CLINICAL PROJECT 2: PROBING THE PATHOPHYSIOLOGY OF ME/CFS The underlying reason for the profound fatigue, pain, cognitive difficulties, and the peculiar response to physical effort of ME/CFS patients, is not understood. One reason that many ME/CFS patients are unable to work is that their maximal ability to produce energy, measured by cardiopulmonary exercise testing (CPET), is extremely low. For many ME/CFS patients an increase in physical or mental activity results in an increase in symptoms termed post-exertional malaise. Prior work by Cornell Center investigators found that most ME/CFS patients who undergo CPET cannot reproduce, a day later, one or more objective measures such as oxygen consumption at maximal threshold intensities, and/or exhibit abnormalities in blood pressure and/or heart rate response. The cause of post-exertional malaise, like the cause of ME/CFS, is not understood, although it is one of the most debilitating symptoms of ME/CFS sufferers. Our central hypothesis is that the study of postexertional malaise provides an exciting opportunity to obtain new insights into the etiology of ME/CFS. We will obtain samples from patients at several different time points, prior to an exercise challenge, when they are experiencing their usual levels of symptoms, and at a time of heightened symptoms, post exercise, enabling deeper insight into abnormal biological functioning associated with the disease. By analyzing, in conjunction with physiological data, metabolites, circulating inflammatory molecules, and extracellular vesicle (EV) cargo in blood samples from before and after exercise sessions, we aim to uncover markers and mechanisms of post-exertional malaise in ME/CFS. Our broad survey of possible molecular responses to exercise will include inflammatory proteins and immunogenic mitochondrial DNA (mtDNA) fragments, targeted and untargeted metabolomics of blood serum, and a detailed proteomic and metabolomic characterization of EVs. EVs are released into the circulation during exercise and could therefore contain biomarkers or contain cargo that plays an active role in mediating the abnormal response to physical activity in ME/CFS. EVs cargo includes signaling proteins, lipids, hormones, and RNAs that can influence the growth, metabolic activity, and gene expression in target cells with which they fuse. We expect that the metabolomic analyses of serum and EVs have particularly high potential to detect ME/CFS-specific changes in the response to exercise, because the metabolome integrates downstream effects from almost any physiological pathway. Therefore, in addition to conventional targeted metabolomics, we will introduce untargeted metabolomic analyses as a powerful approach toward discovery of new or unexpected ME/CFS-associated changes in primary metabolism or the production of signaling molecules such as hormones, prostaglandins, or neurotransmitters. As a primary outcome of this study, we expect to provide comprehensive data on metabolomic and proteomic changes associated with post-exertional malaise in ME/CFS that will enable identifications of novel markers and mechanisms associated with this disease.