A Closed Loop Control System with Live Cells in the Loop

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A CLOSED LOOP CONTROL SYSTEM WITH LIVE CELLS IN THE LOOP This work proposes to develop and apply, for the first 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 significant 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 fluorescence microscopy and an Amnis ImageStream Mark II Imaging Flow Cytometer, available at the University of New Mexico (UNM) Autophagy, Inflammation, 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 flow cytometry and multispectral imaging to detect intracellular AVs, providing an accurate count of total AVs (the output) within individual cells. A perfusive fluidic delivery system to deliver drugs (the inputs) in time and in a controlled manner will be developed as part of the proposed effort. 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 effects of several PRs on the formation of AVs. Our control decisions will be explicated at two levels: first, 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 inflammatory 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 significant, as it has considerable potential to advance scientific knowledge at the nexus of engineering and life sciences, and lead to the development of optimized therapies that could positively affect the well-being of millions of patients worldwide. 1