New tools are revolutionizing biomedical research, enabling an exponential growth in the acquisition of data regarding genes, metabolites, proteins, and their structures and functions in normal and diseased states. Among these advances, the ability to monitor profiles of genes on a large scale is notable. However, given the inherent complexity of biological systems, it is difficult to correlate trends with the underlying biologica mechanism. In an effort to understand the underlying mechanisms behind metabolic responses to external perturbations, I propose the use of a rigorous systems identification process using microfluidics in which stimuli for different temporal dynamics such as periodic sine waves, step functions, and square waves are used to probe the underlying biological response. The proposal seeks to develop a new functional genomics approach for studying gene and protein expression: the use of primary cells for the simultaneous temporal expression profiling of multiple genes and proteins and downstream protein capture techniques in a highly parallel, high throughput format.
The specific aims are: (1) to determine the activity of transcription factors, which play a key role in the regulation of inflammation and metabolism through transfection of primary rat hepatocytes with dual-reporter plasmid constructs; (2) to simultaneously determine patterns of key molecules secreted by primary hepatocytes in a microfluidic system that has capability to dynamically control the input stimulus; and (3) to determine the effect dynamics of inflammatory cytokine signal transduction has on establishment of different metabolic states such as hypermetabolism and obesity. My mentors are experienced investigators - Dr. Martin Yarmush (MGH), who is an expert in bioengineering and quantitative cell and molecular biology will be the primary mentor. Dr. Ronald Tompkins (MGH, Shriners) will co-mentor in investigating metabolic and inflammatory responses. Dr. Mehmet Toner, Director of the BioMEMs Resource Center at MGH, will advise on the development of cellular microfabrication system. Dr. Marianna Bei (MGH, HMS), an expert in transcriptional factor regulation, will advise me on the cell and molecular biology as well as the transfection biology. Dr. Korkut Ugyun (MGH, HMS), an expert in liver metabolism and metabolic pathways, will advise in developing system identification techniques as well as handling large data sets. The advisory team has a very strong collaborative record of accomplishment and set of prior accomplishments that will make this partnership function seamlessly and successfully.
Metabolic disorders, including hypermetabolism and obesity, constitute a significant burden on United States economy and the life quality of patients. The results of this study are expected to directly improve public health by identifying a dynamic link between inflammation and metabolism on a transcriptional and cellular secretome level, thus enable rational engineering of therapeutics aimed at mitigating the detrimental effects of metabolic disorders.
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