Mechanistic studies on the degradation of gasoline oxygenates by advanced oxidation technologies and the reaction of 4-methyl-1,2,4-triazoline-3,5-dione with tetracyclopropylethylene

Gasoline oxygenates (MTBE, methyl tert-butyl ether; DIPE, di-isopropyl ether; ETBE, ethyl tert-butyl ether; TAME, tert-amyl ether) are added to gasoline to boost octane and enhance combustion. The combination of large scale use, high water solubility and only minor biodegradability has now resulted in a significant gasoline oxygenate contamination occurring in surface, ground, and drinking water systems. Combination of hydroxyl radical formation and the pyrolytic environment generated by ultrasonic irradiation (665 kHz) leads to the rapid degradation of MTBE and other gasoline oxygenates in aqueous media. ^ The presence of oxygen promotes the degradation processes by rapid reaction with carbon centered radicals indicating radical processes involving O 2 are significant pathways. A number of the oxidation products were identified. The formation of products (alcohols, ketones, aldehydes, esters, peroxides, etc) could be rationalized by mechanisms which involve hydrogen abstraction by OH radical and/or pyrolysis to form carboncentered radicals which react with oxygen and follow standard oxidation chain processes. ^ The reactions of N-substituted R-triazolinediones (RTAD; R = CH 3 or phenyl) have attracted considerable interest because they exhibit a number of unusual mechanistic characteristics that are analogous to the reactions of singlet oxygen (1O2) and offer an easy way to provide C-N bond(s) formation. The reactions of triazolinedione with olefins have been widely studied and aziridinium imides are generally accepted to be the reactive intermediates. ^ We observed the rapid formation of an unusual intermediate upon mixing tetracyclopropylethylene with 4-methyl-1,2,4-triazoline-3,5-dione in CDCl 3. Detailed characterization by NMR (proton, 13C, 2-D NMRs) indicates the intermediate is 5,5,6,6-tetracyclopropyl-3-methyl-5,6-dihydro-oxazolo[3,2- b][1,2,4]-triazolium-2-olate. Such products are extremely rare and have not been studied. Upon warming the intermediate is converted to 2 + 2 diazetidine (major) and ene product (minor). ^ To further explore the kinetics and dynamics of the reaction activation energies were obtained using Arrhenius plots. Activation energies for the formation of the intermediate from reactants, and 2+2 adduct from the intermediate were determined as 7.48 kcal moll and 19.8 kcal mol−1 with their pre-exponential values of 2.24 × 105 dm 3 mol−1 sec−1 and 2.75 × 108 sec−1, respectively, meaning net slow reactions because of low pre-exponential values caused by steric hindrance. ^

Three-dimensional brain fiber tracking modeling in diffusion tensor imaging

This dissertation establishes the foundation for a new 3-D visual interface integrating Magnetic Resonance Imaging (MRI) to Diffusion Tensor Imaging (DTI). The need for such an interface is critical for understanding brain dynamics, and for providing more accurate diagnosis of key brain dysfunctions in terms of neuronal connectivity. ^ This work involved two research fronts: (1) the development of new image processing and visualization techniques in order to accurately establish relational positioning of neuronal fiber tracts and key landmarks in 3-D brain atlases, and (2) the obligation to address the computational requirements such that the processing time is within the practical bounds of clinical settings. The system was evaluated using data from thirty patients and volunteers with the Brain Institute at Miami Children's Hospital. ^ Innovative visualization mechanisms allow for the first time white matter fiber tracts to be displayed alongside key anatomical structures within accurately registered 3-D semi-transparent images of the brain. ^ The segmentation algorithm is based on the calculation of mathematically-tuned thresholds and region-detection modules. The uniqueness of the algorithm is in its ability to perform fast and accurate segmentation of the ventricles. In contrast to the manual selection of the ventricles, which averaged over 12 minutes, the segmentation algorithm averaged less than 10 seconds in its execution. ^ The registration algorithm established searches and compares MR with DT images of the same subject, where derived correlation measures quantify the resulting accuracy. Overall, the images were 27% more correlated after registration, while an average of 1.5 seconds is all it took to execute the processes of registration, interpolation, and re-slicing of the images all at the same time and in all the given dimensions. ^ This interface was fully embedded into a fiber-tracking software system in order to establish an optimal research environment. This highly integrated 3-D visualization system reached a practical level that makes it ready for clinical deployment. ^

Investigation of Mantle Dynamics from Platinum Group Elements and Rhenium-Osmium Isotope Systematics of Mantle Xenoliths from Oahu, Hawaii

Intraplate volcanism that has created the Hawaiian-Emperor seamount chain is generally thought to be formed by a deep-seated mantle plume. While the idea of a Hawaiian plume has not met with substantial opposition, whether or not the Hawaiian plume shows any geochemical signal of receiving materials from the Earth’s Outer Core and how the plume may or may not be reacting with the overriding lithosphere remain debatable issues. In an effort to understand how the Hawaiian plume works I report on the first in-situ sulfides and bulk rock Platinum Group Element (PGE) concentrations, together with Os isotope ratios on well-characterized garnet pyroxenite xenoliths from the island of Oahu in Hawaii. The sulfides are Fe-Ni Monosulfide Solid Solution and show fractionated PGE patterns. Based on the major elements, Platinum Group Elements and experimental data I interpret the Hawaiian sulfides as an immiscible melt that separated from a melt similar to the Honolulu Volcanics (HV) alkali lavas at a pressure-temperature condition of 1530 ± 100OC and 3.1±0.6 GPa., i.e. near the base or slightly below the Pacific lithosphere. The 187Os/188Os ratios of the bulk rock vary from subchondritic to suprachondritic (0.123-0.164); and the 187Os/188Os ratio strongly correlates with major element, High Field Strength Element (HFSE), Rare Earth Element (REE) and PGE abundances. These correlations strongly suggest that PGE concentrations and Os isotope ratios reflect primary mantle processes. I interpret these correlations as the result of melt-mantle reaction at the base of the lithosphere: I suggest that the parental melt that crystallized the pyroxenites selectively picked up radiogenic Os from the grain boundary sulfides, while percolating through the Pacific lithosphere. Thus the sampled pyroxenites essentially represent crystallized melts from different stages of this melt-mantle reaction process at the base of the lithosphere. I further show that the relatively low Pt/Re ratios of the Hawaiian sulfides and the bulk rock pyroxenites suggest that, upon ageing, such pyroxenites plus their sulfides cannot generate the coupled 186Os- 187Os isotope enrichments observed in Hawaiian lavas. Therefore, recycling of mantle sulfides of pyroxenitic parentage is unlikely to explain the enriched Pt-Re-Os isotope systematics of plume-derived lavas.

The potential role of plant oxygen and sulphide dynamics in die-off events of the tropical seagrass, Thalassia testudinum

1 Oxygen and sulphide dynamics were examined, using microelectrode techniques, in meristems and rhizomes of the seagrass Thalassia testudinum at three different sites in Florida Bay, and in the laboratory, to evaluate the potential role of internal oxygen variability and sulphide invasion in episodes of sudden die-off. The sites differed with respect to shoot density and sediment composition, with an active die-off occurring at only one of the sites. 2 Meristematic oxygen content followed similar diel patterns at all sites with high oxygen content during the day and hyposaturation relative to the water column during the night. Minimum meristematic oxygen content was recorded around sunrise and varied among sites, with values close to zero at the die-off site. 3 Gaseous sulphide was detected within the sediment at all sites but at different concentrations among sites and within the die-off site. Spontaneous invasion of sulphide into Thalassia rhizomes was recorded at low internal oxygen partial pressure during darkness at the die-off site. 4 A laboratory experiment showed that the internal oxygen dynamics depended on light availability, and hence plant photosynthesis, and on the oxygen content of the water column controlling passive oxygen diffusion from water column to leaves and belowground tissues in the dark. 5 Sulphide invasion only occurred at low internal oxygen content, and the rate of invasion was highly dependent on the oxygen supply to roots and rhizomes. Sulphide was slowly depleted from the tissues when high oxygen partial pressures were re-established through leaf photosynthesis. Coexistence of sulphide and oxygen in the tissues and the slow rate of sulphide depletion suggest that sulphide reoxidation is not biologically mediated within the tissues of Thalassia. 6 Our results support the hypothesis that internal oxygen stress, caused by low water column oxygen content or poor plant performance governed by other environmental factors, allows invasion of sulphide and that the internal plant oxygen and sulphide dynamics potentially are key factors in the episodes of sudden die-off in beds of Thalassia testudinum . Root anoxia followed by sulphide invasion may be a more general mechanism determining the growth and survival of other rooted plants in sulphate-rich aquatic environments.

Fish population dynamics in a seasonally varying wetland

Small fishes in seasonally flooded environments such as the Everglades are capable of spreading into newly flooded areas and building up substantial biomass. Passive drift cannot account for the rapidity of observed population expansions. To test the ‘reaction–diffusion’ mechanism for spread of the fish, we estimated their diffusion coefficient and applied a reaction–diffusion model. This mechanism was also too weak to account for the spatial dynamics. Two other hypotheses were tested through modeling. The first—the ‘refuge mechanism’—hypothesizes that small remnant populations of small fishes survive the dry season in small permanent bodies of water (refugia), sites where the water level is otherwise below the surface. The second mechanism, which we call the ‘dynamic ideal free distribution mechanism’ is that consumption by the fish creates a prey density gradient and that fish taxis along this gradient can lead to rapid population expansion in space. We examined the two alternatives and concluded that although refugia may play an important role in recolonization by the fish population during reflooding, only the second, taxis in the direction of the flooding front, seems capable of matching empirical observations. This study has important implications for management of wetlands, as fish biomass is an essential support of higher trophic levels.

Pressure induced structural changes and gas diffusion pathways in monomeric fluorescent proteins

Fluorescent proteins (FPs) are extremely valuable biochemical markers which have found a wide range of applications in cellular and molecular biology research. The monomeric variants of red fluorescent proteins (RFPs), known as mFruits, have been especially valuable for in vivo applications in mammalian cell imaging. Fluorescent proteins consist of a chromophore caged in the beta-barrel protein scaffold. The photophysical properties of an FP is determined by its chromophore structure and its interactions with the protein barrel. Application of hydrostatic pressure on FPs results in the modification of the chromophore environment which allows a systematic study of the role of the protein-chromophore interactions on photophysical properties of FPs. Using Molecular Dynamics (MD) computer simulations, I investigated the pressure induced structural changes in the monomeric variants mCherry, mStrawberry, and Citrine. The results explain the molecular basis for experimentally observed pressure responses among FP variants. It is found that the barrel flexibility, hydrogen bonding interactions and chromophore planarity of the FPs can be correlated to their contrasting photophysical properties at vaious pressures. I also investigated the oxygen diffusion pathways in mOrange and mOrange2 which exhibit marked differences in oxygen sensitivities as well as photostability. Such computational identifications of structural changes and oxygen diffusion pathways are important in guiding mutagenesis efforts to design fluorescent proteins with improved photophysical properties.

Combination of 16S rRNA variable regions provides a detailed analysis of bacterial community dynamics in the lungs of cystic fibrosis patients

Chronic bronchopulmonary bacterial infections remain the most common cause of morbidity and mortality among patients with cystic fibrosis (CF). Recent community sequencing work has now shown that the bacterial community in the CF lung is polymicrobial. Identifying bacteria in the CF lung through sequencing can be costly and is not practical for many laboratories. Molecular techniques such as terminal restriction fragment length polymorphism or amplicon length heterogeneity-polymerase chain reaction (LH-PCR) can provide many laboratories with the ability to study CF bacterial communities without costly sequencing. The aim of this study was to determine if the use of LH-PCR with multiple hypervariable regions of the 16S rRNA gene could be used to identify organisms found in sputum DNA. This work also determined if LH-PCR could be used to observe the dynamics of lung infections over a period of time. Nineteen samples were analysed with the V1 and the V1_V2 region of the 16S rRNA gene. Based on the amplicon size present in the V1_V2 region, Pseudomonas aeruginosa was confirmed to be in all 19 samples obtained from the patients. The V1 region provided a higher power of discrimination between bacterial profiles of patients. Both regions were able to identify trends in the bacterial population over a period of time. LH profiles showed that the CF lung community is dynamic and that changes in the community may in part be driven by the patient's antibiotic treatment. LH-PCR is a tool that is well suited for studying bacterial communities and their dynamics.

Structural flexibility and oxygen diffusion pathways in monomeric fluorescent proteins

Fluorescent proteins are valuable tools as biochemical markers for studying cellular processes. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. The naturally occurring fluorescent proteins with emission peaks in this region of the spectrum occur in dimeric or tetrameric forms. The development of mutant monomeric variants of RFPs has resulted in several novel FPs known as mFruits. Though oxygen is required for maturation of the chromophore, it is known that photobleaching of FPs is oxygen sensitive, and oxygen-free conditions result in improved photostabilities. Therefore, understanding oxygen diffusion pathways in FPs is important for both photostabilites and maturation of the chromophores. We used molecular dynamics calculations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric mFruit variants, and its GFP homolog citrine. We employed implicit ligand sampling and locally enhanced sampling to determine oxygen pathways from the bulk solvent into the mCherry chromophore in the interior of the protein. The pathway contains several oxygen hosting pockets, which were identified by the amino acid residues that form the pocket. We calculated the free-energy of an oxygen molecule at points along the path. We also investigated an RFP variant known to be significantly less photostable than mCherry and find much easier oxygen access in this variant. We showed that oxygen pathways can be blocked or altered, and barrel fluctuations can be reduced by strategic amino acid substitutions. The results provide a better understanding of the mechanism of molecular oxygen access into the fully folded mCherry protein barrel and provide insight into the photobleaching process in these proteins.

Pressure Induced Structural Changes and Gas Diffusion Pathways in Monomeric Fluorescent Proteins

Fluorescent proteins (FPs) are extremely valuable biochemical markers which have found a wide range of applications in cellular and molecular biology research. The monomeric variants of red fluorescent proteins (RFPs), known as mFruits, have been especially valuable for in vivo applications in mammalian cell imaging. Fluorescent proteins consist of a chromophore caged in the beta-barrel protein scaffold. The photophysical properties of an FP is determined by its chromophore structure and its interactions with the protein barrel. Application of hydrostatic pressure on FPs results in the modification of the chromophore environment which allows a systematic study of the role of the protein-chromophore interactions on photophysical properties of FPs. Using Molecular Dynamics (MD) computer simulations, I investigated the pressure induced structural changes in the monomeric variants mCherry, mStrawberry, and Citrine. The results explain the molecular basis for experimentally observed pressure responses among FP variants. It is found that the barrel flexibility, hydrogen bonding interactions and chromophore planarity of the FPs can be correlated to their contrasting photophysical properties at vaious pressures. I also investigated the oxygen diffusion pathways in mOrange and mOrange2 which exhibit marked differences in oxygen sensitivities as well as photostability. Such computational identifications of structural changes and oxygen diffusion pathways are important in guiding mutagenesis efforts to design fluorescent proteins with improved photophysical properties.