Browsing by Subject "molecular engineering"
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Item Mutational analysis of the West Nile Virus NS4B protein(2007-01-31) Jason Alan Wicker; Alan D.T. Barrett, Ph.D.; Stephen Higgs, Ph.D.; Richard M. Kinney, Ph.D.; Norbert J. Roberts, M.D.; James C. Lee, Ph.D.West Nile virus (WNV) is a member of the genus Flavivirus in the family Flaviviridae. The WNV genome is a positive-sense RNA molecule approximately 11kb in length encoding a single polyprotein that is cleaved by a combination of viral and host proteases to produce three structural and seven nonstructural proteins. The NS4B protein is a small hydrophobic protein approximately 27kD in size that is hypothesized to participate both in the viral replication complex and evasion of host innate immune defenses. The objective of this dissertation was to investigate the role of the NS4B protein in viral cell multiplication and mouse virulence phenotypes by studying recombinant mutant viruses encoding amino acid substitutions of selected residues within the NS4B protein. The first aim of this project used protein modeling and phylogenetic analysis of the NY99 WNV NS4B protein in comparison to NS4B proteins from other flavivirus and WNV strains to identify amino acid residues with a theoretical probability of contributing to the function of NS4B. The second aim utilized site-directed mutagenesis of a WNV NY99 infectious clone to introduce amino acid substitutions into the NS4B protein primarily targeting a highly conserved N-terminal domain, the variable central hydrophobic region, and the four cysteine residues. Out of fourteen recombinant viruses encoding engineered substitutions, two highly attenuated mutant viruses were identified (C102S and P38G/T116I viruses) that exhibited temperature-sensitive and mouse attenuation (greater than 10,000,000-fold compared to wild-type) phenotypes. The third aim investigated the putative underlying molecular mechanisms responsible for the attenuation of the C102S and P38G/T116I viruses. Both NS4B mutants exhibited reduced multiplication kinetics both in mice and in murine macrophage and dendritic cell types critical for mediating the antiviral immune response. In addition, preliminary data identified a series of genes by DNA microarray analysis that exhibited differential expression in wild-type WNV-infected cells compared to C102S mutant-infected cells that may be involved in viral manipulation of cellular processes. This study has for the first time demonstrated the role of the NS4B protein as mediator of WNV temperature-sensitive and mouse attenuation phenotypes and has led to the identification of putative molecular mechanisms that may be involved.Item Theoretical-Experimental Molecular Engineering to Develop Nanodevices for Sensing Science(2012-07-16) Rangel, Norma LuciaMolecular electrostatic potentials (MEPs) and vibrational electronics (?vibronics?) have developed into novel scenarios proposed by our group to process information at the molecular level. They along with the traditional current-voltage scenario can be used to design and develop molecular devices for the next generation electronics. Control and communication features of these scenarios strongly help in the production of ?smart? devices able to take decisions and act autonomously in aggressive environments. In sensor science, the ultimate detector of an agent molecule is another molecule that can respond quickly and selectively among several agents. The purpose of this project is the design and development of molecular sensors based on the MEPs and vibronics scenarios to feature two different and distinguishable states of conductance, including a nano-micro interface to address and interconnect the output from the molecular world to standard micro-technologies. In this dissertation, theoretical calculations of the electrical properties such as the electron transport on molecular junctions are performed for the components of the sensor system. Proofs of concept experiments complement our analysis, which includes an electrical characterization of the devices and measurement of conductance states that may be useful for the sensing mechanism. In order to focus this work within the very broad array between nanoelectronic and molecular electronics, we define the new field of Molecular Engineering, which will have the mission to design molecular and atomistic devices and set them into useful systems. Our molecular engineering approach begins with a search for an optimum fit material to achieve the proposed goals; our published results suggest graphene as the best material to read signals from molecules, amplify the communication between molecular scenarios, and develop sensors of molecular agents with high sensitivity and selectivity. Specifically, this is possible in the case of sensors, thanks to the graphene atomic cross section (morphology), plasmonic surface (delocalized charge) and exceptional mechanical and electrical properties. Deliverables from this work are molecular devices and amplifiers able to read information encoded and processed at the molecular level and to amplify those signals to levels compatible with standard microelectronics. This design of molecular devices is a primordial step in the development of devices at the nanometer scale, which promises the next generation of sensors of chemical and biological agents molecularly sensitive, selective and intelligent.