Traditional phylogenetic analyses rely on sequence divergence to infer evolutionary relationships among species. Here, we take a complementary approach by quantifying the distribution of protein domains across vertebrate proteomes. Using domain abundance profiles, we reconstructed a species distance tree with UPGMA, identifying four major clusters: fish, birds, mammals, and a heterogeneous assemblage comprising cartilaginous fish, amphibians, and reptiles. We hypothesize that lineages within this heterogeneous group exhibit relatively lower rates of protein domain turnover. To test this, we will incorporate ancestral reconstruction of domain counts across the vertebrate phylogeny to estimate lineage-specific rates of domain gain and loss. In contrast, ray-finned fish, birds, and mammals may display greater divergence in domain composition, consistent with elevated rates of proteome remodeling. Functional analysis of domains with high inferred gain rates may further reveal lineage-specific innovations. Together, these results provide a domain-centric perspective on vertebrate genome evolution, demonstrating how variation in domain turnover rates shapes proteome diversification beyond what is captured by sequence-based comparisons.

How genetic information replicated before the emergence of complex protein enzymes is a central question in "RNA world" research. The Virtual Circular Genome (VCG) model proposes that short, overlapping oligonucleotides can collectively map to a circular sequence, enabling indefinite replication cycles through configurational shuffling. This project investigates the thermodynamic stability of VCGs and their selective partitioning into model protocells. Using a DNA-based model, we verified the spontaneous, stepwise assembly of higher-order complexes via a controlled thermal annealing protocol. Structural validation through agarose gel electrophoresis and competition assays confirmed the formation of topologically closed circular structures. Quantitative analysis demonstrated high assembly efficiency. Encapsulation studies in Oleic Acid vesicles yielded an efficiency of 4.3% for single-stranded DNA, while larger VCG assemblies exhibited complex elution profiles during size-exclusion chromatography, suggesting significant interactions between genome architecture and membrane confinement. This work is significant as it provides the first quantitative biophysical evidence for the VCG model. By demonstrating that lipid membranes can influence the organization of multi-component genomes, we establish a structural framework for understanding how physical confinement could have driven the evolution of genetic complexity in early life.

Antibody drug conjugates (ADCs) are antibodies specific to cancer cell targets linked to a cytotoxic payload, designed to specifically deliver therapies to cancer cells. Our team has previously developed MMAE-conjugated ADCs targeting the cell surface protein DLK1, however, the emergence of resistance remains a potential limitation for clinical efficacy. This study aimed to investigate mechanisms underlying acquired resistance to DLK1-targeted ADCs, and explore methods to reverse resistance. We developed ADC resistant JR and COR-L297 cell lines (>10x IC50), and quantified changes in the DLK1 target and pumps from the ABC transporter family, implicated in intracellular efflux of MMAE, via flow cytometry and western blotting. Next, resistant cells were cultured in ADC-free media to investigate the reversibility of resistance, and concentration of cleaved DLK1 in media. While we observed a decrease in quantity of DLK1 target in resistant cells and their media, in line with our predicted mechanism of target downregulation, we also observed minimal changes in the quantity of cellular pumps, implying a lack of overexpression. Notably, COR-L279 cells grown in ADC-free media re-expressed DLK1 at normal levels, while JR cells did not, indicating distinct and potentially irreversible resistance mechanisms between cell lines. Overall, findings from this project could provide insight to resistance pathways in DLK1-targeted ADCs and broader application to ADCs in general, thus creating a more effective therapy in clinical settings.

Human pluripotent stem cells (hPSCs) hold significant promise for a wide range of cell therapies. However, their clinical translation is limited by the risk of tumorigenicity. To address this, previous groups have engineered a FailSafe system into donor cells as a safety switch by transcriptionally linking the herpes simplex virus thymidine kinase (HSV-TK) suicide gene to cyclin-dependent kinase 1 (CDK1). Upon administration of the clinically approved prodrug ganciclovir (GCV), proliferating cells expressing CDK1 also express HSV-TK, which phosphorylates GCV into its toxic triphosphate form, leading to DNA chain termination and apoptosis. Here, we evaluate the efficacy of this FailSafe suicide gene system in hPSCs, hPSC-derived skeletal muscle progenitor cells (SMPCs) in vitro, and in mouse engraftment models in vivo. We demonstrate that GCV-mediated activation of the FailSafe system enables robust and selective elimination of proliferating cells. These findings support the potential of this strategy as a critical safety mechanism to mitigate tumorigenic risk and advance stem cell-based therapies toward clinical application.