SUMMARY Inflammatory Bowel Disease (IBD) is a debilitating condition that contributes to high morbidity and poor quality of life. A risk factor for the development of IBD is a ‘leaky gut’ phenotype where elevated amounts of microbially- derived antigenic material traverse the gut epithelium into sub-epithelial compartments provoking a dysregulated inflammatory loop. Therefore, maintaining a strong intestinal epithelial barrier is vital to avoid overt gut inflammation. By extension, identifying the molecular mechanisms that function in preserving gut epithelial barrier integrity is critical for understanding optimal intestinal health. There is mounting evidence that bioactive metabolites generated by the gut microbiome exert profound influence on gut epithelial barrier integrity. However, we know little about how these bioactive metabolites mechanistically influence host biology. Employing mass spectrometry-based metabolomics platforms for analysis of small molecules, our research group demonstrated remarkable differences in the metabolite composition of germ-free and conventional mice, and identified novel small molecules of microbial origin. The most discriminative molecule was δ-valerobetaine (VB). VB structurally resembles γ-butyrobetaine, the immediate biosynthetic precursor to carnitine, which is required for mitochondrial fatty acid oxidation, suggesting a role for VB in controlling energy metabolism in the mitochondria. We also confirmed that VB is undetectable in germ-free mice and their mitochondria, but present in conventionalized mice and their mitochondria. In vivo and in vitro studies showed that VB inhibits mitochondrial fatty acid oxidation through decreasing cellular carnitine levels. Importantly, the intestinal stem cell (ISC) niche is tightly regulated by numerous host-derived and luminal-derived factors, while the plasticity of the ISC niche is associated with cellular metabolism and mitochondrial function. In addition, some gastrointestinal diseases such as IBD are characterized by modifications in mitochondrial function. In preliminary data, we show that VB administration to germ-free mice induces mitochondrial biogenesis in the gut epithelium, and induces cell proliferation in the intestinal crypt. We hypothesize that VB derived from the microbiome can influence mitochondrial bioenergetics in cells within the intestinal epithelium, and functions as a central integrator whereby the microbiota influences gut cell homeostasis, gut epithelia barrier integrity, and tissue restitution following injury. I will test this hypothesis by the following specific aims, 1) to characterize the effect of VB on mitochondria function in gut tissue homeostasis, and 2) to determine the impact of VB on gut epithelial restitution in murine injury models. These aims will be carried out using a variety of methods in which I will be trained, including 3D ex vivo organoid models, mouse models of colitis and epithelial restitution, and gnotobiotic mice...