PROJECT SUMMARY: Significance: Our tissues are comprised of populations of cells that are constantly interacting. One such cellular interaction crucial for tissue health is the balance between stem cell proliferation and differentiated cell death, while imbalance of these events can lead to overproduction of cells and cancer or loss of tissue integrity. During aging, there is a progressive decline in the ability of tissues to maintain homeostasis and regenerate due to impaired cell interactions. How the equilibrium between stem cells and mature cells changes during aging is not well understood. Moreover, much remains unknown about the signals that establish the feedback between stem cells and differentiated cells. This Pathway to Independence Award proposal seeks to understand the biochemical and mechanical stimuli that regulate the balance of stem cells and cell death, and how they are affected during aging. Candidate and environment: The candidate for this Pathway to Independence Award, Dr. David Castillo- Azofeifa, is committed to leading an independent research group at the interface of aging, cell biology, and regenerative medicine. Dr. Castillo-Azofeifa was trained in cell biology, stem cells and development in the laboratory of Dr. Linda Barlow at University of Colorado Anschutz Medical Campus, where he uncovered mechanisms required for tongue epithelium regeneration. During his postdoctoral studies at UCSF in the laboratory of stem cell and developmental biologist Dr. Ophir Klein, he has developed genetic and imaging tools, while using the intestine as a system to study aging. As described in this proposal, he will apply his in vivo and in vitro tools to understand how molecular (Aim 1) and mechanical (Aim 2) cues regulate intestinal stem cells during aging. Career development: During the mentored period, the candidate will gain training in new techniques, including single-cell RNA sequencing, microdroplet injection, atomic force microscopy, and micropipette pulling. The candidate will work with experts in aging biology at UCSF and the Buck Institute for Research on Aging to build the necessary scientific skills to propel his research in this field. He will also undertake a program of training to support his professional development. Combining studies of biochemical signaling and mechanobiology in aging presents a unique and powerful platform for his independent group to apply common tools and approaches to reveal unifying features of regeneration, as well as to identify key aspects of age-specific physiology. The execution of this proposal will equip the candidate with a formidable skillset and a robust platform to launch his independent research career.