This abstract has been withheld from publication.

DNA nanotechnology has been employed as a cheap, simple way to create complex biological structures with the simple base pairing of nucleotides. The molecular architecture of these strands can be used to form polymer networks and condensates of differing sizes and morphologies based on the flexibility, valency, and design of the monomers. We employ our group’s previously characterized nanostar condensate and nanotube filament to determine the interactions of the two structures in confined protocells. We annealed 3 armed nanostars and single-tile nanotubes and suspended them in an MgCl2 solution to promote the nucleation and growth of these structures. I then employed a water-in-oil droplet emulsion method to confine them in an oil-surfactant solution. I used variations in nanotube and nanostar concentration across three concentrations of MgCl2. We discovered that within the droplets, both structures were able to form regularly and with very little biochemical interaction between the filaments and condensates due to the orthogonality of the sticky ends that promote nucleation. At higher nanostar concentrations, the degree of colocalization between the two structures decreased significantly and promoted the nucleation of nanotube filament networks to happen more rapidly. The ability to develop synthetic protocells is a critical field in bioengineering, employing novel technologies to mimic a cell. This project is the first step in creating higher-order protocells for future use in industry and medical technology.

Current barriers remain for researchers seeking to study diseases found in inaccessible cell types. No techniques exist allowing researchers to access cell types located in nervous tissue without implementing highly invasive techniques or placing subjects at risk. While existing technologies allow us to reprogram human cell types by first transforming them into stem cells, this process “resets cell age” essentially erasing informative age-related markers. This especially presents an issue in age-related diseases, significantly limiting the scope of neurodegenerative disease research and studying the vulnerability of elderly populations. Direct cell reprogramming techniques, however, allow for the conversion of human fibroblasts into specialized cell types whilst maintaining aging signatures, but with an efficiency rate under 5%. Current data from the lab indicates that fusing alpha crystalline domains (ACDs), a type of oligomerizing protein, to transcription factors enhances transcription factor binding. We hypothesized that increased binding and recruitment may produce improved transcription factor functionality and, therefore, higher direct cell reprogramming efficiency. To test this hypothesis, we fused 5 ACD proteins with varying oligomerizing strengths to a transcription factor, and assessed whether this enhanced its functionality. Comparing the effects of proteins across a spectrum of aggregation strengths allows us to pinpoint which ACDs most effectively boost on-target transcription factor binding and expression.

Human fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs), which is a remarkable discovery for the future of therapeutics and disease modeling. Yet, reprogramming efficiency—the proportion of somatic cells that complete reprogramming—remains low and varies significantly across donors. During reprogramming, cells undergo a metabolic shift from oxidative phosphorylation to glycolysis, a transition that may be aided by a decrease in mitochondria. Thus, one possible contributing factor is mitophagy, a process in which cells selectively degrade mitochondria. This study investigates whether mitophagy plays a critical role in the early stages of iPSC reprogramming. In the first phase, a pharmacological FG/BL treatment was established to stimulate mitophagy, with digital droplet PCR (ddPCR) used to confirm increased mitophagy activity. In the second phase, two donor-derived fibroblast lines were treated with FG/BL and reprogrammed over three weeks using the CytoTune-iPS 2.0 Sendai virus kit. In the third phase, reprogramming efficiency was assessed by counting iPSC colonies through visual inspection and immunofluorescence microscopy. While this study is ongoing, preliminary findings suggest that mitophagy stimulation via FG/BL may enhance reprogramming efficiency, especially in donor lines with low baseline success rates. These results point to mitophagy as a modifiable factor in cellular reprogramming and highlight its potential to improve consistency and scalability in iPSC generation for therapeutic use.