Regulatory Mechanisms of a Bacterial Multi-Kinase Network

Cells sense and respond to their environment through signaling pathways which often require processing several signals prior to implementing a biological response. Bacterial signaling pathways are responsible for processes such as virulence, biofilm formation, survival, and symbiosis that are of research interest for medical, environmental, and industrial advancements. Emerging discoveries suggest that the systems that control these responses are more complex and intertwined than the previously understood two-component systems. Proteins such as scaffolds and pseudokinases regulate the localization, activity, and timing of the phosphotransfer reactions that dictate cellular decisions. This dissertation describes regulatory mechanisms of a multi-kinase network that controls asymmetric division in the model bacterium C. crescentus. It has been proposed that the novel pseudokinase DivL reverses signal flow by exploiting conserved kinase conformational changes and protein-protein interactions. Chapter 2 describes the development and characterization of a series of DivL-based modulators to synthetically stimulate reverse signaling of the network in vivo. I propose that synthetic stimulation and sensor disruption provide strategies to define signaling circuit organization principles for the rational design and validation of synthetic pathways. In Chapter 3, I further dissect the roles of each DivL domain on subcellular localization and downstream activity. While not catalytically active, pseudokinases have been repurposed to serve functions including complex signal recognition, integration, competition, and intermolecular allostery. I provide a refined model detailing how DivL plays each of these parts within its broader network. The work in Chapter 3 also revealed multiple scaffolding interactions that orchestrate the multi-kinase network in time and space. In Chapter 4 I identify factors that lead to the accumulation of two biochemically distinct signaling hubs at opposite cell poles to provide the foundation for asymmetry. I also provide evidence that a scaffold not only recruits a key signaling protein to the correct location but mediates its switch between kinase and phosphatase activities that drives the cell cycle. In each chapter, I discuss questions that remain and suggest future directions for study. Overall, this dissertation contributes strategies that can be used to interrogate other relevant multi-kinase networks in bacteria

Treating Fair Use as an Easement on Intellectual Property

Copyright holders have run with the copyright-as-property analogy to strengthen their rights, to the detriment of the public. There are few barriers to copyright holders locking all content behind paywalls regardless of the mixed public domain nature of the content or the fair use intentions of the public. If fair use is treated as an easement, fair use applies even if a law doesn’t explicitly invoke it, the public’s fair use rights cannot be eliminated, and copyright holders may be enjoined if they completely block fair use rights. In his 2016 article “Copyright Easement,” Jason Mazzone argues copyright easements are a way authors can reserve rights when assigning their works to publishers, but Mazzone does not equate fair use with an easement. Others have hinted at the possibility of fair use as an easement, but none has developed it. Making fair use an easement rebalances the property analogy to strengthen the public’s interest in copyrighted works and provides a theoretical and case-law foundation to push back against copyright holders’ intellectual land grab. This Note is the first paper to fully advocate treating fair use as an easement on copyright

The short-term effect of a balanced defecit diet on resting energy expenditure in overweight and obese males and females.

There is variability in the pattern of weight change in response to a weight loss intervention (i.e., slowing of subsequent weight loss, cessation of weight loss, weight regain). A reduction in resting energy expenditure (REE) may partially explain the observed variability. Few studies have examined the effect of initial weight loss on change in REE. PURPOSE: To examine the change in REE in response to weight loss across a 4-week period in overweight and obese males and females. METHODS: Thirty-seven subjects (body mass index 25.0-39.9 kg/m2; males = 14, females = 23) participated in a 4-week intervention with random assignment to an Experimental Group or a no treatment Control Group. The experimental group was instructed to reduce energy intake to 1200-1500 kcal/d and participate in 100 min/wk of moderate aerobic exercise. The control group was instructed to maintain current eating and exercise behaviors. Assessments of body weight, body composition, and REE were conducted at 0 and 4 weeks. REE was expressed as absolute REE (kcal/d), REE relative to body weight, (kcal/kg/d), REE relative to lean body mass (kcal/kgLBM/d). RESULTS: Thirty-five subjects completed the study (94.6%). There were significant differences (p<0.05) for change in outcomes between the experimental and control groups for body weight (-3.3+1.7 vs. 0.6+1.1 kg) lean body mass (-0.6+0.9 vs. 0.2+0.9 kg), absolute REE (-205.8+193.0 vs. -11.4+ 140.6 kcal/d), and REE relative to lean body mass (-3.3+2.9 vs. -0.4+2.6 kcal/kgLBM/d). There was a trend toward a significant difference between the groups (p = .07) for REE relative to body weight (-1.4+1.9 vs. -0.3+1.5 kcal/kg/d). When the groups were combined, there was a correlation between change in REE and change in body weight was r=0.41 (p<.05), and change in REE and change in lean body mass was r=0.44 (p<.01). CONCLUSIONS: Results indicate that absolute and relative REE are significantly reduced in response to weight loss over a 4-week period. The modest correlations between change in REE and both body weight and lean body mass may suggest that additional physiological mechanisms influence REE during the acute phase of weight loss

Improving the quality and quantity of commercial loans for today\u27s lenders

The purpose of this project is to create financial models and techniques that will streamline the loan process as well as improve the risk quality

Non-Equilibrium Molecular Dynamics Study of Ion Permeation through the Biological Ion Channel alpha-Hemolysin

Ion permeation properties and current-voltage (I-V) characteristics of the ion channel alpha-hemolysin (a-HL) have been calculated using non-equilibrium molecular dynamics (NEMD). In the simulation setup for our calculations, the channel was embedded in a layer of dummy atoms, which serve as an artificial membrane, and the channel structure was frozen, or held motionless throughout the simulation. This setup served to significantly reduce computational load while testing to see if realistic permeant and I-V properties for the system were maintained, by comparison to both experimental data as well as I-V data calculated using Poisson-Nernst-Planck (PNP) methodology. Additionally, diffusion constant values for both ion types inside the channel pore region were calculated using mean square displacement (MSD) methodology and compared to results for bulk solution, yielding a reduction in the diffusion constants inside the channel for each ion type of approximately one half their bulk values. While our preliminary results have produced qualitatively reasonable data, we concluded that the simulation would be more accurate if a portion of the channel structure, specifically those residues found at the protein-solvent interface, is allowed to move freely for future calculations

The Hydrogen Peroxide Catalase Treatment of Milk for Swiss Cheese Manufacture

Various temperatures and concentrations of the hydrogen peroxide catalase treatment of milk were studied in an attempt to discover the optimum temperature and concentration that would destroy unfavorable organisms and yet allow favorable ones to grow. The Federal Food and Drug Administration in their November, 1959, Definitions and Standards of Cheeses and Cheese Products states that the amount of the hydrogen peroxide solution used shall be such that the weight of the hydrogen peroxide added thereby does not exceed 0.05 percent of the weight of the milk treated. Within the maximum level allowed, it was found that in day old manufacturing milk treated with 0.05 percent peroxide for 10 minutes at either 32 C or 49 C, more than 64 percent of all microorganisms present were destroyed. Coliform organisms were very sensitive to peroxide, exhibiting a 92 percent kill at 32 C, and a 100 percent kill at 49 C. Lactic acid producing organisms were next in sensitivity to bacterial destruction, showing an 80 and 83 percent kill respectively for the same temperatures. Spore-forming organisms showed a 42 and 73 percent kill respectively at the above temperatures, but due to the refractory nature of spores to peroxide, the difference was not significant (p .05). In addition to the foregoing, related studies were conducted on the hydrogen peroxide treatment of milk without the addition of catalase. Storage milk was treated over night at 4 C for 16 hours with two peroxide concentrations, 0.025 and 0.05 percent. At the lower concentration the kill was not satisfactory, while at the higher concentration 50 percent of the spore-formers and 99 percent of the coliforms were killed. There was, however, a peroxide residue which would have to be eliminated to meet Federal Food and Drug Administration standards

Clinical evaluation of wetting angles in gas permeable contact lenses

A clinical evaluation of the wetting angle of six gas permeable contact lenses was made. The wetting angles were measured from actual contact lenses instead of buttons to. better simulate clinical conditions. Each lens was subject to five different conditions. after each condition the wetting angles were measured. We found that five of the six lenses tested had lower wetting angles after being polished than they had coming directly from the lab. The only lens that failed to improve its wetting angle after the polish procedure was the GP II lens

Computational Studies of Biological Ion Channels

Structural and functional characteristics of three biological ion channels were studied. First, current-voltage characteristics were calculated using non-equilibrium molecular dynamics (NEMD), Brownian Dynamics (BD), and Poisson-Nernst-Planck theory (PNP) for the ion channel alpha-hemolysin, comparing and contrasting the results among each other and experimental values. Results show that all methods produce qualitatively accurate results in terms of selectivity, where quantitave accuracy increases with more atomistic detailed simulation methodology. Results from NEMD simulations show that a specific location within the pore may account for selectivity of the channel, and point mutation of one residue (lys147) would likely result in a change in selectivity. The residue was mutated to serine, structural viability was tested with all-atom molecular dynamics, and PNP and BD calculations of the mutated structure show that selectivity is changed via this mutation. Second, pH dependence of current-voltage characteristics of alpha-hemolysin were studied using PNP and compared to experimental data, applying pH-dependent charge states determined from calculated pKa values for all titratable residues in the structure. Results indicate that altered charge states of both internal and external residues most accurately described experimental data. Third, Poisson-Boltzmann and (PNP) calculations were performed to determine the functional state of the crystallographic structure of the mitochondrial channel VDAC1, finding that the current-voltage properties indicated that structure represents the open conformation of the channel. Calculations were repeated using mutant channel structures, reflecting experimental results showing changes in selectivity. Two proposed gating motions of the channel were explored, with calculated current-voltage results from the gated structures not reflecting experimental changes in current-voltage properties, suggesting that the two proposed gating methods were not correct for this channel. Last, Poisson-Nernst-Planck calculations were performed of the influx of ferrous ions (Fe2+) into human H-ferritin protein. All-atom molecular dynamics simulation was used to determine both the equilibrium pore structure as well as the diffusion constant profile through the channel, using Force-Force Autocorrelation Function methodology. Results show relatively slow (compared to other channels) transit of Fe2+ ions through the channel due to greatly reduced internal diffusion constants (from bulk values) within the ferritin pore as well as low physiological concentration of Fe2+

The effect of size and shape of targets upon the near lateral phoria and duction measurements

The problem was to determine the effect of size and shape of targets upon the near lateral phoria and duction measurements