Project Abstract Small molecule and protein signals provide a rich vocabulary for cellular communication. The production and consequences of these signals are exquisitely sensitive to cellular context and microenvironment. Dissecting the molecular dialogue between cell types is challenging, and new methods are required to address fundamental questions: What is the downstream biological function of each signaling molecule? How is the biological function different when molecules are present in mixtures or when different cell types are present in the microenvironment? How do microbes—like the bacteria and fungi present in our bodies—affect the molecular landscape? Our lab is developing new tools to probe these questions including (1) microscale co- and multiculture methods that enable precise positioning of cell types to study cell signaling, (2) specialized culture platforms for complex human-bacteria-fungal multikingdom culture, (3) integration of microbial co- and multiculture systems with volatile organic compound (VOC) sampling to study how volatiles mediate microbial dialogue, and (4) at-home biofluid sampling and stabilization platforms to probe the human immune response over time. The parent R35 award has three stated goals: 1) Develop novel microscale co- and multi-culture platforms to study soluble factor signaling and use these tools to elucidate paracrine signaling mechanisms. 2) Develop and validate new readouts for inflammation (e.g., fibrosis, vasodilation) and apply these methods to identify key effector molecules and signaling pathways in inflammation. 3) Develop new analytical methods to stabilize, isolate, and study inflammatory signals. Under the parent R35 award Goal 3, we developed a novel platform that enables at-home blood sampling and RNA stabilization, homeRNA. The homeRNA kit contains a commercially available at-home blood collection device and a custom ‘stabilizer tube’ that our lab engineered to contain a stabilizing solution (e.g., RNAlater to stabilize RNA in blood). Importantly, homeRNA enables longitudinal studies within an individual to capture temporal changes in gene expression signatures resulting from exposures, disease flares, or in response to treatment. homeRNA enables evaluation of mechanistic hypotheses in human populations, providing a complement to our lab’s in vitro microfluidic platforms, which use cell lines or primary cells. In this supplement, my lab will build on our prior U3 Research Supplement (awarded in 2022, prior to renewal of the parent R35 grant), where we established a cohort of U3 women (n=39) who used homeRNA to sample themselves at home during an active COVID-19 infection. We will conduct surveys and interviews to better understand their experiences in the study, ways that future studies can be improved to facilitate inclusion of diverse populations, and ways that remote/telemedicine can increase access to healthcare for U3 women. We will also analyze the samples collected to evaluate RN...