QCD structure of nuclear interactions

The research presented in this dissertation investigated selected processes involving baryons and nuclei in hard scattering reactions. These processes are characterized by the production of particles with large energies and transverse momenta. Through these processes, this work explored both, the constituent (quark) structure of baryons (specifically nucleons and Δ-Isobars), and the mechanisms through which the interactions between these constituents ultimately control the selected reactions. The first of such reactions is the hard nucleon-nucleon elastic scattering, which was studied here considering the quark exchange between the nucleons to be the dominant mechanism of interaction in the constituent picture. In particular, it was found that an angular asymmetry exhibited by proton-neutron elastic scattering data is explained within this framework if a quark-diquark picture dominates the nucleon’s structure instead of a more traditional SU(6) three quarks picture. The latter yields an asymmetry around 90o center of mass scattering with a sign opposite to what is experimentally observed. The second process is the hard breakup by a photon of a nucleon-nucleon system in light nuclei. Proton-proton (pp) and proton-neutron (pn) breakup in 3He, and ΔΔ-isobars production in deuteron breakup were analyzed in the hard rescattering model (HRM), which in conjunction with the quark interchange mechanism provides a Quantum Chromodynamics (QCD) description of the reaction. Through the HRM, cross sections for both channels in 3He photodisintegration were computed without the need of a fitting parameter. The results presented here for pp breakup show excellent agreement with recent experimental data. In ΔΔ-isobars production in deuteron breakup, HRM angular distributions for the two ΔΔ channels were compared to the pn channel and to each other. An important prediction fromthis study is that the Δ++Δ- channel consistently dominates Δ+Δ0, which is in contrast with models that unlike the HRM consider a ΔΔ system in the initial state of the interaction. For such models both channels should have the same strength. These results are important in developing a QCD description of the atomic nucleus.

Proton nuclear magnetic resonance investigation of the native and modified active site structure of heme proteins

Hemoproteins are a very important class of enzymes in nature sharing the essentially same prosthetic group, heme, and are good models for exploring the relationship between protein structure and function. Three important hemoproteins, chloroperoxidase (CPO), horseradish peroxidase (HRP), and cytochrome P450cam (P450cam), have been extensively studied as archetypes for the relationship between structure and function. In this study, a series of 1D and 2D NMR experiments were successfully conducted to contribute to the structural studies of these hemoproteins. ^ During the epoxidation of allylbenzene, CPO is converted to an inactive green species with the prosthetic heme modified by addition of the alkene plus an oxygen atom forming a five-membered chelate ring. Complete assignment of the NMR resonances of the modified porphyrin extracted and demetallated from green CPO unambiguously established the structure of this porphyrin as an NIII-alkylated product. A novel substrate binding motif of CPO was proposed from this concluded regiospecific N-alkylation structure. ^ Soybean peroxidase (SBP) is considered as a more stable, more abundant and less expensive substitute of HRP for industrial applications. A NMR study of SBP using 1D and 2D NOE methods successfully established the active site structure of SBP and consequently fills in the blank of the SBP NMR study. All of the hyperfine shifts of the SBP-CN- complex are unambiguously assigned together with most of the prosthetic heme and all proximal His170 resonances identified. The active site structure of SBP revealed by this NMR study is in complete agreement with the recombinant SBP crystal structure and is highly similar to that of the HRP with minor differences. ^ The NMR study of paramagnetic P450cam had been greatly restricted for a long time. A combination of 2D NMR methods was used in this study for P450cam-CN - complexes with and without camphor bound. The results lead to the first unequivocal assignments of all heme hyperfine-shifted signals, together with certain correlated diamagnetic resonances. The observed alternation of the assigned novel proximal cysteine β-CH2 resonances induced by camphor binding indicated a conformational change near the proximal side.^

Structure-based modeling of head -related transfer functions towards interactive customization of binaural sound systems

One of the most popular techniques for creating spatialized virtual sounds is based on the use of Head-Related Transfer Functions (HRTFs). HRTFs are signal processing models that represent the modifications undergone by the acoustic signal as it travels from a sound source to each of the listener's eardrums. These modifications are due to the interaction of the acoustic waves with the listener's torso, shoulders, head and pinnae, or outer ears. As such, HRTFs are somewhat different for each listener. For a listener to perceive synthesized 3-D sound cues correctly, the synthesized cues must be similar to the listener's own HRTFs. ^ One can measure individual HRTFs using specialized recording systems, however, these systems are prohibitively expensive and restrict the portability of the 3-D sound system. HRTF-based systems also face several computational challenges. This dissertation presents an alternative method for the synthesis of binaural spatialized sounds. The sound entering the pinna undergoes several reflective, diffractive and resonant phenomena, which determine the HRTF. Using signal processing tools, such as Prony's signal modeling method, an appropriate set of time delays and a resonant frequency were used to approximate the measured Head-Related Impulse Responses (HRIRs). Statistical analysis was used to find out empirical equations describing how the reflections and resonances are determined by the shape and size of the pinna features obtained from 3D images of 15 experimental subjects modeled in the project. These equations were used to yield “Model HRTFs” that can create elevation effects. ^ Listening tests conducted on 10 subjects show that these model HRTFs are 5% more effective than generic HRTFs when it comes to localizing sounds in the frontal plane. The number of reversals (perception of sound source above the horizontal plane when actually it is below the plane and vice versa) was also reduced by 5.7%, showing the perceptual effectiveness of this approach. The model is simple, yet versatile because it relies on easy to measure parameters to create an individualized HRTF. This low-order parameterized model also reduces the computational and storage demands, while maintaining a sufficient number of perceptually relevant spectral cues. ^

Exotic meson decay widths using lattice quantum chromodynamics

Most experiments in particle physics are scattering experiments, the analysis of which leads to masses, scattering phases, decay widths and other properties of one or multi-particle systems. Until the advent of Lattice Quantum Chromodynamics (LQCD) it was difficult to compare experimental results on low energy hadron-hadron scattering processes to the predictions of QCD, the current theory of strong interactions. The reason being, at low energies the QCD coupling constant becomes large and the perturbation expansion for scattering; amplitudes does not converge. To overcome this, one puts the theory onto a lattice, imposes a momentum cutoff, and computes the integral numerically. For particle masses, predictions of LQCD agree with experiment, but the area of decay widths is largely unexplored. ^ LQCD provides ab initio access to unusual hadrons like exotic mesons that are predicted to contain real gluonic structure. To study decays of these type resonances the energy spectra of a two-particle decay state in a finite volume of dimension L can be related to the associated scattering phase shift δ(k) at momentum k through exact formulae derived by Lüscher. Because the spectra can be computed using numerical Monte Carlo techniques, the scattering phases can thus be determined using Lüscher's formulae, and the corresponding decay widths can be found by fitting Breit-Wigner functions. ^ Results of such a decay width calculation for an exotic hybrid( h) meson (JPC = 1-+) are presented for the decay channel h → πa 1. This calculation employed Lüscher's formulae and an approximation of LQCD called the quenched approximation. Energy spectra for the h and πa1 systems were extracted using eigenvalues of a correlation matrix, and the corresponding scattering phase shifts were determined for a discrete set of πa1 momenta. Although the number of phase shift data points was sparse, fits to a Breit-Wigner model were made, resulting in a decay width of about 60 MeV. ^

A nanoliter volume nuclear magnetic resonance (NMR) system using tunneling magneto-resistive (TMR) sensors to recognize biomolecules

The need to incorporate advanced engineering tools in biology, biochemistry and medicine is in great demand. Many of the existing instruments and tools are usually expensive and require special facilities.^ With the advent of nanotechnology in the past decade, new approaches to develop devices and tools have been generated by academia and industry. ^ One such technology, NMR spectroscopy, has been used by biochemists for more than 2 decades to study the molecular structure of chemical compounds. However, NMR spectrometers are very expensive and require special laboratory rooms for their proper operation. High magnetic fields with strengths in the order of several Tesla make these instruments unaffordable to most research groups.^ This doctoral research proposes a new technology to develop NMR spectrometers that can operate at field strengths of less than 0.5 Tesla using an inexpensive permanent magnet and spin dependent nanoscale magnetic devices. This portable NMR system is intended to analyze samples as small as a few nanoliters.^ The main problem to resolve when downscaling the variables is to obtain an NMR signal with high Signal-To-Noise-Ratio (SNR). A special Tunneling Magneto-Resistive (TMR) sensor design was developed to achieve this goal. The minimum specifications for each component of the proposed NMR system were established. A complete NMR system was designed based on these minimum requirements. The goat was always to find cost effective realistic components. The novel design of the NMR system uses technologies such as Direct Digital Synthesis (DDS), Digital Signal Processing (DSP) and a special Backpropagation Neural Network that finds the best match of the NMR spectrum. The system was designed, calculated and simulated with excellent results.^ In addition, a general method to design TMR Sensors was developed. The technique was automated and a computer program was written to help the designer perform this task interactively.^

Nuclear magnetic resonance spectroscopic and computational investigations of chloroperoxidase catalyzed regio- and enantio-selective transformations

Chloroperoxidase (CPO) is the most versatile heme-containing enzyme that catalyzes a broad spectrum of reactions. The remarkable feature of this enzyme is the high regio- and enantio-selectivity exhibited in CPO-catalyzed oxidation reactions. The aim of this dissertation is to elucidate the structural basis for regio- and enantio-selective transformations and investigate the application of CPO in biodegradation of synthetic dyes. ^ To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the dissertation explored the structure of CPO-indole complex using paramagnetic relaxation and molecular modeling. The distances between the protons of indole and the heme iron revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. This provides the first experimental and theoretical explanation for the "unexpected" regioselectivity of CPO-catalyzed indole oxidation. Furthermore, the residues including Leu 70, Phe 103, Ile 179, Val 182, Glu 183, and Phe 186 were found essential to the substrate binding to CPO. These results will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications. ^ To understand the origin of the enantioselectivity of CPO-catalyzed oxidation reactions, the interactions of CPO with substrates such as 2-(methylthio)thiophene were investigated by nuclear magnetic resonance spectroscopy (NMR) and computational techniques. In particular, the enantioselectivity is partly explained by the binding orientation of substrates. In third facet of this dissertation, a green and efficient system for degradation of synthetic dyes was developed. Several commercial dyes such as orange G were tested in the CPO-H2O 2-Cl- system, where degradation of these dyes was found very efficient. The presence of halide ions and acidic pH were found necessary to the decomposition of dyes. Significantly, the results revealed that this degradation of azo dyes involves a ferric hypochlorite intermediate of CPO (Fe-OCl), compound X.^

Incorporating variable peak -to -daily ratios in the equilibrium traffic assignment procedure of the Florida Standard Urban Transportation Modeling Structure (FSUTMS)

The standard highway assignment model in the Florida Standard Urban Transportation Modeling Structure (FSUTMS) is based on the equilibrium traffic assignment method. This method involves running several iterations of all-or-nothing capacity-restraint assignment with an adjustment of travel time to reflect delays encountered in the associated iteration. The iterative link time adjustment process is accomplished through the Bureau of Public Roads (BPR) volume-delay equation. Since FSUTMS' traffic assignment procedure outputs daily volumes, and the input capacities are given in hourly volumes, it is necessary to convert the hourly capacities to their daily equivalents when computing the volume-to-capacity ratios used in the BPR function. The conversion is accomplished by dividing the hourly capacity by a factor called the peak-to-daily ratio, or referred to as CONFAC in FSUTMS. The ratio is computed as the highest hourly volume of a day divided by the corresponding total daily volume. ^ While several studies have indicated that CONFAC is a decreasing function of the level of congestion, a constant value is used for each facility type in the current version of FSUTMS. This ignores the different congestion level associated with each roadway and is believed to be one of the culprits of traffic assignment errors. Traffic counts data from across the state of Florida were used to calibrate CONFACs as a function of a congestion measure using the weighted least squares method. The calibrated functions were then implemented in FSUTMS through a procedure that takes advantage of the iterative nature of FSUTMS' equilibrium assignment method. ^ The assignment results based on constant and variable CONFACs were then compared against the ground counts for three selected networks. It was found that the accuracy from the two assignments was not significantly different, that the hypothesized improvement in assignment results from the variable CONFAC model was not empirically evident. It was recognized that many other factors beyond the scope and control of this study could contribute to this finding. It was recommended that further studies focus on the use of the variable CONFAC model with recalibrated parameters for the BPR function and/or with other forms of volume-delay functions. ^

Nuclear Magnetic Resonance Spectroscopic and Computational Investigations of Chloroperoxidase Catalyzed Regio- and Enantio-Selective Transformations

Chloroperoxidase (CPO) is the most versatile heme-containing enzyme that catalyzes a broad spectrum of reactions. The remarkable feature of this enzyme is the high regio- and enantio-selectivity exhibited in CPO-catalyzed oxidation reactions. The aim of this dissertation is to elucidate the structural basis for regio- and enantio-selective transformations and investigate the application of CPO in biodegradation of synthetic dyes. To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the dissertation explored the structure of CPO-indole complex using paramagnetic relaxation and molecular modeling. The distances between the protons of indole and the heme iron revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. This provides the first experimental and theoretical explanation for the "unexpected" regioselectivity of CPO-catalyzed indole oxidation. Furthermore, the residues including Leu 70, Phe 103, Ile 179, Val 182, Glu 183, and Phe 186 were found essential to the substrate binding to CPO. These results will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications. To understand the origin of the enantioselectivity of CPO-catalyzed oxidation reactions, the interactions of CPO with substrates such as 2-(methylthio)thiophene were investigated by nuclear magnetic resonance spectroscopy (NMR) and computational techniques. In particular, the enantioselectivity is partly explained by the binding orientation of substrates. In third facet of this dissertation, a green and efficient system for degradation of synthetic dyes was developed. Several commercial dyes such as orange G were tested in the CPO-H2O2-Cl- system, where degradation of these dyes was found very efficient. The presence of halide ions and acidic pH were found necessary to the decomposition of dyes. Significantly, the results revealed that this degradation of azo dyes involves a ferric hypochlorite intermediate of CPO (Fe-OCl), compound X.

Transcription-Coupled DNA Supercoiling in Escherichia Coli: Mechanisms and Biological Functions

Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA both in vivo and in vitro. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In E. coli cells, transcription-induced topological change of chromosomal DNA is expected to actively remodel chromosomal structure and greatly influence DNA transactions such as transcription, DNA replication, and recombination. In this study, an IPTG-inducible, two-plasmid system was established to study transcription-coupled DNA supercoiling (TCDS) in E. coli topA strains. By performing topology assays, biological studies, and RT-PCR experiments, TCDS in E. coli topA strains was found to be dependent on promoter strength. Expression of a membrane-insertion protein was not needed for strong promoters, although co-transcriptional synthesis of a polypeptide may be required. More importantly, it was demonstrated that the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. These phenomenon can be explained by the “twin-supercoiled-domain” model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA. Additionally, in order to explore whether TCDS is able to greatly influence a coupled DNA transaction, such as activating a divergently-coupled promoter, an in vivo system was set up to study TCDS and its effects on the supercoiling-sensitive leu-500 promoter. The leu-500 mutation is a single A-to-G point mutation in the -10 region of the promoter controlling the leu operon, and the AT to GC mutation is expected to increase the energy barrier for the formation of a functional transcription open complex. Using luciferase assays and RT-PCR experiments, it was demonstrated that transient TCDS, “confined” within promoter regions, is responsible for activation of the coupled transcription initiation of the leu-500 promoter. Taken together, these results demonstrate that transcription is a major chromosomal remodeling force in E. coli cells.