The design, development and construction of a low cost multi-purpose computer/interface system for on-line experimentation: applications to time averaging of NMR spectra and other chemical instrumentation problems

The project goal was the development of a low cost multipurpose microprocessor interfacing system that would be adaptable to a number of commonly encountered chemical instruments (such as NRM, GC, IR, and UV-VIS.) for the purpose of control, data acquisition processing. The Altair Microprocessor with 4K random access memory was constructed from kit form; it functions as the computer portion of the system. The digital to analog and analog to digital signal conversion elements as well as timing, control and other necessary logic functions are incorporated in the electronic interface portion of the system. The computer, interface, principles of time averaging, and the philosophy of interfacing in general, are the subjects of this report. The Perkin Elmer R-24A NMR spectrometer is presently attached to the computer/ interface system so that signal time averaging on low concentration samples may be accomplished. A by-product of this particular system is a library of spectra stored on binary coded paper tape which can be used to generate spectra without having to make up samples. Future applications of the system are also considered in this thesis

Interspecies hybridization on DNA resequencing microarrays: efficiency of sequence recovery and accuracy of SNP detection in human, ape, and codfish mitochondrial DNA genomes sequenced on a human-specific MitoChip

Background: Iterative DNA "resequencing" on oligonucleotide microarrays offers a high-throughput method to measure intraspecific biodiversity, one that is especially suited to SNP-dense gene regions such as vertebrate mitochondrial (mtDNA) genomes. However, costs of single-species design and microarray fabrication are prohibitive. A cost-effective, multi-species strategy is to hybridize experimental DNAs from diverse species to a common microarray that is tiled with oligonucleotide sets from multiple, homologous reference genomes. Such a strategy requires that cross-hybridization between the experimental DNAs and reference oligos from the different species not interfere with the accurate recovery of species-specific data. To determine the pattern and limits of such interspecific hybridization, we compared the efficiency of sequence recovery and accuracy of SNP identification by a 15,452-base human-specific microarray challenged with human, chimpanzee, gorilla, and codfish mtDNA genomes. Results: In the human genome, 99.67% of the sequence was recovered with 100.0% accuracy. Accuracy of SNP identification declines log-linearly with sequence divergence from the reference, from 0.067 to 0.247 errors per SNP in the chimpanzee and gorilla genomes, respectively. Efficiency of sequence recovery declines with the increase of the number of interspecific SNPs in the 25b interval tiled by the reference oligonucleotides. In the gorilla genome, which differs from the human reference by 10%, and in which 46% of these 25b regions contain 3 or more SNP differences from the reference, only 88% of the sequence is recoverable. In the codfish genome, which differs from the reference by > 30%, less than 4% of the sequence is recoverable, in short islands ≥ 12b that are conserved between primates and fish. Conclusion: Experimental DNAs bind inefficiently to homologous reference oligonucleotide sets on a resequencing microarray when their sequences differ by more than a few percent. The data suggest that interspecific cross-hybridization will not interfere with the accurate recovery of species-specific data from multispecies microarrays, provided that the species' DNA sequences differ by > 20% (mean of 5b differences per 25b oligo). Recovery of DNA sequence data from multiple, distantly-related species on a single multiplex gene chip should be a practical, highly-parallel method for investigating genomic biodiversity

Mass Spectrometry-based Methods for Phosphorylation Site Mapping of Hyperphosphorylated Proteins Applied to Net1, a Regulator of Exit from Mitosis in Yeast

Prior to anaphase in Saccharomyces cerevisiae, Cdc14 protein phosphatase is sequestered within the nucleolus and inhibited by Net1, a component of the RENT complex in budding yeast. During anaphase the RENT complex disassembles, allowing Cdc14 to migrate to the nucleus and cytoplasm where it catalyzes exit from mitosis. The mechanism of Cdc14 release appears to involve the polo-like kinase Cdc5, which is capable of promoting the dissociation of a recombinant Net1·Cdc14 complex in vitro by phosphorylation of Net1. We report here the phosphorylation site mapping of recombinant Net1 (Net1N) and a mutant Net1N allele (Net1N-19m) with 19 serines or threonines mutated to alanine. A variety of chromatographic and mass spectrometric-based strategies were used, including immobilized metal-affinity chromatography, alkaline phosphatase treatment, matrix-assisted laser-desorption post-source decay, and a multidimensional electrospray mass spectrometry-based approach. No one approach was able to identify all phosphopeptides in the tryptic digests of these proteins. Most notably, the presence of a basic residue near the phosphorylated residue significantly hampered the ability of alkaline phosphatase to hydrolyze the phosphate moiety. A major goal of research in proteomics is to identify all proteins and their interactions and post-translational modification states. The failure of any single method to identify all sites in highly phosphorylated Net1N, however, raises significant concerns about how feasible it is to map phosphorylation sites throughout the proteome using existing technologies

Dynamic Certification for Autonomous Systems

Autonomous systems are often deployed in complex sociotechnical environments, such as public roads, where they must behave safely and securely. Unlike many traditionally engineered systems, autonomous systems are expected to behave predictably in varying "open world" environmental contexts that cannot be fully specified formally. As a result, assurance about autonomous systems requires us to develop new certification methods and mathematical tools that can bound the uncertainty engendered by these diverse deployment scenarios, rather than relying on static tools