Application of Liquid Chromatography-Tandem Mass Spectrometry Techniques to the Study of Two Biological Systems

Quorum sensing is a type of bacterial cell-cell communication that uses diffusible signaling molecules to allow the regulation of gene expression based on cell density. The two types of signaling molecules discussed here are autoinducer-2 (AI-2) and a class of signaling molecules of the N-acylhomoserine lactone type (AHL) also known as autoinducer-1 (AI-1). The biosynthesis of both of these molecules has origins in a metabolic pathway. Although undisputed in some cases, the role of AI-2 and AHLs in bacterial systems has come into question. Here, the functionalities of these molecules were investigated by utilizing isotope-labeled versions of both AI-2 and AHLs in conjunction with liquid chromatography tandem mass spectrometry in order to quantify the natural abundance of these molecules in various bacterial cultures. Presented in this thesis are, a synthesis for doubly-deuterated AHLs as well as chromatographic and spectrometric methods for the detection and quantitation of these molecules. Additionally, a series of relevant biological studies which effectively and prolifically utilize these synthetic and analytical techniques are presented here.

The selective destruction of beta-cell mass in the Langerhans of the pancreas is known to cause Type 1 diabetes mellitus (T1DM). Currently, many key aspects of this autoimmune disease remain unclear, including the exact mechanism of beta-cell death. In a collaborative project with Dr. J. Jason Collier, we sought to test the hypothesis that different mechanisms of cell death will present discrete phenotypic profiles which can be distinguished by a specific metabolic response in response to the appropriate stimuli. A second project presented in this thesis is the development and implementation of a method to profile the metabolic signatures of two types of pancreatic beta-cell death using tandem mass spectrometry techniques. Using 832/13 rat insulinoma cells, the metabolite pools of cells exposed to either pro-inflammatory cytokines or known apoptosis inducers, such as camptothecin, were analyzed. In this investigation, it was found that this method was effective in defining reproducible metabolic differences in each sample tested. Taken together with complementary methods used in the Collier lab, the results collectively demonstrate that pancreatic beta-cells undergo apoptosis in response to camptothecin, but not pro-inflammatory cytokines.