Cytokine analysis in Atlantic bottlenose dolphins: Molecular characterization of IL-4, IL-6, and IL-10

The health status of wild and captive Atlantic Bottlenose dolphins ( Tersiops truncatis) is difficult to ascertain. Mass strandings of these animals have been attributed to pollutants, as well as bacterial infections. Using human Enzyme Linked Immuno-Assays (ELISA) for immunological cytokines, I measured soluble cytokine levels with respect to their health status. In a retrospective analysis of dolphin sera, there was a trend of higher cytokine levels in “sick” animals. I cultured dolphin lymphocytes in the presence of a mitogen (PHA), a super antigen (Staph-A), Lipopolysaccharide (LPS), and a calcium flux inducer (PMA). Levels of messenger RNA, from these cultured cells, were assayed with Polymerase Chain Reaction (PCR) using primers for the human cytokines IL-2, IL-4, IL-6, IL-10, Tumor Necrosis Factor, and Interferon gamma. Only IL-4, IL-6, and IL-10 messages were obtained, inferring similar nucleotide homology to the human primer sequences. The PCR products were sequenced. Sixteen IL-4 sequences, twelve IL-6 sequences and seven IL-10 sequences were obtained and analyzed. Each cytokine exhibited the same nucleotide sequence in all dolphins examined. There was no difference in the cytokine profile in response to the various stimuli. The derived amino acid composition for each of the dolphin cytokines was used for molecular modeling, which showed that dolphin IL-4, IL-6, and IL-10 were structurally similar to the corresponding proteins of Perissodactyla. ^

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.^

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.

Computational modeling of biodegradable starch based polymer composites

Purpose. The goal of this study is to improve the favorable molecular interactions between starch and PPC by addition of grafting monomers MA and ROM as compatibilizers, which would advance the mechanical properties of starch/PPC composites. ^ Methodology. DFT and semi-empirical methods based calculations were performed on three systems: (a) starch/PPC, (b) starch/PPC-MA, and (c) starch-ROM/PPC. Theoretical computations involved the determination of optimal geometries, binding-energies and vibrational frequencies of the blended polymers. ^ Findings. Calculations performed on five starch/PPC composites revealed hydrogen bond formation as the driving force behind stable composite formation, also confirmed by the negative relative energies of the composites indicating the existence of binding forces between the constituent co-polymers. The interaction between starch and PPC is also confirmed by the computed decrease in stretching CO and OH group frequencies participating in hydrogen bond formation, which agree qualitatively with the experimental values. ^ A three-step mechanism of grafting MA on PPC was proposed to improve the compatibility of PPC with starch. Nine types of 'blends' produced by covalent bond formation between starch and MA-grafted PPC were found to be energetically stable, with blends involving MA grafted at the 'B' and 'C' positions of PPC indicating a binding-energy increase of 6.8 and 6.2 kcal/mol, respectively, as compared to the non-grafted starch/PPC composites. A similar increase in binding-energies was also observed for three types of 'composites' formed by hydrogen bond formation between starch and MA-grafted PPC. ^ Next, grafting of ROM on starch and subsequent blend formation with PPC was studied. All four types of blends formed by the reaction of ROM-grafted starch with PPC were found to be more energetically stable as compared to the starch/PPC composite and starch/PPC-MA composites and blends. A blend of PPC and ROM grafted at the ' a&d12; ' position on amylose exhibited a maximal increase of 17.1 kcal/mol as compared with the starch/PPC-MA blend. ^ Conclusions. ROM was found to be a more effective compatibilizer in improving the favorable interactions between starch and PPC as compared to MA. The ' a&d12; ' position was found to be the most favorable attachment point of ROM to amylose for stable blend formation with PPC.^

Tool cutting force modeling and wear estimation of micro -end-milling operations

The applications of micro-end-milling operations have increased recently. A Micro-End-Milling Operation Guide and Research Tool (MOGART) package has been developed for the study and monitoring of micro-end-milling operations. It includes an analytical cutting force model, neural network based data mapping and forecasting processes, and genetic algorithms based optimization routines. MOGART uses neural networks to estimate tool machinability and forecast tool wear from the experimental cutting force data, and genetic algorithms with the analytical model to monitor tool wear, breakage, run-out, cutting conditions from the cutting force profiles. ^ The performance of MOGART has been tested on the experimental data of over 800 experimental cases and very good agreement has been observed between the theoretical and experimental results. The MOGART package has been applied to the micro-end-milling operation study of Engineering Prototype Center of Radio Technology Division of Motorola Inc. ^

Fluorescence Characteristics of Size-Fractionated Dissolved Organic Matter: Implications for a Molecular Assembly Based Structure?

Surface freshwater samples from Everglades National Park, Florida, were used to investigate the size distributions of natural dissolved organic matter (DOM) and associated fluorescence characteristics along the molecular weight continuum. Samples were fractionated using size exclusion chromatography (SEC) and characterized by spectroscopic means, in particular Excitation-Emission Matrix fluorescence modeled with parallel factor analysis (EEM-PARAFAC). Most of the eight components obtained from PARAFAC modeling were broadly distributed across the DOM molecular weight range, and the optical properties of the eight size fractions for all samples studied were quite consistent among each other. Humic-like components presented a similar distribution in all the samples, with enrichment in the middle molecular weight range. Some variability in the relative distribution of the different humic-like components was observed among the different size fractions and among samples. The protein like fluorescence, although also generally present in all fractions, was more variable but generally enriched in the highest and lowest molecular weight fractions. These observations are in agreement with the hypothesis of a supramolecular structure for DOM, and suggest that DOM fluorescence characteristics may be controlled by molecular assemblies with similar optical properties, distributed along the molecular weight continuum. This study highlights the importance of studying the molecular structure of DOM on a molecular size distribution perspective, which may have important implications in understanding the environmental dynamics such materials.

Tool cutting force modeling and wear estimation of micro-end-milling operations

The applications of micro-end-milling operations have increased recently. A Micro-End-Milling Operation Guide and Research Tool (MOGART) package has been developed for the study and monitoring of micro-end-milling operations. It includes an analytical cutting force model, neural network based data mapping and forecasting processes, and genetic algorithms based optimization routines. MOGART uses neural networks to estimate tool machinability and forecast tool wear from the experimental cutting force data, and genetic algorithms with the analytical model to monitor tool wear, breakage, run-out, cutting conditions from the cutting force profiles. The performance of MOGART has been tested on the experimental data of over 800 experimental cases and very good agreement has been observed between the theoretical and experimental results. The MOGART package has been applied to the micro-end-milling operation study of Engineering Prototype Center of Radio Technology Division of Motorola Inc.

Multi-objective optimization of the molecular structure of refrigerants

The aim of this work was to develop a new methodology, which can be used to design new refrigerants that are better than the currently used refrigerants. The methodology draws some parallels with the general approach of computer aided molecular design. However, the mathematical way of representing the molecular structure of an organic compound and the use of meta models during the optimization process make it different. In essence, this approach aimed to generate molecules that conform to various property requirements that are known and specified a priori. A modified way of mathematically representing the molecular structure of an organic compound having up to four carbon atoms, along with atoms of other elements such as hydrogen, oxygen, fluorine, chlorine and bromine, was developed. The normal boiling temperature, enthalpy of vaporization, vapor pressure, tropospheric lifetime and biodegradability of 295 different organic compounds, were collected from open literature and data bases or estimated. Surrogate models linking the previously mentioned quantities with the molecular structure were developed. Constraints ensuring the generation of structurally feasible molecules were formulated and used in commercially available optimization algorithms to generate molecular structures of promising new refrigerants. This study was intended to serve as a proof-of-concept of designing refrigerants using the newly developed methodology.

Modeling of a Heat-Induced Buckling of Plates Using the Mesh-free Method

In the process of engineering design of structural shapes, the flat plate analysis results can be generalized to predict behaviors of complete structural shapes. In this case, the purpose of this project is to analyze a thin flat plate under conductive heat transfer and to simulate the temperature distribution, thermal stresses, total displacements, and buckling deformations. The current approach in these cases has been using the Finite Element Method (FEM), whose basis is the construction of a conforming mesh. In contrast, this project uses the mesh-free Scan Solve Method. This method eliminates the meshing limitation using a non-conforming mesh. I implemented this modeling process developing numerical algorithms and software tools to model thermally induced buckling. In addition, convergence analysis was achieved, and the results were compared with FEM. In conclusion, the results demonstrate that the method gives similar solutions to FEM in quality, but it is computationally less time consuming.

Wildfire Assessment Using FARSITE Fire Modeling: A Case Study in the Chihuahua Desert of Mexico

The Chihuahua desert is one of the most biologically diverse ecosystems in the world, but suffers serious degradation because of changes in fire regimes resulting in large catastrophic fires. My study was conducted in the Sierra La Mojonera (SLM) natural protected area in Mexico. The purpose of this study was to implement the use of FARSITE fire modeling as a fire management tool to develop an integrated fire management plan at SLM. Firebreaks proved to detain 100% of wildfire outbreaks. The rosetophilous scrub experienced the fastest rate of fire spread and lowland creosote bush scrub experienced the slowest rate of fire spread. March experienced the fastest rate of fire spread, while September experienced the slowest rate of fire spread. The results of my study provide a tool for wildfire management through the use geospatial technologies and, in particular, FARSITE fire modeling in SLM and Mexico.