Human Immunodeficiency Virus (HIV) is a rapidly evolving pathogen with no effective vaccine for eliciting broad protection against HIV infection. The HIV Envelope protein (Env) is a trimeric glycoprotein that is responsible for host-cell membrane fusion and infection initiation. As the only protein on the HIV virion surface, Env is the sole target for neutralizing antibodies. Characterizing the local structural dynamics of Env provides valuable insight into HIV host-virus interaction mechanisms. HDX-MS is an excellent tool for determining structural dynamics by measuring local backbone amide solvent accessibility. Generally, less structured protein regions uptake deuterium more rapidly compared to buried regions or those that are stabilized by secondary structure. We can use mass spectrometry to measure the kinetics of deuterium uptake for peptides throughout the Env protein. HDX-MS provides a detailed portrait of local structural dynamics and order, effectively identifying switching between completely closed prefusion and more open conformational states. A particular HIV Env isolate, A4, is of interest due to its unusually dynamic nature compared to other well-studied Env isolates, such as BG505. Dynamic Env exhibit more conformational flexibility, allowing them to sample various intermediary conformations between open and closed. We hypothesize that this attribute could increase HIV resistance to broadly neutralizing antibodies (bnAbs) that selectively target the closed Env conformation to prevent virus entry in immune cells. We may be able to correlate antibody binding to local dynamics measured in A4 versus BG505 Env trimers to verify this hypothesis. Biolayer interferometry will be applied to quantify antibody association and dissociation rates, as well as binding affinities. These studies will advance existing knowledge in Env-based vaccine therapeutics to improve immune responses to HIV.

Organs-on-a-chip (OOAC) are biomimetic structures that replicate the physiological environments of human organs. OOACs are growing in popularity as they provide control of parameters including shear stress, concentration gradient, and biological interactions between cells and biofluids; they can be used in pharmacokinetic, physiological, and toxicological studies. The Kelly-Yeung lab works with kidney OOACs to study toxicology and pharmacokinetics. An important component of the chips is the hydrogel that provides a tubular scaffold and biological substrate for the kidney cells. The hydrogel mixture typically consists of decellularized kidney matrix, Collagen I (Col-I), and two types of cell culture media (PTEC and 199x). The matrix mimics the kidney microenvironment, the Col-I is a stabilizer and tissue regeneration agent, and the two cell culture media are used to mimic the extracellular fluids. More matrix in the hydrogel is ideal since it will better mimic a kidney. However, the matrix by itself is not structurally stable, hence the need for a stabilizing agent. The goal of this project is to maximize the ratio of matrix to Col-I while maintaining a stable hydrogel. In order to determine the optimal ratio, stiffness testing of the hydrogels will be performed via AFM (atomic force microscopy) and a Parallel Plate Rheometer to find out how much matrix can be used before the stiffness of the hydrogel composition is compromised. At this stage, we have started testing the collagen hydrogel with the rheometer to gain base measurements before adding the kidney matrix. We aim to incorporate the kidney matrix, achieving measurements closely mirroring those obtained with the collagen hydrogel alone, while supporting healthy cell growth. Creating a more accurate OOAC based on optimized kidney extracellular matrices will help improve the models and recapitulate the effects of drugs, toxins, and diseases on human kidneys more precisely.

Heschl’s gyrus (HG) is a region of the brain containing the primary auditory cortex. The extent of folding within the HG shows high morphological variability across individuals. Interestingly, increased HG folding is more likely to be found in expert than amateur musicians, suggesting a possible role of auditory experience in shaping HG gyrification. In my research, I examined HG folding in blind individuals—another population with extensive auditory experience. I hypothesized that, if experience alters HG structure, then individuals with early-onset blindness might have increased HG gyrification compared to those with late-onset blindness or those who are sighted. I analyzed T1-weighted images collected from previous MRI studies at the University of Washington, University of Pennsylvania, and Oxford University. The combined dataset included 6 anophthalmia (individuals born without eyes), 48 early blind, 18 late blind, and 28 sighted control participants. I created hand-drawn HG regions of interest for each participant in both hemispheres and measured HG gyrification in two ways: 1) by visually categorizing the extent of HG folding (single, partial, or complete duplication), and 2) by obtaining continuous metrics (gyrification index and curvedness index) using FreeSurfer. A chi-squared test revealed that the degree of HG folding was not different across the four groups. A linear mixed-effects model (controlling for the effects of age, hemisphere, and scan location), similarly showed no effects of group on the gyrification index or the curvedness index. To conclude, my findings show that blindness does not affect HG gyrification. The results challenge the idea that auditory experience alters HG structure and offer important insights into previous findings in professional musicians. Our results suggest that the prevalence of duplicated HG in musicians may be the result of individuals with larger processing capacity within the auditory cortex being more likely to take up music as a profession.

Chronic kidney disease of unknown etiology (CKDu) is a pervasive condition not prompted by diabetes or hypertension but instead by environmental stimuli and occupational associated risks. Often detected only in advanced stages, CKDu necessitates interventions such as dialysis and kidney transplant, significantly burdening healthcare systems globally and disproportionally affecting rural populations. Ochratoxin-A (OTA), an abundant, ubiquitous, natural contaminant found in food products and is among the postulated risk factors for (CKDu). While animal studies indicate dysregulation of mitochondrial dynamics and production of superoxides via redox cycling as potential mechanisms of OTA-associated nephrotoxicity, its exposure risk in humans and kidney health remain poorly understood. This study investigates the biological pathways that contribute to the toxicity of OTA in the proximal tubule, leading to CKDu. Luminescence-based imaging approach evaluated the occurrence of OTA-induced oxidative stress in human proximal tubular epithelial cells (PTEC) by detecting cytoplasmic reactive oxygen species (ROS). Preliminary mRNA transcriptomic analysis has indicated the down-regulation of glutathione pathways, a major antioxidant pathway removing cellular oxygen. I probed OTA-treated PTECs with a reduced glutathione detection reagent, ThiolTracker Violet, to investigate the cell’s response capability for oxidative stress and detoxify xenobiotics. To verify oxidative stress mediated by OTA in live cells, CellROX Green Reagent probe treated PTECs were brought under confocal microscopy to visualize mitochondrial phenotype. The results of this investigation seek to reveal the metabolic response to OTA cytotoxicity in the human kidney and elucidate its role in CKDu progression to address diagnostic challenges and confront unmet medical needs.

The use of pesticides in the Pacific Northwest is essential in the process of safeguarding public health, most notably by mitigating pests, protecting our food supply, and aiding in distribution to supermarkets, restaurants, and our homes. However, long-term exposure to pesticides can result in illness for those handling the substances as well as their families. Prior research has shown that current pesticide application methods play a role in accelerating illness. Newer methods, such as aerial drone spraying and “smart” sprayers, involve the use of emerging technologies that are poised to change the landscape of the agricultural industry and health outcomes of farmworkers. Under the supervision of the Pacific Northwest Agricultural Health and Safety (PNASH) Center, my project will be assessing thoughts regarding adoption of these technologies. Through the creation of an electronic REDCap survey, I will be obtaining a variety of responses from agricultural workers, farm decisionmakers, and others involved in the application of pesticides on farms. Once the survey is deployed, I will analyze responses both quantitatively and qualitatively using Dedoose and R statistical methods, respectively. From these responses, I will work with the PNASH team to evaluate the adoption of current and emerging pesticide technologies among Northwest fruit growers, as well as their impacts on occupational health and safety. Through this project, I hope to collect a wide range of perspectives and thoughts regarding the implementation of new pesticide application technologies, particularly unique opinion points (positive and negative) I did not otherwise consider in my initial research with the PNASH Center. The main objective of my research project is to capture the attitudes of the pesticide application technologies to inform policy, regulations, and decision-making regarding their uses.

My field of research holds significant promise for advancing our understanding of the intricate interplay between immune cells and hormonal regulation, with broader implications for both basic science and clinical applications. In this study, I am advancing in vitro models crucial for toxicology and fertility research, specifically addressing the understudied role of resident macrophages within the testis niche. The research question aims to explore the avenues of generating M2 testicular macrophages in vitro from peripheral blood mononuclear cells (PBMCs). M2 macrophages are anti-inflammatory and are associated with wound healing and repair, unlike pro-inflammatory M1 macrophages. I hypothesize that adding macrophage colony-stimulating factor (M-CSF) combined with exposure to testosterone-rich conditioned media from a primary testis cell culture will induce the polarization of monocytes towards the M2 phenotype. The methodology involves the in vitro culture of primary PBMCs obtained from male Sprague-Dawley rats, with assessments based on surface marker expression (CD68 and CD163) and the evaluation of pro-inflammatory interleukin-6 and anti-inflammatory (interleukin-10) cytokine gene expression. M-CSF growth factor is added to monocytes to induce change to macrophages. The conditioned media for macrophages will be generated from primary neonatal rat testis cells cultured with 500 mIU/mL luteinizing hormone (LH). The media contains a higher LH concentration than in vivo, which helps produce a relevant testosterone concentration in the macrophage in vitro culture. RNA for RT-qPCR (reverse transcription–quantitative PCR) is extracted. Cells are fixed for ICC immunocytochemistry of markers (CD68/163). The implications of this research are substantial, ranging from the ability to study inflammation mechanisms in vitro to achieving a more accurate representation of the testicular hormonal environment. Furthermore, the study sheds light on the intracrine function of immune cells, emphasizing their capacity to generate sex steroids and derivatives that mediate intracrine, autocrine, and paracrine effects.