Browsing by Author "Nathan, Noah"

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    Assessment of Polymer Wafer Aided Gene Transfection Efficiency and Cell Viability
    (2018-08) Nathan, Noah; Hanson, Samuel; Wang, Chun
    DNA transfection of eukaryotic cells has been explored using non-chemical, chemical and viral methods. Mucoadhesive, water-soluble and neutral polymer wafers offer a novel sublingual DNA delivery method. Transfection efficiency and viability of GFP/PEI polyplexes in various polymer delivery vehicles into murine fibroblasts (NIH3T3) was studied using flow cytometry, fluorescence microscopy and MTT colorimetric assay. Polyvinyl alcohol (PVA) and hydroxypropyl cellulose (HPC) wafers in HEPES buffer had an increased transfection efficiency compared to polyplexes delivered in HEPES buffer only. DNA/PEI polyplexes delivered in PVA or polyethylene oxide (PEO or PEG) had the highest viability of all the transfection methods.
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    Development of High-Throughput and High-Content Analysis Assays for Neurodegeneration-Related Intrinsically Disorderd Proteins
    (2020) Nathan, Noah
    We developed a FRET-based protein-protein biosensor of Fused in Sarcoma (FUS), an Amyotrophic Lateral Sclerosis and Frontotemporal Dementia related protein. The FUS biosensor had a robust signal, yielding a FRET efficiency of 7.61% with low signal to noise ratio. Based on high-throughput FRET measurements, we determined a standard deviation of 0.0158 ns for the donor/acceptor fluorescent lifetime. In future drug screens for compounds that modulate FUS aggregation, the threshold for hits will be set at 2.45 ± 0.0474 ns (3 SD). In addition, we implemented a MATLAB script that quantifies the ratio of cytoplasmic to nuclear FUS-rich stress granules from fluorescent images of FUS-GFP-expressing N2a cells. We showed that sorbitol, which has been shown to cause FUS mislocalization via hypertonic stress, caused a shift in the cytoplasmic to nuclear ratio of FUS as compared to untreated cells. We implemented a second MATLAB automated algorithm that quantifies total neurite outgrowth from neurospheres expressing the Parkinson's disease-related protein alpha-synuclein. We found that the mutant aSyn A53T caused a reduction of 41% in total neurites as compared to WT aSyn-expressing neurospheres. This result was validated by counting neurites manually with ImageJ, which yielded a reduction in neurites of 49%.