Multimode Analysis of Nanoscale Biomolecular Interactions

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical calculations have been developed for the analysis of biomolecular interactions. Human neuroglobin (hNgb) and cytochrome c from equine heart (Cyt c) proteins have been used to optimize the customized SPR instrument. The obtained Kd value (~13 µM), from SPR results, for Cyt c-hNgb molecular interactions is in general agreement with a previously published result. The SPR results also confirmed no significant impact of the internal disulfide bridge between Cys 46 and Cys 55 on hNgb binding to Cyt c. Using SPR, E. coli topoisomerase I enzyme turnover during plasmid DNA relaxation was found to be enhanced in the presence of Mg2+. In addition, a new theoretical approach of analyzing biphasic SPR data has been introduced based on analytical solutions of the biphasic rate equations. In order to develop a new label free method to quantitatively study protein-protein interactions, quartz nanopipettes were chemically modified. The derived Kd (~20 µM) value for the Cyt c-hNgb complex formations matched very well with SPR measurements (Kd ~16 µM). The finite element numerical simulation results were similar to the nanopipette experimental results. These results demonstrate that nanopipettes can potentially be used as a new class of a label-free analytical method to quantitatively characterize protein-protein interactions in attoliter sensing volumes, based on a charge sensing mechanism. Moreover, the molecule-based selective nature of hydrophobic and nanometer sized carbon nanotube (CNT) pores was observed. This result might be helpful to understand the selective nature of cellular transport through transmembrane protein pores.

The analytical determination of the uniqueness and persistence of the volatile components of human scent using optimized collection methods

The Locard exchange principle proposes that a person can not enter or leave an area or come in contact with an object, without an exchange of materials. In the case of scent evidence, the suspect leaves his scent in the location of the crime scene itself or on objects found therein. Human scent evidence collected from a crime scene can be evaluated through the use of specially trained canines to determine an association between the evidence and a suspect. To date, there has been limited research as to the volatile organic compounds (VOCs) which comprise human odor and their usefulness in distinguishing among individuals. For the purposes of this research, human scent is defined as the most abundant volatile organic compounds present in the headspace above collected odor samples. ^ An instrumental method has been created for the analysis of the VOCs present in human scent, and has been utilized for the optimization of materials used for the collection and storage of human scent evidence. This research project has identified the volatile organic compounds present in the headspace above collected scent samples from different individuals and various regions of the body, with the primary focus involving the armpit area and the palms of the hands. Human scent from the armpit area and palms of an individual sampled over time shows lower variation in the relative peak area ratio of the common compounds present than what is seen across a population. A comparison of the compounds present in human odor for an individual over time, and across a population has been conducted and demonstrates that it is possible to instrumentally differentiate individuals based on the volatile organic compounds above collected odor samples. ^

Segmental body composition determination through a bioelectrical very large scale integration (VLSI) system

The purpose of this investigation was to develop new techniques to generate segmental assessments of body composition based on Segmental Bioelectrical Impedance Analysis (SBIA). An equally important consideration was the design, simulation, development, and the software and hardware integration of the SBIA system. This integration was carried out with a Very Large Scale Integration (VLSI) Field Programmable Gate Array (FPGA) microcontroller that analyzed the measurements obtained from segments of the body, and provided full body and segmental Fat Free Mass (FFM) and Fat Mass (FM) percentages. Also, the issues related to the estimate of the body's composition in persons with spinal cord injury (SCI) were addressed and investigated. This investigation demonstrated that the SBIA methodology provided accurate segmental body composition measurements. Disabled individuals are expected to benefit from these SBIA evaluations, as they are non-invasive methods, suitable for paralyzed individuals. The SBIA VLSI system may replace bulky, non flexible electronic modules attached to human bodies. ^

Integrated surface-ground water modeling in wetlands with improved methods to simulate vegetative resistance to flow

In topographically flat wetlands, where shallow water table and conductive soil may develop as a result of wet and dry seasons, the connection between surface water and groundwater is not only present, but perhaps the key factor dominating the magnitude and direction of water flux. Due to their complex characteristics, modeling waterflow through wetlands using more realistic process formulations (integrated surface-ground water and vegetative resistance) is an actual necessity. This dissertation focused on developing an integrated surface – subsurface hydrologic simulation numerical model by programming and testing the coupling of the USGS MODFLOW-2005 Groundwater Flow Process (GWF) package (USGS, 2005) with the 2D surface water routing model: FLO-2D (O’Brien et al., 1993). The coupling included the necessary procedures to numerically integrate and verify both models as a single computational software system that will heretofore be referred to as WHIMFLO-2D (Wetlands Hydrology Integrated Model). An improved physical formulation of flow resistance through vegetation in shallow waters based on the concept of drag force was also implemented for the simulations of floodplains, while the use of the classical methods (e.g., Manning, Chezy, Darcy-Weisbach) to calculate flow resistance has been maintained for the canals and deeper waters. A preliminary demonstration exercise WHIMFLO-2D in an existing field site was developed for the Loxahatchee Impoundment Landscape Assessment (LILA), an 80 acre area, located at the Arthur R. Marshall Loxahatchee National Wild Life Refuge in Boynton Beach, Florida. After applying a number of simplifying assumptions, results have illustrated the ability of the model to simulate the hydrology of a wetland. In this illustrative case, a comparison between measured and simulated stages level showed an average error of 0.31% with a maximum error of 2.8%. Comparison of measured and simulated groundwater head levels showed an average error of 0.18% with a maximum of 2.9%. The coupling of FLO-2D model with MODFLOW-2005 model and the incorporation of the dynamic effect of flow resistance due to vegetation performed in the new modeling tool WHIMFLO-2D is an important contribution to the field of numerical modeling of hydrologic flow in wetlands.

Reliability analysis of encapsulation methods for lead -based paint through an accelerated life testing

The adverse health effects of long-term exposure to lead are well established, with major uptake into the human body occurring mainly through oral ingestion by young children. Lead-based paint was frequently used in homes built before 1978, particularly in inner-city areas. Minority populations experience the effects of lead poisoning disproportionately. ^ Lead-based paint abatement is costly. In the United States, residents of about 400,000 homes, occupied by 900,000 young children, lack the means to correct lead-based paint hazards. The magnitude of this problem demands research on affordable methods of hazard control. One method is encapsulation, defined as any covering or coating that acts as a permanent barrier between the lead-based paint surface and the environment. ^ Two encapsulants were tested for reliability and effective life span through an accelerated lifetime experiment that applied stresses exceeding those encountered under normal use conditions. The resulting time-to-failure data were used to extrapolate the failure time under conditions of normal use. Statistical analysis and models of the test data allow forecasting of long-term reliability relative to the 20-year encapsulation requirement. Typical housing material specimens simulating walls and doors coated with lead-based paint were overstressed before encapsulation. A second, un-aged set was also tested. Specimens were monitored after the stress test with a surface chemical testing pad to identify the presence of lead breaking through the encapsulant. ^ Graphical analysis proposed by Shapiro and Meeker and the general log-linear model developed by Cox were used to obtain results. Findings for the 80% reliability time to failure varied, with close to 21 years of life under normal use conditions for encapsulant A. The application of product A on the aged gypsum and aged wood substrates yielded slightly lower times. Encapsulant B had an 80% reliable life of 19.78 years. ^ This study reveals that encapsulation technologies can offer safe and effective control of lead-based paint hazards and may be less expensive than other options. The U.S. Department of Health and Human Services and the CDC are committed to eliminating childhood lead poisoning by 2010. This ambitious target is feasible, provided there is an efficient application of innovative technology, a goal to which this study aims to contribute. ^

High energy three -body breakup of three -nucleon systems

The main goal of this dissertation was to study two- and three-nucleon Short Range Correlations (SRCs) in high energy three-body breakup of 3He nucleus in 3He(e, e'NN) N reaction. SRCs are characterized by quantum fluctuations in nuclei during which constituent nucleons partially overlap with each other. ^ A theoretical framework is developed within the Generalized Eikonal Approximation (GEA) which upgrades existing medium-energy methods that are inapplicable for high momentum and energy transfer reactions. High momentum and energy transfer is required to provide sufficient resolution for probing SRCs. GEA is a covariant theory which is formulated through the effective Feynman diagrammatic rules. It allows self-consistent calculation of single and double re-scatterings amplitudes which are present in three-body breakup processes. The calculations were carried out in detail and the analytical result for the differential cross section of 3He(e, e'NN)N reaction was derived in a form applicable for programming and numerical calculations. The corresponding computer code has been developed and the results of computation were compared to the published experimental data, showing satisfactory agreement for a wide range of values of missing momenta. ^ In addition to the high energy approximation this study exploited the exclusive nature of the process under investigation to gain more information about the SRCs. The description of the exclusive 3He( e, e'NN)N reaction has been done using the formalism of the nuclear decay function, which is a practically unexplored quantity and is related to the conventional spectral function through the integration of the phase space of the recoil nucleons. Detailed investigation showed that the decay function clearly exhibits the main features of two- and three-nucleon correlations. Four highly practical types of SRCs in 3He nucleus were discussed in great detail for different orders of the final state re-interactions using the decay function as an unique identifying tool. ^ The overall conclusion in this dissertation suggests that the investigation of the decay function opens up a completely new venue in studies of short range nuclear properties. ^

Integrated Surface-Ground Water Modeling in Wetlands With Improved Methods to Simulate Vegetative Resistance to Flow

This dissertation focused on developing an integrated surface – subsurface hydrologic simulation numerical model by programming and testing the coupling of the USGS MODFLOW-2005 Groundwater Flow Process (GWF) package (USGS, 2005) with the 2D surface water routing model: FLO-2D (O’Brien et al., 1993). The coupling included the necessary procedures to numerically integrate and verify both models as a single computational software system that will heretofore be referred to as WHIMFLO-2D (Wetlands Hydrology Integrated Model). An improved physical formulation of flow resistance through vegetation in shallow waters based on the concept of drag force was also implemented for the simulations of floodplains, while the use of the classical methods (e.g., Manning, Chezy, Darcy-Weisbach) to calculate flow resistance has been maintained for the canals and deeper waters. A preliminary demonstration exercise WHIMFLO-2D in an existing field site was developed for the Loxahatchee Impoundment Landscape Assessment (LILA), an 80 acre area, located at the Arthur R. Marshall Loxahatchee National Wild Life Refuge in Boynton Beach, Florida. After applying a number of simplifying assumptions, results have illustrated the ability of the model to simulate the hydrology of a wetland. In this illustrative case, a comparison between measured and simulated stages level showed an average error of 0.31% with a maximum error of 2.8%. Comparison of measured and simulated groundwater head levels showed an average error of 0.18% with a maximum of 2.9%.

High energy three-body breakup of three-nucleon systems

The main goal of this dissertation was to study two- and three-nucleon Short Range Correlations (SRCs) in high energy three-body breakup of 3He nucleus in 3He(e, e'NN)N reaction. SRCs are characterized by quantum fluctuations in nuclei during which constituent nucleons partially overlap with each other. A theoretical framework is developed within the Generalized Eikonal Approximation (GEA) which upgrades existing medium-energy methods that are inapplicable for high momentum and energy transfer reactions. High momentum and energy transfer is required to provide sufficient resolution for probing SRCs. GEA is a covariant theory which is formulated through the effective Feynman diagrammatic rules. It allows self-consistent calculation of single and double re-scatterings amplitudes which are present in three-body breakup processes. The calculations were carried out in detail and the analytical result for the differential cross section of 3He(e, e'NN)Nreaction was derived in a form applicable for programming and numerical calculations. The corresponding computer code has been developed and the results of computation were compared to the published experimental data, showing satisfactory agreement for a wide range of values of missing momenta. In addition to the high energy approximation this study exploited the exclusive nature of the process under investigation to gain more information about the SRCs. The description of the exclusive 3He(e, e'NN)N reaction has been done using the formalism of the nuclear decay function, which is a practically unexplored quantity and is related to the conventional spectral function through the integration of the phase space of the recoil nucleons. Detailed investigation showed that the decay function clearly exhibits the main features of two- and three-nucleon correlations. Four highly practical types of SRCs in 3He nucleus were discussed in great detail for different orders of the final state re-interactions using the decay function as an unique identifying tool. The overall conclusion in this dissertation suggests that the investigation of the decay function opens up a completely new venue in studies of short range nuclear properties.