A CLOSED LOOP CONTROL SYSTEM WITH LIVE CELLS IN THE LOOP This work proposes to develop and apply, for the ?rst time, control theory-derived strategies to regulate autophagy in live cells using an innovative experimental testbed with sensing and actuating capabilities. Combinations of pharmacological interventions are used extensively for the treatment of many diseases. Therefore, there is signi?cant potential for control theory- based treatments to enhance the well-being of patients through careful tuning of dosage delivery of multiple pharmacological reagents (PRs). Intracellular measurements of the formation of autophagy vesicles (AVs) in response to the application of various PRs will be conducted using traditional ?uorescence microscopy and an Amnis ImageStream Mark II Imaging Flow Cytometer, available at the University of New Mexico (UNM) Autophagy, In?ammation, and Metabolism in Disease Center of Biomedical Research Excellence (AIM Center), the only autophagy-focused CoBRE in the nation and in which PI Sorrentino is an Associate Member. This state-of-the-art instrument uses ?ow cytometry and multispectral imaging to detect intracellular AVs, providing an accurate count of total AVs (the output) within individual cells. A perfusive ?uidic delivery system to deliver drugs (the inputs) in time and in a controlled manner will be developed as part of the proposed e?ort. This experimental testbed will provide a proof-of-concept realization of closed-loop control of autophagy in live-cell experiments. In addition, an integrated mathematical model will be developed to describe the regulation of autophagy in live cells by considering the e?ects of several PRs on the formation of AVs. Our control decisions will be explicated at two levels: ?rst, the choice of PRs to use in a combination study out of a set of available PRs; and second, the dosage function for each PR in the combination, i.e., the rate at which the PR is supplied as a function of time, with time being a continuous function. An innovative multi-scenario optimal controller will also be developed and tested to optimize the control actions in the presence of limited experimental information and uncertainty about the state of the system. The UNM AIM Center hosts a team of specialists who conduct groundbreaking research into the role of autophagy in in?ammatory diseases and infection, and who provide mentoring opportunities for junior faculty. As an Associate Member of the AIM Center, PI Sorrentino is in a unique position within a specialized, local environment to meet with success. The long-term impact of the proposed research will be signi?cant, as it has considerable potential to advance scienti?c knowledge at the nexus of engineering and life sciences, and lead to the development of optimized therapies that could positively a?ect the well-being of millions of patients worldwide. 1
This research will infuse new and innovative ideas, techniques, and perspectives from control engineering and mathematics to create an integrated model encompassing a cell signaling network known to be central in autophagy, and a set of pharmaceutical reagents that interact with this network. An innovative experimental setup with sensing and actuating capabilities will be developed and used to apply control theory-derived strategies to regulate autophagy in live cells. The long- term impact of this work will advance scientific knowledge and have significant and important consequences for optimized combination drug therapies that will positively affect the well-being of patients worldwide. 1
