Although biochemical networks are receiving increased attention in the post-genomic era because of their role in linking genotype to phenotype, little is known about the forces that lead to the selection or maintenance of a specific mechanism for the regulation of a given set of genes in a particular organism. This knowledge is important for understanding the evolution of gene regulation. It also is important in redirecting normal expression for biotechnological purposes or in correcting pathological expression for therapeutic purposes. The long-term objective of this project is to relate function, design, and evolution of integrated biological systems to their underlying molecular determinants. The specific aims of this proposal are to: (1) Determine essential differences in function of altenative designs for signal transduction mechanisms, (2) Develop and apply quantitative methods for charterizing biochemical network robustness to small perturbations and tolerance to large changes, (3) Formulate, test and refine computational models for the highly conserved pyridine nucleotide network involved in oxygen-stress and age-related diseases, (4) Identify functional implications of alternative designs for gene regulation by small non-coding RNAs, and (5) Relate amino acid composition of an organism's proteome to dynamic variations in its amino acid environment. The methodology emphasizes mathematical and computer-assisted analyses because of their unique ability to relate integrated function and design of complex systems to their molecular elements. The general outline for the analysis in each case is as follows: Specific models based on known or suspected molecular elements and interactions are formulated;their integrated behavior is analyzed and compared according to several criteria for functional effectiveness;results are interpreted in terms of optimal designs for specific functions;and, finally, the biological significance is addressed and predictions are made for experimental testing. Accurate predictions of this nature will be necessary if we are to understand more fully the normal systemic processes of homeostasis, growth and development, or their pathological manifestations as metabolic diseases, cancer and birth defects. A focus on prokaryotic organisms is important not only because they serve as model systems for understanding higher organisms, but also because of their role in the pathogenesis of infectious diseases.