Estrogen, a multi-functional steroid hormone, binds within the nucleus through estrogen receptor alpha (ERα), initiating transcription and translation to result in dynamic physiological changes. Given current methods of estrogen signaling detection, factors including tissue permeability, variation, and the timeline of receptor signaling make it difficult to accurately and consistently detect when and where estrogen signaling occurs. To combat this issue, we created Cre-independent and dependent DNA vectors made of estrogen response elements and fluorescent proteins, allowing estrogen to bind to the ERE expressed in the cell along with the vector. Following activation and estrogen binding to the ERE, fluorescent proteins would show the location of estrogen signaling through protein expression. To create a more flexible version of the Cre-dependent constructs, we aim to use molecular cloning to produce Cre-independent versions. We hypothesize that the tissue cell cultures transfected with these vectors will display increased fluorescence upon receiving ERα compared to the Cre-dependent vectors. The cloning process began with a double restriction enzyme digest, where the promoter region of our Cre-independent vector and vector regions from the Cre-dependent vectors were extracted and ligated together, then underwent bacterial transformation and purified into DNA. If performed, this research gives insight into estrogen signaling across a range of tissue types, providing a greater understanding of estrogen’s role.

Oxidative Phosphorylation (OxPhos) is a key component of energy homeostasis and helps maintain neuronal integrity, limiting the progression of neurodegenerative diseases. OxPhos dysregulation is seen in Alzheimer’s Disease (AD) alongside early Amyloid Beta (Aꞵ) misfolding, and is shown to further disrupt mitochondrial function leading to neurodegeneration. Aꞵ42 plaques are a hallmark of AD, so it is important to investigate genes that can prevent plaque accumulation and inhibit disease progression. Our lab identified the Drosophila gene Atossa (Atos)–a key regulator of OxPhos and ATP production. The human AtosA is also linked to AD-associated cognitive decline. I used Elav-GS, a pan-neuronal driver, and expressed APP.C99, a precursor to Aꞵ plaque formation. I then crossed this to an Atos-RNAi knockdown (KD) or control-RNAi. Using these lines, I performed locomotive and olfactory assessments to evaluate how Atos coordinates cognitive function. I then performed immunohistochemistry and quantified the accumulation of toxic amyloid beta species in the brains of adult flies using the same lines. I also incorporated necrosensor and apoptosis reporters into these lines and detected an increase in cell death signaling in Atos KD compared to the control, which we visualized using confocal imaging of fixed brains. My results demonstrate a difference between Atos KD compared to control in the presence of human truncated amyloid, supporting the hypothesis that Atos, and potentially human AtosA, can regulate Alzheimer's pathogenesis.

Understanding cell migration pattern is key to advancing our knowledge of human brain development. Current in-tissue studies of RNA and protein marker already revealed the migratory pathway of inhibitory neurons from the ventral developing brain (i.e. ganglionic eminence) to the dorsal cortex. But without a high throughput in situ sequencing method, tracking regulatory activity at spatial-temporal single-cell resolution remains challenging. By developing a new photonic-index sequencing (pi-seq) technique for cell barcoding, this research will map inhibitory cell migration in developing mammalian brains to reveal epigenomic regulatory landscape that guides the migratory process. By applying pi-seq on mouse brain tissue samples from different developmental stages, this research aims to create spatial-temporal mapping of neuronal migration, enable the barcoding of 100,000 targeted cells within tissue, each receiving a unique oligo barcode attached to open chromatin regions of the cell’s DNA. The acquired information will extend the capabilities of current low-throughput RNA/protein analysis to epigenomic data modalities, enhance our understanding of the regulatory mechanisms for mammalian brain development, enabling future discoveries into how disruptions in these processes may contribute to schizophrenia, autism, and other neurodevelopmental disorders.

The project explored the potential role of IGF2BP3 in multiple myeloma by assessing IGF2BP3 expression in myeloma cell lines and evaluating the impact of IGF2BP3 knockdown on cell growth. IGF2BP3 is a growth protein previously studied in our lab for its effects on amplifying cell growth in MLL-AF4 leukemia and a subsequent decrease in cell growth after generating an IGF2BP3 knockout. Three myeloma cell lines were chosen for their reported varying expression of IGF2BP3: L-363, JJN-3, and MM1.S, from overexpressed to nearly zero expression based on publicly available RNA expression data. An RT-qPCR was conducted to analyze the IGF2BP3 RNA expression in the three cell lines, with Western blot analysis to assess IGF2BP3 expression on the protein level. Simultaneously, IGF2BP3 knockdowns were developed to help investigate how knockdown affects cell growth differentially to the cell lines expressing IGF2BP3 to investigate if IGF2BP3 has a similar effect on cell growth in multiple myeloma cells as in MLL-AF4 leukemia. From the RT-qPCR, scattered differences in IGF2BP3 expression suggested low levels of RNA in the three cell lines, which will be re-evaluated to confirm no errors via comparisons with MLL-Af4 leukemia SEM cell lines’ expression, as a well-researched cell line with high RNA levels. Western blot analysis demonstrated IGF2BP3 protein expression in both JJN-3 and L-363 but not in the MM1.S cell line that is believed to have no IGF2BP3 RNA and protein expression.

The incidence of wildfires is rising globally, driven in part by climate change. Wildfire smoke exposure has been associated with increased cardiopulmonary morbidity and mortality and is known to exacerbate pre-existing respiratory conditions such as asthma and chronic obstructive pulmonary disease (COPD). While clinical associations are well-established, the cellular mechanisms underlying these effects remain poorly understood. One step to addressing this involved human sinonasal samples being collected before and after the 2025 Los Angeles wildfires. Histological analysis using hematoxylin and eosin (H&E) staining revealed significant epithelial damage and disruption of normal tissue architecture, including complete sloughing of the surface epithelium, highlighting the need to investigate the cellular mechanisms underlying wildfire-induced airway injury. Motivated by these findings, we developed an in vitro model using air-liquid interface (ALI) cultures derived from primary human basal cells to recapitulate the structure and function of the human tracheal epithelium. To simulate wildfire smoke exposure in the lab, a custom-built exposure chamber was engineered to deliver controlled, reproducible pine smoke to the ALI cultures. Cells were exposed for 3 minutes a day for 5 consecutive days. Following exposure, ALI cultures were analyzed using high-speed video microscopy, which showed complete ciliary paralysis after a single 15-minute smoke exposure. Immunofluorescent staining was used to assess epithelial proliferation, cell death, and differentiation, providing insight into how smoke affects tissue remodeling and cell turnover One such change was a decrease in staining for SCGB1A1 and MUC5B resulting in changes in the mucin secretion and inflammation rates within the lungs. Together, these findings provide a multi-model view of how wildfire smoke disrupts airway epithelial function in both human tissue and a controlled cell culture system, helping to uncover the mechanisms by which wildfire smoke contributes to respiratory disease.