Increased mitochondrial oxidative stress causes fatigue and metabolic dysfunction in muscle tissue. It is unclear whether the oxidative stress is due to elevated production or impaired consumption of reactive oxygen species (ROS). The purpose of this study is to test whether the capacity of the antioxidant defense system is impaired or the mitochondrial ROS production rate is elevated in response to chronic changes in mitochondrial oxidative stress. To experimentally manipulate mitochondrial oxidative stress, we use an inducible mouse model to knockdown superoxide dismutase 2 (SOD2) in skeletal muscle and heart to increase oxidative stress, and exercise training to decrease oxidative stress. Knockdowns (KD) or littermate controls (CON) performed a six-week voluntary wheel running (EX) or sedentary control intervention (SED). Following completion of the intervention, I isolated heart and skeletal muscle mitochondria using differential centrifugation. I measured mitochondrial hydrogen peroxide (H₂O₂) production rate and tested the antioxidant capacity by treating isolated mitochondria with Auranofin (AFN) or 1-chloro-2,4-dintrobenzene (CDNB), which inhibit the thioredoxin and glutathione S-transferase components of the mitochondrial antioxidant defense system, respectively. KD heart and skeletal muscle had similar absolute H₂O₂ production rates compared to CON, but normalized to oxygen consumption the KD had significantly higher H₂O₂ production. Since absolute H₂O₂ production under vehicle conditions was not different, this suggests that the antioxidant capacity adapts to meet the changes in mitochondrial H₂O₂ production. We will collect data from the exercise-trained cohort next month. I expect to see an increase in H₂O₂ production rate and antioxidant capacity in both groups due to the increased mitochondrial biogenesis from exercise training. These results demonstrate that chronic increases in mitochondrial oxidative stress decrease mitochondrial H₂O₂ production capacity from skeletal muscle.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is a fatty acid β-oxidation disease associated with severe hypoglycemia and sudden death. MCADD is caused by biallelic pathogenic variants in the ACADM gene, which codes for a dehydrogenase specific to carnitines C6 through C12. MCADD is included in newborn screening (NBS) and characterized by elevated medium-chain acylcarnitine fats, annotated as C6, C8, and C10:1. Diagnostic testing is performed in plasma. Acylcarnitine results in dried blood spots have been described to differentiate carriers from affected individuals to reduce false positive NBS. Disease sensitivity and specificity, particularly to differentiate carriers from true positive cases, has not been well described in plasma. We posit that MCADD diagnosis will be more accurate if additional ratios beyond the primary disease markers, C6, C8, and C10:1 acylcarnitines, are considered. This study is a retrospective data review of NBS cases that were positive for possible MCADD and diagnostic testing was performed at Seattle Children’s Hospital. Cases are sorted into four groups: MCADD with homozygous disease variants, compound heterozygous MCADD, carriers of MCADD, or true negative. In collaboration with the biostats core service, linear regression models and receiver operator characteristic curves will compare acylcarnitine species and ratios by group. Preliminary results demonstrate that the primary markers associated with MCADD, C10/C2, C10/C6, C8/C2, C8/C10, and C8/Free carnitine, clearly discriminate affected individuals from control cases in plasma. Analysis is in process to compare the different genotypes of affected MCADD from carriers. Uncovering the diagnostic accuracy of plasma acylcarnitine profiles may influence future testing plans and improve the cost-effectiveness of healthcare services. DNA testing remains costly and slow. Additional biomarkers that provide a conclusive diagnosis of MCADD without requiring genetic testing may lead to more equitable patient care.

Tumor cell survival and recurrence remain a leading cause of death among cancer patients, and it is likely that the residual tumor cells that form the secondary tumor have distinct phenotypes from the primary tumor. The transcription factor NRF2 is thought to play a role in tumorigenesis, metabolic reprogramming, and recurrence in breast cancer. Emerging evidence suggests that NRF2 also intersects with the circadian rhythm, the 24-hour oscillatory clock present in all cells. My project investigates how NRF2 interacts with circadian rhythm genes, and how this interaction affects cancer cell growth. Mouse cell lines NMuMG, EMT6, 66Cl4, and 4T1 were cultured, treated with dexamethasone for synchronization of cellular clock, and harvested over three days. Cell pellets were collected every eight hours after synchronization, for a total of seven timepoints across 48 hours. I performed RNA extraction, cDNA synthesis, and RT-qPCR to analyze gene expression of  NRF2 (Nfe2l2), NRF2 target genes (Nqo1, Slc7a11, G6pdx, Gpx2, Txn1), and circadian rhythm genes (Bmal1, Clock, Per2, Cry1, Per1, Nr1d1) at each timepoint. 66Cl4 cells were further used to perform a CRISPR knockout screen for NRF2 target genes, to investigate which genes are essential for tumor cell viability. I cultured and infected cells with Cas-9 enzyme and sgRNA corresponding to 30 NRF2 targets using lentivirus, then allowed them to proliferate to 14 population doublings over the course of the screen. After the screen had completed, cells were sent for genomic sequencing to identify hit genes. Though these experiments are ongoing, we aim to identify 4-5 hit genes through the screen to direct future research on how NRF2 promotes tumor cell survival and proliferation. My data on NRF2 and circadian clock will also shed light on the intricate role of NRF2 in the cell, and open the door for new therapeutic targets.

Dementia, a growing global health concern, affects the nervous system and leads to severe cognitive impairment, with Alzheimer’s disease (AD) being the most common form, currently impacting nearly 7 million Americans. As life expectancy increases, the prevalence of dementia increases in corresponding fashion, driving research efforts like those of the Darvas Lab, where we study AD and other related dementias using adeno-associated viruses (AAVs) to induce neuropathologic changes. The TDP43 protein is involved in neuropathologic changes such as those in Frontotemporal Dementia (FTD) and in Amyotrophic Lateral Sclerosis (ALS), a primary motor neuron disorder. TDP43, primarily localized in the nucleus, plays a crucial role in regulating gene expression and RNA metabolism. TDP43 pathology in neurons involves the presence of TDP43 in the cytoplasm and its accumulation in cytoplasmic inclusions. To better understand the role of TDP43 in neurodegeneration, we use a mouse model where TDP43 proteins are introduced via AAV, a genetically engineered viral vector commonly used in research. This approach allows control over the timing of neuropathologic changes. Our prior AAV constructs included the Synapsin I promoter, which led to a severe ALS-like motor phenotype due to its expression in spinal motor neurons. However, this model could not be used to study the more subtle effects of dementia due to the extreme nature of the physical pathology. Therefore, our goal is to produce a new model to overexpress TDP43 using an AAV that is exclusive to the cortical brain regions relevant to FTD by instead including the CamKIIα promoter, which exclusively drives expression in the forebrain. I assessed behavioral phenotypes in our mouse model by conducting a Y-maze to evaluate effects on short-term memory, and analyzing neurological scoring to evaluate neuromuscular dysfunction. The development of a more dementia-focused TDP43 model will allow us to more specifically investigate its neuropathology.

Microglia are innate immune cells in the brain that play an important role in maintaining homeostasis, carrying out immune surveillance, and modulating synaptic plasticity. Through the secretion of cytokines, along with physical functions like synaptic pruning and network refinement via phagocytosis, microglia support the health of neurons and help establish a functional neuronal network. In previous experiments, our lab demonstrated that when cultured with microglia, neurons exhibit more robust morphology and greater synaptic activity. However, it remained unclear whether the beneficial effects of microglia on neurons occur during physical contact between the two cell types, or if the supportive factors secreted by microglia are sufficient to drive this change. To investigate this, I compared the morphology and function of neurons directly co-cultured with microglia, to neurons treated with media conditioned by microglia. In the microglia-conditioned media treatment, I conditioned the microglia media for 24, 48, and 72 hours prior to treating the neurons to determine the optimal conditions for establishing a healthy neuronal environment. To evaluate these results, I conducted immunofluorescence staining for microtubule-associated protein tau (MAPT) and microtubule-associated protein 2 (MAP2), both of which are indicators of neuronal health. I analyzed fluorescence intensity and neurite length to quantify morphological differences in neurons between conditions. To investigate differences in synaptic activity, I carried out micro-electrode array recordings of neurons in each condition. Using data from these recordings, I analyzed the coordinated and overall electrical activity to measure the functionality of the synaptic network in each condition. Given that microglia perform both secretory and physical functions, I expected that neurons directly co-cultured with microglia would exhibit more robust phenotypic and functional characteristics. These experiments provide insight into healthy neuron-microglia interactions and reveal possible avenues towards their dysregulation in Alzheimer’s disease (AD), potentially guiding the development of therapeutics targeting such interactions.

My research project focuses on age-related changes in muscle function. We have previously designed and used novel young and naturally aged in vitro three-dimensional engineered muscle tissues (3D-EMTs) using donated myoblasts from the Study of Muscle, Mobility, and Aging (SOMMA) to investigate this. A question raised in this research is the how closely force measured in 3D-EMTs correlates to in vivo force of intact skeletal muscle. To address this, I stimulated young and aged mice's gastrocnemius muscles to contract (Aurora Instruments) measuring maximum force, contraction/relaxation kinetics, and fatiguability. Mice were then sacrificed and hindlimb muscles dissociated to isolate skeletal muscle myoblasts for cell culture. Myoblasts were amplified and used to generate young and aged rodent 3D-EMT. We tested in vitro 3D-EMT muscle mechanics using a Magnetometric Analyzer for engiNeered Tissue ARRAY (MantARRAY, Curi Bio). In vitro muscle force data was compared to in vivo force data from the same mouse. Results generated by this project helped identify the correlation between in vivo and in vitro force measurements and how they are impacted by age. This study also allowed us to bank multiple cell lines for future high throughput studies to utilize these rodent 3D-EMT models to study the progressive loss of muscle mass and function known as sarcopenia. The results from this project and the cellular models created will be used in the future to investigate potential targets for therapeutic interventions to treat sarcopenia in an ever-expanding aging population.

Despite the development of effective COVID-19 vaccines, countries with high rates of HIV infection still have limited vaccine access and inadequate access to antiretroviral medications essential for controlling HIV. Studying COVID-19 vaccination in people living with HIV (PLWH) is needed to improve vaccination strategies in immunosuppressed populations. Previously, in pigtail macaques (PTM) we showed that COVID-19 repRNA vaccination elicits durable and protective immunity against SARS-CoV-2 infection. Here we utilized the simian immunodeficiency virus (SIV)-infected PTM model of HIV/AIDS to test the hypothesis that COVID-19 repRNA vaccination is less durable during immunosuppression. Nine PTM were enrolled into the SIV naive (n=4) and experimental SIV-infected (n=5) cohorts. SIV-infected PTM were infected with 10,000 units of SIVmac293M seven weeks prior to the first vaccination. All PTM were given 2-4 COVID-19 repRNA immunizations, encoding the SARS-CoV-2 WA.1 Spike protein, to reach maximal immunogenicity and monitored for 26 weeks for vaccine durability. Vaccine recall was evaluated by administering a booster immunization after immunity responses waned. Blood and bronchoalveolar lavage samples were collected every 2-4 weeks. SIV-infected animals were monitored for SIV disease progression, including measuring CD4 counts in peripheral blood. Enzyme-linked immunosorbent assays (ELISAs) were used to quantify longitudinal Spike-specific binding IgG antibodies. Preliminary data shows that COVID-19 repRNA vaccination elicited robust anti-Spike IgG antibodies in both PTM groups, with diminished responses in some animals within the SIV-infected group.  Anti-SARS-CoV-2 neutralizing antibodies were also generated in both groups and interestingly, more durable in SIV-infected animals. Ongoing analysis includes evaluation of IgM and IgA binding antibodies. Collectively, this study suggests SIV-associated immunosuppression impacts vaccine-induced humoral memory, which could, in turn, impact long-term protection from COVID-19. These findings are crucial for improving vaccine regimens for PLWH and other immunosuppressed individuals.

Merkel cell carcinoma (MCC), a rare skin cancer, is mostly driven by integration of the Merkel cell polyomavirus which encodes T-antigen (T-Ag) proteins. Previous research has shown that T & B cells target T-Ag. Indeed, patients with virus-driven MCC produce T-Ag-specific antibodies that are useful to track disease progression. These antibodies do not play a direct role in MCC immunity as T-Ag proteins are intracellular. Our group has recently found that in tumors, T-Ag-specific B cells with germinal center or antibody-secreting phenotypes strongly predict improved MCC outcomes. These intratumoral B cell phenotypes reflect a robust cancer-specific T cell response. In contrast, T-Ag-specific B cells in the blood of MCC patients are predicted to predominantly have a memory or naive phenotype, and it is unknown if they contribute to anti-tumor immunity. We used fluorescently labeled T-Ag-proteins and flow cytometry to assess B cell responses in blood at the time of MCC diagnosis. In total, we analyzed samples from 23 patients whose MCC recurred within 3 years of diagnosis and 24 samples from stage- and age-matched MCC patients whose disease did not recur. We found no difference in the frequency of all circulating B cells (regardless of T-Ag-specificity) between patients who did and did not develop MCC recurrence. In contrast, higher frequencies of total memory B cells (CD27+IgD-IgM-) were associated with an increased risk of disease recurrence (HR 3.67 [1.58- 8.55], p=0.003). Intriguingly, T-Ag-specific memory B cells were also more abundant in the blood of patients who ultimately developed MCC recurrence (HR 2.82 [1.22- 6.53], p=0.012). Together, our results demonstrate that higher frequencies of circulating memory B cells associate with worse MCC outcomes. These findings suggest that the functional state of total and T-Ag-specific circulating B cells reflect their immune response within MCC tumors.

Recent research shows the lux operon utilized with in-vivo bioluminescence imaging to detect infectious diseases in animal models. Modifications to this operon led to the development of enhanced bioluminescence in Escherichia coli cells. However, expression of this operon has not been optimized for expression in other bacteria, such as Staphylococcus aureus. This study aims to optimize the lux reporter gene expression for Staphylococcus aureus, so luminescence is bright enough to register without specialized equipment. To date, the research has explored Gibson Assembly for cloning the gene sequences into a shuttle vector and efforts to modulate gene expression to reduce toxicity in E. coli.

Tau protein is highly expressed in neurons and other neural cells, including astrocytes. The accumulation of hyperphosphorylated tau aggregates, known as neurofibrillary tangles (NFTs), is a hallmark of neurodegenerative diseases, particularly Alzheimer’s disease (AD). While tau aggregation is thought to advance AD through toxic gain of function, the loss of tau physiological function may also contribute to the adverse progression of the disease. However, how the lack of tau physiological functions in neurons contributes to AD progression remains understudied.  Studies have shown that tau depletion results in minimal phenotypic differences and may even mitigate cognitive decline in AD mouse models. Here, we investigate the molecular consequences of tau loss in both neurons and astrocytes. Using CRISPR/Cas9 gene editing, we generated tau knockout (Tau KO) human induced pluripotent stem cell (hiPSC) lines, which were subsequently differentiated into neurons and astrocytes. We first focused on assessing how Tau depletion affects the hiPSC-derived neurons. Our findings indicate that tau depletion does not impair neuronal differentiation or increase cytotoxicity and cellular stress. However, preliminary data suggest that Tau KO neuronal cultures—composed of 95% neurons and 5% other neural cells—exhibit reduced synaptic firing activity and network burst frequency. These results suggest that tau loss in neurons and glial cells negatively impacts neuronal activity, providing new insights into the functional consequences of tau depletion in AD pathology. To gain deeper insight into how tau depletion negatively impacts neuronal activity, we performed transcriptomic analysis on Tau KO hiPSC-derived neurons using RNA sequencing (RNA-seq). We are currently analyzing and validating the results, which may further elucidate the molecular mechanisms underlying tau loss-of-function in neuronal regulation and AD pathology. In the second phase of this investigation, we will also differentiate the Tau KO hiPSC into astrocytes and assess how tau depletion impacts astrocytic viability and functions.

Syphilis, caused by the bacterium Treponema pallidum, remains a major global public health concern despite the availability of curative treatment. Cases in the U.S. have increased by nearly 80% since 2018, and congenital cases have skyrocketed by 937% since 2014. Currently, a variety of treponemal and non-treponemal tests exist for syphilis diagnostics. Still, they can be limited by high costs, false positive and negative results, and an inability to distinguish between current and prior infection, depending on the test. Further, the fragility and low protein content of T. pallidum’s outer membrane, coupled with its nature as an obligate pathogen, exacerbates the difficulty of conventional approaches to proteome characterization. Current assays are ultimately incapable of characterizing a high-resolution immune response to T. pallidum in humans. Here, we introduce a phage display and immunoprecipitation sequencing (PhIP-Seq) platform capable of identifying antibody epitopes across the entire T. pallidum proteome. This platform allows for the profiling of antibodies that bind to linear B-cell epitopes. This can further the current understanding of antigenic proteins within T. pallidum, their ability to elicit an immune response in humans, and reveal antigens with the potential as a diagnostic. Utilizing 40 single-draw serum samples from syphilis-infected patients in Peru and Seattle, we characterize how antibody responses differ based on syphilis stage, HIV status, and strain of the infection, and have identified four proteins - TP0136, TP0969, tprK, and arp - as being highly enriched across all patients.

Heart attack survivors experience elevated risk of subsequent events and death, as such there is a clinical need for regenerative techniques to rebuild damaged myocardium and reduce risk. Transplantation of human-induced stem cell-derived cardiomyocytes (hiPSC-CMs) into non-human primate hearts has been shown to significantly improve contractile function after heart attack, however, transplanted hearts were also shown to be at elevated risk for developing potentially lethal arrhythmias. Researchers developed a line of hiPSC-CMs to correct this arrhythmia-promoting behavior by inducing a series of four gene edits to prevent the hiPSC-CM spontaneous beating behavior known as "automaticity", these gene edits spawned the cardiomyocyte cell line dubbed MEDUSA (Modification of Electrophysiological DNA to Understand and Suppress Arrhythmia). I have conducted an in-vitro study of the electrophysiological effects of the MEDUSA edits, knockouts of the sodium-calcium exchanger NCX1, the hyperpolarization-activated pacemaker current channel HCN4, the low voltage Calcium ion channel Cav 3.2, as well as overexpression of the potassium channel Kir 2.1. I have employed immunofluorescence microscopy to analyze sarcomere structures, used lentiviral transduction of calcium-sensitive green fluorescent protein to quantify calcium handling, and constructed engineered heart tissue casts to measure contractile force, to compare the electrical and physiological characteristics of MEDUSA CMs and their wild-type counterparts. Characterizing the MEDUSA cell line is essential for identifying issues that could compromise the cells' ability to function in grafts while uncovering its potential for use in regenerative treatments. Here I show that the MEDUSA gene edits create arrhythmia-resistant cardiomyocytes without compromising the integrity of their structure or function, supporting the development of a regenerative therapy future for the field of cardiology.

TDP43 is an RNA/DNA binding protein that forms pathological aggregates in most amyotrophic lateral sclerosis (ALS) and half of frontotemporal lobar degeneration (FTLD) cases. Knockout of TDP43 in animal models leads to neurodegeneration and motor deficits, but overexpression of wildtype TDP43 leads to the same events; therefore, TDP43 protein homeostasis is critical to prevent ALS/FTLD. To achieve this homeostasis, TDP43 autoregulates its own mRNA splicing, resulting in multiple TDP43 isoforms. Although some of these isoforms go through nonsense mediated decay, other isoforms result in unique proteins with differing C-termini. This leads to variable cellular localization. It is unknown if these alternative, protein-coding isoforms are predominantly associated with ALS/FTLD or if aging changes the frequency of these isoforms. To determine how TDP43 overexpression yields different isoforms and interacts with aging and ALS-like symptoms, the Darvas Lab created a novel approach to overexpress human TDP43 (hTDP43) via Adeno-Associated Virus (AAV) delivered through retro-orbital injection, leading to ALS-like motor deficits. We tested this AAV in young and old mice cohorts. Then, to determine if Tardbp alternative splicing is linked to ALS-like symptoms and aging, I designed and validated primers and protocols to measure the nine Tardbp mRNA isoforms in mice via quantitative real-time polymerase chain reaction (qRT-PCR). I have started to determine if hTDP43 overexpression leads to differential splicing compared to mice injected with a sham-control AAV in these old and young mice. Once this is done, we will clone the most interesting differentially spliced isoform in an AAV and inject that AAV and a full-length TDP43 AAV into mice to see if the spliceform causes increased toxicity, manifesting in worsening motor deficits and mortality.

Plasma cell neoplasms are characterized by the secretion of large amounts of monoclonal antibodies into circulation. Monoclonal gammopathy of undetermined significance (MGUS) is a precancerous plasma cell neoplasm that can develop into a cancer called multiple myeloma. Glycosylation of the antigen-binding (Fab) light chain fragment of monoclonal antibodies is a risk factor for MGUS progression to myeloma. However, heavy chain Fab glycosylation may also occur and be a risk factor for progression, but this has not yet been investigated. In this study, I identify whether heavy chain Fab glycosylation occurs on monoclonal antibodies isolated from MGUS and myeloma patient serum, to help determine whether it should also be investigated as a risk factor of disease progression. I isolate intact antibodies to optimize antibody fragmentation and dissect them into light chain, heavy chain, total Fab, heavy chain Fab, and fragment crystallizable region (Fc) fragments. To do this, I purify IgG from serum from patients with plasma cell neoplasms. I either reduce these purified IgG samples using dithiothreitol into heavy chain and light chain fragments, or cleave them using IdeZ enzyme into total Fab and Fc fragments. I separate and purify reduced heavy chain and light chain fragments using high pressure liquid chromatography, and the cleaved total Fab and Fc fragments using an affinity matrix.  I buffer exchange the fragments through dialysis or using filters, and assess fragmentation and purity using SDS Page gel electrophoresis. After optimizing these protocols, I will isolate pure antibody light chain, heavy chain, total Fab, heavy chain Fab, and Fc fragments from MGUS and myeloma patients and healthy controls. This will enable subsequent glycosylation analysis and characterization. We anticipate that our results will lead to an improved understanding of antibody glycosylation in plasma cell neoplasms and provide insight into their potential role as risk factors for disease.

Huntington’s disease (HD) is a progressive autosomal-dominant neurodegenerative disease due to the expansion of a CAG-repeat in the huntingtin (HTT) gene. CAG-repeat lengths less than 36 are not associated with disease phenotype, however HD with CAG-repeats greater than 39 causes full disease penetrance, characterized by motor, cognitive, and behavioral symptoms. The onset age of HD symptoms and severity of the disease correlates with the length of the CAG repeat, although there is compelling variability in length and age of onset. Somatic instability (SI) in HD is the occurrence of faulty DNA repair that causes CAG-repeat expansion and continues to lengthen with age. There has been significant research on treating HD but not a treatment addressing both SI and lowering mutant HTT protein. Our lab utilizes multiple therapeutic methods to investigate the relationship between SI and mutant HTT protein with mouse genetic models. More specifically, we devise a CRISPR/Cas9-mediated approach to excise the proximal promoter region in Htt^Q111/+ mice, which have their mutant HTT protein knocked down. My roles in this project include measuring the amount of transcription of the HTT gene from these mutant HTT CRISPR-treated mice to determine whether the amount of HTT gene transcription affects somatic instability. We devised a specially designed qPCR assay to measure the pre-mRNA of the HTT gene. Since these CRISPR-treated mice have been found to have lower somatic instability, we expect the amount of transcription of the HTT gene will directly affect the amount of somatic instability.

Alzheimer’s disease (AD) is the most common cause of dementia in the aging population and is characterized pathologically by the presence of amyloid plaques and tau neurofibrillary tangles in the brain. However, other co-pathologies are often present along with AD, such as TDP-43 pathology. TDP-43 pathology, which was first described in other forms of neurodegenerative disease, has more recently been observed as a common co-pathology in AD, particularly in older individuals. The pathology is characterized by aggregates of hyperphosphorylated TDP-43 in the same brain regions as the tau pathology of AD, including the hippocampus. The combination of AD and TDP-43 pathology is associated with accelerated cognitive decline, greater brain atrophy, and increased AD pathological burden, particularly tau. In past studies, it has been suggested that there may be a potential synergistic relationship between tau and TDP-43 co-pathology in model systems. However, there is limited data on the relationship between quantitative tau and TDP-43 in human post-mortem tissues. This project explores the correlation between tau protein and TDP-43 in the aged brain using a quantitative neuropathological approach. We identified brain donors from the University of Washington BioRepository and Integrated Neuropathology (BRaIN) lab with pathology-confirmed high levels of AD pathology and varying degrees of TDP-43 co-pathology, along with a matched group of donors with high AD pathology and no TDP-43 co-pathology (n=8 per group). We use immunohistochemistry to stain the frontal cortex and hippocampus of each donor for hyperphosphorylated TDP-43 and different forms of pathologic tau. We quantify pathologic protein burden on digitized slides using the image analysis platform HALO and assess the relationship between tau and TDP-43 burden and cognitive function. This work will expand our understanding of the relationship between tau and TDP-43 pathology and ultimately provide new avenues for potential diagnostic and therapeutic approaches.

In 2023, the World Health Organization (WHO) estimated that there were 263 million malaria cases and 597,000 deaths globally. Parasites of the genus Plasmodium are the causative agent of malaria, deposited into the dermis of a human host through the bite of a female Anopheles mosquito carrying infected sporozoites (spz). From the dermis, spz migrate through the bloodstream and into the liver where they infect hepatocytes, producing potentially thousands of merozoites from a single hepatocyte which then enter the symptomatic erythrocytic stage of the disease. Higher numbers of CD8+ T cells per infected hepatocyte have been associated with Plasmodium clearance and because eliminating all infected hepatocytes during the pre-erythrocytic stage prevents malaria onset, identifying causes of CD8+ T cell recruitment provides critical insights for malaria prevention. The liver is one of the most sexually-dimorphic organs in both mice and humans, leading us to utilize immunohistochemical light microscopy to observe CD8+ cells in inflammatory foci, defined as abnormal concentrations of hepatic nuclei including at least one CD8+ cell. Using digital pathology software, we quantified these in female, male, and orchiectomized male (ORX) BALB/cJ mice that were either unvaccinated or repeatedly vaccinated with radiation-attenuated spz allowing us to assess the role of androgens in this recruitment. We found that following challenge with the rodent malaria wild-type parasite Plasmodium yoelii spz, vaccinated mice had more inflammatory foci and CD8+ cells than unvaccinated mice while intact male mice had fewer CD8+ cell and inflammatory foci than ORX or females of similar vaccination status. These findings suggest that androgens reduce recruitment of CD8+ T cells to inflammatory foci, providing a potential explanation for the reduced parasite clearance in male mice compared to their female counterparts. Further studies should explore the mechanism behind this reduced recruitment to inform important decisions in malaria vaccinology and translational medicine.

Urine cultures are the primary method for urinary tract infection diagnosis. Like most culturing applications, these tests often require days to yield conclusive results, causing harmful treatment delays. Additionally, hospitals waste substantial time and resources processing negative patient samples. Predicting culture outcomes before their final result can accelerate patient care and improve diagnostic efficiency by minimizing resource allocation towards culturing negative samples. The goal of this study is to build machine learning models to predict urine culture outcomes and help optimize test order protocols. Using clinical data from the UW Medical Center, containing urine culture results, blood test results, and demographics from over 88,000 patients, I trained Random Forest models, Support Vector Machines, and XGBoost models to predict overall culture positivity and specific infection types (E. coli, Klebsiella, etc.). Predictive parameters included common clinical laboratory tests (complete blood counts, metabolic panels, etc.), as well as demographics (age, sex, and race). To evaluate the predictive performance of these features at different points in the culturing timeline, the data was divided into three subsets: (1) patients with blood work up to 30 days prior to sample collection, (2) patients with blood work up to 30 days prior to their first culture result, and (3) patients with blood work predating their final culture result. Tree-based ensemble models (RF and XGBoost) trained on the latter two subsets yielded the most promising results. The Random Forest model’s AUC (area under the curve), a value between 0 and 1 that measures a model’s ability to distinguish between two classes, was 0.87. An AUC closer to 1 indicates more accurate classifications. The results show that ML models can feasibly predict culture results to optimize operations and enable earlier treatment. Further development of this data pipeline will allow for detailed predictions of specific infection types and concurrent infections.

Nearly all forms of cardiac disease are characterized by cardiac fibrosis, which contributes to heart failure and arrhythmias due to the accumulation of collagen deposits. Collagen, a crucial extracellular matrix (ECM) protein, is secreted by cardiac fibroblasts—the primary cell type responsible for generating this stiff scar tissue known as fibrosis. Fibroblasts are highly plastic cells that can transition between quiescent and activated states. The Davis Lab has developed a minimally invasive intermittent injury model to cyclically stress cardiac fibroblasts in vivo, allowing for a deeper investigation into the role of cellular memory in regulating the fibrotic response. Notably, we can reduce fibrotic remodeling in this model by inhibiting p38 gene function in the activated population, thereby encouraging a shift back to a quiescent state. My work aims to determine whether the once-activated population is proliferating at the second injury stimulus as well, or if a new population of fibroblasts is proliferating with repeat injury. To address this, I am utilizing genetic lineage tracing and Click-iT EdU technology, which allows for precise biolabeling while also preserving cell morphology and integrity by integrating into the cell's DNA. I am also performing immunohistochemistry staining to detect other proteins of interest that will serve as proliferation markers as well. Based on prior findings in the Davis Lab, we hypothesize that once-activated fibroblasts will go on to activate again when exposed to repeated disease stimuli, but there will be no second wave of proliferation as there was no change in total fibroblast number. 

Critical care outcomes vary significantly across healthcare settings, and resource-limited environments often lack high-quality data-driven insights that can improve patient care. In particular, there is a strong need to understand whether insights derived from high-resource settings such as the US can be readily applied to more resource-constrained healthcare settings. To this end, here I systematically compared ICU populations between hospitals in India and the University of Washington Medical Center (UWMC) to assess differences in laboratory test dynamics and clinical outcomes (mortality, sepsis, blood transfusions, etc.). Using ICU datasets containing >100,000 patients, I applied clustering algorithms to segment patient populations based on lab test time series data. Clustering was performed across stratifications of age, sex, and admitting diagnosis to capture variations in population health. Resultant clusters demonstrated up to a threefold stratification in mortality rate (3% to 15%), with similar stratifications for other outcomes. Additionally, dimensionality reduction techniques (Uniform Manifold Approximation and Projection [UMAP], t-Distributed Stochastic Neighbor Embedding [t-SNE], etc.) were used to visualize population differences. To identify the strongest predictors of clinical outcomes, I used Cox proportional hazards model to quantify the impact of individual lab tests on patient outcomes. The analysis revealed distinct differences in lab test significance between UWMC and Indian ICU populations. Key lab tests such as Blood Urea Nitrogen and Hemoglobin were strong predictors of adverse outcomes across both cohorts, but their relative importance varied between the two settings, suggesting differences in disease progression, healthcare practices, and marker utility. These findings highlight the need for region-specific risk models and emphasize the importance of integrating time-series lab data into ICU decision-making. Understanding these differences can inform the development of more generalizable risk stratification models and improve critical care strategies in resource-limited settings, ultimately advancing global healthcare equity.

Alphaviruses such as chikungunya virus (CHIKV) pose a significant threat to global health, yet specific antiviral therapies remain unavailable. In this study, we evaluated combinations of three approved oral directly acting antiviral (DAA) drugs (sofosbuvir (SOF), molnupiravir (MPV) and favipiravir (FAV)) against CHIKV, Semliki Forest virus (SFV), Sindbis virus (SINV), and Venezuelan Equine Encephalitis virus (VEEV) in vitro and in vivo . In human skin fibroblasts, synergistic antiviral effects were observed for the drug combinations MPV + SOF and FAV + SOF against CHIKV, and for FAV + SOF against SFV. In human liver Huh7 cells, the combinations of FAV + MPV conferred additive to synergistic activity against VEEV and SINV strains, while SOF synergized with FAV against SINV strains. In a mouse model of CHIKV arthritis, MPV improved CHIKV-induced foot swelling and reduced systemic infectious virus titers. Combination treatment with suboptimal doses of MPV and SOF significantly reduced foot swelling and decreased infectious virus titers in serum as compared to single doses of each drug. Sequencing of CHIKV RNA from mouse joint tissue revealed that MPV caused dose-dependent increases in mutations in the CHIKV genome. Upon combination therapy of MPV with SOF, the number of mutations was significantly lower compared to single treatment with several higher doses of MPV. In summary, combining approved oral nucleoside analogs confers potent suppression of multiple alphaviruses in vitro and in vivo with enhanced control of viral genetic evolution in the face of antiviral drug pressure. These drug combinations may ultimately lead to the development of potent combinations of pan-family alphavirus inhibitors.

The goal of this project is to better understand a driver of immune dysregulation in people with Down syndrome, who experience a complex interplay between genetics and immunity, leading to a higher risk of autoimmune diseases. Despite advances in research, the mechanisms driving this heightened susceptibility remain largely unexplored. In the U.S., approximately 1 in every 700 babies are born with Down syndrome each year. Understanding these differences is crucial for developing targeted therapies to improve health outcomes and quality of life. In the Khor lab, we are focused on understanding these mechanisms and how to treat autoimmune diseases common in people with Down syndrome. Preliminary data shows that RNA expression of Duffy, a gene encoding the Duffy antigen receptor for chemokines, is much higher in blood from people with Down syndrome. We want to determine if the protein expression of Duffy follows this same pattern. The Duffy antigen, located on red blood cells, binds chemokines released during inflammation, attracting immune cells to sites of damage. In our experiment, we will use flow cytometry to detect Duffy on RBCs. We expect to find that RBCs from people with Down syndrome express higher levels of the Duffy protein. Our data may reveal a new mechanism of immune dysregulation in Down syndrome and provide insights into how this gene affects the severity of malaria, as the Duffy protein is a receptor for it. This study can serve as a strong foundation for future research on immune dysregulation and infectious diseases like malaria in people with Down syndrome.

Clinical ventilation methods - where a patient’s breathing is supported by a cannula, a mask, or in severe cases, tracheal intubation – are often used to support patients in ICUs (intensive care units). Given the high risks associated with intubation, a key question is whether a patient's level of care can be de-escalated. This question is crucial in the context of newborn children (or neonates) who are especially fragile.  To address this question, we developed machine learning models to predict ventilatory support outcomes in neonates. Using a multi-center dataset of 6,538 neonates from ICUs (Intensive Care Units) across 121 Indian cities, we extracted time-based snapshots of each patient’s clinical status at 6-hour, 12-hour, and 24-hour intervals. For each snapshot, we utilized features related to vitals (heart rate (HR), respiratory rate (RR), etc.), demographics (e.g., highest HR, highest RR, etc.), and ventilation states (e.g., room air, nasal cannula, noninvasive ventilation, intubation). We trained Random Forest models, with hyper-parameters optimized via 5-fold cross-validation, using a 70-30 train-test split, to predict three outcomes: (1) mortality, (2) escalation of ventilatory support (“step-up”), and (3) successful discharge. Model performance was evaluated using the area under the receiver-operating-characteristic curve (AUROC). Predicting at 6, 12, and 24 hrs, the best models achieved out-sample AUROC values of 0.79–0.87 for mortality, 0.81–0.85 for step-up, and 0.68–0.70 for discharge. The most predictive variables for mortality, escalation, and safe discharge were the highest past FIO2, and the presence of a cannula for escalation and safe discharge, respectively. These findings suggest that machine learning can provide early warnings of clinical deterioration, reducing unnecessary intubations and improving neonatal respiratory care. By enhancing risk stratification, particularly in resource-limited settings, this approach may guide more timely and judicious use of tracheal intubation and contribute to improved neonatal outcomes.

The axon initial segment (AIS) plays a crucial role in maintaining neuronal excitability and action potential initiation. It is structurally and functionally plastic, adapting to pathological conditions such as traumatic brain injury (TBI). While microglia, the resident immune cells of the central nervous system, are known to respond to injury and influence neuronal function, their interactions with the AIS remain underexplored. This study aims to investigate whether microglia associate with and alter the AIS before and after TBI, contributing to potential changes in excitability. Using a transgenic mouse model with GFP-labeled microglia, brain tissue is stained for neurons (Nissl), microglia (GFP), and the AIS (Ankyrin G) followed by confocal microscopy to obtain high-resolution images to visualize microglial interactions with the AIS. Image J is utilized to quantify AIS length, fluorescence intensity, and microglial proximity. I hypothesize that TBI induces structural changes in the AIS, including shortening or fragmentation, and that microglial interaction may play a role in these alterations. Preliminary data suggest an increased microglial presence near the AIS after injury, potentially indicating a role in either AIS disruption or repair. By identifying how microglia interact with the AIS, this research contributes to our understanding of neuroinflammatory responses following TBI. These findings may have implications for therapeutic strategies aimed at preserving neuronal function after injury. Further studies will explore whether microglia mediate AIS remodeling through direct contact or secreted factors, offering insights into potential interventions for TBI-related neurological dysfunction.

Malaria, caused by the Plasmodium parasite, remains a relentless and destructive infectious disease, disproportionately affecting children in Sub-Saharan Africa, due in part to the absence of a highly effective, widely deployable malaria vaccine. Lipid nanoparticle (LNP) vaccines are a promising approach for vaccine development, especially against pathogens such as Plasmodium, which have proven historically difficult to vaccinate against. When coupled with the glycolipid adjuvant 7DW8-5 at a 5ug LNP to 0.5ug adjuvant ratio, malaria-targeting LNP formulations confer protection in mouse models. However, the optimal vaccine-to-adjuvant ratio and the mechanisms underlying 7DW8-5-mediated protection remain unclear. Here, we present a study that aims to refine dosing strategies and elucidate the role of CD8+ T and NKT cells in adjuvant-induced protection in a human-translatable mouse model. Different groups of mice will be vaccinated with varying LNP-to-adjuvant ratios, and immune response will be assessed via ELISPOT 28 and 56 days post-vaccination. Furthermore, we will use ELISA to reveal variations in innate immune response between groups 3 hours after vaccine administration. In parallel, we will investigate the necessity of CD8+ T cells and/or NKT cells in protecting from malaria challenge. Mice will be vaccinated using the standardized LNP-to-adjuvant ratio and treated with depletion antibodies targeting CD8+T or NKT cells 24 hours before challenge with Plasmodium sporozoites. Protection will be assessed via blood smear analysis. Our findings will reveal optimal dosing strategies for malaria-specific LNP vaccines and provide insight into the immunological mechanisms behind 7DW8-5-driven protection. This research will contribute to the development of effective nanoparticle-based malaria vaccines — a necessary innovation to help relieve the global malaria burden.

The lung microbiome plays an important role in immunity where any shifts within the microbial community can affect the immune response. Tropheryma whipplei, a bacterium that causes Whipple’s disease primarily in the human gastrointestinal tract, can also reside in the human lung microbiome of both healthy and immunocompromised individuals. Tropheryma is more commonly found in lungs of individuals with pneumonia, those who smoke, or in people living with HIV. Tropheryma is also found in the lung microbiome of certain non-human primate species, where the dominance of Tropheryma is associated with shifts in pulmonary immune cells. Similarly, we found that Tropheryma is highly prevalent and dominant in the pigtail macaque (PTM) lung. However, little is known regarding the factors contributing to the establishment and dominance of Tropheryma in the non-human primate lung. Here, we test the hypothesis that Tropheryma dominance and microbial diversity (beta diversity indexes) in the PTM lung are similar in co-housed animals. Bronchoalveolar lavage (BAL) and housing metadata were collected from PTM (n = 50). Genomic DNA was extracted using the QIAgen PowerFecal Pro DNA kit and the V3-V4 region of the 16S rRNA subunit was amplified and sequenced. The QIIME2 bioinformatics platform was used to evaluate the composition of the lung microbiome and to determine the dominance index and the beta diversity of the sample set. Expected findings will show similar lung microbial compositions across co-housed animals. Results from this study will help us determine the specific environmental factors contributing to the emergence and colonization of Tropheryma in the lung microbiome of PTM. This will lay the groundwork for further research into the role of Tropheryma in the immune response against respiratory diseases, ultimately guiding the development of targeted therapies for lung infection.

Aging is a significant risk factor for many chronic diseases. Understanding longevity interventions can help prevent these illnesses. When mitochondria fail to function correctly, energy production decreases, leading to diseases and shorter lifespans. This study investigates a potential longevity intervention and utilizes Ndufs4-/- mice, a model for mitochondrial disease resembling a human condition called Leigh Syndrome. Mice carrying this mutation have shortened lifespans and neurological impairments. This study aims to determine whether the Sirt3 gene is required for an extended lifespan when using Adefovir Dipivoxil (ADV) injections in a Ndufs4-/- mouse model. In prior experiments, ADV has been shown to increase the expression of genes involving fatty acid oxidation, allowing cells to break down fats for energy. This increase in energy production has been shown to extend the lifespan of Ndufs4-/- mice. ADV is hypothesized to work through a similar pathway as Rapamycin to influence fatty acid oxidation. Rapamycin inhibits the mTOR complex, which influences the CEBP-Beta complex to produce a high LAP/LIP ratio. This shift in the LAP/LIP ratio enhances fatty acid oxidation, extending lifespan. However, in Ndufs4-/- mice lacking Sirt3, a mitochondrial protein, Rapamycin fails to extend their lifespan. This study determines whether ADV works through the same pathway as Rapamycin, specifically if it requires Sirt3 to exert longevity effects. To investigate this, Ndufs4-/- mice are crossed with Sirt3-/- or Sirt3 +/+ and given ADV injections starting at 10 days of age, continuing until the end of their lifespan. I am responsible for genotyping experimental animals and ensuring the correct genetic profile before enrolling them in the study. My role includes administering ADV injections, tracking weight changes, and monitoring the onset and progression of disease.

HCoV-OC43 is a member of the viral family Coronaviridae, commonly known as coronavirus, and is known to cause respiratory infections in humans. HCoV-OC43 is therefore categorized as a human coronavirus, which includes the virus SARS-CoV-2, known to cause COVID-19. Previous studies showed that human coronavirus infections, specifically HCoV-OC43 and SARS-CoV-2, activate the IRE1α component of the unfolded protein response, leading to a splicing of XBP1 mRNA, which then encodes for a transcription factor. Additionally, the IRE1α and XBP1 host factors were found to be necessary for ideal viral replication. However, the specific genes upregulated by XBP1 that contribute to viral replication remain unknown. Given data suggesting XBP1 regulates genes involved in lipid metabolism, our research aims to explore whether Acetyl-CoA Carboxylase (ACC), an enzyme involved in fatty acid synthesis, is upregulated by IRE1α and involved in human coronavirus replication. We used quantitative reverse transcription polymerase chain reaction (qRT-PCR) to measure relative gene expression of ACC after HCoV-OC43 infection, and in the presence of the IRE1α inhibitor, 4μ8c. We found that activation of IRE1α during HCoV-OC43 infection caused increased expression of the gene encoding ACC, which was blocked by 4μ8c. We then tested the hypothesis that ACC supports viral infection using small molecule inhibitors and found that viral RNA was decreased after inhibition of ACC. Next, we bypassed ACC by adding the downstream product, palmitate, and found restoration of viral RNA. Our results indicate that IRE1α induced splicing of XBP1 mRNA increases ACC transcription, which then promotes optimal viral replication. A greater understanding of the mechanisms behind human coronavirus replication allows for the development of potential therapies targeting these viruses. In our continuation of this research, we plan to expand our knowledge of human coronaviruses by investigating the role of IREα and ACC expression in SARS-CoV-2 infections. 

Turner Syndrome (TS) is a chromosomal disorder caused by the lack of the second sex chromosome, also known as monosomy-X. Individuals with TS are phenotypically female and are likely to exhibit defects of variable severity such as short stature, neurocognitive problems, congenital heart defects, and infertility. Over 95% of TS conceptions do not survive to birth. This fetal lethality and other developmental anomalies are thought to be caused by the reduced dosage of X-linked genes or the complete lack of Y-linked genes, but the exact mechanisms are unclear. Our lab has generated isogenic X0/XY or X0/XX human induced pluripotent stem cell (hiPSC) lines from patients mosaic for TS to study the impact of the lack of a second sex chromosome on the same genetic background. My project is to investigate the effect of monosomy-X on early human development by differentiating our TS derived isogenic hiPSC line pairs into RA-Gastruloids, a stem cell-based embryo model corresponding to week 4 of human development. By performing morphological and gene expression analyses we aim to gain insight into the mechanistic causes of fetal lethality in TS. To date, I have optimized the differentiation conditions to successfully differentiate three pairs of isogenic lines and four independent lines (two X0, one XX, one XY) into RA-Gastruloids. Preliminary results showed no clear evidence of morphological differences among different genotypes. To investigate the cell composition of the gastruloids, I will use quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to measure the expression of cell type-specific markers, as well as immunohistochemistry to detect morphological differences. Due to the severity of TS developmental phenotypes, I expect X0 RA-Gastruloids to have abnormal gene expression and/or cell compositions compared to their XX or XY counterpart. This work will help understand the molecular mechanisms of abnormal development in TS.

Mutations in the RAS gene family are common in various cancers and are estimated to occur in approximately 19% of cancer patients. We utilize the model organism C. elegans to study RAS genes because it sends signals in the worms the same way it does in humans. C. elegans only have one RAS family gene, encoded by let-60, making it simpler to study than the three in humans. The let-60 G13E mutation is a gain of function (gf) mutation also found in cancer patients and is characterized by a glycine to glutamic acid amino acid mutation at residue 13. The mutation is phenotypically marked by neoplasias - pathologically abnormal growths of tissue, effectively constituting tumors. Despite genetic uniformity of C. elegans in the controlled laboratory environment, not all let-60 gf worms develop neoplasias. Preliminary findings show that the penetrance of neoplasias is approximately 81% in the MT2124 strain, which developed the let-60 gf mutation via mutagenesis, and 93% in the ARM219 strain, which developed the mutation via CRISPR technology. Previous reports have identified chaperones as affecting RAS activity, My study aims to identify the effects of heat shock proteins hsp-17/CRYAB  and hsp-70/HSPA5 in C. elegans on the penetrance of neoplasias driven by the let-60 gf worms. Neoplasias shorten lifespan, so I measured their effects on survival in worms with and without the let-60 gf mutation, sorting them by tumor count. I hypothesized that the genetic backgrounds with a lower penetrance and expressivity of let-60 gf will have fewer tumors on average and observe a longer lifespan compared to strains with a higher penetrance of the mutation. Understanding the role of heat shock proteins in neoplasia penetrance could provide insights into potential therapeutic targets for RAS-related cancers.

Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites and in 2023 caused an estimated 597,000 deaths. Although two currently approved malaria vaccines are available, they offer insufficient protection in endemic populations, which prompts the need for new vaccines. Here we tested several lipid nanoparticle (LNP) vaccines and quantified the number of surviving parasites in vaccinated mice challenged with Plasmodium yoelii sporozoites. To quantify surviving parasites, we utilized the Plasmodium 18S rRNA reverse transcription PCR assay, which is a highly sensitive assay that can quantify the amount of Plasmodium parasites in liver or blood samples. The assay works by amplifying and detecting parasite 18S rRNA in a sample through specific primers, probes and quenchers for mouse GAPDH mRNA and pan-Plasmodium 18S rRNA and can be used to quantify the burden of Plasmodium in a sample. Through the 18S assay, we identified LNP formulations that most effectively protected against rodent malaria. Notably, these LNPs required the adjuvant 7DW85 to be protective.  In the absence of the adjuvant, fewer mice vaccinated with LNPs were protected against rodent malaria. Together, we identified our leading LNP vaccines, which we continue to optimize with the goal of attaining sterile protection against rodent malaria. 

Traumatic brain injury (TBI) can occur after experiencing an explosion or any external force to the head. TBIs are exceedingly common and frequently associated with some degree of behavioral and/or cognitive impairment. However, the underlying causes of these impairments are unknown. To bridge this gap in knowledge, our lab examines the pathology in brain regions that account for the nodes of networks important in cognitive and behavioral function, including the default mode/executive control and limbic/salience network respectively, in brain donors with a history of behavioral, cognitive, or mixed decline. Oligodendrocytes are glial cells in the brain that are important to the production of myelin. Injury to the brain can lead to their cell death. We aim to uncover whether TBI donors with cognitive, behavioral, or mixed decline have reduced amounts of oligodendrocyte in brain regions associated with such functions. To investigate this, slides of over 20 regions of the brain are stained with an antibody that marks oligodendrocytes, Olig2. The slides are then scanned with an Aperio slide scanner and imported to Halo image analysis software. Utilizing this software, I annotate the grey matter of these slides, so that the percentage of the area of staining can be determined for pixels in a specific intensity range. Preliminary results in 5 of the brain donors demonstrates no significant difference in the % staining of Olig2 across the brain regions regardless of clinical pattern of decline. Experiments will need to be conducted on controls of donor brains without TBI and on white matter, a region with higher amounts of oligodendrocytes that may function differently than oligodendrocytes in grey matter.

Stress resilience, the ability of cells and tissues to adapt to stimuli, declines with age. Skeletal muscle contraction is a physiological stressor when repeated through exercise training enhances stress resilience and mitigates age-related comorbidities. However, as the body's capacity to mount adaptive responses diminishes with age, the extent to which this decline affects physiological adaptation to stress remains unclear. This would guide future therapeutic strategies surrounding muscular degeneration over the lifespan. The goal of this study is to assess the magnitude of stress response activation across metabolic, oxidative, proteostatic, and heat shock stress response pathways. We use gene expression analysis to evaluate the transcriptional response to controlled in vivo muscle stimulation, providing insight into age-related differences in stress resilience. Young (6mo) and old (23-24mo) male and female mice (C57Bl/6JNia) underwent an in vivo fatiguing muscle stimulation (Stim) or served as an unstimulated control (Unstim). Three hours following the stimulation both right and left limb muscles were collected and processed for gene expression analysis. Following stimulation and collection, I performed tissue processing, RNA extractions, and RT-qPCR assays on muscle tissue. There was a significant increase in PGC1a, HMOX1, TRIM63, and HSPa1a genes in response to muscle stimulation when compared to the unstimulated limb within the same animal. The magnitude of these changes in response to stimulation were not different across age or sex. Analysis of basal changes in unstimulated groups across age and sex is planned for next month. These preliminary results suggest no significant age or sex differences across multiple pathways of stress resilience in skeletal muscle. A strength of this study design is that we use a combined within- and between-animal analysis of both stimulated and unstimulated conditions to control for any potential variations associated with each age, sex, and stimulation condition, increasing confidence in our results.

After traumatic brain injury (TBI), astrocytes can undergo distinct changes in function and morphology, termed astrogliosis. Astrocytes are important glial cells with roles in maintaining neural circuits. This astrogliosis can lead to maladaptive changes, inhibiting proper support of circuitry that might lead to hyperexcitability.  TBI is a risk factor for the development of epilepsy, and we wondered whether increased astrogliosis is present in cases that develop epilepsy compared to TBI without epilepsy. To assess this question, we examined astrogliosis in male brain donors with a remote history of TBI with and without post-traumatic epilepsy, as well as controls in a similar age range. Immunohistochemical staining for glial fibrillary acidic protein (GFAP), an astrocytic cytoskeleton protein, was used to visualize and quantify astrogliosis. The percentage area of staining was determined in both the orbitofrontal cortex (OFC), a region commonly vulnerable to TBI, as well as the thalamus, a region important in seizure spreading in the brain. Morphologic changes in astrocytes were analyzed with immunofluorescence staining for GFAP, using Sholl analysis to determine changes in astrocytic branching patterns in the OFC and the thalamus. Our results demonstrate increased astrogliosis in the thalamus and OFC in the post traumatic epilepsy group but not the TBI without epilepsy group compared controls. This supports our hypothesis that there is an association between post traumatic epilepsy and astrogliosis. Further research is needed to understand how astrogliosis might modify neural circuits to initiate or spread hyperexcitable activity associated with epilepsy.

Down syndrome (DS) is one of the most common genetic conditions and is due to chromosomal anomaly, associated with heightened skin autoimmunity disorders like psoriasis. DS individuals have a significantly altered immune system, often indicated by a higher frequency and severity of infections. A hallmark of this altered immunity is a heightened pro-inflammatory state, characterized by increased signaling by cytokines such as interferon-gamma (IFN-γ). The goal of our project is to investigate the relationship between elevated cytokine signaling and epithelial cell biology, particularly how this dysregulation contributes to inflammation-induced immune responses and dermatological conditions. We hypothesize that elevated signaling of pro-inflammatory cytokines in the serum of individuals with DS alter the biology of epithelial cell function, predisposing them to skin disorders such as psoriasis and vitiligo. To explore this, we are first treating skin samples with serum from individuals with DS to assess its effects on epithelial cells. The serum alone induces a weak transcriptional response, so additional factors may be required to drive significant epithelial cell changes. To better define the impact of IFN-γ, we are treating skin samples with high-dose IFN-γ and comparing the resulting gene expression patterns to known cytokine-induced signatures. This approach will help elucidate the molecular mechanisms underlying epithelial dysfunction in DS. This research may provide insights into targeted anti-inflammatory interventions to preserve normal epithelial function and mitigate skin-related complications like inflammatory skin conditions, barrier dysfunction, slow tissue healing, abnormal cell turnover, and even cancer. Future experimental investigations could  focus on the epithelial microenvironment itself, rather than serum exposure alone, to capture localized skin-specific changes that may provide further mechanistic insights. 

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor long-term survival rates. Cytarabine (Ara-C) is a standard chemotherapy used to treat AML patients. However, many patients relapse due to refractory disease, highlighting the need for new therapeutic strategies. Heparan sulfate proteoglycans (HSPGs) are glycoproteins that regulate key signaling pathways by interacting with growth factors and receptors. HSPG glycan chains are modified by the addition of negatively charged sulfate groups. HS2ST1 and HS6ST1 catalyze sulfate addition at the 2-O and 6-O positions of heparan sulfate chains, respectively. In AML, increased HS6ST1 expression correlates with worse patient survival, while low HS2ST1 expression is linked to adverse outcomes in certain AML subtypes, suggesting distinct roles in disease progression. To investigate the contribution of HS modifications to chemotherapy response, we generated CRISPR-edited (sgHS2ST1, sgHS6ST1, or sgControl) MOLM-13 AML cells. Compared to sgControl cells, sgHS6ST1 cells displayed increased sensitivity to Ara-C, suggesting that 6-O heparan sulfation may contribute to chemoresistance. To test whether MOLM-13 AML cells alter the expression of HS-modifying enzymes in response to chemotherapy, I performed RT-qPCR analysis at 24 and 72 hours after Ara-C treatment. Upon Ara-C treatment, HS2ST1 expression increased by 1.5-fold and HS6ST1 transcript increased by 4-fold at 24- and 72-hours post-treatment. In contrast, sulfatase 2 (SULF2) removes 6-O sulfate modifications at the cell membrane. Strikingly, compared to vehicle treatment, SULF2 expression was increased by sixfold at both time points. Our results highlight HS sulfation as a dynamic regulator of AML chemoresistance and suggest that targeting HS-modifying enzymes could enhance chemotherapy efficacy. In the future, I will create an sgSULF2 cell line to characterize the functional role of SULF2 in AML disease progression and chemotherapy resistance.