Project Summary This K99/R00 application proposes a comprehensive career development plan and a series of investigations, coupled with an outstanding mentoring committee specifically tailored to assist completion of postdoctoral training and establish an independent research program. This proposal is multi-faceted, leveraging previous training in human subjects research to complement current postdoctoral training to investigate the fundamental mechanisms underlying microvascular dysfunction in Type 2 Diabetes Mellitus (T2DM). Autophagic flux is necessary to maintain a healthy microvascular environment. T2DM is associated with reductions in autophagic flux, however whether this underlies the rampant microvascular defects associated with the disease is unclear. During the K99 phase we will examine whether T2DM is associated with a pathological switch in the mechanism of microvascular vasodilation to shear stress in isolated arterioles, and examine whether autophagic flux contributes to cutaneous microvascular function in T2DM using laser Doppler flux coupled with microdialysis. Dr. Hughes will train under the mentorship of a transdisciplinary group of senior scientists with research expertise in mechanisms of microvascular dysfunction, and examination of cutaneous microvascular function within the context of T2DM. The current postdoctoral mentors, Drs. David Gutterman and Andreas Beyer, both of whom have extensive experience mentoring trainees will continue to serve as mentors. They will be complemented by mentoring expertise from Naomi Hamburg and Lacy Alexander. The primary goal is to become an expert in the field of human microvascular physiology within the context of T2DM. This will be achieved through 1) expansion of technical skills including molecular techniques, along with laser Doppler flowmetry coupled with microdialysis; and 2) empirical training in lab and clinical trial management, along with team science and mentoring skills. Achieving these goals will strengthen further scholarly activities, establish important collaborations, and acquire critical data that will ensure a successful transition to independence. The training plan will be strengthened through a sabbatical in the laboratory of Dr. Lacy Alexander, where Dr. Hughes will be trained in investigation of microvascular function in the human cutaneous microvasculature. Obtaining an independent educational experience in one of the world’s most successful laboratories that utilize this technique will strengthen the ability to conduct translational studies. Collectively, this team will provide an outstanding training environment that will fill critical gaps in knowledge and skill set relating to the mechanisms contributing to microvascular dysfunction in T2DM. For the transition to independence phase, the mechanistic contribution of Transcription Factor EB (TFEB), a master regulator of lysosome biogenesis, will be investigated utilizing the skills and techniques learned and mastered...