Manning, Benjamin Michael2015-05-052015-05-052013-07https://hdl.handle.net/11299/172142University of Minnesota Ph.D. dissertation. July 2013. Major: Chemistry. Advisor: Christy L Haynes. 1 computer file (PDF); xv, 145 pages.The principal motivation of this dissertation is to expand the utility of single-cell microelectrochemical methods, specifically carbon-fiber microelectrode amperometry (CFMA), beyond the study of fundamental cellular biophysics and toward applications investigating the cellular signaling networks in inflammation. From an analytical perspective, the inflammatory response presents several technical challenges. The inherent complexity of the immune system makes unraveling the pathogenesis of inflammatory disease a particularly challenging endeavor. An ability to detect and monitor immune cell signaling, at low, physiologically relevant concentrations and in the presence of a complex biological matrix is critical. Furthermore, although the use of bulk in vitro assays are essential for any research in biological systems, the capacity to study important cellular signaling processes at the single cell-level carries several added advantages. For these reasons, CFMA has substantial potential as a unique tool for the study of immune cell signaling. Mast cells, in particular, are an ideal model for this research because 1) they're found in most connective tissues and mucosal surfaces throughout the body, 2) they posses a broad capacity to regulate the immune response and are thought to take part in the progression of many inflammatory diseases, and 3) they release electroactive serotonin, along many other preformed immune-active mediators, via exocytosis which can be monitored by CFMA. The first part of this dissertation consists of several examples wherein CFMA is used to study mast cell degranulation in response to an altered in vivo inflammatory microenvironment, such as the chronic inflammation associated with sickle hemoglobin expression (Chapter 2), chronic in vivo morphine exposure (Chapter 2), and the effects of the endogenous opioid receptor system (Chapter 3). These chapters are followed by research that highlights the advantages of CFMA for the direct comparison of different mast cell stimulation conditions, including a study of chemokine-induced mast cell degranulation to explore the critical interactions between mast cells and airway smooth muscle in asthma (Chapter 4) as well as neuropeptide-induced mast cell degranulation to characterize mast cell function in neurogenic inflammation (Chapter 5). Collectively, this work presents CFMA as a promising technique for the study of cellular signaling in inflammatory disease.enDegranulationElectrochemistryInflammationMast cellsSingle-cell analysisChemistryThesis or Dissertation