Brown, Heather2021-05-172021-05-172019-03https://hdl.handle.net/11299/220125University of Minnesota Ph.D. dissertation.March 2019. Major: Chemistry. Advisor: Edgar Arriaga. 1 computer file (PDF); xvi, 179 pages +4 folders of supplementary files.Autophagy is a degradative intracellular process induced in response to various intrinsic and extrinsic stresses to prevent cell damage and promote cell survival. Dysregulated autophagy has been implicated in a wide range of age-related dysfunction and disease, therefore methods are needed to for the quantification of autophagy flux. In this thesis, techniques are described for the measurement of autophagy flux at the individual cell level during myogenesis and in phenotypically separated cell subsets from young and geriatric murine skeletal muscle using mass cytometry. In addition, multiplexed measurements of individual autophagy-related organelles are discussed. These techniques provide insight to the heterogeneity of autophagy flux at the individual cell and organelle level, which could inform the fundamental understanding of the necessity of autophagy for the maintenance of skeletal muscle mass and function in aging tissues. A method was developed to measure autophagy flux in whole cells by mass cytometry. This method provides accurate quantification of autophagy-related cellular targets in whole cells via post-hoc non-specific binding correction and total antibody loading normalization and was able to detect small changes in autophagy flux induced via pharmacological treatment. Application of this method to heterogeneous cultures of differentiating myoblasts suggest that autophagy flux is increased during myoblast fusion, while specific degradation of mitochondria via mitophagy is increased during myoblast proliferation, expanding current knowledge of the regulation of autophagy flux during myoblast differentiation. Satellite cells, pro- and anti-inflammatory macrophages and fibroadipogenic progenitor (FAP) cells are key players in maintaining the regenerative potential of skeletal muscle. Using the method developed for monitoring autophagy flux in myogenesis above, the contribution of each cell type to the overall decrease of autophagy flux described in the literature was determined. This analysis provides insight into the specific cell types demonstrating decreased autophagy flux with age in skeletal muscle and revealed potential differences in the need for general or selective degradation of cargo via autophagy. Individual autophagy-related organelle heterogeneity was quantified using mass cytometry. Autophagy-related organelles from murine skeletal muscle, brain, and liver were labeled with a lipid membrane stain, DDD-Tb, which enabled detection of individual organelles via mass cytometry. Analysis of specific organelle types revealed organelle associations which can be used to understand the progression of autophagy. In the future, these methods can be expanded and applied to biological models of aging to gain high-resolution insight into the organelle types most directly associated with age-related dysfunction and disease.enThesis or Dissertation