Cancer Cachexia is a condition characterized by muscle wasting, functional impairment and decreased quality of life that leads to 30% of cancer-related deaths. Cytokines, proteins secreted by both the body and tumors that drive inflammatory response, are potential therapeutic targets and biomarkers for tracking disease progression. However, their role in cancer cachexia and relationship with physical function, patient-reported quality of life, and body composition have not been fully characterized. We hypothesized that in cancer patients, inflammatory cytokines would be elevated in patients with weight loss compared to weight-stable patients and inflammatory cytokines would be negatively correlated with muscle function, patient-reported outcomes and body composition. Body composition was assessed by Dual-energy X-ray absorptiometry (DEXA) and X-ray computed tomography (CT). Muscle function was assessed by stair climb power, hand grip strength and one-repetition maximum (1RM) muscle strength of different muscle groups. I assessed 55 cytokine plasma levels with Mesoscale Discovery Assay kits. Preliminary analyses show that in patients with cancer, interleukin (IL)-6 is negatively correlated with CT skeletal muscle mass (r=-0.36, p<0.05, n=53), IL-8 is negatively correlated with body weight (r=-0.40, p<0.01, n=53), body mass index (r=-0.41, p<0.01, n=53), CT skeletal muscle mass and subcutaneous adipose tissue (r=-0.49, p<0.05, n=53). Tumor Necrosis Factor-Alpha was negatively correlated with hand grip strength (r=-0.30, p<0.05, n=53). IL-5 was positively correlated with Anderson Symptom Assessment Scale (ASAS) fatigue (r = 0.40, N = 47, p = 0.005) and ASAS total (r = 0.45, n = 47, p = 0.002). Certain cytokines are linked to worse muscle mass and function, while others correlate with better patient-reported outcomes, suggesting inflammation’s role in cachexia is complex. More research is needed to distinguish these differences for targeted therapies treating specific attributes of cachexia.

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

Cardiac Electrophysiologists (EP) use various devices to diagnose and treat heart rhythm disorders. Unfortunately, these devices can be defective and are subject to recalls by the U.S. Food and Drug Administration (FDA) to protect the patient. This study aims to better understand the rates of these recalls from 2014 to 2024 and thereby evaluate the safety of these devices, compare the safety vis a vis previous decades, and assess the safety of new devices as they are introduced for clinical use. We evaluated the number of FDA advisories related to implantable cardiac devices from 2014 – 2024; total the number of devices recalled under these advisories, determining the most prevalent malfunctions, and analyzing trends compared to rates from 1990-2000. The data shows that Permanent Pacemakers and Implantable Cardioverter Defibrillators (ICD)  have become safer compared to the past, although more units were affected per device advisory. Newer technologies of Leadless Pacemakers, Subcutaneous-ICDs, Implantable Loop Recorders, and Cardiac Contractility Modulation have been impacted by malfunctions and their trend will be critical to follow over the years to come. The findings of this study provide information regarding the safety of these technologies that are valuable to both EP cardiologists and the millions of American patients who live with these devices.

Acute Pancreatitis (AP) is a sudden inflammatory condition of the pancreas that can lead to significant mortality. Despite its rising prevalence and associated healthcare burden, treatment options remain limited to supportive care, with mortality rates in severe cases reaching 30%. Activin A is a key contributor to AP, interacting with the ACVR2A receptor to regulate various pathophysiological processes, including inflammation through immune cell recruitment. This study hypothesizes that activin A binds to ACVR2A to activate the ERK pathway, leading to increased NF-κB expression and elevated production of pro-inflammatory cytokines, including TNF-α and IL-1β. Experiments were designed using the 266-6 immortalized pancreatic acinar cell line and RAW 264.7 macrophages. These cells will be cultured for western blot analysis, ELISA assays, and transwell migration assays following activin A stimulation. Lower ERK phosphorylation and reduced NF-κB expression are expected when cells are treated with ACVR2A inhibitors in combination with activin A, compared to activin A treatment alone. ELISA assays are anticipated to confirm increased TNF-α and IL-1β production in 266-6 cells following activin A treatment. Macrophage migratory capacity is expected to increase when exposed to conditioned media from activin A-treated 266-6 cells. These findings will provide insights into the role of activin A in AP pathophysiology, potentially identifying new therapeutic targets for mitigating pancreatic inflammation and immune cell recruitment.

Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly disease without prognostic tools for early detection or effective therapeutic strategies. Activin A is a cytokine that is upregulated in tumor and stromal cells that surround the tumor in PDAC and acts as a promoter of metastasis. Activin A has also been shown to stimulate the AKT pathway which is proto-oncogenic. Here, we set out to test the hypothesis that activin A drives PDAC development through the AKT pathway. Western blots for proteins of the AKT pathway (phospho-PRAS40 and phospho-β-catenin) and transwell migration assays will be performed on PanC1 pancreatic cancer cells stimulated with activin A. Additionally, inhibitors of the activin A receptor subtype 2A (ACVR2A) and the AKT pathway will be included to delineate receptor-specific effects. Given activin's known role for simulating the AKT pathway, it is expected that activin A stimulation will phosphorylate and trigger increased migratory capacity of PanC1 pancreatic cancer cells. Inhibitor experiments will confirm that these effects are ACVR2A specific. This data will identify if activin A is a novel therapeutic target in late stage PDAC, a disease with limited targeted pharmacological treatments. 

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

Antibiotic resistance is an increasingly critical concern for the treatment of bacterial infections, rendering new therapy options progressively more necessary. Gram positive bacteria are common infectious agents in skin and soft tissue infections, pneumonia, urinary tract infections, bacteremia, and more. A novel antibiotic candidate, MRS-2541 has been demonstrated to inhibit Gram positive methionyl-tRNA synthetase and decrease bacterial loads of both methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pyogenes in mouse thigh infections to the same degree as currently recommended therapy. This study aims to further characterize the activity of MRS-2541 against Gram positive bacteria including Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcus aureus, MRSA, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus salivarius. I conducted this study by first determining the preliminary minimum inhibitory concentration of MRS-2541 in growth media against each of the aforementioned organisms. I then use these results to guide time kill assays that characterize MRS-2541’s synergy with another antibiotic often used to treat Gram positive infections outside of the United States. Preliminary results demonstrate that MRS-2541 inhibits the above-mentioned organisms. Synergy experiments with MRS-2541 and existing antibiotics will be performed and results will be presented at the symposium. These results will further define the spectrum of activity as well as synergy of MRS-2541, allowing new insight into its candidacy for clinical trials. As a novel antibiotic candidate, the development of MRS-2541 will help address the increase in antibiotic resistance among Gram positive bacterial infections.

Obesity is linked to reproductive dysfunction through defects in the hypothalamic-pituitary-gonadal (HPG) axis that arise by unknown mechanisms. Importantly, rodents fed a high-fat diet (HFD) develop similar hypogonadism and reduced ovulatory capacity. During HFD feeding, CNS immune cells (microglia) become activated in the hypothalamus, promoting inflammation, and altering neuronal function indirectly and via cell-cell contacts. Surprisingly, however, genetic ablation of this microglial inflammatory response exacerbates rather than improves HFD-induced HPG axis dysfunction including altering the activity of hypothalamic neurons that express the key reproductive neuropeptide GnRH. Therefore, we hypothesized that increased microglial inflammatory signaling during HFD feeding helps maintain GnRH neuron integrity. To test this hypothesis, we used immunohistochemistry (IHC) to assess microglia-GnRH cell-cell interactions in the hypothalamus of 15-week HFD-fed female mice with microglia-specific deletion of IKKβ (IKKβ-MGKO), a critical regulator of the inflammatory NF-κB pathway. IHC studies using GnRH and Iba1 (microglial marker) revealed fewer cell-cell contacts between GnRH neurons and microglia in the preoptic area of the hypothalamus (POA) of IKKβ-MGKO mice compared with controls. In addition, we found that IKKβ-MGKO mice have reduced levels of Iba1 and total numbers of microglia but no changes in microglial cell morphology as determined by Sholl analysis. These findings suggest that HFD-induced microglial inflammatory signaling promote cell-cell interactions with GnRH neurons that may contribute to maintenance of HPG axis integrity and female reproductive function during diet-induced obesity.

Over a hundred years after the Spanish Flu, the influenza A virus (IAV) remains a leading cause of respiratory infections and mortality worldwide. The proliferation of IAV causes many of the symptoms associated with IAV clinical disease. However, the severity of acute lung injury (ALI) from IAV is primarily driven by the host's immune response to infection. The mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated protein kinase (ERK) signaling cascade is a highly conserved pathway that is activated during lung injury and inflammation in both rodents and humans. Two MEK isoforms, MEK1 (Map2k1) and MEK2 (Map2k2), activate downstream effectors, ERK1 and ERK2, and control critical cellular processes, including the intensity and duration of inflammatory signaling. Our prior research revealed that MEK2-deficient mice exhibited improved weight recovery and overall fitness during IAV infection, suggesting that MEK2 is a host response exacerbating ALI during IAV infection. During IAV infection, excessive pro-inflammatory cytokine production drives immune cell recruitment into the lungs, leading to collateral tissue damage that impairs organ function and exacerbates disease. We hypothesize that MEK2 enhances immune cell recruitment to the lungs, enhancing inflammation, which may occur through exuberant chemokine signaling. To investigate this, we infected MEK2-deficient and wild-type mice with mouse-adapted IAV (H1N1, PR8) and collected cells from bronchoalveolar lavage (BAL) and lung homogenates. Using flow cytometry, we found reduced immune cell recruitment, including decreased numbers of monocytes, dendritic cells, monocyte-macrophages, CD4+ T-cells, CD8+ T-cells, and B-cells. Next, we assessed levels of key chemokines known to attract monocytes and lymphocytes by measuring their gene expression in the lungs and protein levels in the BAL. Investigating MEK2’s impact on chemokine signaling will elucidate the mechanism by which MEK2 perpetuates lung inflammation and injury during IAV infection and will guide the development of future host-directed therapies for IAV-induced lung damage.

Bronchoscopy with bronchoalveolar lavage (BAL) is a common approach to assess the graft in lung transplant recipients. However, bronchoscopy is an invasive approach that carries procedural risks. Identifying plasma biomarkers levels which correlate with those measured in BAL fluid enables less invasive investigations. This study aims to determine the correlation between biomarkers measured from plasma and BAL fluid. We analyze paired plasma and BALF (BAL fluid) samples collected from lung transplant recipients undergoing for-cause bronchoscopy (n = 95 individuals and inclusive of 164 encounters). Researchers collect paired samples from each participant within a two-hour timeframe. Researchers measure a panel of 39 biomarkers in samples using electrochemiluminescence assays. We evaluate plasma/BAL correlation data using Pearson correlation coefficients on log-transformed concentrations. Among participants, the average age is 61 years, 74% are male, 93% are white, with Restrictive lung disease (60%) being the most common pre-transplant pulmonary disease. The median time from transplant to bronchoscopy is 412 days (IQR: 194-741). Results indicate that correlational data is highly variable. The two most highly correlated biomarkers are IL-12/IL-23p40 (r = 0.67) and CXCL10 (r = 0.64). There are no biomarkers that have significant inverse correlations. The levels of 11 out of the 39 measured biomarkers show moderate correlation, ranging from r = 0.20 to r = 0.45. Many of these biomarkers are chemokines related to immune cell migration to the lung (e.g., CCL2, CCL3, CCL4, and CCL22). A key implication of this finding is that researchers should exercise caution when extrapolating alveolar biology from circulating (plasma) samples. Future analyses will test whether the ratio between paired BALF and plasma biomarker levels provides biological or clinical insight beyond using these biomarkers measured in isolation.

In Adoptive Cell Therapy (ACT), a novel modality of cancer therapy, immune cells can be engineered with T cell receptors (TCRs) to aid in targeting specific antigens presented on the surface of cancer cells. However, TCR-T cells often have limited persistence after transfer into patients, which has hampered the effectiveness of this therapy for solid tumors. Last year, our lab identified LSD1 as a target for drug inhibition, which is an enzyme that alters the epigenome of cells via histone modifications. My project aims to understand the mechanism of LSD1 inhibitor drugs, as well as the effect of these drugs on two types of T cells: cytotoxic CD8+ cells and helper CD4+ cells. In addition to understanding how LSD1 drugs work, I also ask exactly how CD4+ cells enhance the function of CD8+ cells in tumor killing. Which receptors on CD8+ cells are activated by helper T cells, what is the signal phosphorylation pathway transducing the "helping" signal from receptors, and what downstream epigenetic regulators play a role in translating the "helping" signal into better function in CD8+ T cells? To assess these interactions, I will generate a diverse population of CD8+ T cells with targeted receptor knockouts, known as a receptor library. Similar libraries will be generated for epigenetic regulators as well as kinases/phosphatases. The performance of T cells will be assessed via coculture assays, where T cells can kill tumor cells but not fully eliminate the tumor because of periodic addition of new tumor cells. At the end of the coculture period, we will assess gRNAs enriched in dysfunctional populations, which will identify genes critical to CD8+ T cell function. This project aims to provide enhanced function of T cells that are better suited for applications in clinic.

The MYH7 gene links closely to the development of hypertrophic cardiomyopathy (HCM), a condition marked by abnormal thickening of the heart’s left ventricular wall and impaired cardiac function. Pathogenic MYH7 single nucleotide variants (SNVs) account for ~40% of HCM cases. However, ~75% of known MYH7 SNVs are variants of unknown significance (VUS). While clinical and computational data can be used to classify the significance of MYH7 variants as pathogenic or benign, these data are sparse and often inaccurate. The Yang lab has established a β-MHC abundance assay that distinguishes clinically known pathogenic and benign MYH7 variants in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), enabling reclassification of many VUS. β-MHC abundance is not a well-established phenotype, whereas existing well-established phenotypes such as impaired contractility are not high-throughput. Therefore to validate the β-MHC abundance assay findings, I aimed to functionally assess a set of variants with normal or abnormal β-MHC abundances using orthogonal assays. Towards this, I generated transgenic hiPSC lines with heterozygous variants p.Ser866Phe and p.Leu863Pro (abnormal β-MHC abundance) and p.Leu881Met and p.Ser851Phe (normal β-MHC abundance) knocked into the endogenous MYH7 locus using CRISPR/Cas9 gene editing. These variants are evaluated for NPPA and NPPB expression using RT-qPCR, cell size using confocal microscopy, and contractility via traction force microscopy, and are compared to a wildtype MYH7 line as a control. VUS with reduced β-MHC abundance are expected to show increased NPPA and NPPB expression, increased cell size, and increased contractility. Conducting these tests could allow for more confident reclassification of these VUS and other MYH7 variants in the future. Pathogenic variants in MYH7 are clinically actionable, meaning that reclassification of VUS will inform earlier medical interventions that improve health outcomes for patients that develop HCM.

Diabetes has emerged as a leading cause of death in America and can affect the kidney, liver, heart, and lung system. Around 34 million Americans, primarily people of color, are currently diagnosed. Diabetes is a leading comorbidity of SARS-CoV-2 fatalities (~15%), highlighting a pertinent need to establish a human diabetic pulmonary model that may unveil dynamic mechanisms behind this phenomena. This project aims to establish a pulmonary model that reflects how diabetic conditions can affect the cellular phenotypes and morphologies of alveolar lung tissue. We utilized a previously established protocol to differentiate human induced pluripotent stem cells (iPSCs) into lung organoids (LOs). These LOs serve as biologically relevant lung models because they share the same complex, 3D cellular structures as human pulmonary systems. In this project, LOs were differentiated over 25 days and subsequently treated with varying glucose concentrations (5, non-diabetic; 10, diabetic; 21, experimental control; and 80, extreme) mM in growth media for 15 days, when morphological differences appear. The LOs were fixed on day 40 and analyzed using immunofluorescence to quantify lung markers. The primary antibody used was ACE2, the receptor for SARS-CoV-2. Additional markers included surfactant protein C (SFPTC), and NKX2.1, a lung progenitor marker, to compare phenotypic differences across the conditions. Our results demonstrated a pattern of upregulation of ACE2 with increasing glucose concentrations, suggesting diabetic conditions enhance susceptibility to SARS-CoV-2 infection, compared to normal glucose levels. Furthermore, SFPTC (Alveolar Type II cells) and ACE2 co-localize, which may play a key role in increased mortality rates amongst diabetic SARS-CoV-2 patients. Further studies, including qPCR analysis, may provide additional insights into these observations. In conclusion, our model highlights the increased vulnerability of diabetic pulmonary systems to SARS-CoV-2, emphasizing the need for targeted therapeutic strategies and investigation of dynamic disease mechanisms.

Discharge planning is an important component of safe and efficient hospital care. We are interested in how patients who live in a baseline unsafe situation conceptualize a “safe” discharge plan. Thus, our study aimed to understand the needs, perspectives, and priorities of patients experiencing homelessness as they prepare to leave the hospital. We conducted qualitative semi-structured interviews in 2023-2024 at a large urban, public, safety-net hospital in the Pacific Northwest. Any adult patient admitted to an acute care general medicine service who was living homeless was eligible. Patients with severe cognitive or mental health disorders that precluded consent process were excluded. This study was approved by our institutional IRB. Patients experiencing homelessness shared their perspectives on discharge planning in three major themes: 1) basic needs, 2) barriers to stability, and 3) role of healthcare systems. Within the theme of basic needs, respondents identified the importance of physical survival: shelter/warmth, protection from violence, and treatment for major injuries or illness. They identified key barriers to stability: loss of property, legal concerns, and financial challenges. While many respondents were hopeful that the role of the healthcare system might be to assist in these issues, they also acknowledged the existence of significant resource constraints. Many reflected on the way that U.S. society views poverty and the impact of policy and funding to what is feasible by healthcare providers. Several participants were aware of interventions that the healthcare system puts into place to achieve better outcomes but noted these may still be out of reach when basic needs are not met. We found that patients experiencing homelessness were overwhelmingly focused on achieving their basic needs, including physical survival and environmental stability. Current discharge processes should be tailored to individual lived experiences, especially with regards to housing status.

Through the tagging and cleaving of DNA sequences in Saccharomyces cerevisiae, we observe changes in MCM (minichromosome maintenance) protein recruitment, loading, and activation. The functions of MCM2-7 are critical to separate and unwind DNA in preparation for replication. In the G1 phase, MCMs are recruited and loaded to replication origins in an inactive state, within G1 cells. S phase follows, in which the CDC7/DBF4 kinase phosphorylates the MCM, allowing it to fire and initiate DNA unraveling for replication. The regulation of licensing and activation through these phases is crucial to ensure appropriate replication timing (early vs. late) in the genome. By tagging either a histone or one of the MCMs with micrococcal nuclease (MNase), I implement ChEC (chromatin endogenous cleavage) sequencing to cleave the DNA specifically where it surrounds the nucleosome or the MCM complexes. This method allows for precise mapping of the location of MCM binding sites and nucleosomes. We expect to see an increase in MCM helicase complex licensing and firing in regions occupied by less nucleosomes, resulting in regions of earlier DNA replication timing.

Alzheimer's disease (AD) is the most common form of dementia. Improper cleavage of amyloid precursor protein by a complex containing presenilin 1 or presenilin 2 (PSEN2) can result in pathological amyloid beta plaques. Recent work from the Valdmanis group found novel PSEN2 RNA isoform variants in AD. Specifically, we identified two PSEN2 3'UTR isoforms - a short (507bp) and a long (3976bp) 3'UTR. The 3'UTR harbors essential regulatory elements such as microRNA binding sites and Alu elements that control transcript maturation, stability, and abundance. Here, we sought to elucidate the functional significance of the PSEN2 3'UTR isoforms. To accomplish this, we completed small RNA sequencing to identify microRNA levels in human AD and control frontal cortex brains and used TargetScan7 to map these reads to the PSEN2 3'UTR isoforms. Our analysis identified 53 miRNAs with significant differential regulation in AD frontal cortex bulk homogenate and 76 miRNAs in purified synaptosomes. One miRNA, miR-34c, was significantly downregulated in both fractions. We identified five different miRNAs with significant regulation changes in AD, including miR-326, miR-346, miR-548p, miR-890, and miR-217. Of note, the long PSEN2 3'UTR had nine miRNA binding sites and two Alu elements, while the short PSEN2 3'UTR only contained one miRNA binding site. We next tested PSEN2 3'UTR isoform localization in human AD and control frontal cortex brain tissue using BaseScope in-situ hybridization. We found a marked decrease in PSEN2 expression in AD samples. To develop in vitro PSEN2 3'UTR isoform models, we designed constructs containing the PSEN2 3'UTR isoforms to overexpress in either HMC3 human microglial or SH-SY5Y human neuroblastoma cell lines. In vitro validation results indicated increased long PSEN2 3'UTR isoform abundance to the short isoform. Determining the functional relevance of the short and long 3'UTR of the PSEN2 transcript will further our understanding of AD pathology.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disease, impacting the protein interactions responsible for muscle contraction. Aficamten (Afi) is a novel myosin inhibitor that is designed to treat the underlying hypercontractility of HCM by reducing the number of myosin heads available for interaction with actin. In this study, I investigate the mechanism of Afi in both unloaded systems where muscles contract without external resistance and loaded muscle systems where contraction works against an external force. ADP release is the rate limiting step in complex/loaded muscle contraction - comparing how myosin level changes in unloaded systems translate to loaded muscle function provides a detailed mechanistic understanding of how Afi affects ADP release, and thus overall cardiac function. To determine ADP release rates in an unloaded system, pcS1 (porcine cardiac subfragment 1) is incubated with fluorescently labeled pyrene-actin and ADP and is rapidly mixed with a large excess of unlabeled ATP. As unlabeled ATP displaces the ADP bound to the actin.myosin complex, ADP dissociates, leading to actin detachment. This detachment results in a measurable decrease in fluorescence over time, allowing for the determination of ADP release rate constants. I expect Afi to slow the rate of fluorescence decay, indicating prolonged ADP release. To probe the effect of Afi in a loaded system, I utilize the demembranated mechanics assay to measure stress in complex muscle tissue. Subjecting muscle attached between a piezoelectric motor and a force transducer to rapid length steps and decreasing concentrations of ATP, I analyze the relationship between [ATP] and stretch response, providing a proxy for ADP release. I also expect Afi to decrease ADP release in complex muscle tissue. Understanding the mechanism of Afi in both unloaded and loaded models will provide protein and tissue level measurements, offering insight into its therapeutic effects on cardiac muscle function. 

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes joint damage, frailty, and potential disability. Its progression is unpredictable, making it difficult to manage in clinical settings. A major challenge in treatment is the lack of reliable clinical indicators or biomarkers to track disease activity and predict long-term outcomes like frailty and joint damage. Growth differentiation factor-15 (GDF-15) has shown promise as a biomarker in other diseases, but its role in RA remains unclear. This study explores whether GDF-15 can predict disease progression, frailty, and joint damage in RA patients. To understand the role of GDF-15 in RA, we measured its levels in both RA patients and healthy individuals using ELISA, which detects specific proteins. We explored how GDF-15 levels are related to disease activity, inflammation, and joint damage. In a group of patients followed for 8 years, we investigated whether GDF-15 levels at diagnosis could predict how the disease might progress. We used various statistical tests to analyze the data. The Mann-Whitney U-test helped compare GDF-15 levels between RA patients and healthy controls, Spearman’s correlation showed the relationship between GDF-15 levels and disease activity, and logistic regression allowed us to evaluate whether GDF-15 levels at diagnosis could predict future RA development. Through this study, we (i) analyzed how GDF-15 levels are linked to disease activity and inflammation in RA, (ii) explored whether measuring GDF-15 levels early on could predict disease progression and (iii) assessed whether GDF-15 could help identify patients at higher risk of developing severe joint damage or other complications. Ultimately, this research could help rheumatologists better understand and predict how RA will progress in patients, leading to more personalized and effective treatments.

Diabetes results in hyperglycemia and elevated lipid levels (diabetic dyslipidemia), both of which contribute to complications such as atherosclerotic cardiovascular disease. Preliminary data from our laboratory suggest that monocytes are lipid-loaded in diabetes, and the Cluster of Differentiation 36 (CD36) receptor mediates monocyte lipid-uptake. My preliminary data indicate that Cd36 mRNA expression increases in monocytes treated with high glucose. Thus, I hypothesize that hyperglycemia augments uptake of the Very-low density lipoprotein (VLDL), a triglyceride-rich lipoprotein (TRL) elevated by diabetes. To address this, bone marrow monocytes will be cultured in low and high glucose, ex vivo. An osmotic control will be included. Following the glucose stimulation, monocytes will be challenged with fluorescently labeled VLDL (Dil-VLDL), and the uptake will be measured by fluorescent microscopy. Furthermore, to verify that CD36 is critical for monocyte VLDL-uptake, bone marrow monocytes from control and CD36-deficient mice will be used. This study will help us clarify the relationship between lipid metabolism and hyperglycemia in diabetes-induced monocyte lipid loading.

Type 1 Diabetes is characterized by a dysfunctional response of the immune system, with our project focusing on CD8+ T-cells. Studying epigenomics provides us with information about differential gene expression, as well as distal enhancers and their targets. Understanding this genetic background enables more efficient means of treatment. In this paper, we look at three different kinds of sequencing: ATACseq, RNAseq, and Hi-C. Our focus up until this point has primarily rested upon the first, as we have used it to analyze chromatin accessibility across the genome in patients with T1D and healthy controls. To do so, we have found peaks within the reads for both demographics, which we then used to examine the individual peaks for each subject and the consensus peaks between each condition to see which are especially prominent. These peaks are then the focus of our differential expression analysis, which will allow us to understand the areas of significance and perform further exploration: variance calling and footprinting. As we continue with this project, we hope that RNAseq and Hi-C will provide us with information on gene expression levels and the physical structure of chromatin, respectively. The former was run and sequenced within our lab, but the latter is pre-existing data we will be drawing from for this analysis. Understanding the regulatory landscape allows for better informed treatments, not just for T1D but for autoimmune diseases as a whole.

Over 38 million Americans have diabetes, and over 90% of people with diabetes have Type 2 Diabetes. Diabetes increases the risk for complications, including Diabetic Kidney Disease (DKD), a disease which impacts filtration of the kidneys. This filtration occurs in the glomerulus, a specialized capillary network lined with a single layer of endothelial cells. The glycocalyx, an extracellular matrix (ECM), produced by the endothelial cells, plays a crucial role in regulating filtration. Injury, reduced function, or changes in the ECM of endothelial cells cause abnormal filtration and kidney disease. Previously generated single-cell RNA sequencing data from our lab and the Kidney Precision Medicine Project indicated an upregulation of genes involved in ECM remodeling, such as Adamts6, in diabetes, both in mice and humans. My hypothesis is diabetes induces degradation of the endothelial cell glycocalyx through increased expression of ECM degrading enzymes, contributing to glomerular endothelial cell dysfunction. To test if diabetes induces ECM degradation, I examined the abundance of glomerular glycocalyx in a mouse model of DKD in Type 2 Diabetes and in nondiabetic mice. Glycocalyx levels were assessed using wheat germ agglutinin staining and quantified through immunohistochemistry and flow cytometry. Diabetes significantly reduced glomerular glycocalyx in diabetic versus nondiabetic mice. Additionally, I investigated the role of hyperglycemia and dyslipidemia individually on ECM degradation in cultured glomerular endothelial cells. Further investigation will focus on the role of ADAMTS6 on ECM remodeling in diabetes by using a siRNA to block ADAMTS6 expression in cultured endothelial cells. Additionally, to further test the role of hyperglycemia in glycocalyx degradation, an SGLT2 inhibitor was given to diabetic mice to reduce blood glucose levels and examine the impact on endothelial cell glycocalyx. ECM remodeling may be induced by increased expression of ECM-degrading enzymes in diabetes, contributing to the glomerular filtration barrier breakdown seen in DKD.

Obesity in youth populations is an increasingly prevalent issue in the U.S., affecting 14.7 million children between ages 2-19. Evidence shows that poor diet quality directly contributes to inflammation in body-weight regulating areas of the brain, which predates the occurrence of obesity. Controlled-feeding studies can improve diet quality and assess health outcomes in children with overweight or obesity but require thoughtful execution and strict participant adherence to the study-prescribed diet. This project will assess the feasibility of a short-term controlled feeding study using two measures: acceptability from children of a high-quality diet menu and caregiver opinion on their child’s participation in a controlled feeding study. In collaboration with the Fred Hutch Nutrition Kitchen, I created a high-quality diet menu to meet study goals including: standard nutritional principles of USDA guidelines, whole foods, and low-fat, as well as age-appropriate and easy to prepare. Furthermore, I developed a child-appropriate taste-test questionnaire using a 5-point Likert scale and contributed to an interview guide in order to capture the qualitative and quantitative data from parents. These tools will be applied in a study of 7-10 families with healthy children aged 9-11. Families will attend a focus group where child caregivers will undergo the structured interview and children will taste-test the menu items. Quantitative survey data from the taste-test will assess menu acceptability to help ensure participant adherence to a study-provided diet. Qualitative data themes from adult caregivers will assess feasibility of child participation in a controlled feeding study by illuminating social and logistical concerns of parents. Understanding child acceptability of a high-quality diet menu and the perceived feasibility of study participation from families will inform the most optimal design of our planned controlled-feeding study in children to test if high-quality diet can acutely reduce inflammation in body-weight regulating areas of the brain.

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

Diabetes is characterized by hyperglycemia, which is exacerbated by the inappropriate storage and mobilization of hepatic glycogen. Incretin drugs, specifically GIP and GLP1 agonists, have been clinically successful. In the islet, GIP stimulates insulin and glucagon, while GLP1 stimulates insulin and inhibits glucagon production. Incretin receptors are not in the liver, suggesting that any effect of incretins on liver metabolism is through indirect islet hormone effects. Preclinical studies show the benefits of increasing hepatic glycogen storage in diabetes models, yet no drugs currently target this important source of carbohydrate metabolism. Therefore, my study aims to investigate how incretin drugs interact with hepatic glycogen stores to modulate islet hormone action. I hypothesized that increased hepatic glycogen will amplify islet hormone action and GIPR agonism will increase glycogen metabolism, while GLP1R agonism will have little effect on hepatic glycogen. I used hepatocyte-specific AAVs to overexpress the PPP1r3b protein in mice (PPP1r3bOE), resulting in a significant increase in hepatic glycogen compared to the control. Post-incretin injection (GIPR agonist, D-Ala2-GIP or GLP1R agonist, Ex4), I measured blood glucose and collected plasma to quantify circulating insulin and glucagon by ELISA. I collected liver to measure glycogen by glucose oxidase reaction and glycogen signaling intermediates by western blot. My results have indicated that GIP is less effective at glucose lowering in PPP1r3bOE compared to controls, while there is no effect on GLP1R-mediated glucose-lowering. This supports that elevated hepatic glycogen is likely altering glucagon action in response to GIP, with no effect on GLP1 action. I am currently analyzing plasma insulin and glucagon and will be assessing post-receptor insulin and glucagon signaling in the liver in response to GIP. This study will provide a better understanding of hepatic glycogen regulation, and offer an opportunity to investigate how incretin action can be optimized as a diabetes treatment.

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

Latines are the largest minority group in the United States but only represent 8% of clinical trial participants. This underrepresentation is due to significant barriers the Latine population faces including, but is not limited to language differences, limited access to insurance coverage, socioeconomic, urban-rural divide, immigration status concerns, and mistrust of research. Increasing Latine participation in clinical trials is an essential step in reducing health disparities. This study aims to explore the impact these barriers have on Latine participation in clinical trials and determine ways to address them. We are utilizing a mixed-method approach to conduct research - we are analyzing existing literature data, conducting qualitative interviews, and gathering survey data. Initial findings have suggested that language barriers are a main contributing factor to lack of representation, as many Latine participants report difficulties with reading and understanding consent forms in addition to other legal documents and speaking with healthcare professionals. Insurance coverage presented another significant barrier, as individuals without insurance are less likely to participate in clinical research due to the burden of out-of-pocket expenses. Additionally, there is also hesitancy with sharing personal information, as immigration status was a major concern, with individuals fearing deportation or detention. In order to address the challenges that impact Latine participation in cancer research it is important to set up culturally competent outreach programs, provide language and community health resources, and advance policy changes to ensure equitable participation. This study emphasizes the urgency for inclusive clinical research methods in order to reduce health inequalities and improve healthcare outcomes for Latine populations.

Platelet transfusions are critical for bleeding patients or patients at risk of bleeding. For this purpose, platelets are either stored at room temperature or in the cold (1-6 degrees C). Cold-stored platelets (CSPs) have a longer shelf life, can reduce bacterial contamination, and may be more effective than room temperature-stored platelets (RTPs). However, CSPs can form aggregates, ultimately making them unusable. What causes these aggregates and how to prevent them is poorly understood. This study aims to identify potential factors related to aggregate formation in CSPs. We obtained CSP units manufactured in plasma between 3/16/22 and 9/25/24 from 88 unique donors. The units were sent from South Texas Blood and Tissue, TX to Swedish Medical Center, WA. Using this data, we analyzed the rates of aggregates among donors of different sex, age, and blood groups in 88 unique samples. The same criteria for aggregates used for RTPs were applied to CSPs. Of the 88 donors in our sample, 36.4% were female and 63.6% were male. Of the 88 unique samples, 47 had formed aggregates. Donor ages ranged from 17 to 85 years. The average donor was 53 years and the median age was 56 years. Our sample consisted of donors with type A- (6.82%), type A+ (70.5%), type O- (1.14%), and type O+ (21.6%) blood. We found no significant difference between donor characteristics and aggregates. We also compared aggregate formation to the time between CSP collection and shipment, time spent at the hospital blood bank, and total time of storage. We found no significant associations between aggregate formation and any of the time variables. In summary, there were no significant differences between our variables and aggregates in CSPs collected in plasma. These findings can be used to explore alternate factors associated with aggregate formation in CSPs.

Alzheimer’s disease (AD) is characterized by microvascular (MV) changes due, in part, to basement membrane (BM) alterations. Collagen IV (Col IV), a key BM structural protein, is often found near amyloid-beta (Aβ) deposition in AD, but their relationship remains unclear. Our project investigates how Aβ binding and removal using lecanemab (mAb158) affects MV structure and Col IV in 5xFAD mice, an AD model with extensive brain Aβ deposition. We hypothesized that Aβ removal disrupts Col IV, increasing MV damage and hemorrhage risk. Six-month-old 5xFAD male mice were treated weekly for eight weeks with mAb158 10mg/kg, mAb158 20mg/kg, or isotype control. A fourth group consisted of wild-type (WT) mice (n=4/group). We stained brain sections with hematoxylin and eosin (H&E) for acute hemorrhages and Prussian blue (PB) for subacute hemorrhages. Additionally, I performed immunohistochemistry using collagen hybridizing peptide (CHP) for Col IV degradation and antibody 6E10 for Aβ deposition. In our preliminary results, H&E and PB indicated no acute or subacute hemorrhages in any of the groups. CHP levels were highest at 10 mg/kg, while 20mg/kg and isotype groups had levels similar to or slightly lower than the WT group. Contrastingly, Aβ decreased at 10 mg/kg but increased at 20 mg/kg, and was consistently higher in the lower cortex than in the upper cortex. All non-WT mice exhibited extensive Aβ deposition, suggesting that the late start to treatment reduced efficacy. Concurrently, our co-investigator observed increased blood-brain-barrier (BBB) leakage at 20 mg/kg, but not at 10 mg/kg. Overall, this pilot informs how Aβ-targeting antibodies affect Aβ deposits, Col IV structure, and BBB integrity during AD treatment. Ongoing studies with younger 5xFAD mice (n=10/group), treated from four-months-old for 12 weeks, will further define effects of Aβ on MV structure.

Globally, men who have sex with men (MSM) are at disproportionate risk of contracting STIs like HIV, particularly in sub-Saharan Africa (sSA). This epidemic is further compounded due to the sexual stigma and heteronormative culture present in countries like Kenya, where male-male sex remains illegal. MSM must maintain discretion surrounding their sexual behaviors, commonly preventing them from accessing sexual health services and disclosing their sexual orientation and activity. Consequently, female partners of men who have sex with both men and women (MSMW) may be at greater risk of STI transmission not only due to physiological causes but also because of social factors, such as being unaware of the same-sex sexual activity that their partners engage in. Kenya is a resource-limited area where preventative STI screening is prohibitively expensive and inaccessible, and the standard of care is syndromic treatment – individuals only seek medical care if they experience STI symptoms, yet over 80% of STIs are untreated due to individuals being asymptomatic. Despite the significance of this issue, few studies have attempted to distinguish the psychosocial characteristics and sexual behaviors of MSMW from those of men who have sex with men exclusively (MSME). This baseline analysis of the Tatu Pamoja Study explores differences in risk factors for STI transmission between MSMW and MSME in Kenya. We hypothesize that, compared to MSME, MSMW will exhibit a higher prevalence of poor mental health and partnership-level sexual risk behaviors, including one-time partners, condomless anal sex, and group sex – all characteristics associated with STI transmission. The findings of this study aim to identify MSMW as a subpopulation of MSM at particularly high risk of STI transmission who may benefit from being offered further preventative interventions – such as doxyPEP and routine STI testing – to prevent onward transmission and reduce the incidence of STIs in sSA.

The global pandemic of obesity has increased the prevalence and burden of metabolic diseases, including type 2 diabetes and cardiovascular disease. Obesity and its comorbidities are frequently associated with hypogonadism (low levels of testosterone (T) in men), and both preclinical and clinical evidence support a causative role of hypogonadism in predisposing individuals to metabolic diseases. However, the mechanisms remain unknown. One potential mechanism arises from our recent discovery that in mice, surgical castration (reducing T levels) amplifies the pro-inflammatory response to consumption of a high-fat diet, specifically leading to activation of astrocytes within the hypothalamus, a brain region critical for regulating whole-body metabolism. Concomitantly, there is a striking reduction of the anti-inflammatory neuropeptide neurokinin B (NKB; encoded by the Tac2 gene) in the same brain region. Therefore, we hypothesized that T limits astrocyte inflammation via enhanced NKB-neurokinin-3 receptor (NK3R) signaling. Using primary astrocytes harvested from newborn mice, we found that T and dihydrotestosterone (DHT; a non-aromatizable androgen) increase the expression of tachykinin genes like Tac2. Further, androgen treatment blunted the proinflammatory response of primary astrocytes to lipopolysaccharide (LPS), a sepsis-inducing bacterial cell wall component. To assess the anti-inflammatory capacity of NK3R signaling, we co-incubated astrocytes with the NK3R agonist Senktide and LPS, finding a significant attenuation of proinflammatory cytokine expression. Together, these data suggested that androgen receptor signaling might constrain astrocyte inflammation through induction of NKB-NK3R. However, the ability of DHT to reduce cytokine expression in response to LPS was preserved in the presence of Osanetant, an NK3R antagonist, indicating that the anti-inflammatory actions of androgens are independent of NK3R signaling. These findings form the foundation for future pharmacologic and genetic interventions in obese mouse models to further clarify the role of astrocyte T and NK3R signaling in hypogonadism-associated metabolic diseases.

Asthma is a chronic respiratory disease characterized by airway inflammation and remodeling. One key feature of airway remodeling is the thickening of the subepithelial basement membrane zone (BMZ) beneath the airway epithelium, which has been identified in severe asthma relative to milder severity asthma and other airway diseases. We aim to characterize the relationship between BMZ thickness, airway physiology, and airway immune cell populations. I am utilizing design-based stereology to precisely measure BMZ thickness in endobronchial biopsies obtained from 30 individuals with asthma and 10 healthy individuals. These individuals underwent extensive characterization for asthma airway physiology, profiling of airway immune cell populations, and airway inflammatory gene expression. Stereology provides unbiased thickness estimates that have greater reproducibility and overcome the limitations of two-dimensional measurements. I am measuring BMZ thickness using the orthogonal intercept method, which involves averaging the lengths of lines extended perpendicularly from the epithelial surface across the thickness of the BMZ at systematically sampled points. I am correlating BMZ thickness with clinical characteristics (allergic sensitization), airway physiology (baseline lung function, measurements of airway hyperresponsiveness), densities of both mast cells and eosinophils in the airway wall, and gene expression profiles obtained from airway epithelial brushings. I hypothesize that individuals with asthma patients will have more BMZ thickening in comparison to healthy controls. I also anticipate that there will be a positive correlation between the thickness of the BMZ and the expression of type-2 (T2) inflammatory genes (IL4, IL5, IL13). Finally, I hypothesize that there will be a positive correlation between BMZ thickness and the density of mast cells in the airway epithelial compartment. This research study provides new insights into the potential mechanisms responsible for airway remodeling in individuals with asthma and how they connect with airway inflammatory endotypes, which may guide further development of targeted therapeutics. 

Estrogen plays a key role in metabolic regulation including fat distribution and glucose homeostasis. Less understood are the differences of estrogen metabolism in different fat depots, and the conversion mechanisms that underlie the effects we see between two prominent isoforms of estrogen, Estradiol (E2) and Estrone (E1). E2 dominates during reproductive years, shifting to an increase in E1 post-menopause. These hormonal changes contribute to a switch in fat storage from subcutaneous to visceral depots, elevating the risk of metabolic diseases. My research investigates how differences in estrogen metabolism, mediated by cytochrome P450 aromatase (CYP19A1) and 17β-hydroxysteroid dehydrogenase (HSD17B1), influence glucose uptake in inguinal (IWAT) and epididymal (EWAT) white adipose tissue. Using ex vivo explants from C57BL6/J male and female mice, I treated IWAT and EWAT with E1, E2, an agonist of CYP19A1, and an HSD17B1 inhibitor. Glucose uptake was measured at baseline, after 24 hours, following insulin stimulation. Additionally, I performed RT-qPCR to quantify depot-specific expression of CYP19A1, HSD17B1, and related metabolic genes. Preliminary results show that IWAT exhibits higher baseline expression of CYP19A1 than EWAT correlating with IWAT also demonstrating greater insulin-stimulated glucose consumption. E1 treatment decreased glucose uptake in both depots, while E2 had minimal effect in IWAT. Most interestingly, E1 and the forskolin + HSD17B1 inhibitor combination significantly decreased glucose consumption. This suggests depot-specific metabolic adaptations driven by differences in estrogen metabolism. The differences between IWAT and EWAT in estrogen-mediated glucose regulation offers new opportunities to better understand the metabolic impact of E1 and E2 in estrogen metabolism and glucose uptake. Understanding these mechanisms could inform strategies for targeting adipose tissue to mitigate impacts of insulin resistance and obesity, especially for postmenopausal women. My contributions include conducting tissue treatments, measuring glucose uptake, and analyzing gene expression data.

Hyperphosphorylation and aggregation of the microtubule protein tau is a notable feature of diseases such as Alzheimer’s Disease, Frontotemporal Dementia and Chronic Traumatic Encephalopathy. However, the mechanism by which this causes diseases is still unclear. One avenue of interest is sphingolipid metabolism because various genes in sphingolipid metabolic pathways have been implicated in both tau toxicity and disease development. Sphingolipids are critical for cell membrane structure and stability, and play major roles in cell signaling pathways. This project will investigate the effects of mutations in gba-3, sphk-1, asah-1 (corresponding to GBA, SPHK1 and ASAH1 in humans), and various genes implicated in similar pathways, on a C. elegans model of tau toxicity. Forward mutagenesis screening identified a mutation of unknown function in gba-3. Previous investigation of loss of function gba-3 mutants showed no suppression of tau toxicity, so I intend to generate the identified mutation again using CRISPR. sphk-1 and asah-1 are known to be critical for the proper catabolism of sphingosine-1-phosphate (S1P), which plays a role in calcium regulation and neural differentiation and health. Improper catabolism of S1P has been implicated in the aggregation of hyperphosphorylated tau in neurons. This project will analyze the role of these key genes using various approaches, including locomotive assays, protein and lipid quantification, and the measurement of resistance to oxidative stress. Results from these three initial genes will help guide the subsequent investigation of specific metabolites implicated in disease development and may even highlight potential therapeutic targets.

Sarcopenic obesity (SO) is characterized by muscle weakness, atrophy, and an increase in body fat with age. While there is currently no FDA-approved treatment for this condition due to its complex pathogenesis involving chronic low-grade inflammation, impaired mitochondrial function, and a significant shift in muscle fiber quantity, function, and composition. However, previous studies have demonstrated an association between AMPK, an enzymatic mediator of cellular energy homeostasis, and aging. Thus, we aim to evaluate AMPK's viability as a therapeutic target by investigating its role in muscle mass maintenance, body composition, and mitochondrial function in aged mice. We used muscle-specific AMPKα2i transgenic (α2 D157A mutant, TG) mice and compared them to wild-type (WT) mice. Young (4-6 month) and old (20-24) female and male TG and WT mice were evaluated for body composition, grip strength, endurance, and muscle mass. We then used immunohistochemical and histochemical techniques on the collected muscle samples to analyze muscle fiber composition and mitochondrial activity, respectively. As shown in our previous studies, when compared their WT counterparts, young TG mice only demonstrated a decrease in endurance; old TG mice also had decreased muscle mass, greater body weight and fat mass, and more fatigable muscles. We expect to see consistent results when investigating mitochondria in AMPKα2i mice, namely a decrease in mitochondrial activity and density. AMPK is crucial for maintaining endurance in young mice, as well as retaining muscle mass and strength while attenuating obesity in old mice. Therefore, AMPK serves as a promising therapeutic target for prevention and treatment of SO.

Individuals with diabetes experience a unique set of challenges as metabolic disease impairs the proper regulation of glucose homeostasis. Glycogen stores in the liver are mobilized in response to islet hormones, insulin and glucagon, to address changes in circulating blood glucose levels. Individuals with diabetes are known to have lower hepatic glycogen levels and repairing these levels in preclinical mouse models of metabolic disease improves the diabetic state, suggesting hepatic glycogen storage may be a rational therapeutic target. With the use of a mouse model of increased hepatic glycogen by overexpression of a protein called Ppp1r3b ( Ppp1r3bhepOE), I explored the hypothesis that increasing hepatic glycogen levels affects the hormonal response and hepatic post-receptor signaling in response to nutrient feeding. Using oral gavage mixed-nutrient meals (consisting of varying glucose and/or alanine), I monitored plasma glucose and hormone levels from mice under different feeding conditions. I collected blood glucose and plasma samples from the tail vein and used ELISA to quantify circulating insulin and glucagon levels. In comparison to a control AAV group, the Ppp1r3b OE mice showed significantly elevated glycogen levels and following an overnight fast increased blood glucose levels. Furthermore, after conducting a 5 hour fast, the Ppp1r3b OE group had lower insulin levels without changes in glucose, signifying increased insulin sensitivity.  Yet, after an insulin tolerance test, Ppp1r3bhepOE mice did not decrease blood glucose to the same extent as controls, perhaps due to increased liver-derived glucose output. Lastly, to measure post-receptor signaling I administered either insulin or glucagon to control and Ppp1r3bhepOE mice and measured the glycemic response and activation of relevant hepatic signaling intermediates. My preliminary evidence reveals the importance of hepatic glycogen in energy metabolism and lays the foundation for future studies investigating how the alteration of glycogen storage could optimize energy expenditure in metabolic disease.