The tools of modern biology are revolutionizing biomedical research, enabling an exponential growth in the acquisition of data regarding genes, proteins, and their structures and functions in normal and diseased states. Among these advances, the ability to monitor profiles of genes, and to a lesser extent, protein expression accurately, and on a large scale are notable. However, it is often not easy to correlate the trends and relationships observed in normal or abnormal states to the phenotype resulting from the gene expression profile. For physiological states involving metabolic derangements, gene expression profiling does not fully explain the complex molecular mechanisms involved. Thus, in order to develop a comprehensive understanding of metabolic states, it is essential to understand both the gene expression events as well as the cytoplasmic events that control changes in metabolites. The proposed research seeks to accomplish the following: (1) To determine the genes whose expression is altered by molecular mediators of the stress response and to generate green fluorescence protein (GFP)-tagged expression constructs of these genes; (2) To use microfabricated and microfluidic techniques for developing a living cell array systems where differentiated cells can be cultivated and exposed to multiple inputs; (3) To obtain temporal gene expression profiles using the living cell array that has been exposed to combinatorial mixtures of stress mediators that closely mimics the physiological stress response and to use this information to predict the molecular events that determine the cell's progression to recovery or failure during stress. This proposal is an interdisciplinary project that integrates scientific inputs from biology, engineering, and computational methods. Each element of the proposal is integral to the success of the overall project and we anticipate that this will provide excellent training to the postdoctoral fellow and graduate students working on this project. The results should provide valuable new information on the molecular mechanisms governing metabolic states, which will be disseminated via peer-reviewed publications and presentations at national conferences. In summary, the proposed work seeks to provide fundamental research and a base of personnel equipped to solve problems in fields such as metabolic engineering where complex biological phenomena are under investigation.
