The absorption of a photon by a photoreactive opsin pigment triggers the photoisomerization of its retinaldehyde chromophore (11-cis-retinal to all-trans-retinal). To restore light sensitivity to the resulting apo-opsin, the photobleached chromophore must undergo chemical isomerization back to its photoactive state. This pigment reactivation is carried out by multistep enzyme pathways in the retina and the retinal pigment epithelium (RPE) called the visual cycles. In this study, I identified that zebrafish dihydroceramide desaturase-1 (DEGS1) is a retinol isomerase. Zebrafish DEGS1 isomerizes 11-cis-retinol as a substrate into multiple retinol isomers that serve as precursors for 11-cis-retinal. To study the activity of this protein, zebrafish DEGS1 cDNA was cloned into a plasmid with a mammalian expression promoter to allow expression in human embryonic kidney (HEK) cells. DEGS1 zebrafish plasmid was transformed into E. coli, amplified, purified, and subsequently transfected into HEK cells. Transfected HEK cells were used as a protein source to perform homogenate assays with 11-cis-retinol. Results reveal that zebrafish DEGS1 has retinol isomerase activity. This finding improves our understanding of the role that DEGS1 plays in the metabolic pathway for the regeneration of visual chromophore in zebrafish.

Gene therapy has long been a promising therapeutic candidate for many genetic and chronic diseases. However, effective nucleic acid delivery is limited by the efficiency of vectors. Both viral and non-viral vectors have limitations that decrease the likelihood of successful gene delivery. Viral vectors have a limited cargo capacity, are costly, and highly immunogenic. Non-viral vectors have larger cargo capacity for genetic material, and are less immunogenic. However, they have a much lower efficiency than viral vectors, thought to be due, in part, to limited endosomal escape, which leads to degradation of the vector and cargo by the lysosome. In nature, pathogens produce enterotoxins that incorporate multiple cell-penetrating peptide/antimicrobial peptide (CPP/AMP)-like domains to remodel the endosomal and facilitate endosomal escape. Our lab hypothesized that we could create “pathogen-inspired” cationic polymers, which include motifs that mimic CPPs/AMP (cationic and hydrophobic) to interact with the endosomal membrane and maximize endosomal escape.Through reversible addition-fragmentation chain transfer (RAFT) polymerization, we have created a series of cationic polymer segments of different lengths and ratios of cationic and hydrophobic monomers. We hope that these pathogen-inspired vectors improve upon efficiency of traditionally non-viral vectors while retaining their safety.

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by accumulation of toxic amyloid-beta (Aβ) plaques and hyperphosphorylation-induced tau neurofibrillary tangles (NFTs), which ultimately contribute to neuronal cell death and cognitive decline. Soluble amyloid precursor protein alpha (sAPPα) is a neuroprotective, proteolytic amyloid precursor protein (APP) fragment that inhibits accumulation of both Aβ and phosphorylated tau (p-tau) by inhibiting BACE1 and GSK3β, respectively. F03 is a small molecule shown to increase sAPPα and decrease the p-tau/total-tau (t-tau) ratio in two AD mouse models. My research focuses on optimizing F03 and identifying novel chemical entities through synthetic modifications. The goal is to develop F03 analogs with improved potency (EC50 < 0.5 μM) and enhanced oral brain bioavailability, capable of increasing sAPPα expression and reducing p-tau. In addition to synthesizing new analogs, I conduct assays to determine ADME properties of these compounds, including kinetic solubility and microsomal stability. In-vitro assays, performed by other team members, assess the potency of these analogs in increasing sAPPα expression and reducing p-tau, quantified via ELISA and AlphaLISA, respectively. Preliminary in-vitro results show that several analogs, DDL-33, 34, 36, and 38, significantly enhance sAPPα levels, offering a promising foundation for in-vivo studies and guiding future analog development through identification of key functional group modifications.

The Nava lab is interested in the development of ligands that mimic the primary coordination environments of zeolites, the most industrial used catalyst, but models do not faithfully replicate the primary coordination environment of zeolites due to silanol flexibility and condensation reactions. We have identified the truxenetriol platform as a suitable organic scaffold to build zeolite mimics, however, the synthesis and isolations of these compounds is challenging due to regioisomers. 4-tBuphenyl-3-truxenetriol has been synthesized in moderate yield by mixing PhMgBr with truxenetrione in THF but the product is composed of two regioisomers: the desired syn isomer with all hydroxy groups on the same face, and the anti isomer with two out of the three hydroxy groups on the same face. The anti isomer is statistically favored and produced in ~75% yield. Through a novel workup procedure, I have been able to isolate the syn isomer in one step in very high purity without the need of column chromatography thereby enabling the Nava lab’s future efforts towards studying zeolite chemistry. The precursor, truxenetrione, and both regioisomers were characterized using a combination of spectroscopic techniques including 1H, 13C NMR spectroscopy, HPLC, and UV-Visible spectroscopy. Extension of this work include improving the yield of the desired syn-isomer from the expected ratio and to turn the triols into silanols which will serve as zeolite mimetic ligands.

Infertility affects 17.5% of the global adult population and continues to burden society due to scientific ambiguity. Central to this ambiguity is the limited understanding of oogenesis, or egg development. Proteomics is a powerful tool for probing cellular mechanisms, revealing protein expression across developmental stages. We use Drosophila melanogaster, a genetically tractable model, to study egg development. However, proteomic studies in Drosophila oogenesis are limited by sample preparation challenges. Bulk ovary sampling obscures stage-specific signals due to the ovary’s heterogeneous structure. Separating thousands of egg chambers to produce stage-specific samples is not feasible. Single-cell proteomics and Data-Independent Acquisition (DIA) will help us overcome these limitations. Single-egg-chamber sampling reduces dissection time, while DIA enhances protein identification, enabling richer profiles from smaller samples. To perform DIA, a spectral library must first be created to define elution windows. This requires high-fractionation Data-Dependent Acquisition (DDA) data. I have worked to optimize a medium-scale sample preparation method, SP3, significantly enhancing protein retention, and increasing potential proteome coverage in Drosophila. I am also developing protocols for hydrophilic interaction liquid chromatography (HILIC), to improve the quality of the spectral library.