The actin cytoskeleton plays a vital role in various biological processes such as cell migration, morphogenesis, and intracellular trafficking. The polymerization of actin filaments at membranes provides the force for generating dynamic actin structures such as protrusions and invaginations that drive these processes. In filopodia, which are finger-like protrusions comprised of bundled actin filaments, actin regulatory proteins are believed to assemble a distal 'tip complex' which stimulates actin nucleation at the membrane. However how these regulators collectively behave in a macromolecular complex still remains poorly understood. To understand the macromolecular nature of these complexes, I investigated the dynamic properties and spatial organization of actin regulatory factors, using an in vitro reconstitution assay for filopodia-like structures (FLS) utilizing artificial lipid bilayers and Xenopus laevis egg extracts. FRAP analysis of seven actin regulatory factors (Toca-1, N-WASP, GTPase-binding domain, Ena, VASP, Diaph3, Fascin) revealed that the FLS tip complex has both dynamic and stable properties, with different proteins displaying distinct dynamics. Further analyses on the membrane-binding protein Toca-1 showed that its dynamic turnover is controlled by interactions with actin and exchange of molecules with solution. Single-molecule localization microscopy resolved the nanoscale organization of Toca-1, showing its arrangement into flat plaque-like and narrowly elevated tubular substructures. Plaque-like structures showed similarities to phase-transition patterns, while tubule-like structures closely resembled those previously found to decorate membrane tubules in vitro, which are thought to be involved in endocytic membrane remodeling. Endocytic accessory proteins such as SNX9 and Dynamin2 were also found to localize to FLS tips. This work provides new insights into the dynamics and organization of protein ensembles at actin nucleation sites, and proposes a novel link between endocytosis and filopodia formation, which is relevant to understanding how cells decide when and where to assemble actin at the membrane.

Synapses are the communication junctions of the nervous system and contain protein machinery necessary for cognitive functions such as learning and memory. Postsynaptic density protein-95 (PSD-95) is a key scaffolding molecule at the PSD of synapses, yet its sub-synaptic organisation in the mammalian brain remains poorly understood. This thesis presents the use of genetically labelled PSD-95 with super-resolution imaging to resolve its nano-architecture in the mouse brain. To visualize PSD-95, two knock-in mouse lines were generated where the fluorescent proteins eGFP or mEos2 was fused to the carboxyl terminus of the endogenous PSD- 95 protein (PSD-95-eGFP or PSD-95-mEos2). Methods were developed by which fixed tissue sections of PSD-95-eGFP mice were examined using gated-stimulated emission depletion (g-STED) microscopy and PSD-95-mEos2 sections were examined with photoactivatable localisation microscopy (PALM) and quantitative image analysis was developed for both methods. From these platforms it was demonstrated that PSD-95 has a two tiered organisation: it is assembled into nanoclusters (NCs) approximately 140 nm diameter, which form part of the greater envelope of the PSD within synapses. Synapse subtypes were observed as characterised by the number of NCs per PSD. Using double colour g- STED microscopy. It was then asked whether PSD-95 nano-architecture remained the same across different sub-regions of the brain. A survey of PSD-95 was performed from seven different sub-regions of the hippocampus, quantifying ~110,000 NCs within ~70,000 PSDs from across the two super-resolution platforms. It was found that synapses displayed structural diversity both within and between different brain subregions as a function of the number of NCs per PSD. PSD-95 NCs were structurally conserved across the hippocampus, but showed molecular diversity in the abundance of PSD-95 molecules within. The findings of this thesis are: 1) genetic labelling of endogenous proteins combined with super-resolution microscopy is a powerful tool to study synaptic protein organisation in tissue. 2) Synaptic structural diversity in the brain is underlined by the number of PSD-95 NC units per synapse 3) PSD-95 NCs are structurally conserved but molecularly diverse synaptic units of synapses throughout the brain. These findings suggest that cognitive processing at the synapse is based upon a conserved, fundamental, molecular architecture.

This study developed new concepts and interpretative methods for mapping reservoir heterogeneity and connectivity of a fault controlled Wilcox clastic reservoir in Texas, USA. The application of high-resolution seismic enhancement in this study allows for better delineation of subsurface geologic features, detailed mapping of reservoir heterogeneities and more accurate identification of depositional, structural, and stratigraphic characteristics that control reservoir connectivity and fluid flow. Seismic enhancement in this study pertains to amplitude preserving neural network implementation of the Volterra integral equation of the first kind from a plane-wave solution of poro-viscoelasticity (Sun, et al., 2003). This enhancement amounts to an advanced spiked deconvolution of post-stack seismic data that broadened the dominant seismic frequency from 16Hz for the conventional seismic to 65Hz for the enhanced seismic. Bed resolution is improved from 175ft to 45ft and fault offset resolution is improved from 80ft to 20ft. High-resolution seismic interpretation was validated through synthetic seismograms, stratigraphic surface comparisons, and most importantly using a comprehensive model-based knowledge of regional tectonics and depositional environments. Stratigraphic features that were not resolvable in conventional seismic data can now be interpreted using the enhanced seismic data. An Upper Wilcox reservoir was identified as a transgressive sheet sand overlaying a progradational deltaic seismic facies. An Upper Middle Wilcox reservoir was identified as a probable lobate gravity flow, and a Middle Wilcox reservoir was identified as a transgressive sheet sand with over and underlying progradational deltaic seismic facies. Geobody extraction from seismic inversion volumes delineates reservoir compartments and flow units. Reservoir connectivity analysis performed on the Middle Wilcox reservoir determined the probable drainage area for a producing well by comparing estimates of compartmentalized hydrocarbon volumes with production information. The methodology developed could help extract connected geobodies defined by sand, porosity, permeability, and hydrocarbon indicators, to map in detail the internal structure of produced reservoir and to locate new development prospects. Enhanced seismic may thus enable us to find bypassed hydrocarbons and to provide better methods for improving recovery in the studied and other mature fields.

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: Gonzalo Arce, Dept. of Electrical & Computer Engineering. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Zugl.: Berlin, Humboldt-Universiẗat, Diss., 2009.

Vertical cable seismic methods are becoming more relevant as we require high quality and high resolution seismic data in both land and marine environments. Our goal in this thesis is to demonstrate the impacts of vertical cable surveying in these areas. Vertical cable methods have been applied to the marine environment with encouraging results. Data quality is similar to that of traditional towed-streamer data, without the long, cumbersome towed-streamers which are difficult to maneuver in congested areas. The current marine vertical cable processing schemes tend to use primaries and receiver ghosts of primaries for imaging. Therefore, we demonstrate the ability of the current multiple attenuation algorithms developed by Ikelle (2001) to preserve either primaries or the receiver ghosts of primaries. As we focus on land acquisition, we discover that vertical cable surveying can overcome many of the traditional problems of land seismics. In fact, our investigations lead us to believe that problems such as ground roll, guided waves and statics can be avoided almost entirely using vertical cable acquisition methods. Furthermore, land vertical surveying is naturally suited for multi-component acquisition and time-lapse surveying. To fully analyze the applicability of vertical cable surveys in marine and land environments, we also investigate the problem of cable spacing and sampling within each cable. We compare the resolution of vertical cable data and horizontal data by calculating the maximum angular coverage of each acquisition geometry and measuring the occurrence of each angle within this coverage, such that more occurrences means better resolution. From our investigations, we find that by using vertical cables of no more than 500 m in length at 500 m intervals, we can acquire higher resolution seismic data relative to horizontal surface methods for an image point, horizontal reflector or a dipping reflector. The key tool used in these investigations is fully elastic finite-difference modeling. We chose this technique based on its ability to properly and accurately model the full wavefield through complex models, all the while preserving amplitudes and the phase of reflected, diffracted and converted wavefields.

No description available.

No description available.

Enantiopure epoxides are versatile compounds in the production of single enantiomer drugs, and are of high value as building blocks and intermediates in the preparation of more complex single enantiomer pharmaceuticals and agrochemicals. Epoxide hydrolases, ubiquitous enzymes in nature, can be versatile tools in the biocatalytic production of these single enantiomer epoxides due to their capability of selectively hydrolysing one enantiomer of a wide range of these compounds, and thus rendering an enantiopure epoxide and diol. The value of epoxide hydrolases for the kinetic resolution of epoxide compounds are dependant on factors such as availability, ease of production, long term stability, activity and the displayed enantioselectivity. The first objective of this study was to investigate and optimise the growth media and time for the production of two red yeasts, Rhodotorula glutinis and Rhodospondium toruloides, and their epoxide hydrolysing enzymes. Maximum and minimum epoxide hydrolase (EH) activity for R. glutinis was respectively observed with the YMvit (0,26 mM.min"1) and malt (0,17 mM.min"1) media, while peak biomass production was observed from the YM medium (64,9 mg.mL"1). For R. toruloides, the highest biomass was produced in the YM (130,8 mg.mL"1) medium, with similar epoxide hydrolase activities (average c = 0,75 ± 0,01) displayed for the YM, YMvit and malt grown biocatalysts. With varying the YM medium glucose concentration (0,5 - 2,0 %) the most biomass was produced for R. glutinis with the addition of 1,5 % glucose (60,0 + 0,9 mg.mL"1), with a slight drop in the biomass observed with the addition of 2,0% glucose (56,0 + 1,7 mg.mL"1). No significant differences in epoxide hydrolase activity was observed for the lower glucose additive concentrations (0,5 - 1,5 %), while 2,0 % (m/v) rendered a biocatalyst with almost 20 % higher activity (0,29 mM.min"1). For R. toruloides an increase in the glucose concentration lead to a significantly higher biomass production while the time needed to attain the stationary phase increased progressively from 40 to 96 hours. Almost equal activity was observed for the top three glucose concentrations (average c = 0,82 ± 0,01) at 36 hours growth time, but in all cases a decrease in the EH activity was observed during the stationary phase, with the most pronounced decrease for the 2,0 % (m/v) glucose concentration, that showed a drop in conversion of almost 62 % at 144 hours growth time. The second objective was to synthesise meta and para nitro-, methyl- and methoxystyrene oxides and the successive production of their single enantiopure epoxides through R. glutinis EH mediated kinetic resolution, and the determination of the absolute configuration of the pure residual enantiomers through VCD analysis. R. glutinis selectively hydrolysed the whole range of styrene oxide derivatives, with the highest activity displayed towards the meta substituted derivatives in the order of methyl > methoxy > nitro. m-Methylstyrene oxide reached a % e.e. of >98 within 60 minutes, with an exceptionally high yield of 42,5 %. The absolute configuration of the residual epoxide enantiomers of /n-nitro, m-methyl and m-methoxystyrene oxides were determined to be of the (S)-configuration, indicating that R. glutinis EH preferentially hydrolyses the (R)-epoxides. Thirdly, we attempted to increase the R. glutinis EH activity through the addition of hydroxypropyl-p-cyclodextrin (HPB) and to correlate the rate of chemical and R. glutinis EH mediated enzymatic hydrolysis, and the enzyme's enantioselectivity to the electronic properties of their substituents and the spatial arrangement of the substrates in relation to the EH catalytic triad of the EH active site. An increase in the HPB concentration (0 - 20 % w/v) lead to a substantial increase in both the solubility as well as enzyme activity for p-N02 (para-nitrostyrene oxide) with a significant increase in the solubility of between 2,89 and 6,28 times for the substrate range with the addition of 5 % HPB in comparison to the buffer solution. The acid induced chemical and R. glutinis EH mediated enzymatic reaction rate was correlated to both the Hammett constant as well as the Mulliken charge distributions. The Mulliken charge distribution over the protonated epoxides was correlated to the acid induced chemical hydrolysis rates, while the Mulliken charge distribution over the neutral epoxides could be correlated to the enzymatic reaction rates. An increase in the electron-donating properties of the styrene oxide substituent groups was correlated to an increase in both the chemical as well as the R. glutinis EH mediated hydrolysis reaction rates of the styrene oxide derivatives. Docking of the possible conformers of the (R)- and (S)-enantiomers of these meta and para substituted styrene oxides into the EH binding site of the closely related Aspergillus niger displayed a closer and more preferential fit of the (R)-epoxides which is the faster reacting enantiomerfor both A. niger and R. glutinis EHs. The proven relationship between R. glutinis EH activity and selectivity and the electronic properties of substituent groups, as well as the relationship between spatial arrangement of the epoxide hydrolase binding site and the enantioselectivity of the enzyme, could open up the possibility to correctly predict both the enantioselectivity as well as the activity of R. glutinis EH, and possibly other red yeasts, towards more complex epoxide substrates without the need of time consuming screenings. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.