Thursday, November 21, 2024
11/21/2024
Abstract Circadian clocks throughout the body drive rhythmic expression of thousands of genes, resulting in rhythms in biochemistry, physiology and behavior. Disruption of circadian clocks through genetics or environmental perturbations such as jet lag or shift-work, can have profound negative consequences and has been linked to obesity, diabetes, cancer, cardiovascular disease and mental illness. In particular, circadian clocks exert control over nearly every major metabolic pathway, allowing optimal utilization of typically cyclic availability of nutrients. Our work is focused generally on understanding the detailed molecular mechanisms of the mammalian circadian clock machinery and the mechanisms by which these clocks control rhythmic metabolism. In this Equipment Supplement request, we seek funds to purchase an integrated plate reader system that allows determination of fluorescence and luminescence signals both in a typical plate reader fashion (total signal per well) and also as high quality and high-resolution images due to the integrated spinning disk confocal microscope. Because of the built-in CO2 incubator with temperature control and shaker, this piece of equipment will allow us to measure circadian rhythms of gene expression or metabolites continuously for many days – a requirement for assessing circadian periodicity. Our studies on the central clock mechanism require careful determination of circadian period length, amplitude and phase following different experimental manipulations and this reader will allow these measurements in multi-well format. The sophisticated time-resolved fluorescence and fluorescence polarization detection will allow the quantitative analysis of protein-protein interactions within the clock mechanism. In addition, our work on circadian control of metabolism is focused on the control of NAD(H) and NADP(H) by the circadian NADP(H) phosphatase Nocturnin. Nocturnin is present in two forms, a mitochondrial form and a cytosolic form that is myristoylated and associates with ER and other intracellular membranes. In order to determine the role Nocturnin plays in controlling these metabolites, it is necessary to examine their levels in different intracellular compartments. Because this plate reader system has spinning disk confocal imaging capabilities, this will allow us to use NADP(H) fluorescent sensors to follow the flux of NADP(H) and other associated metabolites over time in different organelles and subcellular locations over time and in response to perturbations in the clock. These types of experiments are currently impossible since the NADP(H) measurements cannot be done on a normal confocal microscope and require a spinning disk (we do not have access to a spinning disk confocal within our department). Furthermore, the length of the experiments and the need for high throughput require a dedicated instrument. In conclusion, this piece of equipment will have a major impact on both of the main objectives of our R35-funded work.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
