domain. RAD then antagonizes DIV activity in the dorsal regions, leading to radial symmetry. In other flowering plants, duplications in CYC have been associated with changes in floral symmetry, but the role of RAD and DIV remain unstudied. A number of transitions to and reversals from radial symmetry have occurred throughout Rhododendron from a bilaterally symmetric ancestor. Focusing on RAD and DIV, the goal of this study is to better understand the evolutionary history of these floral symmetry genes in rhododendrons, and their correlation to floral symmetry changes. I sampled eight different species throughout Rhododendron in addition to various outgroups and reconstructed phylogenies of these genes. I obtained sequences from genomic and transcriptomic databases, and used these sequences to design primers for amplifying and sequencing these genes from laboratory samples from wild and cultivated specimens. I found two distinct copies resulting from one duplication, in both RAD and DIV. In future studies, we will expand our sampling of species to investigate the phylogenetic placement of the origin of these duplications in tandem with changes in flower symmetry, to determine whether duplications in RAD or DIV are associated with symmetry changes as has been shown for CYC.

The exceptional biodiversity in the Coral Triangle is partially attributed to the elevated rates of evolution that occur in the shallow reefs of the Indo-Pacific Ocean. Its current geographical location and complex oceanographic history have facilitated rapid speciation in many lineages of coral reef fishes. The processes causing these unique evolutionary patterns can be closely studied in Eviota (Gobiidae), a widespread lineage of rapidly diverging marine fishes. Their restricted dispersal capabilities, short generation time, and specific habitat preferences have facilitated repeated exploitation of novel niches and thus catalyzed their high species diversity. Here, we examine morphological and genetic diversity in relation to biogeography in the Blackbelly Dwarfgoby, the Eviota atriventris species complex. This study analyses E. atriventris from nine localities across its range spanning the Indo-Australian Archipelago to determine whether recent speciation has occurred at fine scales across the Coral Triangle. Our combined morphological and molecular phylogenetic analysis examines differences in meristic, coloration, morphometrics, and gene sequence data from seven mitochondrial and nuclear genes. Results show strong divergence in mitochondrial DNA sequences in Milne Bay, Papua New Guinea and Solomon Islands (eastern) populations, as compared with a western haplotype from Indonesia. This suggests that lineages within E. atriventris are geographically and genetically isolated. However, evidence from nuclear gene sequences show few differences between groups, and morphological data to support the separation of these populations are inconclusive. These similarities imply that eastern and western haplotypes may be in the early stages of speciation. Due to phylogenetic evidence and lack of geographic overlap, these lineages may be considered separate species under some species concepts. This study illustrates challenges in separating and defining species concepts for closely related species with conserved morphology.

The North American beaver (Castor canadensis) is a keystone species and ecosystem engineer, capable of modifying the landscape and creating diverse habitats for other species. Through dam-building, beavers impound streamflow and create ponds and wetland complexes behind dams. The presence of beavers has been shown to increase biodiversity, reduce downstream flooding, and improve water quality. While beavers alter landscapes greatly, often in ways beneficial to native habitats and species, their dam-building can also create conflict with human land-use and infrastructure. One way to reduce human-beaver conflict is to adopt non-lethal approaches such as the use of pond leveler devices. Pond levelers, consisting of a pipe through the beaver dam with a cage around the inlet, control the water level of the pond by establishing a desired level while allowing beavers to persist, thereby reducing flooding. Little observational evidence exists on whether pond leveling devices create fish barriers or otherwise impede fish passage for migrating salmon. The objective of this research was to evaluate whether migrating salmon can pass through or around beaver dams fitted with standard pond leveling devices, under high flow conditions. We tested this by observational analysis collected from five pond levelers on Big Spring Creek, King County, Washington during the 2017 autumn salmon migration. Target migrating species include Chinook (Oncorhynchus tshawtscha) and Coho salmon (Oncorhynchus kisutch). During prolonged precipitation events it was determined that stream stage overtopped dams, providing unhindered passage in our study areas. Additionally, during the 2017 migration period, over-dam stream velocity and through-dam velocity (i.e. within the pipe) were passable by salmonids during 100% of the flows we observed.

Aerosol particles, solid or liquid particles suspended in the air, can affect Earth’s climate by scattering and absorbing sunlight. PM_2.5, particles less than 2.5 micrometers in diameter, are largely responsible for degraded visibility and easily traverse respiratory system pathways causing adverse health effects in humans. Often, a majority of PM_2.5 is composed of organic matter formed through photo-chemical reactions of hundreds of volatile organic compounds (VOC). This process depends on nitrogen oxide radical (NO_X = NO + NO₂) concentrations in the atmosphere, now predominantly contributed by anthropogenic emissions. NO_X and VOC react to produce organic nitrates, thought to be significant contributors to PM_2.5, but for which measurements are generally lacking. My project involves developing a concrete assessment of the role of organic nitrates as contributors to PM_2.5. I am developing an analytical method to thermally desorb organic nitrates from atmospheric particles collected on filters and promptly decompose them into nitrogen dioxide (NO₂). The resulting NO₂ is measured with a Cavity Attenuated Phase Shift (CAPS) spectroscopy instrument. I am establishing whether there is a direct correlation between the concentration of organic nitrates in PM_2.5 and desorbed NO₂. The goal is to quantify organic nitrates without individually measuring each of the likely hundreds present in the ambient atmosphere. Moreover, the thermal desorption process provides information on the physical properties of organic nitrates, such as effective saturation vapor pressure, needed for air quality computer models to accurately simulate their contribution to PM_2.5. As a result, I will help develop a better understanding of how natural and anthropogenic emissions affect the composition of the atmosphere by allowing assessments of how PM_2.5 levels have changed in response to NO_X emission reductions by the Clean Air Act.

Patients with cerebral palsy tend to have altered muscle activations during walking compared their unimpaired peers. Quantifying these changes is important for clinicians to be able to proscribe treatment protocols. Every brain injury with cerebral palsy is unique, requiring patient specific measures of motor control. Muscle synergies are used to quantify a patient's motor control by using the electrical activity of individual muscles during walking. The resulting electrical activity of the muscles, electromyography, are processed using matrix factorization algorithms. These algorithms identify weighted groupings of muscles which are commonly activated during activities such as walking. However, across clinical institutions differences in data gathering protocols and resources, as well as muscle selection can make the interpretation of synergies challenging. Moreover, calculation of synergies requires programing and data processing expertise not uniformly available to clinicians. The aim of this research was to design a web application that could intuitively represent synergy results for clinicians and patients based upon the data they collect at their institution. This web application uses processing and factorization scripts in Python and integrates the results into JavaScript and HTML for an accessible and flexible display. The application allows the user to select the muscles they want to include in the analysis, as well as how many synergies they want calculated. Each synergy solution is displayed by presenting the weighting of every muscle in each synergy. Additionally, the application shows how accurately the synergies reconstruct the electromyography signals. Generated results from the application can be exported as a report for the patient and clinician to refer to. Because of the differences in data gathering practices across institutions, this research also looked to identify if there are specific ways to quantify how individual muscles impact synergy results so that synergy results can be more translatable across institutions.

Homo naledi is a recently-discovered species of hominin that lived in South Africa at 280 ka, around the same time that anatomically modern humans were evolving in east Africa. While the lower body of Homo naledi shares many characteristics with us that aid bipedality, the upper body shows a more primitive morphology related to climbing. Did H. naledi have climbing abilities more like apes, or tool-making and throwing abilities more like modern humans? To answer this question, we focus on the distal humerus because it is one of the more common limb bones found in the archaeological record, making it excellent for multi-species comparisons. We collected coordinates of landmarks indicating distinctive locations from 3D scans of distal humeri from seven Homo naledi specimens, as well as distal humeri from great apes, modern humans, Neanderthals, and other extinct hominins. We analysed the landmarks using geometric morphometry, which uses quantitative representations and analyses of morphological shape using geometric coordinates instead of linear measurements. The variation in the landmarks' coordinates was analyzed with statistical clustering techniques such as principal component analysis and partial least squares analysis using the R programming language to identify and visualize similarities and differences among the taxa in our sample. The results improve our understanding of upper limb mechanics and the role of climbing in the locomotion of Homo naledi, as well as sexual dimorphism among Homo naledi, and clarify their phylogenetic relationship with other hominins.

Antibiotic resistance is a growing threat across the planet. This resistance, coupled with a dearth of new antibiotics, makes development of new antibiotics of critical importance. Antibiotics derive from microbial pathways that synthesize complex natural products, and engineering these pathways to produce unique products is an exciting prospect. We will use such an engineering strategy to produce variants of the antibiotic Microcin J25 (MccJ25) in E. coli. MccJ25 belongs to a unique set of compounds known as lasso peptides. It is produced by three biosynthetic enzymes that modify the precursor peptide, McjA, and then export the mature lasso. MccJ25 functions by blocking the nucleoside triphosphate (NTP) uptake channel of RNA polymerase, interfering with transcription. Moreover, a single amino acid change in RpoC, one component of RNA polymerase, confers resistance to MccJ25. Extensive studies on the MccJ25 biosynthetic pathway suggests that the biosynthetic enzymes are tolerant of mutations in McjA. We plan to identify variants of MccJ25 that overcome resistance due to rpoC mutation. We will construct a library of protein variants of McjA and transform this library into a strain of E. coli carrying the rpoC T931I mutation. After induction of the MccJ25 biosynthesis pathway, cells with biologically active variants of MccJ25 should drop out of the population. By deep sequencing the population before and after selection, we can infer which variants successfully inhibited growth. This strategy of altering a precursor peptide to create novel antibiotic structures may provide a framework for future antibiotic engineering efforts.

Symbiotic relationships are vital to the survival of a vast array of organisms. A well-documented set of symbiotic relationships in the marine environment are between bivalve mollusks and the variety of epibionts that encrust their shells. Marine sponges are some of the most frequently observed epibionts of bivalves, and can confer both positive and negative effects on their hosts. The Puget Sound sponge species Myxilla incrustans lives almost exclusively on the valves of the scallops Chlamys rubida and Chlamys hastata. Studies have found that this symbiosis is mutualistic, with both the scallop and the sponge acting as predator deterrent for the other, among other interactions. Additional research has shown that presence of sponge does not significantly affect the swimming distance of scallops, however little has been done to look at the effect of different predators on scallop swims, as different scallop species may invest different amounts into protecting their sponge symbionts. This study aimed to evaluate the responses of two species of scallops to two species of predatory sea star to determine if they react differently and if one species of scallop is a better host to the sponge. Scallops were placed in tanks with either the sponge predator Pteraster tesselatus or the scallop predator Pycnopodia helianthoides and the scallop-sea star interactions were recorded. Time and distance of swims were measured for each scallop swim and were categorized by what initiated the swim. Distance and duration of swims were used to evaluate the response of the scallops to different predators to determine if there was a difference between the two species.

Microhabitat association is a key factor contributing to the diversification of coral reef fishes. For species with specific microhabitat associations, a change in host organism (a coral, sponge, or other organism) on a reef could enable sympatric speciation, where a new species evolves in the same geographic area as the ancestral species. The tusked goby, Risor ruber, is a poorly studied obligate sponge-dwelling coral-reef fish. Preliminary genetic data on R. ruber indicate that there are eight genetically distinguishable lineages based on mitochondrial DNA sequence data and that these linages often live on specific host sponges, pointing to a possibility of ecologically-driven speciation. This study aims to uncover potential connections between morphology, microhabitat, and genetics, by determining if the morphological variation of the recurved canines and other morphological features, such as body length versus depth, correspond with the genetic lineages identified and their host sponge preferences. Measurements will be taken from CT scans and preserved specimens. Fin counts, vertebral counts, and teeth will be examined on cleared and stained specimens. Results of this study may lead to new species descriptions within the genus Risor.