Face Value: Integrative Project Thesis

School of Art and Design: Integrative Project ThesisArt and Design, School ofUniversity of MichiganUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/60476/1/IPThesis_Fagerburg.pd

Single-Molecule Insights into PcrA-Driven Disruption of RecA Filaments

Homologous recombination (HR) plays a critical role in many important cellular processes, including the resolution of stalled replication forks. HR must be highly regulated within the cell because aberrant recombination can introduce gene deletions as well as structural barriers to genetic replication and repair; various families of proteins have evolved in different organisms to achieve this regulation. The bacterial DNA-binding protein RecA is one such prototypical agent that promotes HR. It forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) that act as HR loci. The assembly and disassembly of RecA filaments are dynamic, and depend on the ATPase cycle of the protein. Both processes are subject to modulation and regulation by other factors. RecA filaments can be actively removed from DNA by non-replicative helicases such as PcrA (present in Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumoniae) and UvrD (present in Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa), and deletion of either of these leads to dysregulation of HR, which suggests that they play an important role in regulating HR via the removal of RecA filaments. We used single-molecule FRET (smFRET) to further investigate this removal and discovered that the ATPase activity of RecA is required for it to occur. The exquisite sensitivity of the single molecule technique allowed us to observe individual, short RecA filaments on ssDNA, as well as how PcrA disrupts them. The work described in this dissertation highlights a novel mechanistic component in the regulation of RecA, namely the crucial role that its ATPase activity plays in filament removal by PcrA

Adjustable Power Supply with Smart Battery Interface

This disclosure describes a programmable smart battery emulator that is usable to test laptops or other computing devices during a design phase. The smart battery emulator is operable from a user terminal that can be utilized to adjust various parameters associated with battery parameter emulation as well for emulation of communication protocols between a computing device under test (DUT) and a smart battery. The smart battery emulator includes a microcontroller that enables communication with the DUT and to regulate the output of the bench supply to a specified voltage. A software application transmits commands to the smart battery emulator. The commands can include the settings for the battery to be emulated such as manufacturer name, model, open-circuit voltage, capacity, and charge/discharge capabilities. Commands can be issued from the workstation to set the state of charge for the battery and enable a corresponding battery voltage output at the smart battery emulator

Steric exclusion and wrapping of the excluded DNA strand occurs along discrete external binding paths during MCM helicase unwinding

The minichromosome maintenance (MCM) helicase complex is essential for the initiation and elongation of DNA replication in both the eukaryotic and archaeal domains. The archaeal homohexameric MCM helicase from Sulfolobus solfataricus serves as a model for understanding mechanisms of DNA unwinding. In this report, the displaced 5′-tail is shown to provide stability to the MCM complex on DNA and contribute to unwinding. Mutations in a positively charged patch on the exterior surface of the MCM hexamer destabilize this interaction, alter the path of the displaced 5′-tail DNA and reduce unwinding. DNA footprinting and single-molecule fluorescence experiments support a previously unrecognized wrapping of the 5′-tail. This mode of hexameric helicase DNA unwinding is termed the steric exclusion and wrapping (SEW) model, where the 3′-tail is encircled by the helicase while the displaced 5′-tail wraps around defined paths on the exterior of the helicase. The novel wrapping mechanism stabilizes the MCM complex in a positive unwinding mode, protects the displaced single-stranded DNA tail and prevents reannealing

The relationship between consumer ethnocentrism, cosmopolitanism and product country image among younger generation consumers: the moderating role of country development status

Although the differences between developed and developing countries have been extensively studied in the context of globalization strategies, few studies have so far been conducted on the relationship between country development status and the possession by countries of a favorable (or unfavorable) product country image (PCI). Moreover, the results of such studies to date have been inconclusive. The purpose of this paper is to investigate the moderating role of country developmental status on PCI coupled with two antecedents of PCI, namely consumer ethnocentrism and cosmopolitanism. The paper also distinguishes between the PCI of the home and foreign country images of respondents. We test a new model that incorporates these constructs with a sample of 2655 younger generation consumers. The results show that country development status moderates some relationships but does not moderate others. These findings have significant implications for international companies from both developed and developing countries when developing global strategy

Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission

Pressure Tests in Flood-Control Conduit, Melvern Dam, Kansas

Source: https://erdc-library.erdc.dren.mil/jspui/Prototype tests were conducted at Melvern Dam, Kansas, to determine pressures on the flood-control conduit walls and to obtain the hydraulic (piezometric) grade lines of the system. No critical pressures were observed during the operation of the tests. Resistance coefficients and intake losses were also obtained. The findings were comparable to those obtained from tests at similar projects