A software architecture for autonomous spacecraft

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1997.Includes bibliographical references (leaf 47).by Jimmy S. Shih.M.Eng

A designed protein interface that blocks fibril formation

Protein fibril formation is implicated in many diseases, and therefore much effort has been focused toward the development of inhibitors of this process. In a previous project, a monomeric protein was computationally engineered to bind itself and form a heterodimer complex following interfacial redesign. One of the protein monomers, termed monomer-B, was unintentionally destabilized and shown to form macroscopic fibrils. Interestingly, in the presence of the designed binding partner, fibril formation was blocked. Here we describe the complete characterization of the amyloid properties of monomer-B and the inhibition of fiber formation by the designed binding partner, monomer-A. Both proteins are mutants of the betal domain of streptococcal protein-G. The free monomer-B protein forms amyloid-type fibrils, as determined by transmission electron microscopy and the change in fluorescence of Thioflavin T, an amyloid-specific dye. Fibril formation kinetics are influenced by pH, protein concentration, and seeding with preformed fibrils. Under all conditions tested, monomer-A was able to inhibit the formation of monomer-B fibrils. This inhibition is specific to the engineered interaction, as incubation of monomer-B with wild-type protein-G (a structural homologue) did not result in inhibition under the same conditions. Thus, this de novo-designed heterodimeric complex is an excellent model system for the study of protein-based fibril formation and inhibition. This system provides additional insight into the development of pharmaceuticals for amyloid disorders, as well as the potential use of amyloid fibrils for self-assembling nanostructures

Computational design and experimental verification of a symmetric protein homodimer

Homodimers are the most common type of protein assembly in nature and have distinct features compared with heterodimers and higher order oligomers. Understanding homodimer interactions at the atomic level is critical both for elucidating their biological mechanisms of action and for accurate modeling of complexes of unknown structure. Computation-based design of novel protein–protein interfaces can serve as a bottom-up method to further our understanding of protein interactions. Previous studies have demonstrated that the de novo design of homodimers can be achieved to atomic-level accuracy by β-strand assembly or through metal-mediated interactions. Here, we report the design and experimental characterization of a α-helix–mediated homodimer with C2 symmetry based on a monomeric Drosophila engrailed homeodomain scaffold. A solution NMR structure shows that the homodimer exhibits parallel helical packing similar to the design model. Because the mutations leading to dimer formation resulted in poor thermostability of the system, design success was facilitated by the introduction of independent thermostabilizing mutations into the scaffold. This two-step design approach, function and stabilization, is likely to be generally applicable, especially if the desired scaffold is of low thermostability

A computationally engineered RAS rheostat reveals RAS-ERK signaling dynamics.

Synthetic protein switches controlled with user-defined inputs are powerful tools for studying and controlling dynamic cellular processes. To date, these approaches have relied primarily on intermolecular regulation. Here we report a computationally guided framework for engineering intramolecular regulation of protein function. We utilize this framework to develop chemically inducible activator of RAS (CIAR), a single-component RAS rheostat that directly activates endogenous RAS in response to a small molecule. Using CIAR, we show that direct RAS activation elicits markedly different RAS-ERK signaling dynamics from growth factor stimulation, and that these dynamics differ among cell types. We also found that the clinically approved RAF inhibitor vemurafenib potently primes cells to respond to direct wild-type RAS activation. These results demonstrate the utility of CIAR for quantitatively interrogating RAS signaling. Finally, we demonstrate the general utility of our approach in design of intramolecularly regulated protein tools by applying it to the Rho family of guanine nucleotide exchange factors

Competing biosecurity and risk rationalities in the Chittagong poultry commodity chain, Bangladesh

This paper anthropologically explores how key actors in the Chittagong live bird trading network perceive biosecurity and risk in relation to avian influenza between production sites, market maker scenes and outlets. They pay attention to the past and the present, rather than the future, downplaying the need for strict risk management, as outbreaks have not been reported frequently for a number of years. This is analysed as ‘temporalities of risk perception regarding biosecurity’, through Black Swan theory, the idea that unexpected events with major effects are often inappropriately rationalized (Taleb in The Black Swan. The impact of the highly improbable, Random House, New York, 2007). This incorporates a sociocultural perspective on risk, emphasizing the contexts in which risk is understood, lived, embodied and experienced. Their risk calculation is explained in terms of social consent, practical intelligibility and convergence of constraints and motivation. The pragmatic and practical orientation towards risk stands in contrast to how risk is calculated in the avian influenza preparedness paradigm. It is argued that disease risk on the ground has become a normalized part of everyday business, as implied in Black Swan theory. Risk which is calculated retrospectively is unlikely to encourage investment in biosecurity and, thereby, points to the danger of unpredictable outlier events