Post-Traumatic Stress Disorder (PTSD) is one of the most common mental disorders in the world. People with PTSD often have difficulty managing time or have witnessed a traumatic event in the past. PTSD is also associated with memory issues which are characterized by intrusive memories that can cause re-experience aspect of the traumatic event. Through the use of computational models, we aim to investigate the differences in memory retention in spaced vs massed practice presentations. We hypothesize that mass practice will lead to less accuracy in recognition and less intrusive thoughts during retention. Through recruiting healthy adult participants with no history of PTSD or other stress-related psychiatric disorders we get a base data avoiding discrepancies. Participants view visual stimuli as images, either neutral or triggering which are presented in pseudo-order and are asked to rate them based on their valence ranging from neutral to extremely negative. The images are presented under two conditions: mass and space presentation. For spaced presentation, different versions of the triggering image category are presented in no particular order with neutral and filler images in between. For massed presentation, all versions of the triggering image category are presented one after the other with no neutral or filler images in between. 24 hours after the initial presentation of the images, participants are tested on memory retention in the form of image recognition. Participants are asked about the difficulty of recalling the image and how often they think about the image. Having collected data during AUT 24, we were able to understand there is a correlation present between memory retention in mass and spaced recognition in terms of traumatic and non-traumatic events. By collecting data through WIN 25, we will have greater accuracy in terms of significant data.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that affects millions of people. Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive stimulation typically used in psychiatric conditions such as depression and anxiety. rTMS works by using an electric current to generate a transient magnetic field, depolarizing neurons in a target region and creating lasting changes in brain connectivity via synaptic plasticity. Patients with AD show disruptions in the Default Mode Network (DMN), a network of brain regions typically active during rest and crucial for memory consolidation. We hypothesize that strengthening the DMN through rTMS targeted at the left Brodmann 8AV region, selected for being an easily accessible node of the DMN, will improve memory in AD patients. To test this hypothesis, we are conducting a single-blind, single-arm, randomized cross-over trial of rTMS on early-stage AD patients over a 12 week period with week 1 where we scan for the 8AV region via MRI, during week 3 and 8 being the placebo or treatment week. We measure our primary outcome of the participants’ speed of forgetting —a novel index of memory function—through an individualized, adaptive memory test. To eliminate potential confounding variables, we also measure depression and anxiety symptoms during the 1st, 8th and 12th week of the study. Additionally, functional MRI scans will be analyzed for potential structural or functional differences caused by treatment. Preliminary results from our initial participants have shown promising improvements, and we are hopeful that similar outcomes will be observed in the remaining participants. Successful results would provide a novel target for AD treatment using rTMS, and support further investigation of rTMS as a viable treatment option.

Soluble peptides from the HIV-1 (Human Immunodeficiency Virus) envelope heptad repeat-2 domain, known as HIV fusion inhibitors, can inhibit viral entry by blocking formation of the gp41 6-helix bundle required for membrane fusion and infection. However, this treatment is unfeasible because it requires twice-daily subcutaneous injections with high risk and cost. The Lieber Lab is working to engineer hematopoietic stem and progenitor cells (HSPCs) to express HIV fusion inhibitors in vivo, potentially offering sustained protection against HIV. In my work I used SIVmac239 (Simian Immunodeficiency Virus) challenged Rhesus Macaques sera and developed viremia (from another study by Lieber lab). My goal was to test whether anti-gp41 antibodies from these animals cross-reacted with synthetic gp41-derived fusion inhibitor peptides, specifically C46-v2o, C34-SFT, and Enfuvirtide(T20). If antibodies interfered with fusion inhibitors, their therapeutic effect would be severely compromised. In my project, I developed an Enzyme-Linked Immunosorbent Assay (ELISA) to measure antibody titers. These peptides were coated, then blocked with 3% bovine serum albumin, and incubated with diluted Macaque serum to allow antibody binding. I used anti-monkey immunoglobulin-G conjugated with Horseradish Peroxidase for detection of antibody binding. I optimized the serum dilution to 1:200 to reduce background signal and concluded SIV-challenged Macaques had detectable antibody levels against C46-v2o and C34-SFT, but not T20. Ongoing work will determine more detailed IC50 antibody titers in serum samples. Notably, animals with high viral loads exhibited higher levels of antibodies against HIV fusion inhibitors. T20 is a promising candidate for sustained HIV inhibition, as no detectable antibodies means it’s less susceptible to pre-existing immune responses. These findings provide valuable insights into how fusion inhibitors interact with the immune system and help refine strategies for HSPC-based HIV therapies, bringing us closer to a long-term, self-sustaining approach for HIV prevention. 
 

Changing patterns of human land use near coastal zones have increased the abundance of aquatic habitats where mosquito and midge larvae develop. Salt-tolerant species, such as Aedes togoi mosquitoes and Dicrotendipes enteromorphae midges, have evolved distinct anatomical and physiological mechanisms that allow them to thrive in saltwater environments while also being able to develop in freshwater. While much is known about how freshwater-confined species maintain salt and water homeostasis (i.e. osmoregulation) in dilute freshwater, these processes remain largely unexplored in salt-tolerant species. The main goal of this research was to describe and compare the osmoregulatory strategies of Ae. togoi and D. enteromorphae larvae reared in seawater and freshwater, focusing on how they maintain ion and water balance in saline conditions. Based on information from other salt-tolerant insects, I hypothesized that the rectum of these species is the main organ that excretes salts and will have higher expression of ion pumps compared to other osmoregulatory organs of the larvae. This increased expression would support the secretion of hyperosmotic (salty) urine, a critical adaptation for survival in saltwater. Using immunohistochemistry and fluorescence microscopy, I localized key ion-transport pumps within the rectum and other osmoregulatory organs of Ae. togoi and D. enteromorphae larvae. I identified a high expression of Na⁺/K⁺-ATPase (sodium/potassium pump), and V-type H⁺-ATPase (proton pump) enriched in the anal gills, renal tubules, and the rectum. My findings suggest that the coordinated function of many osmoregulatory organs, and not just the rectum, allows for salt-secretion by marine insects, and this establishes an initial framework of cellular mechanisms among marine insects. This research has implications for predicting future species distributions with rising sea levels, as well as the potential to identify novel targets to control salt-tolerant mosquito populations to mitigate disease transmission in coastal regions.

Neotrypaea californiensis, or burrowing shrimp, is a native pest for oyster growers in Washington. The shrimp create networks of interconnected burrows that displace and liquify sediment, suffocating oysters. The pesticide carbaryl was used for decades to effectively control shrimp populations. More recently, the Environmental Protection Agency (EPA) deemed carbaryl an environmental hazard, leading to a vital need for a shrimp control method with minimal non-target effects. Very little is known of the physiology of shrimp that allows them to tolerate environmental extremes. My study aims to understand the tolerance of Netotrypaea californiensis to high concentrations of salts that challenge their ability to regulate internal ions, pH, and metabolic wastes. Using a laboratory-based system whereby shrimp burrow in 6 inch deep sediment in seawater, my research findings revealed that the addition of high concentrations of sodium bicarbonate (NaHCO₃) caused shrimp mortality while high concentrations of regular sea salts did not. This suggested that an ionic imbalance—high levels of sodium and reduced levels of chloride—may be causing shrimp death. To investigate, I collected the gills, a critical organ that regulates systemic ion levels, of shrimp after NaHCO₃ exposure and quantified the abundance and localization of key ion pumps using Western blotting, immunohistochemistry and confocal microscopy. I found a significantly higher expression of Na⁺/K⁺ pumps in the gills with NaHCO₃ exposure, suggesting a major ion-regulatory disturbance caused by NaHCO₃. These data will expand our understanding of how salts that cause ionic imbalance in seawater can disrupt the internal levels of ions that are critical for most biological processes. Findings will be disseminated to Washington oyster growers to aid in the management of their crop.

The external fertilization and transparent embryos of zebrafish make them an informative model of vertebrate embryonic development from the 1-cell stage. In this study, we examine the impact of de novo GTP synthesis on the formation of the embryonic somites, which are embryonic cells which develop into segmented blocks of muscle that run the length of the body. We hypothesize the de novo GTP synthesis is required for the correct patterning of somite borders in zebrafish embryos, and that this process facilitates the formation of a vertebrate body plan. Inosine monophosphate dehydrogenase 2 (IMPDH2) is the enzyme which catalyzes the conversion of inosine monophosphate (IMP) towards the de novo synthesis of GTP instead of ATP. To test the impact of de novo GTP synthesis on somite formation, we inhibited IMPDH2 function with mycophenolic acid (MPA) both before and after somite formation began. MPA caused stronger defects in the somite morphology and embryonic body shape when added to embryos before somite formation began, earlier in development. We performed in situ hybridization against xirp2a to assess the effect of inhibiting IMPDH2 function on the formation and patterning of the somite borders. MPA treatment decreased the definition of somite borders we could observe in the posterior tail. Inhibiting IMPDH2 with MPA produced somites with smooth, round borders instead of the chevron-shape typical of zebrafish. We next conducted immunohistochemistry against IMPDH2 to examine the expression and localization of this enzyme in embryonic cells when GTP conditions are low. In MPA-treated embryos, we observed increased expression of IMPDH2 across the entire embryo. We will next explore how GTP abundance affects activity of the clock, a mechanism which synchronizes gene expression of embryonic cells.

In vivo genome editing of hematopoietic stem cells (HSCs) offers a promising approach for treating hemoglobinopathies and HIV/AIDS. The Lieber Lab has developed helper-dependent adenoviral (HDAd) vectors that preferentially transduce primitive HSCs in mobilized CD46-transgenic mice, humanized mice, and rhesus macaques following intravenous injection. However, off-target transduction (including other blood cell lineages and various organs) remains a critical challenge, potentially compromising safety. Moreover, the comparison of PGK and the relatively strong Ef1α promoters revealed that the editor expression level influences editing outcomes, especially in multiplex editing approaches. To address these limitations, the project’s goal is to engineer a highly active HSC-specific promoter that maximizes on-target gene editing while minimizing off-target effects, improving both the safety and efficacy of HDAd-based therapies. I first generated GFP reporter plasmids containing roughly 2 kb of proximal promoter sequence from five genes highly expressed in HSCs: CD164, cKit, DSG2, PROM1, and PROCR. These constructs were introduced into human CD34⁺ cells via nucleofection, and the cKit and PROCR promoters showed the strongest GFP expression in the HSC-enriched (CD34⁺/CD45RA⁻/CD90⁺) subset. To further enhance promoter activity, we linked the top-performing promoters to distal HSC enhancers that, according to ENCODE/Hi-C analyses from Dr. David Hawkin’s Lab, regulate cKit (2 enhancers), CD164 (4 enhancers), and PROM1 (2 enhancers). Engineered promoter-enhancer constructs yielded 3- to 4-fold higher GFP expression than Ef1α in CD34⁺/CD45RA⁻/CD90⁺ cells, with the cKit promoter + CD164-3 and PROM1 enhancers showing the highest activity. Building on these findings, we have incorporated these HSC-specific promoter–enhancers into helper-dependent adenoviral (HDAd) vectors driving an ABE8e-base editor for γ-globin reactivation. Ongoing work is focused on evaluating the specificity and efficacy of these HDAd vectors in humanized mice and CD46/βYAC-transgenic mice, with the ultimate goal of achieving safer, more effective in vivo genome editing in HSCs.

Hyalella azteca are freshwater amphipod crustaceans abundantly found throughout North America and are frequently used in toxicology for water and sediment toxicity testing. As freshwater bodies change due to anthropogenic climate change, understanding chronic, sublethal impacts to aquatic life is critical. Amphipods are known to have a higher tolerance to heavy metals (i.e. Cu²⁺) and road salt (i.e. NaCl) compared to other aquatic invertebrates, however, these contaminants may have subtle, sublethal consequences on their ability to smell and detect chemical cues for survival and reproduction. This research identifies the combined impacts of environmentally relevant levels of Cu2+ and NaCl, on the olfactory system and olfactory-related behaviors of H. azteca. H. azteca underwent acute 96 hour exposure to control, Cu2+ (30 µmol/L), and combined Cu2+ (30 µmol/L) and NaCl (5 ppt) contaminated freshwater. I recorded daily measurements of survival, and utilized qPCR to examine changes in the expression of key olfactory genes that we predicted would be modulated in response to these multiple stressors. I assessed the olfactory-associated foraging behavior to determine changes in their detection of an attractive food cue when exposed to Cu2+ and NaCl, which would indicate olfactory impairment. Preliminary results show that amphipod survival is not impacted by Cu2+ and NaCl contamination but changes to their olfactory system occur. Therefore, U.S. Environmental Protection Agency water quality standards for heavy metals and salt may not mitigate long-term, sublethal effects on aquatic animal populations as it relates to this important sensory modality.

Certain species exhibit the remarkable ability to regenerate their appendages, a process that requires complex metabolic pathways to facilitate the cellular proliferation needed to regrow tissue. Among these species, Xenopus tropicalis, the Tropical Clawed Frog, serves as a great model for regeneration studies because of its transient regenerative capacity. X. tropicalis tadpoles exhibit the ability to regenerate their tails, but this capability is gradually lost after metamorphosis. This unique characteristic allows for direct comparison of regenerative and non-regenerative processes within the same species. Previous work from the Wills lab indicates that genes encoding the enzymes of the pentose phosphate pathway (PPP), which generates precursors of biosynthetic molecules such as nucleotides and lipids, are highly expressed during tadpole tail regeneration. Although tail regeneration has been well studied, the variation in hind limb regenerative capacity across developmental stages and the metabolic pathways involved remains unclear. Hence, I performed a live imaging study to determine the developmental progression of hind limbs and assess their regenerative potential. This data suggests a decrease in success as the tadpole gets older. Immunohistochemistry staining of mitotic cells in developing limbs shows that proliferating cells decline as regeneration competency decreases. I hypothesized that genes for the PPP enzymes would also be expressed during successful limb regeneration, which was confirmed by in-situ hybridization. Together, these findings indicate that the regenerative capacity in limbs of X. tropicalis is stage-dependent and that PPP genes are expressed during all stages of regenerative capacity. This provides insights into the role of metabolic reprogramming in appendage regeneration, with the potential for translating it into non-regenerative species like mammals.

Overgeneral Autobiographical Memory (OGM) is a common symptom of depression and Post-Traumatic Stress Disorder (PTSD). Instead of remembering specific details, individuals with OGM can only describe past events from their lives in general terms. The "trauma hypothesis" suggests that OGM emerges because individuals suffering from PTSD tend to reduce the number of details they retrieve about their memories to avoid remembering their trauma. However, this hypothesis does not fully explain how this avoidance is learned, or why avoidance spreads from traumatic memories to all autobiographical memories. To this end, we propose a computational model of OGM that integrates theories of memory retrieval and trauma with reinforcement learning. In this model, multiple episodic memories are nodes in an interconnected network, and memories are retrieved when visiting that node in the network. The more nodes that are visited, the more detailed that autobiographical recall will be. On the other hand, visiting more nodes comes with an increased risk of encountering a traumatic memory, which comes with negative emotional valence. The decisions about whether to visit another node or terminate the retrieval process are made using reinforcement learning, which takes actions based on predicted outcomes. By obtaining a greater understanding of OGM through this model, we hope to improve treatments for PTSD that specifically targets its effects on memory. 

The proximal tubule (PT) and glomerulus are vital blood-filtering components of the nephron, the functional unit of the kidney. The components’ micro-scale sizes and intricate three-dimensional structures are critical to kidney function, although accurate in vitro modeling has proven difficult. Limitations in fabrication techniques have forced size scaling and imprecise morphology in models. In this study, we addressed fabrication limitations using multiphoton ablation to etch intricate, three-dimensional proximal tubule and glomerulus vessels in collagen hydrogels. We sought to demonstrate model viability by introducing human proximal tubular epithelial cells (hPTECs) and human umbilical vein endothelial cells (HUVECs), respectively, through cell perfusion. However, we encountered a significant challenge: due to the small diameter and high curvature of the micro-scale channels, the cells tended to aggregate, disrupting cell profusion and cellularization throughout the vessels. Cell aggregation was especially prominent in the glomerulus model due to the more tortuous and complex geometry. While our cellularization trials on native-scale models proved it is feasible to perfuse cells throughout the vessel, we still need to refine cellular profusion and cellularization. To improve cellular profusion and cellularization, we are first studying a 1.5-scale glomerulus model. The scaled model's increased vessel diameter and lower curvature demote cell aggregation and enhance the ease of cell profusion. We anticipate that cellularizing the 1.5-scale model will provide a deeper understanding of the variables facilitating cell profusion that we can use to improve native-scale vessel cellularization. Fabricating native-scale, accurate in vitro PT and glomerulus models is crucial for developing a deeper understanding of hemodynamic influence on kidney function. These findings contribute to the fabrication of more biomimetic in vitro PT and glomerulus models that will pioneer therapeutics and the understanding of kidney physiology and pathology.

Adoptive cell therapy with CAR-T cells has shown promise in hematological malignancies, but efficacy in solid tumors remains a challenge in part due to CAR-T cell exhaustion and antigen heterogeneity. However, the vast majority of preclinical models do not recapitulate the tumor-immune interactions that produce these barriers. To study CAR-T therapy in a rigorous model that recapitulates tumor-immune barriers, we adapted a Kras^LSL-G12D/+;P53^f/f (KP) genetically engineered mouse model (GEMM) of lung adenocarcinoma. However, adapting the KP-GEMM model for various target antigens, genetic drivers of disease, or interfacing with the vast array of powerful genetic mouse models is resource intensive which prohibits widespread utility. Here, we propose a defined, modular system for generating GEMM for CAR-T preclinical studies using the Sleeping Beauty (SB) transposon system. The proposed system uses polyethylenimine (PEI) to deliver SB transposon encoding oncogenic Kras^G12D and P53^R175H dominant alleles as well as our target antigen hROR1, in vivo to wild-type mice. We demonstrate that in vitro PEI successfully introduces genetic cargo into lung epithelial cell lines, while SB transposons mediate stable integration and expression. Next, we will test this in vivo. This system affords the induction of tumors with specific oncogenic driver mutations and specific tumor antigens on any genetic background. Ultimately, we expect that this approach will streamline preclinical use of GEMM in preclinical research. 

Previous studies have established that galaxy shape and structure, otherwise known as morphology, correlate with environmental density: elliptical galaxies are more prevalent in high-density regions, and spiral galaxies are more prevalent in low-density environments. However, recent studies suggest that stellar mass may primarily drive this trend. In this work, we analyze around 3 million galaxies observed by the Hyper Suprime-Cam survey to reassess the correlation of morphology with large-scale environmental density from a quantitative perspective. The morphological measurements for our galaxies were done using the Bayesian machine learning framework Galaxy Morphology Posterior Estimation Network (GaMPEN). Our analysis employs a Monte Carlo-based framework to account for uncertainties in structural parameter measurements while investigating the correlation between bulge-to-total light ratio, the proportion of light emitted from the center of a galaxy, and environmental density. Leveraging the statistical power of our large dataset, we conclusively demonstrate that the morphology-environment correlation disappears when controlling for stellar mass. Thus, the observed trend arises predominantly because denser environments preferentially host more massive galaxies, making stellar mass the key driver of the morphology-environment relationship. Our results mark a significant advance in addressing this long-standing debate. Furthermore, the methodological framework presented provides a versatile tool for probing the interplay between galaxy properties and the large-scale structure of the universe, which will be particularly valuable in light of ongoing and forthcoming large surveys that supply high-resolution data needed to examine this relationship across extensive cosmic volumes.

The Democratic Republic of Congo (DRC) owns abundant natural resources like minerals and oil, yet they struggle to translate this wealth into sustainable economic growth and prosperity. This research explores the question: What strategies can the DRC implement to achieve sustainable economic growth while fostering prosperity for its population? The study hypothesizes that embracing resource-driven economic diversification, improved governance, and international partnerships can transform the DRC’s economic trajectory. This research is a secondary research because there are many study cases for many countries in which they had faced economic challenges due to the non-transparency of their government. One example is Brazil; they are also land-rich and rich in natural resources. However, unlike the DRC, Brazil has managed to achieve a significantly higher GDP. This research indicates that a major factor behind the DRC’s hardship is the lack of transparency in its government, which reduces foreign investor confidence and limits economic growth. To foster their prosperity, we suggest that global organizations, like the International Monetary Fund (IMF), communicate with the DRC’s government based on the information from the World Bank and IMF for their openness to trade and transparent management to confide foreign investors’ investment in the country. If these, transparent government and confiding foreign investors strategies are well-implemented, this project could greatly impact many nations. It could lift millions of people out of economic hardship and turn the DRC into an example for other nations with similar challenges, especially in the African continent. The accomplishments made by the transparent government and confiding foreign investors policy in DRC may act as a model for nations worldwide on how to develop an economy that is sustainable, equitable, and advantageous for every world citizen.

As the sensitivity and capabilities of modern synchrotron facilities continue to develop, so does the field of computational material sciences in an effort to meet the demand for analysis of new properties in various systems. 3d transition metals are of special interest due to their wide range of conductive and optical properties. Traditionally, local bonding environments are characterized in terms of group symmetries, but this has limitations in complex systems. Linearly polarized emission of x-rays from these 3d materials can provide information about local anisotropy, and valence-to-core (VtC) x-ray emission spectroscopy (XES) is especially sensitive to oxidation state, ligand environment, and bond length. The purpose of this project is to use local geometric and electronic information to formulate a measure of local anisotropy. This metric is evaluated against real-space Green's function calculations of linearly polarized XES, where we apply a supervised machine learning approach trained on this metric to predict differences in the polarized spectral shapes. Polarized spectroscopy techniques are critical for a wide range of applications including the development of microelectronics, nanostructure characterization, analyzing anisotropy within quantum dots, and studying the polarization sensitivity of non-linear optics. An accurate formulation of this continuous anisotropy parameter will provide researchers with quick and inexpensive computational insight. For the development of new functional materials, this metric can be used for searching databases efficiently, allowing researchers to select the candidates that will provide a more ideal signal of any polarization dependent properties.

Climate change has affected every part of the world. However, nowhere is affected more than the Arctic. More and more arctic ice melts every season, and while the environmental implications are disastrous, it may open up positive new opportunities for trade and recreation, bringing life into small, dwindling Arctic economies. One area of the Arctic that will see increased use in the future is the Northwest Passage (NWP). The once icelocked and barely usable trade route has the potential to influence the economy of all territories it touches, primarily Alaska. Current data indicates that the melting ice along the NWP will have a positive impact on the Alaskan economy. This Literature Review predicts the possible quantitative impact on the region. Strategically positioned Alaskan communities, such as Nome, have fragile infrastructures which will have to adapt to increased commercial demand from cruise ships and recreational vessels. While it is unlikely that trade will have much of an impact economically as most of the ships will not stop along the route, it is likely that the increase in cruise ships will stimulate forced economic and infrastructural growth for these communities. Additionally, as this region of Alaska is mostly undisturbed, there needs to be more study into the environmental impacts of economic growth in the area. Considering the possibility of the NWP becoming a well traveled route is essential in order to help prepare small towns for potential economic booms. An overview of the research suggests that while trade in general may not impact the overall Alaskan economy, the potential increase in tourism via cruise ships and other recreational vessels has the potential to overwhelm the infrastructure of smaller Alaskan cities, even as it jump-starts their economies.

Pseudomonas aeruginosa (Pa) is an opportunistic pathogen that infects the airways of people with cystic fibrosis, a genetic disease that increases susceptibility to lung infections. Pa uses an intercellular communication system called quorum sensing (QS) that allows bacteria to sense cell density and coordinate behaviors among the population, including regulation of virulence. In the laboratory strain PAO1, there are three complete QS systems in Pa that are regulated by the transcription factors LasR, RhlR, and PqsR. PAO1 QS is organized hierarchically with LasR regulating RhlR, and the hierarchy is influenced by the transcription factor MexT that delays RhlR activity. However, it is unknown if QS hierarchy is found widely in Pa strains. My project tested whether the QS hierarchy exists in clinical isolates of Pa. We obtained 3 clinical isolates with intact lasR, rhlR, and mexT genes and created lasR and mexT knockout mutants for each strain to test the effects on RhlR activity compared to wild-type. To measure RhlR activity, we transformed each strain with a RhlR reporter plasmid. We found that a PAO1 mexT mutant shows greater RhlR activity compared to wild-type, while each clinical isolate showed similar RhlR activity between wild-type and the mexT mutant. We observed lower RhlR activity in clinical-isolate lasR mutants compared to wild-type, demonstrating LasR-dependent QS like PAO1. In PAO1, a ∆lasR∆mexT double knockout mutant restored RhlR activity. Interestingly, in clinical isolates, we observed no change in RhlR activity in these ∆lasR∆mexT double knockout mutants as compared to the lasR mutant, indicating MexT is not regulating QS hierarchy in these clinical isolates. Altogether, the clinical isolates displayed a LasR-dependent QS architecture similar to PAO1, but this was not dependent on MexT. Thus, my work points to undiscovered factors that influence QS architecture and highlight the diversity of QS regulation in strains of Pa.

Patient-provider communication impacts healthcare outcomes, but assessing the quality of interactions manually takes time and effort. This project explores the automatic assessment of patient-provider interactions using Language Style Matching (LSM). LSM scores the linguistic similarity of function words between conversational partners (e.g.,  pronouns, articles) from 0 (low matching) to 1 (perfect matching), reflecting how in-sync partners are. Past research establishes LSM as a marker for the quality of interpersonal communication that predicts how likely romantic relationships are to last, but has not been explored for clinical interactions. We (CL, NE) applied LSM to investigate how well patients and providers matched each other's speaking styles for insights into the quality of clinical interactions. We used Linguistic Inquiry and Word Count (LIWC), a software program for LSM analysis of transcripts. Using LIWC, we analyzed the transcripts of 108 simulated visits between 54 primary care providers and four standardized patients. We used descriptive statistics to characterize LSM across visits. Our initial findings show that LSM scores range from 0.77 to 0.94 ( mean=0.86, SD=0.03) which is similar to prior research where most verbal conversations fall between 0.83 and 0.94. These findings show that on average providers and patients tend to match each other in their speaking style at a level similar to typical conversations. However, we identified some outliers that fall below 0.83 threshold. Opportunities for future work include thin-slice analysis of the transcripts to understand how LSM scores change throughout a visit and comparing LSM scores to self-reported survey data about visit quality. ​​We hope to further investigate this efficient marker of conversational quality as LSM has the potential to characterize the quality of clinical interactions without the time and effort required of traditional manual approaches.

The resistance of castration-resistant prostate cancer (CRPC) to androgen receptor signaling inhibitors (ARSIs) continues to be a significant clinical problem. Translation inhibitors are being researched as a potential treatment for AR-independent CRPC after our laboratory discovered that elevated mRNA translation as one of the major contributing factors. We screened pharmaceutical firms' known mRNA translation inhibitors in three human LuCaP models of advanced prostate cancer: AR-low prostate cancer (LuCaP 176), castration-resistant prostate cancer (LuCaP 35CR), and AR-intact castration-sensitive prostate cancer (LuCaP 35CS). We discovered that only a unique eukaryotic translation initiation factor 4E (eIF4E) 5' cap-binding domain inhibitor was able to efficiently target LuCaP 176, whereas the majority of inhibitors were able to stop the growth of LuCaP 35CS/CR. 5' cap binding inhibition increased the efficacy of enzalutamide in AR-low cells by inducing basal to luminal lineage plasticity and post-transcriptionally downregulating basal keratins. Furthermore, in the AR-low basal LuCaP 176 PDX (patient derived xenograft) model, enzalutamide effectiveness was enhanced by eIF4E cap binding domain suppression. These results are consistent with patient data indicating that people with elevated eIF4E levels have faster resistance to ARSI. Overall, our preliminary data demonstrates that translation inhibitors targeting the eIF4E cap binding domain can reverse lineage plasticity through a translation-based mechanism and sensitize AR-low prostate cancers to ARSIs. To further investigate these mechanisms and assess their implications in patient specimens, we are determining how eIF4E cap binding domain inhibition regulates translation of basal keratins and cellular plasticity along with impacting CRPC growth. Our study shows a promising method for reducing ARSI resistance in deadly metastatic CRPC which is to target eIF4E cap-binding. In order to optimize patient outcomes, this study lays the groundwork for eIF4E cap binding domain inhibition as a possible therapeutic and incorporates a cap-binding assay as a biomarker for individualized treatment.

The project aims to engineer a new µMASS sensor with increased baseline fluorescence and improved dynamic range by applying linker optimization and subsequent high-throughput screening. µMASS is a genetically encoded fluorescent indicator that, when bound to an opioid, changes conformation, causing an increase in fluorescence intensity. µMASS is a tool that enable real-time imaging of opioids in the brain, allowing researchers to study neural pathways involved in addiction. While the sensor can detect opioid ligands in vitro, it requires optimization for use in vivo to study real-time opioid release. Linker optimization is a technique that involves introducing mutations into the linker region of the sensor. I hypothesize that mutating the linker residues will enhance the conformational change observed in µMASS-5-HT upon opioid binding while retaining enhanced baseline fluorescence.

In the Berndt lab, we develop genetically encoded fluorescent indicators (biosensors) by attaching a naturally occurring sensing domain to a fluorescent protein. When the ligand of interest, such as dopamine or calcium, interacts with the sensing domain, the protein will undergo a conformational change that induces a fluorescent response. The change in fluorescence can be measured and used to quantify biochemical activity. Applications of these biosensors span a wide range of research topics in neuroscience and behavior, providing insights into the neuronal network activity correlated with addiction, pain perception, emotion, and reward signaling. The current project that I am working on is optimizing the red dopamine sensor, GRABrDA2m. I developed a genetic library, mutating the linkers that connect the sensing domain and fluorescent protein. The behavior of proteins is highly dependent on structure and orientation, which is why I have chosen the linkers as a target region to explore. I have cloned in degenerate codons that randomize the nucleotides at specific positions on these linkers, with the linker locations having been recently identified in published literature. After sequencing the DNA to validate that the sites of interest were mutated appropriately and that the remainder of the sensor is intact, I will transfect these plasmids into human embryonic kidney 293 (HEK293) cells and screen for promising variants by employing OptoMASS, an cell array technique developed in the Berndt Lab that allows for the testing of hundreds of mutations simultaneously. I will pick out the cells whose sensors performed better than the parental variant, looking for improvements in baseline fluorescence and sensitivity to dopamine, then conduct reverse-transcriptase polymerase chain reactions to extract the sequences of the high-performing sensors.

Tame knots, which are equivalent to a polygonal knot with a finite number of sides, have well-studied invariants; conversely, wild knots that exhibit infinite and pathological behavior are difficult to study and classify. Knot mosaics, introduced by Lomanoco and Kauffman, are an example of a complete invariant for tame knots. Our project aims to expand the existing formal system of knot mosaics to develop an invariant for wild knots. We define n-singular mosaic tangles, the mosaic analog of tangle insertions in pseudoknots and singular knots, and we formalize a system of infinite insertion that generates a wild mosaic to represent certain wild knots. We also intend to define wild mosaic equivalence moves to capture the notion of wild knot equivalence in the mosaic setting. This gives insight to many wild knots explored in existing literature and provides methods to generate and classify new examples.

This study investigates the Namonuito region of Micronesia to determine whether the many guyots and atolls in this region contain specific slope conditions suitable for cobalt crust growth. This project also investigates whether there is a relationship between suitable zones and general bathymetry. The primary research focused on NAM-2 Atoll and Enterprise, Essex, and Namonuito Guyots. After collecting multibeam and sub-bottom data, the slopes were analyzed for slope angle, optimal slope distribution and composition. Then the results were compared with each other to determine if there are any similarities or correlations. Among all the guyots it was found that almost all the optimal zones were found in gullies, ridge slopes, and at the bottom of areas that experience slope failures. However, there isn’t a clear correlation between mean slope angle, optimal slope distribution and slope face orientation. In addition, sub-bottom data showed that almost all the guyots northern slopes were covered by a single thick pelagic layer, while the southern slopes of Namonuito and Enterprise contained thin pelagic layers with zones of exposed hard substrate. NAM-2 Atoll also was entirely covered in a single thick pelagic layer. Overall, by conducting a geomorphology and sub-bottom comparison test it is possible to narrow down locations of interest that can be further surveyed. The implications of cobalt crust research are that cobalt crusts are another natural source of cobalt which is becoming increasingly difficult to obtain on land. If the Namonuito region contains a large cobalt crust and the cobalt crusts can be extracted, it could have a huge impact of global technology and manufacturing industries.

Estradiol, a steroid hormone, plays a crucial role in bone density, cardiovascular function, and neuroprotection. It signals through Estrogen Receptor α (ERα), a nuclear receptor that, upon estradiol binding, undergoes a conformational change, translocates to the nucleus, and regulates gene transcription. While ERα's role in gene regulation is well established, the real-time kinetics of estradiol signaling remain poorly understood. To address this, I have been developing and optimizing a fluorescent biosensor, ER_mNG, to enable real-time monitoring of estradiol levels in living cells. ER_mNG consists of ERα’s ligand-binding domain (LBD) inserted within the mNeonGreen fluorescent protein. Estradiol-induced conformational changes in ERα alter mNeonGreen’s fluorescence, providing a readout of estradiol dynamics. To improve the sensor’s dynamic range, I have employed linker optimization, a structure-guided protein engineering approach. I designed and cloned ER_mNG variants with modified linker lengths and amino acid compositions using site-directed mutagenesis and in vivo assembly (IVA) cloning. These variants were transiently expressed in HEK293 cells via lipofection, and their fluorescence response to estradiol stimulation was quantified using live-cell fluorescence microscopy. By systematically modifying the sensor’s structure, I aim to develop an improved ER_mNG variant with a significantly enhanced dynamic range, enabling more precise measurements of estradiol signaling. This tool has the potential to advance our understanding of estradiol’s role in health and disease.

Thermodynamic properties of conventional s-wave superconductors (i.e. superconductors with an isotropic gap Δ) are insensitive to weak disorder.  In unconventional superconductors with a p-wave and d-wave symmetry of the order parameter, disorder strongly suppresses superconductivity. Experiments indicate that the disorder improves the superconducting properties of aluminum, an s-wave superconductor with a significant gap anisotropy. This project aims to study the effect of a single impurity on the density of quasiparticle states in an s-wave superconductor with a strong gap anisotropy Δ→Δ(n). The density of quasiparticle states is expected to migrate from smaller to greater energies. By using numerical methods, I can reveal how the density of states changes. Understanding the behavior of the quasiparticle density of states can allow further exploration into several types of s-wave superconductors without the need to assume isotropy.

Humans have limited regenerative capabilities, providing incentive to study other natural models of regeneration to make advances in the field of regenerative medicine. In response to injury, species including Xenopus tropicalis employ cellular mechanisms to replenish lost tissue, a process that has high metabolic demands. Depending on their developmental stage, X. tropicalis tadpoles exhibit different regenerative capabilities after tail amputation, posing them as a unique model system. Three-day-old tadpoles (NF stage 41) are able to regenerate their tails completely after injury, but transiently lose this ability during what is known as the refractory period. However, they soon regain regenerative capabilities in the tail and in the developing hind limb before permanently losing them during metamorphosis. Previous work by the Wills lab has determined that the pentose phosphate pathway (PPP) is required for successful tail regeneration in stage 41 tadpoles, but leaves open the question of whether the PPP remains significant at subsequent regenerative stages and structures. Here I test the hypothesis that the PPP continues to facilitate appendage regeneration in post-refractory tadpoles. To functionally test the requirement of the PPP in post-refractory tail regeneration, I performed pharmacological inhibition of g6pd, a key enzyme in the PPP, during tail regeneration. To assess regeneration quality, I developed a pipeline using FIJI ImageJ and R to quantify metrics of regenerative success such as tail area and length. Using this framework, I found that post-refractory tadpoles had diminished regenerative success under PPP inhibition similar to stage 41 tadpoles. These results suggest that the PPP is required at all stages of tadpole tail regeneration and will provide a more comprehensive understanding of metabolism during regeneration, a potentially beneficial insight for research in wound-healing initiatives in mammals. 

Sequencing of deoxyribonucleic acid (DNA) is important for a variety of biological and medical research. Nanopore sequencing is a fast and effective way to sequence DNA, and can be used for DNA with genetic alphabets that go beyond the four naturally occurring nucleobases (adenine, guanine, cytosine, thymine). Our group has used nanopore sequencing on synthesized eight-letter “hachimoji” DNA, which contains four artificial nucleotides (called P, Z, S, and B) in addition to the four nucleotides of natural DNA. Expanding sequencing efforts is critical in furthering biotechnological applications of such artificial DNA. Nanopore sequencing requires a motor enzyme to control the translocation of the DNA through the pore. Here, I analyzed the interactions between the Hel308 helicase and hachimoji DNA, specifically the time that Hel308 spends at a step along the DNA (known as the dwell time) and the tendency for Hel308 to step backwards (known as the back step probability). I compared my results to previous work done by our group using natural DNA, and found sequence-dependent behavior at similar sites in the enzyme for both the natural and artificial nucleotides. Studying the kinetics of Hel308 offers deeper insight into its mechanisms and role in genetic processes, as well as its use for other bioengineering applications.