In recent years, the United States has seen an increasing number of opioid overdoses, causing many research studies to focus on opioid drugs of abuse. However, we still lack a fundamental understanding of the opioid receptor that these drugs bind to. The mu-opioid receptor (MOR) is involved in both the pain-relieving and euphoric effects of opioid drugs. The Ventral Tegmental Area (VTA), known to be a major source of dopamine, contains a significant amount of inhibitory gamma-aminobutyric acid (GABA) neurons that express MORs. Previous research has shown that activity at the MOR in the VTA is rewarding and that dopamine projections from the VTA to the Nucleus Accumbens (NAc) are activated to reinforce behavior. However, whether the expression of MORs on GABA neurons in the VTA is important for influencing VTA to NAc dopamine activity during reward seeking behavior is unclear. To investigate the role of the MORs in reward seeking behaviors, I knocked out MORs in the VTA and used dLight, a fluorescent dopamine sensor, to measure dopaminergic release from synapses in the NAc during Operant and Pavlovian conditioning tasks in the absence of these receptors. Pavlovian conditioning utilizes chambers in which a house light turns on and a sucrose pellet is delivered via food hopper to food-restricted mice. In the operant conditioning task, mice must learn to complete a nose poke in order to receive the sucrose pellet reward. These tasks allow me to gain a more complete understanding of how the absence of MORs in the VTA changes behavior and dopamine activity during reward-seeking. This research furthers our understanding of how the MOR affects natural reward and motivated behaviors and is crucial in helping us understand how opioids of abuse alter existing brain circuitry to cause opioid use disorder.

Plants recognize herbivore-associated molecular patterns (HAMPs) during herbivory that activates signaling to induce immune defenses. Caterpillar oral secretions contain Inceptin 11 (In11) which is a HAMP recognized by legumes such cowpea via Inceptin Receptor (INR). Thus, In11 and INR are a model system to study proteins involved in HAMP induced defenses, including Kunitz trypsin inhibitors (KTIs). It is known that KTIs are serine protease inhibitors with anti-herbivore activity; however, the precise role of In11 induced KTIs and the effect of cysteine content variation in cowpea KTIs remains unknown. Here, we show that selective removal of cysteines has a negative effect on KTI function in cowpea experiencing herbivory from the fall armyworm (Spodoptera frugiperda). We found that cowpea KTIs act as antiherbivore proteins against the fall armyworm when expressed in Nicotiana benthamiana, as we saw reduced weight gain on larvae feeding on leaves expressing wildtype KTI. Furthermore, we found that KTI function was negatively affected by the removal of cysteines, and larvae fed leaves expressing any of the mutant gained more weight than those feeding on wildtype. We hypothesize that these findings are due to reduced protein stability because we did not detect mutant KTIs in frass samples by westernblot. Understanding KTI protein structure and how it influences protein function is important for designing and selecting antiherbivore proteins to be used for plant defense in agriculture.

Existing studies show the importance of migration in ensuring both the livelihood and safety of migrants around the world, and that factors such as ethnicity and caste have a significant impact on an individual’s ability to migrate. My study aims to further explore the association between caste/ethnicity and the order of starting, intermediate, and most recent destinations a migrant passes through. To do so, I perform a sequence analysis on places traveled to by participants of the Chitwan Valley Family Study (CVFS) dataset. CVFS focuses on the Chitwan Valley region in Nepal, a country that hosts a variety of ethnic groups and has a wide range of destinations that its inhabitants migrate towards, with a much higher rate of human migration than other countries of similar population size or GDP. Using the dataset, I also search for common factors between caste groups, such as the kinds of intermediate locations they may travel to before heading to a more permanent destination, and how long certain castes take to migrate either due to legal issues, their own caution or the resources available to them. I expect people of lower caste to face more difficulty in migrating due to how caste profoundly influences social life in the region. This transforms how we view the impact of caste or ethnicity on migration, not just isolated to the Chitwan region but across any region involving the migration of multiple ethnic groups. It is also relevant to note that sequence analysis has little pre-existing use in migration sociology, especially when focusing on migration across multiple continents. Therefore, this study presents a new method of studying migration along with a better understanding of what goes on between the assumed starting and most recent point of a migrant’s journey.

Dark matter is a theoretical form of matter that doesn’t interact with light or conventional matter despite its large expected abundance in our universe. One potential candidate for dark matter, predicted in models, is weakly-interacting long-lived particles (LLP). The ForwArd Search ExpeRiment (FASER), located in the Large Hadron Collider (LHC) at CERN, uses detectors to search for LLP produced in proton-proton collisions. Upon hitting the detectors, particles generate electronic hit signals that are used to reconstruct the decay products of LLPs. Analyzing these tracks may offer insights into the properties and characteristics of LLP. The FASER detector is made of four tracking stations: 1, 2, 3, and interface tracker, each comprising of three layers with eight Semiconductor Tracker modules in each layer. Due to the imprecise installation of these tracking stations, misalignment occurs preventing accurate track reconstruction. To address this issue, I aim to execute an iterative local Chi-square alignment test to determine alignment parameters for each station individually and collectively using previously collected FASER data. I hypothesize that modules will have improved residual values and sensitivity after alignment.

In the face of a global crisis, the poaching of pangolins (Manis spp) has emerged as a dire threat, with Africa at the epicenter of this illicit trade; these enigmatic creatures, sought after for their scales and meat, face exploitation driven by illegal activities that endanger their populations and disrupt the balance of the African ecosystems. Studying the lineage distributions of pangolins could prove crucial for their protection as it provides valuable insights into their evolutionary history, genetic diversity, and ecological adaptations. This information may be essential for formulating effective conservation strategies, understanding their vulnerabilities to diseases, and identifying key habitats for preservation. We aim to address these gaps, by exploring whether pangolin (Manis temminckii) lineages from Mozambique can be genetically distinguished from those from Southern Africa and by investigating the region of origin for two scale samples seized by law enforcement in Singapore in 2019. We conducted genetic and phylogenetic species delineation by sequencing two mitochondrial genes, Cytochrome b oxidase (cytb) and the Control Region (D-loop), from pangolin skin tissues from 10 villages in 3 provinces in Mozambique to complement existing data from Southern Africa. We will then use RAxML (Randomized Axelerated Maximum Likelihood) for phylogenetic analysis and to estimate phylogenetic trees based on our collected data. This multi-locus approach ensures robust species delineation and provides essential support for species recognition. Our focus on mitochondrial DNA (mtDNA) in Mozambique aims to make a reference map of the distributions of genotypes throughout Southern Africa, and our research contributes valuable genetic insights, offering a nuanced perspective on the spatial dynamics of pangolin populations. Ultimately, these findings play a pivotal role in addressing the complex issue of illegal wildlife poaching in Africa, providing essential resources for conservation efforts and informed management strategies as well as contributing to the field of forensic science for counter wildlife trafficking.

Sudden unexpected death in epilepsy (SUDEP) is the most severe consequence of uncontrolled epilepsy. SUDEP is a multifactorial disease associated with serotonin (5-HT) imbalance and exacerbated neuroinflammation. Unfortunately, current preclinical animal models do not adequately explain all underlying causes of these events or their subsequent effects. Seizures are a common comorbidity in Alzheimer’s disease (AD), especially in patients with genetic variants in amyloid precursor protein (APP) and presenilin 1 (PSEN1) and 2 (PSEN 2). Clinical evidence suggests that seizures in AD patients worsen their cognitive decline and increase mortality rate compared to AD patients without seizures. Our lab demonstrated that 2-month-old mice with an APP/PS1 variant subjected to chronic evoked seizures resulted in premature mortality, heightened neuroinflammation, and altered 5-HT system enzyme expression prior to AD onset. These findings reveal a novel preclinical platform to test potential preventative agents for SUDEP. Given the relationship between seizures and AD, I aim to prevent seizure-induced premature mortality, and define 5-HT and neuroinflammatory changes in APP/PS1 mice treated with 2 investigational agents: lorcaserin, a selective 5-HT receptor agonist, and cannabidiol (CBD), a broad-spectrum anti-inflammatory and 5-HT modulator. I hypothesize that targeting seizure-induced neuroinflammation and the dysregulated 5-HT system with these compounds will decrease premature seizure-induced mortality. To assess this, 2-month-old APP/PS1 mice underwent corneal kindling procedure to evoke investigator-controlled chronic seizures and received lorcaserin (10 mg/kg) or CBD (100 mg/kg) via the intraperitoneal route. Then, I tracked survival during the chronic seizure period and performed molecular analysis to quantify neuroinflammatory proteins and 5-HT system enzyme expression. Our preliminary results show that mice treated with lorcaserin or CBD had a mortality rate of 10% compared to 75% in the untreated APP/PS1 mice. Future directions include using these compounds in other preclinical SUDEP models to confirm the translational potential of these medications to clinical use.

Pain is the number one reason why patients seek medical treatment, yet current pain therapeutics such as opioids have limited efficacy and produce harmful side effects. This has produced a critical need for the development of novel therapeutics for the treatment of acute and chronic pain. Cannabidiol (CBD) shows promise as an analgesic, but the mechanism of action is not well understood as it interacts with several receptors such as cannabinoid receptors CB1 and CB2 and noxious nociceptors TRPA1 and TRPV1. I am investigating how CBD acts on the nervous system to disrupt nociception (pain perception) utilizing the Danio rerio model system and human embryonic kidney cell line 293T (HEK 293T). I use behavioral assays with genetic knockout models to interrogate the molecular mechanism of CBD-mediated analgesia and ratiometric Fura-2 calcium imaging of HEK 293T cells that express TRPA1 or TRPV1 to further elucidate their responses to combinations of CBD, heat, and allyl isothiocynate (AITC, a TRPA1 agonist). My preliminary results indicate that CBD is pronociceptive at low concentrations (10uM) and analgesic at high concentrations (20uM). Recent experiments suggest that CBD’s pronociceptive properties occur via TRPA1 activation, and that this sensitization attenuates CBD-mediated analgesia. I anticipate each CBD receptor knockout will alter CBD-mediated analgesia, with CB1 and CB2 null animals experiencing deficits, while CBD-evoked analgesia may be potentiated in TRPA1 and TRPV1 null animals. I anticipate observing heightened intracellular calcium concentrations when HEK293T cells expressing TRPA1 are perfused with CBD, and increased responses to AITC when cells are perfused with CBD. Importantly, this project creates a platform for the investigation and characterization of minor cannabinoids and other potential therapeutics, using behavioral phenotype based screening to aid in the development of novel, non-opioid analgesics that can revolutionize pain treatment.

The Tacoma-Pierce County Health Department assessed six communities in Tacoma and on the peninsula that are facing limited opportunities for optimal well-being; the objective is to investigate health care quality and efficiency. These communities are called Communities of Focus (CoF). Our work explores state data to evaluate the relationship between hospital length of stay (LOS) and cause-specific hospital readmission rates (CSR) in CoF. The current literature has not reached consensus on understanding the designated relationship. Applying a multivariate logistic regression model, we explored the relationship between length of stay and CSR using data from the Comprehensive Hospital Abstract Reporting System (CHARS). Several covariates including race and gender were included in the regression model. One extra day of hospital stay resulted in a 0.5% higher risk of 30-day CSR [Odds Ratio (OR): 1.005, CI: 1.00–1.01]. The control factor substantially increasing risk of readmission, was age 55-65 years (OR: 3.477, CI: 3.19–3.79). The covariate significantly reducing CSR was being female (OR: 0.816, CI: 0.80–0.83). Individuals of Indian race (OR: 1.120, CI: 1.04–1.20, p <0.05) and Hawaiian race (OR: 1.157, CI: 1.06–1.26, p <0.0001) were more likely to experience CSR compared to White patients. Finally, CoF faced higher risk of CSR (OR: 3.973, CI: 3.77–4.19) relative to other regional patients. CSR is re-admission to the healthcare setting due to a complication or an exacerbation of disease. A single day hospital stay yielding higher risk for CSR suggests that further exploration is needed to understand patient experiences while admitted. Additionally, an investigation into the social, economic, environmental and personal factors placing CoF at risk for CSR is warranted in order to reduce risk. Our research identifies the problem; future work is needed to elucidate the source so that approaches can be identified to lower CSR for CoF.

Staphylococcus aureus is an important human pathogen that has increasingly developed resistance to antibiotics and antimicrobials. It is important to understand how genetic mutations influence antibiotic resistance to anticipate how the organism is able to evolve and combat emerging resistance. My project aims to fully characterize the impact of variants in the norA gene, which is associated with efflux-mediated resistance. The Salipante lab has developed a system for precision genome engineering in S. aureus by constructing a vector that is able to conduct recombineering while suppressing DNA repair, and a separate vector for killing unmodified bacteria using programmed CRISPR/Cas9. Using this system, I will construct a library of genetic mutations in the S. aureus genome that will encompass all possible single mutations in norA and to subsequently test the fitness costs of those mutations in the presence and absence of antibiotics.We will recombineer into S. aureus randomly mutagenized oligonucleotides that encode a silent mutation that renders transgenic bacteria immune to targeted CRISPR/Cas9 cutting. After killing off unmodified bacteria, the transformed population will be composed entirely of different mutants, so that with a large population size, we can statistically ensure that all possible random mutations are represented. We will analyze the gene sequence from the initial population, after the population has expanded in the absence of drugs, and after exposure to different concentrations of drugs, to compare the relative representation of each specific mutation. Changes that are detrimental under a condition will be underrepresented relative to the starting population, while those that are beneficial will be overrepresented. Mutations that are lethal will not be recovered in the first place. Our project has the potential to fully and comprehensively address clinically important questions regarding antibiotic resistance evolution in this gene.

While epilepsy can affect anyone at any age, it is roughly three times more prevalent in people over age 65. Prevalence and incidence of epilepsy will rise as the global population becomes increasingly older. Epilepsy in older people is associated with comorbidities such as neurodegeneration. We have previously demonstrated that administration of the antiseizure medicine (ASM), lacosamide (LCM), to young, corneally kindled mice was associated with increased levels of Ki-67 positive/NeuN-positive neurons relative to vehicle (VEH)-treated kindled or untreated sham-kindled animals in the dentate gyrus of dorsal hippocampus of the brain, suggesting beneficial effects of LCM on neurogenesis in young mice with chronic seizures (Zierath et al, IJMS 2023). We thus hypothesized that LCM administration to aged wild-type mice with chronic seizures could increase the number of Ki-67 positive/NeuN-positive hippocampal neurons, indicating neurogenesis. To test this hypothesis, I randomized mice aged >30 months-old to receive either LCM (4.5 mg/kg) or VEH treatment administered prior to each seizure stimulation. Mice were then kindled with a twice-daily 3 second, 60 Hz transcorneal stimulation over 15 days. After 27 stimulations to evoke consistent behavioral seizures, mice were euthanized, and brains collected for immunohistochemistry. Tissues were labeled for Ki-67, marking for neurogenesis, GFAP for astroglial cells, and NeuN, for mature neurons. Unlike in similarly treated young, kindled male mice, LCM administration significantly slowed male mouse kindling acquisition rate (p=0.0022) compared with VEH-treated littermates. There was no significant effect of LCM administration on kindling rate in aged female mice. Further, LCM was poorly tolerated in aged male but not female mice, revealing significant sexual dimorphism in ASM tolerability with chronic administration to aged mice. Ongoing immunohistochemistry is quantifying Ki-67 expression to further demonstrate whether LCM administration induces hippocampal neurogenesis in aged mice with chronic seizures.

Artificial intelligence has made significant progress in image classification, an essential task for machine perception to achieve high-level image understanding. Despite recent advances in vision-language fields, image classification is still challenging because classes are inherently multimodal. Traditional approaches provide solely images or descriptions of a given class, but this does not properly encapsulate the entire concept. I aim to understand if utilizing multiple modalities when classifying images will allow for more robust and generalizable image classification. Here, I leverage Ensemble Learning to form a weighted sum of the embeddings produced by my classifiers. The ensemble consists of an image-to-text classifier that utilizes generated class descriptions and an image-to-image classifier that uses generated images for prediction. My results show that this improved conceptual representation from the multimodal approach directly results in higher classification accuracy. Additionally, this approach allows for these improvements without any additional context, training, or fine-tuning, meaning this method is completely zero-shot. Improved image classification directly impacts a myriad of fields, and enhances how humans use computing technology. Whether it be more accurate diagnoses in medical imaging or robust crop monitoring in agriculture, improvements in image classification extend across disciplines and aid the quality of life for all.

Traumatic spinal cord injury (tSCI) is a devastating condition that causes sensory and motor dysfunction and permanently impairs normal life. Spasticity is one of the most common complications associated with tSCI that limits independent functional abilities. Spasticity is defined as a velocity-dependent increase in muscle tone, in response to passive movement and it is accompanied by pain and stiffness. Unfortunately, current treatments provide only transient and often incomplete relief of spasticity and individuals often experience long-term adverse effects. Through a collaborative project between three labs, we aim to develop a durable non-invasive electrical stimulation treatment to alleviate spasticity. I participated in preparing the model of spasticity by performing spinal surgeries on the cervical spine of rats. To evaluate spasticity, we studied the loss of Rate-Dependent Depression (RDD) of the H-reflex which is considered the electrophysiological hallmark of spasticity. To do so, I fabricated an electrode nerve cuff that was implanted on the median nerve of the rodent’s forearm to study the H-reflex of the affected muscle in the rat’s forelimb. I then recorded and analyzed the temporal development and change of spasticity. H-reflex results validated the spasticity model by showing RDD reduction in injured rats compared to the uninjured rats. The developed treatment shows promising modulation of the H-reflex and recovery of RDD in injured animals. Additionally, to measure velocity-dependent muscle tone, we developed a robotic device that passively moves the rodent’s forearm at different velocities. Employing this robotic behavioral assessment allows me to objectively assess the effect of stimulation on spasticity in the rodent forelimb. Obtained data reveals the muscle resistance to be three times higher in the injured rodent. This novel therapeutic stimulation protocol could potentially be used for clinical use as a non-invasive therapy, to help patients with spasticity in the hand after suffering from cervical tSCI.

The mental health of family caregivers who care for elders who have passed is often dismissed by healthcare providers and society because of the age of their family members. When an older adult dies, their death is seen as expected and as the natural ending to having lived their life. However, there is still a tremendous grieving process that family members, friends, and caregivers experience with the death of a loved one. After losing a loved one, family caregivers report experiencing higher rates of depression, anxiety, and ambiguous grief. Additionally, many family caregivers find that medical help decreases drastically after the death, thus creating a sense of isolation. Current studies tend to explore one aspect of the experience of caregiver loss. Furthermore, much of these findings are based on data that is homogeneous in race and social status with an overwhelming majority being white and middle class. No existing studies have attempted to untangle the complexity of emotions that come with family caregiving. This systematic literature review starts to fill these gaps by answering the question: What are the psychological impacts experienced by family caregivers following the loss of an elderly loved one they provided care for? The databases used to identify the articles include PubMed, Web of Science, and PsycINFO. Approximately 15 articles have been identified that meet the inclusion criteria. The preliminary findings are themes of support changing before and after death, relationship with caregiving, relationship with family members cared for, psychological adversities (anxiety, depression, and loneliness), and religion as a way to find peace. These findings will help family caregivers see trends in their experience, helping with the loneliness that comes while grieving. It will also be a starting point to show healthcare workers what to look for when determining whether the grieving process is chronic.

The placenta is a crucial fetal organ providing oxygen and nutrients to the developing infant. Placental cell models, which are derived from immortalized or placental cancer cells, are typically used to study the organ. The use of placental cell models is important because human samples are difficult to obtain, and placental physiology is highly species-specific. However, our understanding of these models and how they compare to placental tissue samples is limited. This project aims to determine which placental cell model most directly reflects the gene expression of the human placenta. We obtained twenty-eight RNA sequencing datasets from the HTR-8/SVneo, JEG-3, JAR and BeWo placental cell models as well as human placental villous explants and primary trophoblast cells using the Gene Expression Omnibus database. Fetal sex was determined by quantifying expression of the Y-chromosome for each of the models. From this analysis we identified that HTR-8/Svneo was of female origin, while JEG-3, JAR and BeWo was of male origin. A clustering analysis was also conducted which identified groups of genes that showed similar expression profiles across the groups of cell lines and placenta tissue. This was subsequently used within a pathway analysis to identify which biological pathway defined the cluster. Pathways are chains of reactions leading to products or changes in a cell. The analysis showed at 22 of the 53 clusters were enriched for 1 or more pathways, which helps provide insight into the biological functions of these clusters and indicates biological processes that may be different between these models. With this information we have created an interactive web application. This site allows users to search a given gene and identify the expression data across all the models. This tool aims to provide a resource to the placental biology research community in further investigations of the placenta.

Eukaryotic DNA is wrapped around nucleosomes to be packaged into a cell’s nucleus. Nucleosomes are made up of a combination of canonical histones (H2A, H2B, H3, and H4) and histone variants. Histone variants are evolutionarily derived from their canonical counterparts and can replace canonical histones to perform specialized chromatin functions. Given their crucial and widespread functions, mutations of histone genes are correlated with poor prognosis in cancers. Histones and their variants are typically evolutionarily conserved in sequence and function. Therefore, lineage-specific differences in histone repertoires present unique opportunities to understand their functional consequences on genomic organization and biological processes. Here, we study two such changes in the H2A repertoires of budding yeast and fruit flies. Most eukaryotes including humans have an H2A repertoire of canonical H2A and two variants– H2A.X important for DNA damage response (DDR) and H2A.Z essential for gene regulation. However, in yeast, H2A.X entirely replaced H2A, and in flies, H2A.X and H2A.Z are fused into a single variant H2Av. We take two approaches to discover the adaptive advantages of these changes. First, we are studying the evolutionary origins and diversification of H2A repertoires in yeast. We find that many basally branching fungi have a canonical H2A, suggesting that only some yeast have lost canonical H2A. Second, we are recreating the fly and human H2A repertoire in S. cerevisiae. We find that while yeast with a fly-like H2A repertoire have a DDR, it is dramatically reduced compared to wild-type yeast. This raises the intriguing possibility that a fly-like repertoire might lead to a trade-off of compromised DDR in fly genomes. We are now analyzing how DDR-dependent processes like meiosis are altered in fly-like yeast. By leveraging the power of evolution and yeast genetics our work will reveal the biological consequences of unexpected histone innovation.

Alphaviruses are arthropod-borne viruses that are responsible for febrile illness, chronic arthralgias, and premature deaths worldwide. Yet, there are no existing vaccines or therapeutics for the treatment of alphaviral diseases. Despite the limited size and coding capacity of alphavirus RNA genomes, most alphaviruses can adapt to multiple, evolutionarily divergent vertebrate and insect host species. As such, alphavirus RNA genomes and proteins carry host-specific adaptive features to compensate for the differences between hosts, such as body temperature (28°C in insects vs. 37°C in vertebrates) and methods of host immune response. Previous experiments have shown that continuous passaging of the dual-host alphavirus Sindbis virus (SINV) in Adedes albopictus (C6/36) cells results in a gain of fitness in said cells and a loss of fitness in human embryonic kidney (HEK293T) cells, resulting in a mosquito-adapted SINV (SINV^M). Using a modified long-read sequencing method (MrHAMER), we identified an assortment of fixed mutations that were serially acquired over the course of mosquito cell adaptation. Interestingly, multiple synonymous mutations were mapped to the 5' end of the SINV RNA genome, which is known to adopt functionally important RNA structures necessary for virus replication and genome packaging. Additionally, non-synonymous mutations were acquired within the viral structural genes. In this study, I aimed to understand the functional consequences of these two classes of SINV^M mutations with regards to viral fitness in insect and vertebrate cells. Furthermore, I assessed if the phenotype of these mutants were influenced by host body temperature by incubating the infected HEK293T and Vero cells at both 28°C and 37°C. We envision that studying these mutations will allow us to better understand the selective pressures influencing alphavirus evolution and potentially identify host-specific viral determinants of infection. Ultimately, this knowledge will allow us to identify ways to intervene at different stages of the alphaviral transmission cycle.

 During gestation, a human fetus is fully dependent on the placenta which provides oxygen, nutrients, regulates waste transport, and acts as an immunological barrier. Glial Cells Missing Transcription Factor 1 (GCM1) is a transcription factor (TF) that plays a crucial role in placental development. Our goal is to understand the downstream effects of GCM1 knockdown on genes necessary for placental development. The BeWo choriocarcinoma cell line is a model of placental syncytiotrophoblasts cells which undergo a cell fusion process called syncytialization to form multinucleated cells that help exchange nutrients. BeWo cells were treated with Forskolin (FSK) to induce syncytialization to represent syncytiotrophoblast cells. RNA sequencing was conducted after GCM1 knockdown using siRNA to quantify downstream gene expression changes. BeWo cells were treated in 4 different conditions: FSK, GCM1 siRNA (GCM1 KD), FSK treatment first then GCM1 siRNA treatment (FSK + GCM1 KD), and lastly, GCM1 siRNA treatment first and then FSK treatment (GCM1 KD + FSK). In the GCM1 KD group, a total of 533 genes exhibited significant differential expression (FDR < 0.5), when compared to control BeWo cells, with 236 genes upregulated and 297 genes downregulated. Subsequently, in the FSK + GCM1 KD group, 961 genes were significantly altered, where 386 were upregulated and 575 were downregulated. In the GCM1 KD + FSK group, a notably higher number of genes, 7,165, displayed significant differential expression, with 3527 genes being upregulated and 3638 genes being downregulated. We also identified a number of biological pathways such as focal adhesion and adherens junction that may be crucial to GCM1-induced syncytialization. Overall, our results will improve our understanding of how GCM1 coordinates gene expression in the placenta during pregnancy.

Vertical velocities are a fundamental component of ocean flow and are vital to characterizing global circulation. However, vertical velocities are small compared to horizontal velocities and are thus difficult to measure. Previous studies attempting to estimate them ignore the impacts of topography, mesoscale eddies, internal waves, and spatial variability. Novel estimates from the Argo float array allow for direct estimates of vertical velocities. This project will focus on comparing these new Argo estimates with vertical velocity observations from moorings in the Southern Ocean. The Southern Ocean is an important site of vertical volume transport for mass ocean circulation with global implications, particularly the Antarctic Circumpolar Current which dynamically links many of these interactions. We expect vertical velocity characterized by moorings to maintain coherency with Argo float estimates. Differences may occur, however, due to mismatches in spatial resolution between Argo-based estimates and mooring-based estimates, which rely on mass conservation across larger scales. In comparing novel Argo datasets to known mooring values, we gain a more complete understanding of vertical velocities in the Southern Ocean which have direct implications for data assimilation in models and parameterization of energy pathways.

Science is rapidly evolving, yet its advances do not enter classrooms at the same rate. Systems Education Experiences (SEE) is a program in the Baliga Lab at the Institute for Systems Biology (ISB) that accelerates the transfer of scientific knowledge and practices to classrooms. One active area of Baliga Lab research is elucidating the level of resilience organisms have, when faced with complex environmental changes. My role is to design laboratory experiments that investigate this with the model organism Halobacterium salinarum (Halo) and to connect this to broader rules governing natural systems for use in high school classrooms. My first experiment probes the resiliency of Halo with the introduction of a combination of stressors (salt and hydrogen peroxide) and its recovery after population collapse. The second measures the long term phenotypic changes in the population. I wanted to see if after being exposed to a new environment if there was an advantage to having gas vesicles and if it is an irreversible trait that allows Halo to be resilient across a variety of environmental conditions. This relates to broadly applicable rules governing resilience across many systems. This project serves as a model for how all organisms respond to stress. Combinations of stressors in human lives can make us less resilient. However, strategies to quickly prepare, respond, and react can improve outcomes for individuals and the overall population. This project connects to a goal of K-12 science education which is to not just teach academic concepts but to equip students with knowledge that can be applied to all parts of life. Our knowledge on the mechanisms that control how organisms respond to stress is extremely limited. By understanding the biological stress response we can promote resilience in the earth's most vulnerable systems in the wake of climate change.

In order to design effective countermeasures against HIV, we must first understand the forces that drive it to evolve resistance within hosts. While linkage patterns in genetic data are potentially a powerful tool to quantify the relative contributions of multiple evolutionary forces (mutation, recombination, selection) acting during an HIV infection, the severe viral population bottlenecks accompanying drug therapy complicate these patterns. To interpret genetic linkage in the context of such major changes in population structure (which are themselves driven by specific mutations), we develop a simulation framework for viral evolution in which genetics and population structure influence each other. This framework overcomes limitations from both dynamical modeling, in which patterns of linked variation are ignored, and from population genetic modeling, in which population structure is predetermined. Using few parameters, we are able to reproduce linkage patterns and population bottlenecks that broadly conform to those observed in vivo. As a case study to demonstrate this model’s utility, we consider a recent hypothesis that viral recombination is suppressed during population bottlenecks due to diminished opportunities for coinfection. In simulating populations with and without recombination suppression during population contraction, we show that this effect measurably changes genetic diversity in rebounding populations, but is less visible when examining simulated viral loads or resistance mutations alone. Then, using this model as a null expectation for linkage patterns, we assess if the linkage structure in HIV populations treated with bNAbs is consistent with density-dependent recombination in vivo. Collectively, our work demonstrates that, by generating realistic null expectations of linkage under complex changes in population structure, we can employ linkage patterns as a powerful source of information for evaluating viral evolutionary hypotheses.

Spasticity is an increase in muscle tone (hypertonus) and abnormal muscle stiffness that impedes functional activity. Oftentimes observed among individuals with chronic neurological conditions such as traumatic brain or spinal cord injury (SCI), spasticity develops as a result of damage to the central nervous system (CNS). This damage disrupts the balance of supraspinal inhibitory and excitatory inputs to the spinal cord, which can lead to the loss of inhibitory inputs and hyperexcitation of the spinal reflex arc. The aim of this project is to develop an electrical stimulation protocol that regulates imbalances of supraspinal input and the spinal reflex in order to potentially alleviate spasticity caused by traumatic neural injury in patients. The hyperexcitation associated with spasticity is measured using the Hoffman-reflex (H-reflex). Previous studies have revealed that electrical stimulation of the rat motor cortex can modulate long-term spinal excitability. In this study, behaving noninjured Long Evans rats are implanted with cortical implants to induce stimulation to the motor cortex, grounding electrodes to filter environmental noise, cuff electrodes to evoke the H-reflex, and EMG electrodes to record the H-reflex response. The H-reflex is assessed by stimulating a cuff electrode surrounding the median nerve and measuring the consequent activity of EMG electrodes that are implanted into the flexor, extensor, and tricep muscles before and after electrical stimulation of the motor cortex. Our preliminary results indicate that different frequencies of cortical stimulation can modulate the H-reflex, suggesting that our novel cortical stimulation protocol may reduce spasticity and promote restoration of motor function. In future studies, I plan to assess the efficacy of cortical stimulation for improving spinal excitability in spastic animals following chronic SCI.

Most eukaryotes use histones to package the genome. However, many animals package their sperm genomes using specialized DNA-binding proteins called protamines, which package DNA in sperm more tightly to fit inside the sperm head. Based on the transcriptional silencing role of protamines, we hypothesize that protamines can suppress meiotic drivers, which kill other sperm to bias their own transmission. Previously, we found that one protamine gene, Mst77F, is required to suppress meiotic drivers on the Y-chromosome in Drosophila melanogaster. Since drive is generally deleterious for the transmission of autosomal alleles (due to lower male fertility for example) theory predicts that multiple suppressors of drive will arise in populations; Mst77F may represent just one such suppressor. We hypothesized that multiple natural variants in distinct genetic loci interact with and impact meiotic drive in Drosophila melanogaster. To identify these natural variants, I crossed wildtype flies to knock out flies and generate hemizygous Mst77F flies carrying genetic backgrounds from four different populations. I measured the fertility and drive strength by crossing a single hemizygous male from each cross to five wild type females. In all cases, I found that the sex ratio was skewed to favor male offspring, indicating they all carry X-linked targets. However, I did not identify any dominant genetic variation associated with the drive strength, indicating Mst77F might be the major suppressor of this drive. I am conducting reciprocal crosses to determine whether Y chromosomes from different populations carry the same strength of drive. In the future, I will extend my analyses to other genetic backgrounds. My study contributes to a better understanding of the pervasive effects of meiotic drive in natural populations and unexpected functions of protamines.

Graph workloads are challenging for query optimizers in databases because of query features like larger sizes, frequent joins, and fewer filters. Traditional methods see large errors on queries with more joins, while machine learning methods tend to be complex and slower. We propose a framework to improve estimators by using graph colorings to make compact summaries of a data graph, storing important information about node relations. By modelling cardinality estimation as a subgraph matching problem, we can make use of this summary information and traverse the lifted graph to estimate the number of query graph matches. Additionally, we explore optimizations such as node summation and sampling to enable estimation even for larger queries. After evaluating various designs using this framework, we find improvements up to 100x to cardinality estimation accuracies compared to other recent methods while still maintaining efficient runtimes and memory usage. We discovered that quasi-stable colors, where nodes of one color have similar connections to other colors, result in these improved results when used to build the summary. These findings help improve graph database performance and offer a new application for graph theory.