People with diabetes have an increased risk of developing cardiovascular disease (CVD). Hyperglycemia is the hallmark of diabetes, but diabetic dyslipidemia with increased circulating lipid levels is also present, which is believed to contribute to the augmented CVD seen in diabetes. The Cluster of Differentiation 36 (Cd36) receptor mediates fatty acid and lipoprotein uptake in macrophages. Lipid-loaded macrophages are a key feature of atherosclerosis, the underlying CVD pathology. Preliminary data suggest that monocytes (macrophage precursors) are lipid-loaded via increased Cd36. However, it is unclear what drives the increased Cd36 expression in diabetes. Reduction in blood glucose, but not lipid levels, in diabetic mice reduced monocyte cell surface Cd36 expression. Based on these preliminary data, I hypothesize that hyperglycemia induces increased Cd36 mRna expression in monocytes in diabetes. To address whether glucose or lipids increase monocyte Cd36 mRna expression, I will isolate monocytes from the bone marrow of non-diabetic mice and stimulate them ex vivo. To address if hyperglycemia alters Cd36 expression, I will stimulate monocytes with 4 glucose conditions: 5.6 mM, 15 mM, 30.6 mM D-glucose, and an osmotic control of 5.6 mM D-glucose and 25 mM L-glucose. The 5.6 mM represents non-diabetic blood glucose conditions. To address if dyslipidemia alters monocyte Cd36 expression, I will use the same 4 glucose conditions in conjunction with 50 µg/mL of VLDL, a triglyceride-rich lipoprotein that is elevated in diabetic dyslipidemia. Following a 24-hour stimulation, I will isolate monocyte RNA and use qPCR to determine the amount of Cd36 mRna. If elevated glucose induces an increase in Cd36 expression, this suggests that hyperglycemia stimulates increased Cd36 expression in monocytes in diabetes. However, if the presence of VLDL is required to observe an increase in Cd36 mRna, this indicates that dyslipidemia is needed for increased monocyte Cd36 mRna expression. Results from this study will help us understand the relationship between lipids and hyperglycemia in the context of diabetes-induced monocyte lipid loading.

Over 38 million Americans have diabetes, with 90% of diabetic Americans having Type 2 Diabetes. Diabetes causes an increased risk of complications, including diabetic kidney disease (DKD), a disease that affects kidney filtration. This occurs in the glomerulus, a specialized capillary network of single-layered endothelial cells on one side and podocytes on the other, and their extracellular matrix (ECM). Injury, reduced function, or changes in the ECM of either of these cells cause abnormal filtration and kidney disease. Preliminary data from our group suggests that ECM remodeling driven by endothelial cells is a key feature in DKD in both our mouse model and in humans. Additional preliminary data from our lab indicates that two metalloproteases involved in ECM remodeling, ADAMTS6 and ADAMTS9, are increased in endothelial cells in diabetes via elevated lipids. My hypothesis is that very-low-density lipoprotein (VLDL), a lipid often increased in diabetes, induces increased endothelial cell ADAMTS6 and ADAMTS9 expression, contributing to remodeling the ECM and altering the filtration capacity of the glomeruli. To investigate this, isolated endothelial cells from non-diabetic mice were stimulated with varying VLDL concentrations, alone or with elevated glucose, to simulate diabetes. Following the stimulation, I isolated the RNA from these cells and measured the mRNA expression of Adamts9 and Adamts6 using real-time PCR. To test if endothelial cells isolated from mice with diabetes would respond differently, similar experiments are being carried out in cells isolated from diabetic mice. Western blots are used to verify the altered protein expression I observe. I am utilizing already acquired kidney sections from mice with diabetes with different lipid levels and determining the glomerular endothelial cell ADAMTS9 and ADAMTS6 expression by immunohistochemistry. Results from these experiments will help us understand the mechanisms through which endothelial cells respond to diabetes and thus contribute to DKD.

Treponema pallidum subspecies pallidum (T. pallidum) is growing in incidence in high-income countries like the United States and remains endemic and highly prevalent in low-income countries, primarily sub-Saharan Africa, and South America. Despite being treatable, syphilis is associated with significant fetal and perinatal mortality in low-income settings, due to congenital transmission of the infection. Improving our understanding of syphilis pathogenesis, immunology, and T. pallidum biology, could result in novel measures to curtail syphilis spread, including improved diagnostics, novel therapeutics, and a preventive vaccine. We are exploring the use of a chimeric antigen, composed of a scaffolding/carrier protein of T. pallidum and a series of protective epitopes from other T. pallidum antigens previously described and patented by our laboratory, to be used as a recombinant vaccine. The first step to using this protein as a vaccine scaffolding is determining the most immunogenic sequences of the protein to be used for replacement. Specifically, we used sera from 63 patients with syphilis at different stages, and sera longitudinally collected from rabbits infected with either the Nichols or SS14 isolates of T. pallidum, which represent the model strains for the two known clades of this pathogen. Recognized amino acid sequences were then mapped to the experimentally determined Tp17 structure. Reactive epitopes in both serum groups mapped predominantly to the α-helix preceding the Tp17 soluble β-barrel and to the loops of the barrel. We are currently using the same Enzyme-Linked Immunosorbent Assay (ELISA) to assess reactivity levels in control sera of naive human patients. These results identify sequences of the Tp17 antigen that could be replaced by protective epitopes. Our work provides the basis for future research on the use of this scaffolding to develop a syphilis vaccine able to confer protection from this this serious infection.

Adoptive Cell Therapy (ACT) is a novel modality of cancer therapy, in which immune cells can be engineered with T cell receptors (TCRs) to aid in targeting specific antigens presented on the surface of cancer cells. ACT has already brought curative therapy to many previously treatment-refractory blood cancers. However, TCR-T cells often have limited persistence after transfer into patients, which has hampered the effectiveness of this therapy for solid tumors, which require a sustained anti-tumor response. I evaluated if functional improvements could be induced by a small molecule inhibitor of LSD1 (LSD1i), a histone lysine demethylase that modifies chromosome accessibility, as a strategy to improve long-term activity against tumor cells after infusion into patients. Previous research has also found that “helper” CD4+T cells enhance the functionality of cytotoxic CD8+ cells, and I am now seeking to assess broader activity and benefits of LSD1i on a combined population of CD4+ and CD8+ cells. I first treated CD8+ T cells with a small molecule drug, Bomedemstat (an inhibitor of LSD1) during the in vitro cell expansion process, and demonstrated enhanced function in a co-culture system that models chronic stimulation as would occur in a solid tumor. To assess activity of Bomedemstat on CD4+ T cells, I used lentiviral transduction to insert functional TCR constructs into both CD4+ and CD8+ T cells, and plan to expand these cells with the optimized concentration of Bomedemstat in a second coculture assay, this time with combined CD4+ and CD8+ populations. I expect Bomedemstat will prevent dysfunction similar to our previous results with a LSD1i in CD8+ T cells, as illustrated by improved tumor killing and changes in T cell phenotype and function. Demonstrating applicability of LSD1i pretreatment on combined T cell populations would establish a stronger foundation to advance this epigenetic perturbation to clinical applications.

Dilated cardiomyopathy (DCM), a heart muscle disease characterized by ventricular enlargement and disorganized sarcomeres, stands as a leading cause of heart failure in young adults. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) can be used to study diseases with genetic components such as DCM which can be caused by mutations in structural components of the sarcomere. Missense mutations in the myosin heavy chain 7 gene (MYH7), encoding for the myosin heavy chain beta isoform (β-MHC), are the third most common genetic cause of DCM. However, the mechanism by which MYH7 variants lead to sarcomere disarray is poorly understood. In this study, I developed a novel assay to interrogate sarcomere incorporation and turnover dynamics in hiPSC-CMs. CRISPR/Cas9 genome editing was used to knock-in mEos, a green-to-red photoconvertible fluorescent protein, into the endogenous MYH7 locus of hiPSCs to visualize sarcomeric β-MHC incorporation and turnover. Upon differentiation of transduced hiPSCs, MYH7-mEos fusion protein is expressed and integrated in the sarcomeres of hiPSC-CMs. Pre-photoconversion, these cells exhibit green fluorescent sarcomere striations, transitioning to red fluorescence upon UV irradiation. Tracking green signal recovery (incorporation) and red signal depletion (turnover) over time post-photoconversion allows for quantification of sarcomere kinetics in these hiPSC-CMs. Through preliminary experimentation, I revealed a steady state fluorescence recovery and depletion of 40% in wildtype MYH7-mEos hiPSC-CMs. Future work is aimed towards using this novel strategy to analyze sarcomere dynamics of pathogenic hiPSC-CMs, thus allowing for the discovery of how and which of these rates differ across various MYH7 variants. I hypothesize that in hiPSC-CMs derived from a DCM patient expressing the MYH7 E848G variant, decreased incorporation or increased turnover rates may contribute to sarcomere disarray leading to a disturbance in sarcomere homeostasis among patients with this disease.

Syphilis remains a serious global health concern, underscoring the need for better control strategies. If left untreated, the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), can persist for decades due to its ability to evade the host immune response. Antigenic variation of the surface-exposed outer membrane protein TprK is believed to mediate persistence. TprK contains seven discrete variable (V) regions. TprK antigenic variation occurs via non-reciprocal gene conversion between the variable regions in the tprK expression site and 53 donor cassettes (DCs). We previously engineered a T. pallidum strain impaired of antigenic variation (SS14-DC^KO) by eliminating 51 of the 53 DCs. Rabbits infected with the DC^KO strain developed an attenuated infection phenotype with a reduced burden of T. pallidum cells compared to wild type (WT). Therefore, we hypothesized that if immunosuppressed rabbits are infected with SS14-DC^KO, the disease would undergo similar progression to rabbits infected with the WT strain. In this study, two rabbit groups (n=8) were either immunosuppressed with Depomedrol or untreated. Four rabbits in each group were infected intradermally with SS14-WT or SS14-DC^KO strain on clipped backs. Sera was isolated weekly to measure antibody titers using VDRL and TPPA. In addition, DNA was isolated from lesion biopsies to perform TprK profiling. Based on TprK profiling data, synthetic peptides for the most abundant V5, V6, and V7 variants were used to measure antibody reactivity by ELISA. Antibody titers measured by TPPA and VDRL were similar between immunosuppressed rabbits regardless of infecting strain. TprK profiling shows that the most abundant V sequence at the inoculum decreases over time in lesions as humoral reactivity to these peptides increases. Overall, this research demonstrates the role that TprK plays in the persistence of T. pallidum during syphilis infection and the need for novel control strategies.

Phagocytosis is a primary function of macrophages that facilitates the elimination of pathogens and the clearance of endogenous and exogenous debris from tissues. This function can become impaired by aging or exposure to environmental toxicants, which may contribute to impaired immune responses and wound healing. To determine the involvement of macrophage phagocytosis in different pathophysiological conditions, we developed an in vitro macrophage phagocytosis assay with lung macrophages and brain microglia from CD-1 mice (three male, three female). The tested groups included an untreated control group, a lipopolysaccharide (LPS)-treated positive control group to increase phagocytosis, a cytochalasin D-treated negative control group to inhibit phagocytosis, and an experimental pioglitazone-treated group to stimulate PPAR-g (nuclear hormone receptor and transcription factor that has been shown to stimulate phagocytosis). We used green fluorescent protein (GFP)-labeled dextran beads opsonized with serum to assess the phagocytotic abilities of the macrophages in each treatment group. We are currently analyzing data that will determine the percentage of macrophages that phagocytosed the beads (GFP+ macrophages) and the average number of beads phagocytosed among GFP+ macrophages per treatment. We predict that there will be fewer GFP+ macrophages in the cytochalasin D-treated group vs. controls, indicating that phagocytosis is properly inhibited. We predict that both LPS and the pioglitazone-treated macrophages will present more GFP uptake and GFP+ cells than the control. A comparison of pioglitazone and LPS uptake will indicate the relative efficacy of pioglitazone in enhancing phagocytosis. Because pioglitazone is thought to stimulate phagocytosis without causing a pro-inflammatory response like LPS does, pioglitazone treatment may be a way to improve the helpful functions of macrophages with impaired phagocytic functions, which can occur with aging or following exposures to environmental toxicants.

Alzheimer’s disease and related dementias (ADRD) are neurological disorders that affect cognitive function, including impaired memory, thinking, and reasoning, primarily in older adults (OA). Cultural and linguistic differences can pose additional challenges. The experience of ADRD differs between patients from diverse backgrounds, and language barriers can create additional challenges when communicating with healthcare professionals. Our overall objective is to improve healthcare processes for persons with ADRD and limited English proficiency (LEP) in the United States. In this phase of a larger qualitative study, we are seeking to understand the experiences of older adults experiencing dementia whose primary language is Chinese and their family members. To understand the experiences of this population, we are conducting semi-structured interviews with OA with LEP and ADRD and their family members. I am performing qualitative data analysis along with three other team members. Data gathered from these interviews is transcribed and coded using a codebook developed using the inductive method. In this poster, I present key themes that are emerging in our preliminary analysis. There are significant barriers in patient-provider interactions between healthcare providers, OA with ADRD, and their family members, in the context of LEP. A complex dynamic is formed as providers seek to convey information to the OA and family members who play different roles in an OA’s life, including but not limited to acting as interpreters. OAs struggle to adjust to their progressive symptoms as they lose autonomy over time while navigating life with ADRD. Language barriers, lack of understanding of cognitive decline, and denial can also exacerbate problems. By identifying challenges experienced in patient-provider communication in the context of ADRD and LEP, we hope to work with healthcare providers to equip them with a better understanding of how to serve this demographic.

Influenza A virus causes highly prevalent and pathogenic respiratory infections that regularly rank among the top ten causes of death in the U.S. annually. It propagates by co-opting the host’s Raf/MEK/ERK signal transduction pathway. Pharmacological inhibition of the Raf/ MEK/ERK cascade leads to the retention of viral ribonucleoprotein complexes (RNPs) in the nucleus. Impaired RNP export then results in reduced viral propagation. The protein kinase family MEK predominantly appears in animals as the isoforms MEK1 and MEK2. While it is known that MEK inhibitors interfere with influenza virus propagation, it is unknown whether this is due to the inhibition of MEK1, MEK2, or both isoforms. To investigate the dependency of influenza A virus disease and viral proliferation on MEK2, we infected MEK2-deficient mice and their wild-type controls with a mouse-adapted influenza A virus (H1N1, PR8). We then conducted clinical scoring of mice and monitored their weight daily for two weeks after infection. MEK2-deficient mice demonstrated improved weight loss recovery and higher fitness during the influenza A infection period compared to wild-type mice. Additional studies are currently being conducted to determine if reduced viral loads account for the improved recovery of MEK2-deficient mice compared to wild-type mice. This work has important implications for using MEK inhibitors to treat pneumonia caused by influenza A virus.

Mycoplasma genitalium (MG) is a sexually transmitted bacteria that causes urethritis in men and cervicitis, pelvic inflammatory disease, and infertility in women. MG infections vary in length: some infections are cleared within a few weeks, while others last for years and are difficult to treat due to antimicrobial resistance observed in MG. Previous studies have determined that MG was susceptible to nitroimidazoles. Preliminary analysis of four resistant strains of the MG type strain G37 found mutations in the MG_342 gene which encodes an oxidoreductase hypothesized to react with nitroimidazoles and produce the toxic form of the drug. In order to determine the possible mechanism for nitroimidazole resistance we will amplify the MG_342 region of MG. As MG clinical strains require months to establish cultures in vitro we will develop a sensitive PCR assay so that resistance-associated mutations can be identified directly from patient specimens in future clinical trials. The PCR products will then be sequenced to determine if the MG clinical isolates have mutations that confer resistance to nitroimidazoles. Understanding how these mutations affect nitroimidazole resistance could allow for future studies on nitroimidazoles as a possible treatment for MG.

Patients with Human Immunodeficiency Virus (HIV) are known to have increased risk of cardiovascular complications. High Density Lipoprotein (HDL) is a circulating lipoprotein responsible for removing lipids, such as cholesterol, from the blood and returning them to the liver, and is known to have a large impact on cardiovascular health. HDL is also known to have a protective effect on endothelial cells, which line the blood vessel walls, and it normally stimulates nitric oxide to cause an anti-inflammatory response. However, little is known about whether HDL from HIV patients has unique effects on the function of endothelial cells. I hypothesize that HIV-positive patients have increased inflammation due to impairment of HDL’s protective anti-inflammatory function. To test this, I am determining whether there is an increase in pro-inflammatory cytokines in plasma from HIV-positive patients compared to control patients, using enzyme-linked immunosorbent assays. I am also testing the hypothesis that HDL from HIV patients has a more pro-inflammatory effect on endothelial cells. I am culturing human microvascular endothelial cells (HMEC) and treating them with HDL from HIV-positive and non-HIV patients, along with appropriate control stimuli, followed by in-cell Western assays to measure activation of NFkB protein, a master pro-inflammatory regulator. I am using the same methods to measure activation of Akt, an intracellular signaling protein that can activate the production of nitric oxide via the enzymatic activity of endothelial nitric oxide synthase. I anticipate that HDL from HIV patients will cause increased activation of NFkB and decreased activation of Akt, which could explain, at least in part, the increased inflammation and cardiovascular issues in HIV patients. This research will begin to reveal possible mechanisms by which dysfunctional HDL may contribute to cardiovascular risks in HIV patients, and such findings could ultimately identify novel targets for therapeutic intervention.

During times of increased hospital capacity, unconventional areas (what we have termed “non-standard clinical spaces” such as converted conference rooms, hallways etc.) may be activated for inpatient activities. Studies regarding issues with Emergency Department boarding while awaiting an inpatient bed have been well documented, but the same practices in inpatient spaces have not been well studied. Qualitative semi-structured interviews were conducted between February-April 2023 at a large urban, public hospital to understand how patients experience these spaces. Any adult English-speaking patient admitted to the hospital for at least two days who was physically located in a non-standard clinical space (hallway beds, PACU beds, former Emergency Department bays converted to inpatient spaces) upon approach for interview was eligible to participate. Eight participants ranging in age from 20-70, across diverse demographics were interviewed for the study. Three themes were identified from the interviews: overall experience, unspoken messaging, and impact to care. Poor patient experience was affected by noise related to crowding in their space and a lack of barriers from noise, lack of privacy from close quarters with others, lack of reliable bathroom access, loss of control over their environment, and inability to gain the attention of nursing staff (though, interestingly, this was not universal as some mentioned that they were in closer proximity to their nurses and able to receive more direct interaction). They described perceiving unspoken messaging about being selected for a nonstandard clinical space. Most commented on the kindness and understanding of the nursing staff and did not necessarily perceive a significant impact to their overall care. When these types of spaces are considered for use for inpatient boarding, patient privacy, control, and access should be prioritized. Transparency and communication about the situational context may help mitigate negative effects of these spaces.

Mycoplasma genitalium (MG) is a sexually transmitted bacterial pathogen commonly associated with urethritis in men and cervicitis, endometritis, pelvic inflammatory disease, infertility, and preterm birth among women as it invades the upper reproductive tract. Infections can persist for months to years without effective treatment due to antimicrobial resistance. Current first-line drug choices are only successful in less than half of all patients. Preliminary in vitro data suggests that MG is susceptible to tinidazole (Tdz) and may fill the need for additional treatment options for drug resistant infections as it is already FDA-approved for other indications. As strains can vary in their susceptibility to particular drugs, we aim to identify the minimum inhibitory concentration (MIC), the concentration that inhibits growth by 99%, of Tdz against 10 MG clinical isolates. This data will determine if these strains are susceptible to Tdz, define the range of MICs, and reveal whether current strains have already developed resistance. Twofold dilutions of Tdz and doxycycline (DOX) antibiotics are added to MG clinical strains in 48-well plates and incubated at 37 C/ 5% CO2 for 21-28 days. DOX is one of the first-line drug choices with a known MIC; it is used to confirm that assays are performed correctly and to compare the effectiveness of Tdz. Four of the wells have no drugs to serve as a control to compare the number of genomes against those in the Tdz wells to determine MG inhibition. MG growth in each Tdz dilution is quantified with qPCR by isolating DNA from the wells. Calculations of the percent inhibition will dictate which antibiotic concentration is useful for treating infected patients. As physicians are already beginning to treat MG patients with Tdz, data regarding susceptibility of multiple isolates is crucial in informing these treatment regimens.

Infection from Mycobacterium tuberculosis is the leading cause of death due to infectious disease worldwide, with rates of tuberculosis infection greatest in low and middle-income countries (LMICs). Tuberculous meningitis (TBM) is the most severe form of M. tuberculosis disease with nearly half of all cases resulting in death or neurological consequences. Recent studies in our lab have found that single-nucleotide polymorphisms (SNPs) in MUC5AC, a secretory lung mucin, are associated with increased TBM susceptibility, morbidity, and mortality. The purpose of my study is to identify the functional MUC5AC SNP. Four candidate SNPs were selected within the MUC5AC promoter region based on high linkage-disequilibrium scores across multiple global populations with a SNP in the MUC5AC promoter, rs28737416. I utilized molecular cloning techniques to combine a luciferase-expressing plasmid with isolated regions of the human MUC5AC promoter containing the SNPs of interest and subsequently transformed this recombinant plasmid into competent cells. Next, I performed site-directed mutagenesis at the SNPs of interest and am currently transfecting mutants into HEK293T cells to investigate how genotypic variation in each candidate SNP influences promoter function by measuring luciferase expression. I anticipate variants in at least one SNP of interest will reduce gene expression (measured by luciferase expression), indicating functionality. Characterization of this genetic mutation will provide insight into TBM susceptibility across populations and could inform studies of novel therapeutics to treat TBM.


Human immunodeficiency virus type 2 (HIV-2) is endemic in West Africa and is also found in areas with socioeconomic ties to the region. Historically, advancements in the treatment of HIV-2 infection have been slow compared to HIV-1, and there is an urgent need to identify effective antiretrovirals (ARVs) for people with HIV-2 (PWH2) who harbor drug-resistant virus. Lenacapavir (SUNLENCA®) may help fill this gap in the HIV-2 treatment landscape. Lenacapavir is a long-acting, injectable ARV that targets the HIV-1 capsid protein and is approved for use in adults with multidrug-resistant HIV-1 infection. We recently showed that lenacapavir is highly active against HIV-2 isolates in culture, albeit with a 11- to 14-fold lower potency relative to HIV-1. These data suggest that lenacapavir binds to HIV-2 capsid cores in a manner similar to that observed in HIV-1. To further test this hypothesis, I am using site-directed mutagenesis (SDM) to introduce specific amino acid changes into the putative lenacapavir-binding region of the HIV-2 capsid protein. These changes fall into two categories: 1) mutations that convert the targeted residue in HIV-2 to the corresponding amino acid residue found in HIV-1; and 2) mutations that are associated with the development of lenacapavir resistance in patients with HIV-1 infection. To date, I have constructed a 5.5-kilobase plasmid clone that contains the capsid-encoding region of HIV-2ST (plus downstream flanking sequences) in a pBluescript plasmid vector. I introduced the desired mutations by SDM, and verified that the plasmids are correct via automated Sanger sequencing. My next step is to clone the mutated capsid sequences into a full-length clone of the HIV-2 ST genome for virus production and culture-based drug susceptibility testing. These studies have direct implications for improving HIV-related care and public health in West Africa and other areas where significant numbers of PWH2 reside.

Prostate cancer is the most common type of cancer amongst men in the U.S. It relies on androgens that bind to the androgen receptor (AR), which increases the transcription of genes associated with the growth and proliferation of the prostate cells. For the AR-driven prostate cancer (ARPC), current treatments involve decreasing androgen levels (Androgen Deprivation Therapy) or inhibiting the ARPI (Androgen Receptor Pathway Inhibitors). However, around 15% of patients develop resistance to these treatments, resulting in a type of prostate cancer called neuroendocrine prostate cancer (NEPC). NEPC cells are no longer dependent on AR activity, which makes this subtype difficult to treat with the current treatment options in the clinic. To better understand the biology of NEPC, we focused on gene expression at the protein synthesis level and found that NEPC has a decreased level of a tRNA called Arg-TCT-1-1. Following Arg-TCT-1-1 tRNA overexpression in NEPC, we detected elevated expression of AR downstream targets via qPCR and western blot. NEPC with high Arg-TCT-1-1 also responded to an AR inhibitor called enzalutamide as measured by cell viability assays. To further investigate the role of Arg-TCT-1-1 in prostate cancer, we used shRNA-mediated knockdown of this tRNA in prostate cancer cells with high AR expression and measured changes in gene expression. This study will provide important insights on the role of Arg-TCT-1-1 during the differentiation process from ARPC to NEPC.

Acute pancreatitis is an abrupt and painful inflammation of the pancreas that is often associated with serious complications and high mortality rates. Current diagnostic and therapeutic options remain limited, underscoring the need for novel strategies based on the underlying mechanisms of the disease. Our previous research suggests a key role for activin A, a member of the Transforming Growth Factor β (TGF-β) superfamily, in modulating inflammatory responses. Notably, activin A appears to trigger the c-Jun N-terminal kinase (JNK) pathway, a crucial member in macrophage activation during inflammatory diseases. This study aims to explore the role of activin A and the pJNK signaling pathway in macrophages during the pathogenesis of acute pancreatitis. Our investigation involves cell culture experiments using the RAW264.7 macrophage cell line, qPCR and Western blot analysis to identify protein alterations in activin-stimulated macrophages, and transwell migration assays to examine macrophage’s migratory ability toward chemoattractant after activin stimulation. We predict that activin A-stimulated macrophages would have increased JNK activation, increasing macrophage infiltration in inflamed tissues and polarization toward the pro-inflammatory subtype, potentially exacerbating the severe inflammatory response of acute pancreatitis. Our study is expected to reveal novel pathogenic mechanisms of acute pancreatitis and pave the way for the development of more effective diagnostic and therapeutic strategies for this disease.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disease. Traditional therapies focus on treating the symptoms of the disease and do not directly treat the underlying functional changes. Myosin modulators are a novel class of pharmaceutical agents designed to treat patients with cardiomyopathies by directly modulating cardiac myosin function in the sarcomere. Compounds including Mavacamten (Mava) and Aficamten (Afi) reduce myosin function, measured through its ATPase activity. In this study, I investigate how these small molecules affect the multiple turnover kinetics of the ATPase cycle. Porcine cardiac heavy meromyosin (pcHMM) is rapidly mixed with a two-times excess of fluorescently labeled ATP (mant.ATP) in the presence of actin and fluorescence is measured over time. Calculating the length of time, tau (τ), until the fluorescence has returned to 50% of the peak value estimates the time taken to hydrolyze all the mant.ATP, and is used to calculate the rate constant of ATP hydrolysis (kcat). Both Afi and Mava increase τ and decrease kcat, while a lower concentration of Mava is required to reach similar inhibition as Afi. I also utilize the In Vitro Motility assay to measure the ability of myosin to move actin and compare the effects of Mava and Afi with stopped-flow data. Preliminary results indicate that both Mava and Afi inhibit actin filament velocity, with Mava requiring a lower concentration, similar to stopped-flow. I will discuss how each modulator affects ATP turnover with a direct effect on catalytic activity and extend those results to the functional consequences of these myosin inhibitors. Insight into the impact of these myosin inhibitors on the myosin actin cross-bridge cycle will help provide tailored treatments to patients who are impacted by HCM.

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.

Acute pancreatitis (AP), inflammation of the pancreas in response to injury, is the leading cause of hospitalizations for gastrointestinal disorders in the US. Severe AP is correlated with a 30% mortality rate and treatment options are limited to supportive care. Our lab has previously reported that increasing serum levels of activin A, a TGF-β family member that regulates inflammation, is correlated to severity of AP in humans. Additionally, activin A has an established role in immunomodulation of neutrophils, which are critical innate immune cells driving AP. Our project aims to test the hypothesis that activin A promotes neutrophil activation and infiltration in AP. We performed qPCR on HL-60 cells treated with DMSO and/ or activin A to identify changes in the mRNA expression of immune activation genes, including CD11b, CD66b, and TNFɑ. We will also perform transwell migration assays on HL-60 cells treated with DMSO and/ or activin A to observe differences in neutrophil migration in vitro. We also intend to perform Western blot analyses, probing for pAKT and pERK, to identify the mechanism of activin A-induced neutrophil stimulation. We observed an increase in immunostimulatory mRNA in neutrophils treated with DMSO, which was further increased by activin. Additionally, we anticipate activin A and DMSO stimulation to promote neutrophil immunomodulation via the ERK pathway and increase neutrophil migratory capacity. The results of this project can provide more evidence that activin A can be a biomarker useful for risk stratification of patients with AP and allow for individualized treatments.

The emergence of induced Pluripotent Stem Cells (iPSCs) have allowed researchers to better study the effects of various diseases and mutations on fetal development. One such way of accomplishing this is the breakthrough of the organoid: a complex, iPSC-derived, 3D structure, that provides biologically relevant models for human systems. Lung Organoids (LO) were developed through this technology. However, the current LO models utilize mature lung phenotypes, which do not consider progenitor stages that may be critical for fetal development and the understanding of diseases that may affect this development in utero. The goal of this project is to provide characterization to the early stages of iPSC LO development: the Embryoid Body (EB) and Anterior Foregut (AFE). Using a previously established protocol, the LOs were fixed with 4% paraformaldehyde (PFA) on day 4 (EB stage) and day 6 (AFE stage), then analyzed with immunofluorescence analysis of the corresponding fetal lung (FL) development markers. 135 day old FL tissue sections were used as a positive control. The markers used to establish characterization were SOX17, a marker for the early endoderm germ layer, OCT4, an iPSC marker for pluripotency, and ECAD, a marker for tissue layer separation and cell migration. We hypothesized that all markers would appear in the EB stage, and the AFE stage would experience an upregulation in SOX17 and downregulation in OCT4 and ECAD. My results confirmed an upregulation of 133% for SOX17 and a downregulation of OCT4 by 58% from the EB to AFE stages. Lastly, as hypothesized, ECAD was present in EBs, but not in AFE. In conclusion, the LO stages proved to be similar to developmental stages of in utero development. Further analysis could help with new disease and mutation models for early development in utero, helping prevent devastating outcomes.

Malaria, caused by parasites belonging to the genus Plasmodium, accounts for roughly 600,000 deaths per year, with the most vulnerable population being children under 5 years old. The current available malaria vaccines show limited effectiveness in children. After being deposited by the bite of an infected mosquito, Plasmodium travels through the bloodstream and invades liver hepatocytes, causing an asymptomatic infection. Within hepatocytes, Plasmodium multiplies until the hepatocyte bursts, in which they are released into the bloodstream to cause a symptomatic infection. To develop an effective vaccine for children, it is critical to understand the immune response to liver stage Plasmodium infection in children. However, our current understanding of immune responses in children compared to adults, specifically in the liver, is limited. During Plasmodium infection, CD8 T cells in the liver are able to confer sterilizing protection. The main goal of this study is to identify differences in the spatial location of immune cells in human liver tissue across ages. We are currently utilizing a combination of RNAscope and immunofluorescent assay (IFA) staining to visualize CD8αα+ T cells expressing the promyelocytic leukemia zinc finger (PLZF) transcription factor, which distinguish them from conventional CD8+ memory-like cells as innate-like T cells. These unconventional cells play a key role in autoimmune responses in the liver. We are doing further spatial analysis on Imaris to gather quantitative data on these samples. We hypothesize that children would express higher levels of CD8αα+ T cells expressing PLZF because they do not produce memory-like cells as well as adults, and thus lack an efficient response to infections. The results of this study will help us better understand the differences that age may cause in immune cell types and quantities, and how this can be used to develop a more effective malaria vaccine for children.

Malaria, a disease caused by the Plasmodium parasite, kills approximately 600,000 people per year. This disease consists of two stages, first an asymptomatic liver stage, followed by a symptomatic blood stage. The goal of our lab is to eliminate the transmission and spread of the parasite via the use of genetically modified parasites as vaccines. These parasites die in the liver, preventing blood infection and creating long-term immunity in the liver. Previous research in our group has shown that our vaccine model is effective in mice, with improved efficacy when the type-1 interferon response to the vaccine is disabled. Type-1 interferons are cytokines produced in an early immune response to a variety of pathogens. Yet, the mechanism in which these interferons are activated in a Plasmodium-infected liver cell is unknown. My project's goal is to identify and understand the mechanism in which this early immune response is turned on in response to parasite infection. To do this, I am developing an in-vitro system which can quantify the type-1 interferon response in Plasmodium-infected liver cells in culture. Utilizing various in-vitro techniques, we can identify the sensors, adaptors, and transcription factors that are most important in upregulating this early immune response. This knowledge will be used to inform methods to inactivate the type-1 interferon response, in turn improving our vaccine model. Our lab hopes to eventually use our model in humans for the goal of eradicating malaria from the modern world.

VirScan, a revolutionary technology based on Phage Immunoprecipitation Sequencing (PhIP-Seq), allows the interrogation of antibody responses to all known human viruses using a small blood volume, providing information on an individual's previous viral exposures. This study aims to provide a comprehensive data quality assessment system for VirScan, which will improve its reliability and interpretability by routinely assessing VirScan data quality at both the sample, assay (N=96 samples in replicate), and sequencing batch levels (N=192 samples in replicate). The study focuses on creating standards and thresholds for data quality at all three levels, considering aspects such as aligned reads, read depth, percent of epitopes discovered, and correlation of sequence counts between replicates. The assay/batch-level analysis provides metrics like the mean, median, standard deviation, and range of mapped reads and correlations for count and peptide detection, evaluating consistency, accuracy, and comparability across assays and batches. Further, these criteria can effectively categorize sample quality into Good, Questionable, and Failed, identifying samples that may need to be repeated or excluded from analysis. These quality calls were all encoded within an R Shiny App, enabling a user-friendly and flexible interpretation of VirScan data. Implementing this systematic quality control strategy will considerably improve the usability of VirScan in research and clinical contexts, allowing for more trustworthy interpretations of an individual's viral exposure history while also contributing to a better knowledge of immune response dynamics.

Myotonic dystrophy type 1 (DM1) is a genetic disease that causes many serious health conditions in a variety of tissues including skeletal muscle stiffening and cardiac conduction disorders. This disease affects 1 in 2,300 people worldwide and is the most common form of muscular dystrophy. DM1 is caused by a CTG repeat expansion, which in lay terms means that in a gene, there's a sequence of 10 CTG DNA bases. However, in a specific part of the gene responsible for making messenger RNA (mRNA), the number of CTG repeats increases significantly. This unusual mRNA sequence is linked to the development of the disease. This mutated mRNA (messenger RNA) disables the splicing regulator muscle-blind-like 1 (MBNL1) gene and ultimately causes disease. It does this by sequestering and limiting the MBNL1s critical role in splicing mRNA (figure 1). In my proposed research project, I am focusing on cardiac function when testing adeno-associated viral vector (AAV)-mediated systemic delivery of the MBNL1 gene to increase MBNL1 protein expression in muscle. The lab found that body-wide delivery of AAV vectors with CK8-intron-MBNL1, which expressed MBNL1 only in striated muscle, was toxic in the hearts of mice and caused death (figure 2). Over the last few months, my mentor Matt Karolak and I have learned together methods such as echocardiography and tissue histological techniques to determine whether it is possible to prevent MBNL1 protein production and its damaging effects in the heart while still expressing MBNL1 protein in skeletal muscle for therapeutic disease benefits.

Androgen deprivation therapy (ADT), the pharmacologic reduction of testosterone (T), is a critical treatment for prostate cancer patients, improving cancer-related outcomes but markedly increasing the risk of cardiovascular disease development. Our recent findings suggest that patients with prostate cancer subjected to ADT have increased hypothalamic gliosis (the activation of astrocytes and microglia), a hallmark of central nervous system injury. Similarly, castrated mice fed a high-fat, high-sucrose with added cholesterol diet (HFHS) develop hypothalamic astrogliosis and atherosclerosis, which can be prevented through T replacement at the onset of castration. In this experiment, we tested whether established astrogliosis in hypogonadal mice can be reversed by restoring healthy T levels. Using a gonadotropin-releasing hormone antagonist (acyline) as ADT, we treated 3 groups of HFHS-fed C57Bl/6 wild-type mice with: 1) vehicle, 2) acyline for 4 weeks, and 3) acyline for 4 weeks followed by a 4 week period of reversal to vehicle. Hypothalamic brain sections were used for immunohistochemistry analysis to quantify levels of glial fibrillary acidic protein (GFAP) expression, a marker for astrogliosis, and neurokinin B (NKB) expression, a marker for reproductive function. As anticipated, ADT resulted in a significant reduction in testes weight and NKB expression, serving as surrogate measurements of low T production. ADT also induced a significant increase in hypothalamic GFAP expression, confirming that gliosis is exacerbated in hypogonadal conditions. Following the discontinuation of ADT, testes weight and hypothalamic NKB expression rebounded to normal levels, however, hypothalamic astrogliosis remained significantly elevated. Together these data suggest that once astrogliosis is established, the restoration of T via 1 month of ADT cessation is insufficient to reverse it. This finding is a critical step forward in clarifying the pathways between androgen signaling and cardiometabolic regulation, and raises an important clinical concern given the high incidence of morbidity and mortality associated with ADT.

Androgen Deprivation Therapy (ADT) is the standard treatment for advanced prostate cancer (PCa), although it adversely affects muscle mass, physical function, and quality of life (QOL). It is unknown whether 1) these factors Pre-ADT can predict ADT-induced changes or 2) if non-White, non-Hispanic (NWNH) men experience greater adverse impact on these outcomes than White, non-Hispanic (WNH) men. I hypothesized that 1) greater muscle mass Pre-ADT would be protective against ADT-induced changes in muscle mass, physical function, and QOL and 2) NWNH men would experience worse changes in muscle mass, physical function, and QOL than WNH men. I assessed lean body mass (LBM) [dual energy x-ray absorptiometry], physical function [aerobic capacity (VO2Peak), hand grip strength (HGS), 6-minute walk test (6MWT), stair climb power (SCP)], and QOL [QLQ-C30 questionnaire] in PCa patients (n=59) from the Seattle VA before and 6 months after ADT. I used Pearson correlations to test associations between variables and independent t-tests to compare variables between WNH and NWNH men. Larger LBM (r=0.33, p=0.019, n=50), lower QLQ-C30 Function (r=0.31, p=0.03, n=50), and higher QLQ-C30 Fatigue (r=0.40, p=0.004, n=50) Pre-ADT were correlated with larger 6-month decreases in LBM. NWNH men (n=20) displayed significantly worse Pre-ADT HGS (p=0.038), 6MWT (p=0.037), and SCP (p=0.005) than WNH men (n=39). I anticipate that NWNH men will display worse 6-month changes in LBM, HGS, 6MWT, SCP, and QOL than WNH men in my ongoing analyses. Contrary to my hypothesis, larger LBM Pre-ADT was not protective against muscle loss. Consistent with reports that NWNH men experience greater tumor-related adverse impacts of PCa treatment, worse functional performance Pre-ADT may indicate physical function of these individuals is also disproportionately adversely impacted by ADT. By identifying predictors of adverse ADT outcomes, researchers can develop interventions aimed at preserving muscle mass, physical function, and QOL during ADT for high-risk populations. Additionally, reducing the disproportionate adverse effects of ADT on NWNH men will address disparities within PCa treatment, promoting personalized healthcare optimizing outcomes for all patients. 

Since approval by the FDA in 2019, BPaL/ BPaLM regimens are the favored treatment for multidrug-resistant tuberculosis (MDR-TB). Whereas traditional MDR-TB treatment generally includes an injectable and takes at least 18 months to complete, BPaL/ BPaLM regimens are all oral and typically take 26 weeks, suggesting potential for widespread implementation. Here, I aimed to elucidate patient demographic patterns and outcomes following treatment with BPaL/BPaLM to fill current gaps in knowledge. King County, Washington, with large populations of immigrants, refugees, and asylum seekers arriving from TB-endemic countries, sees high rates of TB incidence each year. Using medical data collected from active TB cases in King County, WA since 1993, I compiled demographics and outcomes (resolution of TB symptoms and treatment adherence) from patients receiving BPaL/BPaLM regimens. Of the 70 patients in our King County cohort, eight were given BPaL or BPaLM therapy to treat MDR-TB. At this time, five (63%) have completed therapy. Four (50%) are from Vietnam, two (25%) from China, one (13%) from Myanmar, and one (13%) is from the Russian Federation. Two (25%) are male; one (13%) was previously diagnosed with TB; and median age was 35 (range of 19-81). All eight had pulmonary disease and were HIV-negative. One patient was a household contact of another identified on contact tracing. Limitations of this analysis include a small BPaL/BPaLM cohort (n=8), and missing patient data. As more follow-up time accumulates, I can compare BPaL/BPaLM to the prior 18-month regimens using relapse rates and treatment completion. Based on my current analysis of King County MDR-TB cases, BPaL/BPaLM appears effective and well-tolerated—conducive to better TB outcomes than seen with prior MDR-TB regimens. Moreover, this study can provide data-driven insight to effectively treat MDR-TB patients from diverse populations across the US.

The majority of people with Alzheimer’s dementia confirmed at autopsy are found to have one or more additional brain pathologies. To address this, we have developed a harmonized brain pathology score (BPS) across autopsy cohots that incorporates multiple forms of postmortem neuropathology. We sought to explore the genetic architecture of BPS using a systems-biology approach to further understanding of mixed pathology. We ran genome-wide association studies (GWAS) of BPS using HRC imputed data from European ancestry participants in each cohort separately, adjusting for age at death, sex, and population substructure. We performed meta-analysis using METAL. We performed gene-wide analysis using the GWAS results which we then integrated into the human protein-protein interaction (PPI) data using a dense module searching (DMS) method to identify network hub genes for BPS. We interrogated the Seattle Alzheimer's Disease Brain Cell Atlas (SEA-AD) dataset on the middle temporal gyrus to determine which cell types both hub genes were expressed in and how they differed across donors with higher degrees of AD pathology. The sample size consisted of 1,848 brain donors where 63% were females and mean age at death was 89.3. The quantile-quantile plot and genomic inflation (λ=1.005) for GWAS meta-analyses showed no bias. Apart from significant SNPs around the APOE region, we identified two candidate loci a) (Chr 9: rs1332179; MAF=0.1; P_meta=8.7×10-8) and b) (Chr 17: rs11078196; MAF=0.34; P_meta=1.9×10-7). The PPI network analysis identified VCP and IQCB1 as hub genes. While both hub genes were expressed broadly across cell types, IQCB1 was specifically higher with higher degrees of AD pathology in Microglia and VCP was lower with higher degrees of AD pathology in several neuronal populations in the SEA-AD dataset. Further functional enrichment analyses of these candidate loci are needed to determine whether these novel loci may identify targets for interventions to ameliorate AD.

Respiratory viruses are a major cause of mortality and morbidity in vulnerable populations. Together, Respiratory syncytial virus (RSV) and Human metapneumovirus (HMPV), are responsible for over ⅓ of serious viral respiratory infections in hematopoietic stem cell transplant (HCT) recipients. Currently, no treatments are available for RSV or HMPV in immunocompromised adults. While monoclonal antibodies (mAbs) show promise as a treatment, challenges arise, including limited efficacy when administered post-infection. Our goal was to enhance the therapeutic efficacy of a newly discovered cross-neutralizing human mAb to RSV and HMPV. We aimed to investigate whether modifying the Fc domain of the antibody could increase its binding to Fcγ receptors (FcγRs) found on different types of immune cells. Activation of FcγRs initiates important cell processes such as clearance of virus-infected cells, also known as Antibody-dependent cellular cytotoxicity (ADCC). This modification potentially makes the antibody a more effective treatment option for RSV and HMPV infections. To do this we looked at the binding kinetics and affinity of modified antibodies to human FcγRIIIa, FcγRIIa and FcγRIIb receptors using Bio-Layer Inferometry (BLI). Our data indicate that certain amino acid modifications or afucosylation of the Fc region can increase the antibody’s binding affinity to different human FcγRs. Since hamsters are an important preclinical model used to determine RSV and HMPV drug efficacy, it was important to examine the binding affinity of our human antibody to hamster FcγR’s. Our data indicate that the wild-type Fc region does bind to the homologous hamster receptors. Moreover, certain modifications in the Fc region led to increased binding to hamster FcγR’s. Together, these data indicate that modifications in the Fc region of human antibodies can increase their binding affinity to both human and hamster FcγRs. This increase in binding affinity could translate to enhanced potency in the preclinical hamster model and in humans.

HIV is characterized by a chronic state of inflammation that may lead to the development of cardiomyopathy, a condition where the heart muscle cannot effectively pump blood. The purpose of this report is to explore the relationship between HIV and the phenotypic expression of hypertrophic cardiomyopathy (HCM). In this patient case study, I analyze the possibility of HIV-related changes in heart structure affecting HCM’s development into heart failure, as well as an increased risk of surgical complications. I examined a 43-year-old female HIV patient who showed symptoms of heart failure and was diagnosed with HCM. She went through various surgical procedures that were complicated by post-pericardiotomy syndrome, an inflammatory response to cardiac surgery. I used the patients' hospital vitals and procedures, as well as current HIV and HCM literature to propose the possible relationship between HCM phenotype and HIV, as well as the role that inflammatory properties play between HIV and the probability of post-surgery complications. HCM phenotype may vary among patients due to its interactions with gender, genetics, and the environment, and can result in heart failure. In this patient, it is possible that there is an increase in severity in HCM phenotype due to the inflammatory factors associated with HIV, further contributing to the development of both heart failure and complications in surgery. The case of this patient illustrates how further research needs to evaluate the specific molecular mechanisms in HIV that could be impacting the expression of HCM, as well as the further development of post-pericardiotomy syndrome.

Tuberculosis is the leading cause of infectious disease death globally, but understanding of the host factors that contribute to immune control are incompletely understood. Mtb-infected macrophages, the primary host cell defense cell and replicative niche of Mycobacterium tuberculosis (Mtb), become lipid-laden, or "foamy," which promotes Mtb growth. Toll-interacting protein (TOLLIP) is a tuberculosis-resistant protein that metabolizes lipids in other cell types. Our study objective was to determine whether TOLLIP was responsible for preventing lipid accumulation and impairing host defense in macrophages. I harvested alveolar macrophages of B6 and Tollip^-/- mice via bronchoalveolar lavage and measured lipid accumulation, cell death, and cytokine production. Macrophages were incubated with the Mtb cell wall lipid mycolic acid and labeled with a fluorescent neutral lipid stain to measure intracellular lipid content. Mycolic acid was also instilled intratracheally into the lungs of mice and lipid content in lung-resident macrophages was measured using flow cytometry. Since lipid accumulation can induce cell death, cellular apoptosis and necrosis were measured via Annexin V and membrane permeability staining. Cytokine responses were determined via enzyme-linked immunosorbent assay (ELISA), and data was gathered through a standard curve. Statistical significance was determined using a two-sided t-test, with a threshold p-value of less than 0.05. Mycolic acid-stimulated Tollip^-/- macrophages developed increased lipid accumulation and cell death in a dose-dependent fashion. Tollip^-/- alveolar macrophages selectively accumulated more lipids than B6 mice (mean B6 0.45%, mean Tollip^-/- 0.68%). Tollip^-/- macrophages secreted increased cytokines (mean B6 131pg/mLvs Tollip^-/- 233 pg/mL, p=0.05) after mycolic acid stimulation. We conclude that Tollip^-/- macrophages accumulate excess lipids and produce more cytokines, which may contribute to its role in Mtb host defense in macrophages. These results suggest that excess cytokine production leads to worsened defense mechanisms and increased lipid production induces macrophage immune response. 

Renal Cell Carcinoma (RCC) is one of the seven most frequent malignant cancers in the United States and is incurable for most patients when metastatic. One potential new treatment for RCC is T-cell immunotherapy, which has proved effective for treating certain blood cancers. To study T-cell interaction with RCC cells, I am utilizing a microphysiological system to model RCC, known as RCC-on-a-chip, which comprises spheroids of RCC cells embedded in a 3D collagen extracellular matrix (ECM). From these studies, I have observed that T cells are better able to infiltrate the collagen matrix at lower collagen concentrations (1.75 mg/mL) than at higher collagen concentrations (2.5 mg/mL). The higher collagen concentration reflects the dense tumor ECM in vivo. This led to my hypothesis that there is a subset of T cells that exhibit enhanced collagen invasion. The approach to test this hypothesis involves 1) Retrieving T cells that have infiltrated the collagen matrix in the chip; 2) Injecting the T cells that have infiltrated the collagen matrix once before into another chip to compare their migration capabilities to unselected T cells; and 3) Quantifying T-cell invasion utilizing novel custom ImageJ/FIJI macros I have written to automate the extraction of data from confocal Z-stack images taken of the chip system. If successful, I aim to retrieve enough of these infiltration-enriched T cells to conduct single-cell RNA sequencing to analyze the transcriptomic profiles of the T cells that are superior infiltrators. The knowledge gained could lead to advanced therapeutic T-cell products with the enhanced ability of targeting solid tumors. These studies demonstrate how use of the RCC-on-a-chip model can facilitate the investigation of T-cell immunotherapy for RCC.

African immigrants are highly disproportionately affected by Human Immunodeficiency Virus (HIV) compared to US-born individuals in the U.S.. In King County, HIV stigma is a significant barrier to HIV testing among African immigrant communities. Our study aims to partner with communities to confront the HIV epidemic among the African immigrant population in King County by decreasing HIV stigma that prevents individuals from utilizing HIV testing. In this formative study, we have partnered with three African community organizations, Ethiopian Health Board, Eritrean Health Board, and the Congolese United Foundation, to deliver community-based HIV testing via health fairs at faith-based organizations and adapt and pilot an existing HIV stigma reduction intervention (Project FAITHH). We are working with faith-based organizations to address religious/moral beliefs that may foster negative social labels towards and further stigmatize people living with HIV. Data collected at health fairs hosted in churches serving each of these communities include HIV stigma scales, perceived barriers faced by individuals and community members that impact HIV testing, and demographics including country of origin, gender, religious affiliation, and primary language(s). Additionally, we have adapted an 8-module, faith-based stigma reduction intervention for these communities, aimed to address inequities in HIV testing perpetuated through HIV stigma, misinformation, and lack of awareness among intersecting, religious identities. The intervention includes activities to explore and address the sources of stigma around HIV in African immigrant communities, as well as information about HIV, including HIV epidemiology in King County. Anticipated findings regarding HIV stigma of participants before and after the intervention will inform our implementation of Project FAITHH among a more diverse group of African immigrant communities in King County. This work furthers the impact of community-based interventions designed to address the disparities in HIV in King County.

The mechanisms underlying the development of neurodegenerative diseases such as Alzheimer's Disease (AD) are not well understood. The characteristic accumulation of pathological protein plaques and tangles has led AD research to focus primarily on abnormal protein function and metabolism contributing to the pathology. Tau is one such protein that forms toxic aggregates in those with AD and various other neurodegenerative diseases. Our lab researches the mechanisms of tau toxicity using the nematode C. elegans. Recent research has implicated improper lipid metabolism as another potential contributor to neurodegeneration. Mutations in a gene known as gba-3, which is critical for lipid metabolism, are risk factors for the development of Parkinson’s Disease (PD), but little is known about the relationship between gba-3 mutations and tau toxicity in AD models. My research project will investigate the role of gba-3 in a C. elegans model of tau toxicity. I have crossed strains with various mutations in the gba-3 gene into two strains with tau toxicity. I will perform motor performance assays, measure the accumulation lipids, quantify the amount of tau, and measure oxidative stress to investigate whether gba-3 mutations modulate tau toxicity. Preliminary results suggest some gba-3 mutations slightly rescue some tau models but not others, but further investigation is needed to validate these findings. Ultimately, this project will provide further insight into the complex mechanisms underlying neurodegenerative diseases such as AD and may guide future development of treatments targeting lipid metabolism.

Alzheimer's disease (AD), the most common form of dementia, is characterized by the improper cleavage of amyloid precursor protein by a complex containing presenilin 1 (PSEN1) or presenilin 2 (PSEN2). Notably, PSEN1 and PSEN2 are strong genetic risk factors for heritable AD. However, 95% of AD cases currently have no known genetic cause. Recent work from the Valdmanis lab found PSEN2 isoform variations at the RNA level in sporadic AD. One such variation was the detection of differential 3'UTR lengths on the PSEN2 transcript. The 3'UTR is an important regulatory region that controls transcript maturation, stability, and abundance and is subject to environmental regulation. The length of this regulatory region is determined by RNA processing machinery during polyadenylation, and differences in this post-transcriptional process lead to differences in the 3'UTR length known as alternative polyadenylation (APA). APA may represent a functional mechanism by which PSEN2 regulation differs in AD. The goal of these studies is to understand the impact of PSEN2 APA on neuronal function. We hypothesize that the length of the 3'UTR on PSEN2 transcript aligns with phenotypic changes associated with AD. To test this hypothesis, we are cloning PSEN2 with short and long 3'UTRs to test the functional differences of PSEN2 APA in vitro. Our goal is to introduce the short and long PSEN2 3'UTR constructs in the cells, specifically, microglia, the brain's immune cells, which are heavily implicated in AD pathology. Then, we will visualize the subcellular location of these transcripts and test for altered amyloid beta processing, which is a pathological hallmark of AD. We anticipate detecting differences in regulation and subcellular localization between the short and long PSEN2 3'UTR transcripts. Elucidating the functional relevance of the short and long 3'UTR of the PSEN2 transcript will further our understanding of APA in AD.