PhD Oral Exam - Joseph Trani, Biology

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

Extracellular vesicles (EVs) mediate intercellular communication across all kingdoms of life, yet mechanisms governing EV cargo selection and delivery remain incompletely understood. This limits our ability to explain fundamental EV biology and rationally engineer EVs for therapeutic applications. Here, I establish Saccharomyces cerevisiae (baker’s yeast) as a genetically tractable platform to uncover evolutionarily conserved mechanisms of EV cargo incorporation and engineer yeast EVs for small-molecule delivery. First, I developed a pooled cloning strategy to express >15,000 human open reading frames tagged to green fluorescent protein (GFP) in S. cerevisiae. EVs released from these humanized yeast pools were intact, lipid-bound, and contained GFP-positive particle populations. Proteomic analysis identified 292 GFP-tagged human proteins in yeast EVs, including canonical human EV proteins. Orthology, Gene Ontology, motif, and protein-interaction analyses showed that many EV-associated human proteins have yeast orthologs, contain predicted KFERQ-like motifs, or connect to ESCRT-associated networks, suggesting that conserved molecular features and biogenesis pathways contribute to selective EV cargo incorporation. Second, I tested whether yeast EVs can be engineered as delivery vehicles for therapeutic small molecules. Doxorubicin was successfully loaded by electroporation into EVs from S. boulardii (a probiotic yeast), but this altered EV size distribution and zeta potential. Despite these changes, detergent-sensitive membrane staining and transmission electron microscopy supported preservation of EV membrane integrity and gross morphology. Doxorubicin-loaded EVs were internalized by triple-negative breast cancer cells and produced greater cytotoxic and anti-proliferative effects than dose-equivalent free doxorubicin, accompanied by a shift toward membrane-compromised cell-death states. In all, this thesis establishes yeast as both a model to study conserved EV cargo-sorting mechanisms and a promising chassis for EV-based drug delivery, first steps towards bridging bench to bedside.