Nanoliter Assay Method Development for Glutamate, Cysteine and Glutathione from Mice and D. Melanogaster
thesisposted on 2016-10-29, 00:00 authored by Srivani Borra
Neurotransmission is a fundamental communication process between two neurons. Most of the synaptic neurotransmission is chemically mediated across the synapse through neurotransmitters. Synthesis, release, reuptake, destruction, and various other aspects of neurotransmitters play a pivotal role in regulating neurotransmission. Chemical analyses of neurotransmitters are indispensable, as they would provide a fundamental understanding of neuropsychiatric symptoms and also help discover pharmacological agents to alleviate disease conditions. Various malignant tumors such as leukemia, lymphomas, pancreatic cancer, and brain cancer have been related to cystine-glutamate transporter (xCT) expression. The xCT is a membrane bound transporter hypothesized to transport extracellular cystine into the cell in exchange for intracellular glutamate thus maintaining extracellular glutamate levels and functioning as a cystine uptake mechanism for intracellular glutathione (GSH) synthesis. Glutamate, a major neurotransmitter, and GSH, a major antioxidant, are clearly important in regulating normal cell functioning. This dissertation illustrates method developments for chemical analysis in xCT model organisms. In vivo sampling methods enabled 10-2000 nL biological fluid collection from xCT mice and fruitfly model organisms, requiring proficient sample handling and preparation techniques. The three nL-scale, low-dilution assays presented here analyzed for glutamate, cysteine, and glutathione content in 500 nL sample volumes of mouse cerebrospinal fluid and 10-60 nL hemolymph volumes of single D. Melanogaster, to understand xCT regulation. In vivo sample collection and handling of the 10-500 nL sample volumes through their preseparation labeling optimizations at nL scale was achieved using defined lengths of fused silica capillaries. Chemical measurements of oxidative stress precursors like cysteine/cystine and GSH/GSSG emerged from these nL assay method developments implying xCT function under oxidative stress. Glutamate and GSH regulation along with gender based differences were found in comparison studies of xCT mutants and their controls. The usage of in vivo sampling methods along with the developed nL assays for chemical component analysis definitely benefit towards understanding cellular homeostasis and neurotransmission related abnormalities.