Surf smelt (Hypomesus pretiosus) are ecologically and culturally important forage fishes that rely on intertidal beach habitat for spawning. However, the combined effects of rising sea level and human modification (e.g. seawalls, bulkheads, riprap) have put this habitat at risk of coastal squeezing, which could reduce available spawning areas along Puget Sound. This research aims to assess the vulnerability of surf smelt spawning beaches to climate change by combining field data collection with quantitative analysis. We evaluated existing risk assessment methods, such as the Coastal Vulnerability Index (CVI) to determine its applicability to Puget Sound. Additionally, we conducted beach surveys at productive spawning beaches to characterize the beach morphology. Our morphodynamic analysis included measurements of beach slope, sediment composition, pH, and water table depth from the high tide line to the waterline. Our hypothesis suggests that spawning beaches with a lower slope, smaller sediments, and a shallow water table will be more resilient to climate change impacts. Findings from this study will improve our understanding of climate-driven and anthropogenic threats to intertidal ecosystems and provide insight into habitat resilience, supporting conservation efforts for surf smelt populations.

Aquacultures are an environmentally responsible way of breeding and harvesting a variety of marine organisms. Challenges to aquacultures include the introduction of nonnative pathogens and parasites to wild fish as well as intense changes to the physical environment. Monitoring the environment for impacts of aquaculture is costly and time-consuming, so finding a cost-effective rapid monitoring solution is important. This project tests the feasibility of monitoring the diversity of meiofauna species present in the sediment via DNA sequencing. Meiofauna species are marine organisms between 0.06 mm and 1 mm in size that live in marine sediment. DNA sequencing is quicker and cheaper than current monitoring practices, and since changes to the physical environment affect meiofauna species diversity, observing the change in species diversity is a potential alternative to monitoring aquaculture health. Through samples taken before and after the introduction of a sablefish net pen aquaculture, analysis of both physical sediment samples and meiofauna samples will determine the feasibility of implementation. By looking at the total organic carbon content and grain size distribution of the sediment samples, we can determine the physical changes to the environment caused by aquaculture. Suppose the DNA sequencing results show shifts in species diversity that match the physical changes. In that case, we can confirm there is evidence supporting the feasibility of implementing monitoring via meiofauna DNA sequencing. 

Transcriptional repression plays a critical role in the regulation of various biological processes, including developmental pathways and disease progression. Corepressors are proteins recruited by partner proteins to negatively influence the transcription of genes. TPL is a corepressor from the model plant Arabidopsis thaliana, and is known to play a pivotal role in transcriptional repression. My project aims to identify other proteins that work with TPL to form a transcriptional repression complex at a single engineered locus. To further understand the function of corepressors, we built a synthetic repressor system, dCas9-TPL, designed to specifically repress the transcription of the RUBY reporter gene. When expressed, the RUBY reporter turns Arabidopsis thaliana pink. In previous phases of this research, we utilized the EMS (ethyl methanesulfonate) mutagenesis protocol to create a population of plants containing many random mutations. Screening these plants for increased RUBY expression, I successfully Identified promising homozygous lines where plants demonstrated bright pink flowers and unique phenotypes such as early flowering, light avoidance, and small stature. Last quarter, I sent five lines to be sequenced and each line displayed distinct mutations that I can further explore to pinpoint the exact TPL interactor responsible for its unique phenotype. I am also investigating known TPL interactors such as SPT4, SPT5, and MED21 by creating transgenic lines within my control dCas9-TPL + RUBY line. Through genetic screening, I have validated the phenotypes among these control experiments. By investigating the intricate network of interactions between these regulatory proteins, I aim to gain a deeper understanding of how gene expression is coordinated across different cell types and how this process controls complex developmental pathways.

Surf smelt (Hypomesus pretiosus) are an ecologically and economically significant forage fish species that spawn in the intertidal zone of beaches throughout the Salish Sea. Despite their importance to marine food webs, the environmental factors influencing their spawning site selection and seasonal distribution remain poorly understood. This project aims to investigate the morphological characteristics of beaches used for surf smelt spawning during different times of the year, comparing morphological and spatiotemporal variables that influence spawning. In order to study these characteristics, we will record sediment grain size, slope, wave energy, beach temperature and the water chemistry at verified winter as well as summer spawning sites identified by the Washington Department of Fish and Wildlife (WDFW). We will also sample non-productive sites in order to identify key differences between them and further establish parameters that enhance spawning success. Preliminary research suggests that these key characteristics strongly influence surf smelt spawning distribution​​​. Optimal surf smelt spawning beaches appear to consist of mixed sand and gravel substrates, low levels of wave action, high amounts of shading, moderate slopes and moderate temperatures. Habitat alterations such as shoreline armoring along with sea-level rise in response to global warming could lead to a drastic decrease in the upper inner tidal ranges where surf smelt usually spawn. Consequently, we expect beaches heavily influenced by these factors to be poor spawning sites​​. The results of this study will contribute to a deeper understanding of the environmental variables driving spawning site selection, egg survival, and seasonal spawning peaks. This research will be instrumental in informing conservation projects and supporting policy initiatives aimed at preserving surf smelt populations and their critical spawning habitats in the Salish Sea.

Syphilis remains a serious global health concern, underscoring the need for better control strategies. In the absence of treatment, the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), can persist for the lifetime of the host and syphilis can progress to its later stages. To combat the increase in syphilis incidence, doxycycline post-exposure prophylaxis (doxy-PEP) can be used to reduce the likelihood of infection with T. pallidum. However, the widespread use of doxy-PEP raises concerns about the possibility that this pathogen might become resistant, as seen in the past when azithromycin was used to treat syphilis. We wanted to explore whether continuous in vitro exposure to doxycycline could induce resistance in T. pallidum. To test our hypothesis, cultures of T. pallidum Nichols or SS14 strain were exposed to either increasing concentrations of doxycycline, azithromycin, or grown without antibiotics. Darkfield microscopy (DFM) was used to quantify the treponemal yield in cultures weekly. DNA was also extracted from T. pallidum cultures to evaluate bacterial presence by PCR, targeting the tp0574 gene. We found no sign of doxycycline resistance in T. pallidum SS14 cultures. Darkfield microscopy counts were detectable for up to three weeks in Nichols, whereas they lasted for five weeks in SS14. DNA extractions and PCR analysis showed no significant differences between strains, suggesting that albeit no strain developed resistance, one might be intrinsically more tolerant to the antibiotic. The results from this research provide encouraging evidence that T. pallidum may not easily develop resistance to doxycycline.

Syphilis, caused by Treponema pallidum (T. pallidum), remains a significant global health concern, with increasing cases worldwide. Doxycycline post-exposure prophylaxis (Doxy-PEP) has emerged as a potential strategy to prevent infection. However, widespread use raises concerns about the possibility that doxycycline-resistant T. pallidum strains might emerge and spread. This issue is alarming since doxycycline is a second-line therapeutic for syphilis and is often used in patients with allergies to beta-lactams or when beta-lactams are unavailable due to shortages. If genetic resistance to doxycycline were to develop in T. pallidum, it could undermine the effectiveness of Doxy-PEP and further narrow the range of treatment options for syphilis. To address this concern, I developed a restriction fragment length polymorphism (RFLP) assay to detect potential doxycycline resistance mutations in T. pallidum. This assay analyzes the 16S rRNA gene region of T. pallidum where most likely mutations could develop based on the analysis of other resistant pathogens. The assay was optimized using three synthetic 16S rRNA gene constructs containing the resistance-associated mutations and DNA from a wild-type T. pallidum strain (Nichols) as controls. The presence of mutations in the amplified control DNA was assessed by restriction digestion with the AluI, RsaI, and SfaNI enzymes, which can selectively cut wild type and mutant sequences and reveal specific mutations. The analysis of 60 archived samples from syphilis patients collected in the US, Madagascar, Argentina, and Sri Lanka is ongoing. Results will provide data on the frequency of doxycycline resistance mutations in T. pallidum, if any are found in this selected group of specimens. Developing a rapid, cost-effective surveillance tool is essential for monitoring potential resistance and preventing treatment failures when doxycycline is used.

Disruption of the Wnt signaling pathway is critical in the emergence of some of the most difficult cancers to treat. Transducin β-like protein 1 (TBL1) forms a complex with β-catenin, a transcription factor that switches ON Wnt target genes (Li & Wang, 2008). The Nemhauser Lab engineered a synthetic repressor circuit, dCas9-TBL1, that targets a constructed constitutive promoter driving GFP expression in human cells. I hypothesize that levels of TBL1 activity will correlate strongly with expression of Wnt target genes. My research uses time course qPCR to test Wnt-induced gene expression in both HEK293 and HCT15 cell lines. HEK293 have normal levels of Wnt signaling, whereas the HCT15 colon cancer cell line is known to have high Wnt activity which contributes to uncontrolled cell growth. Specifically, I will extract RNA from both cell types at 6, 24, and 48 hours after treatment with a control chemical and test for expression levels of  Wnt-target genes such as AXIN2. These experiments will test whether the elevation of downstream Wnt-target gene expression is correlated negatively or positively with TBL1 activity, and will enable further understanding of this route to oncogenesis and future optimization of chemotherapy targets.

Corepressors are an essential element of gene repression – complexes of proteins that keep genes off, yet poised to turn on when needed. Clarifying the mechanism of this repression is key to understanding gene regulation in all eukaryotes in diseased and non-diseased states. My project is implementing a forward genetic screen in Arabidopsis thaliana to identify and characterize proteins that bind to and regulate the conserved plant corepressor TPL. TPL is fully essential to plant development, so to visualize TPL inhibition in living plants, we created an Arabidopsis line containing a synthetic repressor, TPL fused to dCas9(dCas9-TPL), that represses RUBY, a genetic reporter that turns Arabidopsis plants dark pink. Plants with both constructs appear light pink as dCas9-TPL represses RUBY expression. Mutations in proteins needed to maintain TPL-based repression lead to dark pink plants, allowing us to identify mutants to study. Using ethyl methanesulfonate (EMS), we created a pool of seeds with random point mutations and the repressed RUBY construct. My team and I visually screened the mutated pool for pink plants showing inhibited RUBY repression and successfully identified promising homozygous mutants with unique phenotypes including infertility, shade avoidance, and irregular growth patterns. Using whole genome sequencing and computational analysis, I selected specific loci to further investigate. We are currently testing our candidate mutants' sensitivity to the plant hormone auxin, one of the best-understood TPL-regulated pathways. My next steps will be to identify the causal mutation through the following: (1) characterizing additional mutations in the same gene to compare phenotypes using available mutant libraries, (2) testing whether the candidate gene interacts with TPL using assays like yeast two-hybrid, and (3) complementing my mutants with wild-type versions of candidate genes. By uncovering new proteins, I aim to piece together more of TPL's conserved mechanism of repression. 

All cells have a stochastic component to their gene expression, such that even when in the same environment, there will be cell-to-cell differences in gene expression. Studies of this variability in gene expression dynamics have been limited by technological capabilities for measuring gene expression history with single-cell resolution. We have built a history-dependent integrase recorder of gene expression with single-cell resolution in the model plant Arabidopsis thaliana to study the impact of cell-to-cell gene expression variation in two contexts: development of side or lateral roots (LRD) and root regeneration (RR). The recorder uses integrases, proteins from bacteriophages that mediate permanent, heritable DNA changes based on the presence and orientation of a pair of integrase sites. Fluorescent reporter genes within the target construct allows for expression of fluorescent proteins associated with sequential expression of developmental genes. The recorder allows us to tie the switching to expression of developmental genes by expressing integrases with developmental promoters for genes that guide root differentiation. Utilizing our recorder, we are able to illuminate and evaluate variation in the recorder output among roots growing in different contexts. We hypothesize that regeneration leads to more heterogeneity in gene expression than lateral root development, as the latter has more standardized initial conditions and consistent local cues to constrain transcriptional dynamics. We aim to investigate connections between larger scale anatomical variation and underlying cell-to-cell gene expression heterogeneity. This technology will allow us to further understand the dynamics of gene expression during root development and could unlock new avenues for agricultural research and engineering.

The auxin hormone is necessary for many essential plant functions. Corepressors from the TPX family hold auxin response genes (ARGs) OFF unless auxin levels are high. TPX proteins are brought to ARGs through interaction with Aux/IAA adaptor proteins, which can bind to auxin-regulated transcription factors. Plant pathogens interfere with the auxin transcriptional pathway, making a plant more susceptible to infection. Oomycetes, for example, are a common plant pathogen commonly found as a mold growing on ripe tomatoes and strawberries. Oomycetes inject RxLR effector proteins into plant cells to reprogram the immune response. RxL21 is one of these effectors, and it contains a binding site for TPX proteins that is very similar to what is found in the Aux/IAA proteins. We hypothesize that RxL21 competes with Aux/IAA for recruitment of TPX proteins and keeps auxin genes on during an infection. I tested this hypothesis by performing a cytoplasmic split ubiquitin assay (Cyto-SUS), which is a protein-protein interaction assay done in yeast. Through this assay, we detected weaker TPX-Aux/IAA interaction when RxL21 was present, suggesting that competition for TPX protein interaction is occurring. I also tested whether the RxL21 competition would alter transcription of an ARG using a fluorescence-based assay in yeast. I observed much greater fluorescence when RxL21 was present, suggesting that RxL21 competition with Aux/IAA for recruitment of TPX results in increased transcription of ARG. In future experiments, I will further test our hypothesis by expressing RxL21 and other effector proteins in specific cell types in the model plant Arabidopsis thaliana. These experiments will allow me to quantify the impact of the competition for TPX corepressors on a developmental process. The results of this work could guide the design of new, broad-spectrum strategies to protect plants from pathogens.

All organisms regulate genes for proper cell development, healthy environmental response, and prevention of disease. One way to regulate genes is through transcriptional repression, specifically through corepressors that bind to repressors to inhibit expression of genes. The TPL/TPR corepressor family is crucial in Arabidopsis thaliana for regulating auxin-dependent genes during embryogenesis, root and shoot axis formation, differentiation, and environmental responses. Due to functional redundancy among the TPL/TPR gene family, partial mutations in the family do not create full loss of function. However, knocking out multiple family members is lethal. My research aims to induce loss of function for TPL in specific tissue. To achieve this, I started with a plant strain that is mutated for three of the five family members through insertional mutagenesis by T-DNA, leading to a partial loss of function, with two remaining genes remaining functional. Then I constructed a single TPL copy under the control of an integrase-based molecular switch, which when expressed, inverts the promoter of the TPL gene, turning it off. This construct, assembled through Golden Gate cloning, includes a YFP-tagged TPL gene and an mScarlet reporter that allows me to confirm TPL expression (YFP) or its absence (mScarlet) through fluorescence microscopy. I have integrated this construct with the controllable TPL switch into Arabidopsis, and my next goal is to use a CRISPR/Cas9 system to mutate the remaining two TPL genes for full loss of function. I will then utilize the integrase control switch system for specific TPL repression in the lateral roots. Such a study helps synthetic biologists understand the necessity of TPL in specific tissues, avoiding full knockout lethality. With corepressors existing among different eukaryotes, this study has broader implications in understanding human repressors, such as TBL-1 that are linked to dysregulation of gene expression in diseases like cancer.