Bacterial pneumonia is a major cause of morbidity and mortality in critically ill patients. While antibiotics and oxygen supplementation remain the mainstay of therapy, no targeted molecular interventions currently improve patient outcomes. We previously found that activation of BK (large-conductance Ca²⁺-activated K⁺) channels protects against LPS-induced lung injury, but whether these effects translate to live bacterial infections remains unknown. To address this, we developed a murine P. aeruginosa pneumonia model and administered the BK channel activator NS1619 intratracheally 24 hours post-infection as a clinically relevant therapeutic approach. NS1619 treatment significantly improved alveolar-capillary barrier function at 48 hours post-infection, as evidenced by reduced bronchoalveolar lavage fluid (BALF) total protein and IgM levels. In vitro permeability studies using electric cell-substrate impedance sensing (ECIS) technique demonstrated strong protective effects of NS1619 on barrier function in primary human lung microvascular endothelial cells. ECIS measures electrical resistance across a cell monolayer, where decreased resistance reflects impaired barrier integrity. Mechanistically, protein expression and phosphorylation analyses suggest that NS1619-mediated protective effects occur via inhibition of STAT3 signaling. Together, these findings identify endothelial barrier preservation as a central mechanism of BK-mediated protection and support BK channel activation as a promising novel therapeutic strategy.

Glioblastoma is the most aggressive primary brain tumor. Alterations in the epidermal growth factor receptor (EGFR) drive tumor growth in nearly half of all cases, yet clinical responses to EGFR inhibition are only temporary. Preliminary data suggest resistance involves both signaling rewiring and shifts in cell states, but mechanisms linking target inhibition to lineage adaptation remain unclear. I investigated whether reactivation of RAS–MAPK signaling is a central driver of this adaptive response. To test MAPK sufficiency, I expressed constitutively active ERK1 in patient-derived gliomaspheres and measured growth under EGFR inhibition. To assess necessity, I applied genetic pan-RAS inhibition in vitro and in an intracranial xenograft model. I evaluated proliferation by live imaging, while single-cell RNA sequencing was used to examine lineage composition and cell-cycle dynamics. ERK1 overactivation restored spheroid growth despite sustained EGFR inhibition, demonstrating MAPK sufficiency. RAS suppression impaired proliferation in vitro, and prolonged survival in vivo. Single-cell analyses showed that RAS inhibition shifts cells from proliferative progenitor-like states toward differentiated astrocyte-like populations, linking RAS–MAPK activity to maintenance of plastic, cycling lineages. These findings establish RAS–MAPK signaling as sufficient and necessary for adaptive resistance to EGFR-targeted therapy in glioblastoma and support combination or sequential targeting strategies to improve therapeutic effects.

Wound healing is essential for preventing infection and tissue damage. Chronic wounds, which result from a failure to resolve inflammation, affect 7 million Americans and lack effective treatments. Prior work from the Coller Lab showed that autophagy deficiency prolongs inflammation, delays wound healing, and elevates NF-kB activity. Expanding on this, I analyzed wound healing in a mouse model with loss of autophagy in ZBTB46+ cells, which include classical dendritic cells. I found that these mice exhibit significantly delayed healing, identifying dendritic cells as a possible key regulator of inflammatory resolution during tissue repair. To test whether excessive NF-kB signaling drives this phenotype, I treated these mice with JSH-23, a canonical NF-kB inhibitor, or vehicle control. Unexpectedly, JSH-23 further worsened healing. Western blot analysis showed increased IKKα levels after canonical NF-kB inhibition, indicating activation of noncanonical NF-kB signaling. These results suggest autophagy is required to properly regulate NF-kB signaling in dendritic cells during wound healing. Loss of autophagy enhances noncanonical NF-kB activity, prolonging inflammation and delaying repair, while canonical NF-kB inhibition further amplifies this response. These findings reveal a regulatory interaction between autophagy and NF-kB signaling in wound repair, providing mechanistic insight into chronic wound pathology and supporting the development of targeted and novel strategies to promote inflammatory resolution and wound repair.

The Trabecular Meshwork (TM) regulates the drainage of aqueous humor (AH) in the human eye. One of the major modifiable risk factors for primary open angle glaucoma is impaired aqueous humor outflow, which leads to an elevated intraocular pressure (IOP). To mimic glaucoma in vitro, one group of cells was treated for five days with dexamethasone (dex), a steroid used to treat eye inflammation that can also increase IOP and contribute to steroid-induced glaucoma, while another group of received vehicle treatment as a control. RNA sequencing was then used to identify genes that were upregulated and downregulated in response to dex. These gene expression data were analyzed using the L1000 program to identify drugs predicted to reverse or promote the effects of dex. The goal of the study was to identify drugs that could reverse glaucoma-associated gene expression and phenotypes in TM cells. ANGPTL7 was selected as a key marker because it is strongly upregulated in response to dex and has been associated with the extracellular matrix remodeling within the TM, contributing to reduced AH outflow and increased IOP. Vanoxerine, a noncompetitive dopamine antagonist, was tested for its potential to reverse the effects of dex. TM cells from nine different samples were treated with either Vanoxerine or vehicle, and the results showed that increasing Vanoxerine treatment up to 10nM correlated to the decrease of ANGPTL7 expression.

This project focuses on engineering and optimizing the Ras-LOCKR system, a modular protein switch that generates fluorescence in response to active Ras signaling. A key challenge in molecular biosensors is achieving strong, reliable signal readouts, and this work addresses how reporter choice and linker design can improve signal intensity and detection efficiency. The system is dimerization-dependent and remains inactive until GTP-bound Ras binds the key protein, triggering a conformational change that enables cage–key interaction and fluorescence emission. To investigate this, ddGFP and ddRFP Ras-LOCKR constructs were developed using molecular cloning, including Gibson assembly and bacterial transformation. Successful plasmid construction was confirmed through colony growth and sequencing validation using PlasmidSaurus. The GGS linker region was also randomized via degenerate codon PCR to assess its effect on system performance. Following plasmid scale-up, constructs were transfected into HEK293 cells for biosensor expression. These results establish a platform to evaluate how linker variation and reporter selection impact signal output. Next steps include encapsulating cells in picoshells and performing fluorescence-activated cell sorting (FACS) to isolate high-performing variants with improved signal and reduced background. This work advances tunable, high-performance biosensors for studying cellular signaling and enables scalable optimization of protein-based sensing systems.