To help alleviate disease progression and eliminate viral reservoirs within people living with HIV (PLWH), chimeric antigen receptor (CAR) based therapies have been developed using lentiviral vectors (LV) that aid in redirecting immunity against HIV. These LVs contain an anti-HIV CAR expressed on recombinant T cells to aid in recognition and destruction of HIV. Therefore, engineering T cells to clear HIV in infected patients is a promising approach to achieve antiretroviral therapy (ART)-free controlled viremia. Previously, we developed a LV that expresses a CD4-based CAR under a MND promoter that showed best expression of surface CAR expression. Using an optimized MND promoter, we aim to co-express the immunostimulatory cytokine IL-15 to generate better memory differentiation, proliferation, and less exhausted in anti-HIV CAR T cells that will improve anti-HIV responses.

To further advance our understanding on gene expression and developmental processes within echinoderms, this study aimed to determine whether an unknown gene from Strongylocentrotus purpuratus could be successfully identified and cloned using a portion of its coding region. Identification of the gene was found through DNA sequencing using BLASTn, BLASTx, InterPro, and Echinobase sequence analyses. A phylogenetic tree was created to further analyze the gene. Cloning of the gene proceeded with PCR which was used to amplify the gene and was confirmed through gel electrophoresis. The amplified product was purified to remove impurities, ligated into a vector, and then introduced into bacterial cells via transformation. Successful cloning was verified using colony PCR and restriction digest analysis through gel electrophoresis. After a successful separation of the unknown gene insert from the plasmid indicated from the gel, the sample was sequenced and was confirmed that the unknown gene was transcription factor 12. These results confirm the successful identification and cloning of a gene from S. purpuratus and thus suggest a successful tool to aid in the further understanding of gene expression, development, and regulation within echinoderms.

The function of genes in Strongylocentrotus purpuratus genome can be studied to gain insight into their biology. With full genome sequences now available, an unknown gene from S. purpuratus was identified and cloned. BLAST was used to compare the gene with known genes, InterPro was used to identify protein domains, Echinobase was used to obtain gene annotations, and a phylogenetic analysis was done to compare the gene to genes of other species. These program results were used to support the gene’s identity. Results indicated that the gene is a protein atonal homolog 7 (Atoh7), a transcription factor involved in the sea urchin nervous system. PCR was used to amplify the purified and unpurified gene, and validation of amplification was carried out using gel electrophoresis and Nanodrop analysis. Ligation of the gene into a plasmid was performed, followed by bacterial transformation to insert the plasmid into competent cells. Confirmation of successful ligation and transformation was conducted in several ways. Colony PCR and gel electrophoresis was done to check for the presence of the correct insert. Minipreps were used to isolate plasmid DNA from bacterial cultures, followed by restriction digestion and gel to verify the insert size. DNA sequencing was also done for final confirmation. The combined results of the three confirmation experiments confirmed the success of the cloning of the Atoh7 gene. After identifying the gene, a potential future direction is to use fluorescence microscopy to localize the gene.

Microridges are actin-based protrusions found on the surface of mucosal epithelial cells, such as those found in zebrafish periderm. These structures help maintain the epithelial barrier and retain mucus. Previous studies have shown that periplakin, a cytolinker protein, is essential for microridge formation by bridging actin and keratin cytoskeletons. However, how post-translational modifications (PTMs), such as SUMOylation, regulate periplakin’s activity during this process remains unknown. In this project, I aim to examine the role of SUMOylation in microridge morphogenesis by expressing either wild-type or SUMOylation-deficient periplakin in periplakin knockout zebrafish embryos. Using confocal microscopy, I will assess periplakin localization and quantify changes in microridge length across conditions. Since SUMOylation has been shown to affect periplakin’s interaction with keratin in mammalian systems, I expect that disrupting this modification may lead to altered microridge organization and stability. This research will enhance our understanding of the molecular mechanisms governing epithelial cell structure.