Glomeruli are the basic filtration unit of the kidney. The current understanding of its physiology is limited by the partial or 2D analysis of its structural components. The Vaughan Group uses optical super-resolution microscopy in combination with advanced chemical labeling techniques and powerful data analysis approaches to perform high-resolution 3D reconstruction of the whole mouse glomeruli. Overall, the work has the potential to provide a novel understanding of the glomerular structures and how they are altered in aged and diseased conditions. The labeling of the overall morphology of the glomeruli is achieved by chemically labeling the distribution of abundant macromolecules (carbohydrates, amine, and DNA) using Fluorescence Labeling of Abundant Reactive Entities (FLARE). Though we could visualize the general physiology of the sample with FLARE, incorporating specific targeting of molecules with FLARE is still challenging. My role is to optimize the FLARE protocol to add the capability of labeling the distribution of specific molecules using immunolabeling. The most challenging part is that all the fluorophores labeled prior to FLARE will be bleached out by the strong oxidation step while labeling carbohydrates. I am focusing on exploring possible workarounds to incorporate immunostaining with FLARE. The only way to bypass the bleaching fluorophores is to label dyes after the FLARE. However, the FLARE involves the gelation part, and the gel makes antibodies which are linked to fluorophores hard to get into the sample. So, instead of using regular secondary antibodies, I use biotin and then link to the streptavidin dye, which is smaller in size and easier to enter the sample. With this optimization working, we could incorporate whatever target of interest with high resolution on top of three general stains provided by FLARE, giving us an extra degree of information for our 3D reconstructions of glomeruli.

Epigenetic factors, including histone marks, change the patterns of gene expression without altering the DNA sequence. Variations in such marks are known to account for the ability of stem cells to differentiate into various cell types, but a preliminary experiment conducted by a former member of the Vaughan Group, Dr. Marcus Woodworth, has revealed that even in a phenotypically homogeneous, terminally differentiated cell population, the presence of H3K27me3, a repressive histone mark, varies on the HOXC gene of human retinal pigment epithelium (RPE1) cells at single-cell, single-loci level. My role is to evaluate the two possible origins of such heterogeneity: inheritance (histone mark varies due to events that happened during differentiation, or the random drift after differentiation, and the variations are kept within each lineage of cells), and multiple mark co-repression (one histone mark varies, but summing its effect with another histone mark that perform a similar function lead to the observed functional homogeneity), and to validate that such pattern exists among a broader range of genes. To achieve these ends, I profile selected histone marks (H3K27me3 and H3K9me3) on genes that experience different types of regulations during differentiation (HOXC, GAPDH and SIX6), using imaging-based methods, including the time-lapse imaging of live cells to map out lineages, and expansion microscopy (ExM) to capture fluorescently labeled histone marks at single-loci level. If the hypothesized origins are true, a significant difference in the number of histone mark clusters around the genes of interest would be observed between cells of different lineages, and complementary variation patterns would be observed between H3K27me3 and H3K9me3. The study reveals the nuances of histone mark dynamics on the single-cell, single-loci level, and optimizes an imaging-based method that has the potential for multiplexing at high spatial resolution, thereby providing a powerful tool for further studies on epigenetics.

The kidney is composed of thousands of filtration units called nephrons. Within each nephron lies a tuft of capillaries, the glomerulus, that filters from the blood through a filtration barrier. Over time this filtration barrier thickens, ultimately causing decreased blood filtration. A main marker of this age-related degradation are telomeres which comprise the ends of chromosomes and protect the coding DNA from degradation. If telomeres become too short, the coding region of DNA will begin to degrade. To combat this, telomere shortening signals for cells to enter a state of permanent cell cycle arrest, senescence, which prevents replication of cells with degraded DNA. Accurately quantifying telomere length will enable the development of correlations between cell lineage and structural changes within the kidney. I hypothesize that Expansion Microscopy (ExM) and quantitative-Fluorescent In-Situ Hybridization (Q-FISH) will allow me to determine the relationship between physiological changes in the filtration barrier and single-cell telomere length. ExM enables a superresolution cellular view by embedding a tissue sample in a swellable hydrogel, achieving four-fold isotropic expansion. This technique confers greater resolution of Q-FISH signal versus traditional confocal microscopy. To determine telomere length, I developed custom analysis scripts to quantify Q-FISH signal brightness. Preliminary results indicate an increased brightness of younger mice compared to their aged counterparts. Additionally, to receive a base-pair output I compared the Q-FISH signal to the signal of a DNA region of known length, Major Satellites, determining young telomeres to have an average base-pair length of 30 kb. I am validating these results in collaboration with the Miller Lab using next-generation sequencing techniques. Future work includes concurrent application of general physiology stains to identify and measure the glomerular filtration barrier physiology. Results from this method will allow for a wealth of information regarding the relationship of single-cell telomere length and glomerular structural health.

Elementary school educators are primary influences on children’s development by helping shape their behavior with peers and in the classroom. Employees in education are deferred to by their newer coworkers regarding student interactions and set precedents for students’ school environment. New and seasoned educators’ disciplinary practices greatly vary when addressing students exhibiting problematic behaviors which may differently affect student-instructor relationships. I use an existing dataset from a larger study testing the impact of a novel implementation strategy on the implementation of a universal social, emotional, and behavioral program in nine elementary schools from one district. Educator participants in this study completed a survey battery when starting and ending the school year and the district provided quarterly discipline records via a universal prevention program. The findings identify trends in student disciplinary actions between new (<5 yrs) and experienced (>5 yrs) instructors. Understanding the relationship between teacher tenure and the treatment of student misbehavior is crucial to demystifying the manifestation of poor student enrichment and hostile student-instructor relationships. These findings could suggest how differential disciplinary practices by educators based on their years of experience influence student learning.

Teachers play an integral part in a child's development as they are responsible for educating youth. Since COVID-19, schools are reporting rising rates of teacher burnout (52%) and students exhibiting problematic behaviors (e.g., disruptions [59%], disrespectful [52%], rowdiness [49%]). Disruptive classroom behaviors hinder the ability of teachers to adequately implement curriculum which results in a variety of disciplinary actions administered. My research analyzes the correlation between elementary school teachers' burnout rate and disciplinary actions taken. My data comes from a larger study that examined the implementation of a preventative social-emotional behavioral program in nine different schools. Teachers completed an online survey distributed in the middle of the academic year, including a measure on burnout, and the district provided systematically obtained disciplinary records (i.e., September to December) upon completion of the study. Preliminary analysis is ongoing to examine the relationship between teacher burnout and disciplinary actions recorded. This can be a first step towards improving teachers' professional well-being and creating a more prosocial classroom environment for student growth.

Housing First (HF) is low-barrier, permanent supportive housing for individuals experiencing homelessness without the prerequisites of attaining abstinence. Past research shows that in-person harm reduction treatment for alcohol (HaRT-A) within HF facilities are preferred because they focus on client-centered goals and improving quality of life. Unfortunately, COVID-19 caused significant disruptions to in-person service provision, and although they requested remote alternatives, currently no services or best practices for HF residents exist. This ongoing project is adapting HaRT-A into a digital platform (eHaRT-A), which when completed, will be tested among HF residents with alcohol use disorder. Our project aims to examine barriers to technology use among HF residents through user-centered design (UCD) principles to improve the acceptability of eHaRT-A. Semi-structured interviews were conducted with HF residents (n=31) discussing technology literacy, viewpoints on technology use, and HaRT-A. Findings highlighted that only a few residents are comfortable enough to use technology on their own; thus, eHaRT-A will be better implemented as a “telehealth station” that is set up in a “somewhat common,” “but still private” environment. Some residents (n=10) were recruited as consultants on the Community Advisory Board (CAB) to provide ongoing input on the eHaRT-A platform. Over a one-year period, UCD interviews on low- and high-fidelity prototypes were conducted with the CAB to inform iterative changes made to the eHaRT-A platform. Within our team, I took this feedback and adjusted HTML and JavaScript code to reflect what is preferred. Feedback included a preference for a “whimsical” theme, presenting personalized feedback through line graphs to show change over time, and interpolating reported outcome measures (e.g., substance use frequency/related-consequences). These findings offer insight into best practices for implementing eHaRT-A within HF facilities. This project serves as a foundation for future eHaRT-A phases and provides the necessary tools to integrate digitized healthcare for underserved communities.