Cloning and isolation of the flagellin gene, and characterization of the posttranslational modification of the flagellin protein in Pseudomonas aeruginosa

This dissertation was built around a central theme of developing a better understanding of the flagellin filament protein of P. aeruginosa by cloning the flagellin gene, and characterizing a posttranslational modification in the flagellin protein. To detect the flagellin gene within a recombinant clone of the pLAFR1 - PAO1 library, the recombinant clones were triparentally mated into fla- mutants and scored for their ability to complement the mutant. Motility spreading assays were performed determine if complementation had occurred, and confirmed by colony blot assays. Electron microscopy and amino acid analyses showed the structure and make-up of the flagella from the cells containing clones to be nearly identical with wild type flagella. Following isolation of positive recombinant clones, two different approaches were employed to determine which region of the 25 kb PAO1 fragment was necessary for expression of the flagella. First, deletion mapping was utilized. The positive clones were further digested with restriction enzymes and religated and triparentally mated back into P. aeruginosa fla- cells, and complementation was scored. The fla gene is located within a 5.0 kb Xho I fragment. The second approach involved transposon mutagenesis with Tn501. The sites of the transposon insertions was mapped by analyses of restriction digests. A gene replacement technique was utilized, and the loss of flagellar structure was characterized by motility spreading assays and colony blotting. The other major portion of this research was identification and characterization of the posttranslational modification occurring in the flagellin filament of P. aeruginosa. Both a- and b-type purified flagellin from P. aeruginosa grown in radiolabeled phosphate were shown to be phosphorylated. Initial characterization of labeled flagellin was by autoradiograms of SDS-PAGE gels and immunoblots with anti-flagella antibody. Thin layer electrophoresis (TLE) of partial acid hydrolyzed flagella filaments revealed that the radioactivity was in phosphotyrosine. This finding was unusual because a phosphorylated tyrosine is not prevalent in prokaryotes. Intact flagella filaments have been shown to react specifically with antiphosphotyrosine monoclonal antibody. Also, the labeled filaments were susceptible to phosphatase, indicating a monophosphoester linkage. The biological role of these modifications remains unknown

Development of a measurement system optimized for detecting prebreakdown currents in liquid dielectrics

When an insulating fluid is electrically stressed to near its breakdown level, prebreakdown phenomena occur. Currents flowing under these conditions are referred to as prebreakdown currents, and their measurement is important to the understanding of the physical processes leading up to breakdown. Preliminary results indicated that for dc-stressed hexane the currents were pulse-like in nature and that the accuracy with which they could be measured was limited by the bandwidth and noise level of the measurement system. This research comprises a study of fundamental measurement limitations and the development of an optimized measurement system. A typical system used to measure prebreakdown currents consists of a high voltage source, a test cell and electrodes (to contain and electrically stress the fluid), a detection resistor (to convert the currents to a voltage) and a recorder (to capture a record of the currents). Sometimes a preamplifier is included to allow viewing of lower level currents. This research began with a study of the factors limiting the bandwidth and noise level of such a measurement system. A simple circuit model for the test cell was developed and used. The model consists of a current source shunted by a small capacitance. Two potential preamplifier configurations (voltage-sensitive and transimpedance) were considered for use. For the voltage-sensitive configuration, the bandwidth is limited by the test cell capacitance shunting the detection resistor, and the thermal noise of the resistor places a lower bound on the system noise level. Analysis revealed that for the same bandwidth, the transimpedance preamplifier could provide a lower noise level and therefore a lower minimum detectable current. A model describing the transimpedance preamplifier in terms of an open-loop gain function, feedback impedances, source impedances and noise power spectral densities was developed. A simplified version of the model was analyzed algebraically to give equations describing the frequency response and noise performance of the preamplifier. A computer-aided-design tool was developed to perform similar analyses of the complete model numerically. The noise analysis revealed that the input device for the transimpedance preamplifier should have low input noise power spectral density and low input capacity and an FET would be preferred over a bipolar device. A number of different types of FETs were characterized, and it was found that the use of GaAs MESFETs would produce a transimpedance preamplifier with the lowest noise level in a dc to 100 MHz bandwidth. A 90-MHz bandwidth transimpedance preamplifier using a GaAs MESFET input and a 5 kΩ feedback resistor was developed and incorporated into a system consisting of a cell, the preamplifier, two wideband buffer amplifiers, a 200-Msample/s transient digitizer and waveform reconstruction software. The system has an overall bandwidth of 65 MHz, including the digitizer, and a minimum detectable current of less than 250 nA rms for a signal-to-noise ratio of five. Waveform reconstruction software was used to quadruple the effective sampling rate from 5 ns per point to 1.25 as per point. This does not increase the bandwidth, but provides a better representation of the high-frequency components of the digitized waveform than simple linear interpolation does. The measurement system was tested both with artificially generated current pulses and by measuring prebreakdown currents in hexane with either positive or negative high voltage. Pulses with extremely short durations (7 ns fwhm) and amplitudes ranging from tens of μA down to near the noise level of the measurement system were observed. The usual pulse shape appeared to be the system impulse response, which implies that a still greater bandwidth would be required to see the actual shape of the current pulses

Indirect, Non-Thermal Atmospheric Plasma Promotes Bacterial Killing in vitro and Wound Disinfection in vivo Using Monogenic and Polygenic Models of Type 2 Diabetes (Without Adverse Metabolic Complications)

ABSTRACTA novel atmospheric plasma device that uses indirect, non-thermal plasma generated from room air is being studied for its effects on wound disinfection in animal wounds of monogenic and polygenic murine models of type 2 diabetes. As a proof-of-concept report, the goal of this study was to demonstrate the efficacy and safety of the indirect non-thermal plasma (INTP) device in disinfecting polycarbonate filters established with Pseudomonas aeruginosa (PAO1) biofilms as well as wound disinfection in diabetic murine wounds. Dorsal excisional wounds in BALB/c, polygenic TALLYHO, and monogenic db/db mice established with PAO1 infection all demonstrated a 3-log colony-forming unit (CFU) reduction when subjected to a course of 20-min INTP treatments. Importantly, blood glucose and body weights in these animals were not significantly impacted by plasma treatment over the study period. Plasma safety was also analyzed via complete blood count and comprehensive metabolic panels, showing no deleterious systemic effects after 3 consecutive days of 20-min plasma applications. Therefore, the results obtained demonstrated the Pseudomonas aeruginosa isolates were highly sensitive to INTP in vitro, CFU reduction of infectious Pseudomonas in wounds of diabetic mice after INTP treatment is far superior to that of non-treated infected wounds, and the application of INTP shows no indication of toxic effects. Our results are consistent with indirect non-thermal atmospheric plasma as a promising adjunct to disinfecting wounds

An instrument to sychronize Thomson scattering diagnostic measurements with MHD activity in a tokamak

An instrument to synchronize the firing of a ruby laser for a Thomson scattering diagnostic with plasma oscillations was designed, developed, and evaluated. The instrument will fire the laser at a user-selected phase of an input sine or sawtooth wave with an accuracy of ±15°. Allowable frequencies range from 20 to 500 Hz for a sawtooth and from 1 to 30 kHz for a sine wave. The instrument also allows synchronization with a sine wave to be enabled by a preselected sawtooth phase. The instrument uses analog signal processing circuits to separate the signal components, remove unwanted components, and produce zero-phase synchronization pulses. The instrument measures the period between zero-phase pulses in order to produce phase synchronization pulses delayed a fraction of the period from the zero-phase pulses. The laser is fired by the phase synchronization pulse. Unwanted signal components are attenuated by bandpass filters. A digitally controlled self-adjusting bandpass filter is used for sine processing. The instrument was used to investigate the variation of the electron temperature profile with the phase of the X-ray signal from an Impurity Studies Experiment (ISX-B) plasma exhibiting magnetohydrodynamic (MHD) activity

The PHENIX Experiment at RHIC

The physics emphases of the PHENIX collaboration and the design and current status of the PHENIX detector are discussed. The plan of the collaboration for making the most effective use of the available luminosity in the first years of RHIC operation is also presented.Comment: 5 pages, 1 figure. Further details of the PHENIX physics program available at http://www.rhic.bnl.gov/phenix

Divergent stutter bisimulation abstraction for controller synthesis with linear temporal logic specifications

This paper proposes a method to synthesise controllers for systems with possibly infinite number of states that satisfy a specification given as an LTL\◦ formula. A common approach to handle this problem is to first compute a finite-state abstraction of the original state space and then synthesise a controller for the abstraction. This paper proposes to use an abstraction method called divergent stutter bisimulation to abstract the state space of the system. As divergent stutter bisimulation factors out stuttering steps, it typically results in a coarser and therefore smaller abstraction, at the expense of not preserving the temporal ‘‘next’’ operator. The paper leverages results about divergent stutter bisimulation from model checking and shows that divergent stutter bisimulation is a sound and complete abstraction method when synthesising controllers subject to specifications in LTL\◦

Whole-arm obstacle avoidance system conceptual design

Whole-arm obstacle avoidance is needed for a variety of robotic applications in the Environmental Restoration and Waste Management (ER WM) Program. Typical industrial applications of robotics involve well-defined workspaces, allowing a predetermined knowledge of collision-free paths for manipulator motion. In the unstructured or poorly defined hazardous environments of the ER WM Program, the potential for significant problems resulting from collisions between manipulators and the environment in which they are utilized is great. The conceptual design for a sensing system that will provide protection against such collisions is described herein. The whole-arm obstacle avoidance system consists of a set of sensor bracelets,'' which cover the surface area of the manipulator links to the maximum extent practical, and a host processor. The host processor accepts commands from the robot control system, controls the operation of the sensors, manipulates data received from the bracelets, and makes the data available to the manipulator control system. The bracelets consist of a subset of the sensors, associated sensor interface electronics, and a bracelet interface. Redundant communications links between the host processor and the bracelets are provided, allowing single-point failure protection. The system allows reporting of 8-bit data from up to 1000 sensors at a minimum of 50 Hz. While the initial prototype implementation of the system utilizes capacitance proximity sensor, the system concept allows multiple types of sensors. These sensors are uniquely addressable, allowing remote calibration, thresholding at the bracelet, and correlation of a sensor measurement with the associated sensor and its location on the manipulator. Variable resolution allows high-speed, single-bit sensing as well as lower-speed higher-resolution sensing, which is necessary for sensor calibration and potentially useful in control

Decolonisation of MRSA, S. aureus and E. coli by Cold-Atmospheric Plasma Using a Porcine Skin Model In Vitro

In the last twenty years new antibacterial agents approved by the U.S. FDA decreased whereas in parallel the resistance situation of multi-resistant bacteria increased. Thus, community and nosocomial acquired infections of resistant bacteria led to a decrease in the efficacy of standard therapy, prolonging treatment time and increasing healthcare costs. Therefore, the aim of this work was to demonstrate the applicability of cold atmospheric plasma for decolonisation of Gram-positive (Methicillin-resistant Staphylococcus aureus (MRSA), Methicillin-sensitive Staphylococcus aureus) and Gram-negative bacteria (E. coli) using an ex vivo pig skin model. Freshly excised skin samples were taken from six month old female pigs (breed: Pietrain). After application of pure bacteria on the surface of the explants these were treated with cold atmospheric plasma for up to 15 min. Two different plasma devices were evaluated. A decolonisation efficacy of 3 log10 steps was achieved already after 6 min of plasma treatment. Longer plasma treatment times achieved a killing rate of 5 log10 steps independently from the applied bacteria strains. Histological evaluations of untreated and treated skin areas upon cold atmospheric plasma treatment within 24 h showed no morphological changes as well as no significant degree of necrosis or apoptosis determined by the TUNEL-assay indicating that the porcine skin is still vital. This study demonstrates for the first time that cold atmospheric plasma is able to very efficiently kill bacteria applied to an intact skin surface using an ex vivo porcine skin model. The results emphasize the potential of cold atmospheric plasma as a new possible treatment option for decolonisation of human skin from bacteria in patients in the future without harming the surrounding tissue

Design and characterization of the BVX: An 8-channel CMOS preamplifier-shaper for silicon strips

This paper presents the design and characterization of an 8channel preamplifier-shaper intended for use with silicon strip detectors ranging in capacitance from 1 to 20pF. The nominal peaking time of the circuit is 200ns with an adjustment range of {plus_minus}50ns. The circuit has a pitch (width) of 84{mu}channel with a power dissipation of 1.2mW/channel and has been fabricated in 2{mu}m p-well CMOS. The 0pF noise is 330e with a noise slope of 64e/pF. The design approach is presented as well as both test bench and strip detector measurements

IMPROVED BIOMASS UTILIZATION THROUGH REMOTE FLOW SENSING

The growth of the livestock industry provides a valuable source of affordable, sustainable, and renewable bioenergy, while also requiring the safe disposal of the large quantities of animal wastes (manure) generated at dairy, swine, and poultry farms. If these biomass resources are mishandled and underutilized, major environmental problems will be created, such as surface and ground water contamination, odors, dust, ammonia leaching, and methane emission. Anaerobic digestion of animal wastes, in which microorganisms break down organic materials in the absence of oxygen, is one of the most promising waste treatment technologies. This process produces biogas typically containing {approx}65% methane and {approx}35% carbon dioxide. The production of biogas through anaerobic digestion from animal wastes, landfills, and municipal waste water treatment plants represents a large source of renewable and sustainable bio-fuel. Such bio-fuel can be combusted directly, used in internal combustion engines, converted into methanol, or partially oxidized to produce synthesis gas (a mixture of hydrogen and carbon monoxide) that can be converted to clean liquid fuels and chemicals via Fischer-Tropsch synthesis. Different design and mixing configurations of anaerobic digesters for treating cow manure have been utilized commercially and/or tested on a laboratory scale. These digesters include mechanically mixed, gas recirculation mixed, and slurry recirculation mixed designs, as well as covered lagoon digesters. Mixing is an important parameter for successful performance of anaerobic digesters. It enhances substrate contact with the microbial community; improves pH, temperature and substrate/microorganism uniformity; prevents stratification and scum accumulation; facilitates the removal of biogas from the digester; reduces or eliminates the formation of inactive zones (dead zones); prevents settling of biomass and inert solids; and aids in particle size reduction. Unfortunately, information and findings in the literature on the effect of mixing on anaerobic digestion are contradictory. One reason is the lack of measurement techniques for opaque systems such as digesters. Better understanding of the mixing and hydrodynamics of digesters will result in appropriate design, configuration selection, scale-up, and performance, which will ultimately enable avoiding digester failures. Accordingly, this project sought to advance the fundamental knowledge and understanding of the design, scale up, operation, and performance of cow manure anaerobic digesters with high solids loading. The project systematically studied parameters affecting cow manure anaerobic digestion performance, in different configurations and sizes by implementing computer automated radioactive particle tracking (CARPT), computed tomography (CT), and computational fluid dynamics (CFD), and by developing novel multiple-particle CARPT (MP-CARPT) and dual source CT (DSCT) techniques. The accomplishments of the project were achieved in a collaborative effort among Washington University, the Oak Ridge National Laboratory, and the Iowa Energy Center teams. The following investigations and achievements were accomplished: Systematic studies of anaerobic digesters performance and kinetics using various configurations, modes of mixing, and scales (laboratory, pilot plant, and commercial sizes) were conducted and are discussed in Chapter 2. It was found that mixing significantly affected the performance of the pilot plant scale digester ({approx}97 liter). The detailed mixing and hydrodynamics were investigated using computer automated radioactive particle tracking (CARPT) techniques, and are discussed in Chapter 3. A novel multiple particle tracking technique (MP-CARPT) technique that can track simultaneously up to 8 particles was developed, tested, validated, and implemented. Phase distribution was investigated using gamma ray computer tomography (CT) techniques, which are discussed in Chapter 4. A novel dual source CT (DSCT) technique was developed to measure the phase distribution of dynamic three phase system such as digesters with high solids loading and other types of gas-liquid-solid fluidization systems. Evaluation and validation of the computational fluid dynamics (CFD) models and closures were conducted to model and simulate the hydrodynamics and mixing intensity of the anaerobic digesters (Chapter 5). It is strongly recommended that additional studies be conducted, both on hydrodynamics and performance, in large scale digesters. The studies should use advanced non-invasive measurement techniques, including the developed novel measurement techniques, to further understand their design, scale-up, performance, and operation to avoid any digester failure. The final goal is a system ready to be used by farmers on site for bioenergy production and for animal/farm waste treatment