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Methods for Epitope Characterization of Adeno-Associated Virus Type-2 Through Antibody Neutralization Escape MutantsUnknown Date (has links)
The Adeno-Associated Virus has moved to the forefront as a vector for human gene therapy. Vectors have been constructed with AAV to repair many genetic deficiencies such as cystic fibrosis and hemophilia and have demonstrated remarkable success. For the application of a gene therapy vector, the particle must be afforded maximal capability to deliver the therapeutic gene without immunological elimination. Unfortunately, AAV is endemic in the human population and as a result, a large proportion of individuals harbor immunity to AAV leading to rapid elimination upon subsequent exposure. One of the major obstacles for the development of AAV as a vector is the absence of robust epitope data. If such data was in hand, gene therapy vectors could be constructed with modifications to these highly immunogenic sites on the viral particle. Given the non-cytopathic nature of AAV, the virus does not lend itself well to traditional plaque assays. Without such assays in hand, the ability to isolate and recover viable viral clones is severely limited. We set out to map the viral epitope to monoclonal antibody A20 by generating monoclonal antibody neutralization escape mutants in cell culture. The lack of plaque assays had prompted us to focus on the development of methods that would facilitate these selection experiments. Having successfully developed a robust and reliable method for plaquing AAV we were capable of applying it to the preliminary selection experiments. The initial application of these methods did not yield the desired mutant. Presented here are the methods developed, their preliminary applications, analysis of the results and future prospective for escape mutant production. / A Dissertation Submitted to the Institute of Molecular Biophysics in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Summer Semester, 2008. / April 17, 2008. / Mutants, Parvovirus, Escape, Epitope, Antibody, Aav / Includes bibliographical references. / Hengli Tang, Professor Co-Directing Dissertation; Michael Chapman, Professor Co-Directing Dissertation; Hong Li, Committee Member; Kenneth Roux, Committee Member; Robert Reeves, Outside Committee Member.
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Understanding Structural Mechanisms of Endolytic RNA Cleavage EnzymesUnknown Date (has links)
The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (ab)2 family of splicing endonucleases that require two different subunits for splicing activity. N. Equitans splicing endonuclease consists of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here we report the crystal structure of the functional NEQ enzyme at 2.1 Angstroms resolution containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 Angstroms resolution. The functional enzyme resembles previously known a2 and a4 endonucleases but forms a heterotetramer; a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homo-dimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity. / A Dissertation Submitted to the Institute of Molecular Biophysics in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Summer Semester, 2009. / June 26, 2009. / RNA Cleavage, tRNA Splicing Endonuclease, RNA Processing / Includes bibliographical references. / Hong Li, Professor Directing Dissertation; Penny J. Gilmer, Outside Committee Member; Brian Miller, Committee Member; W. Ross Ellington, Committee Member; Branko Stefanovic, Committee Member.
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Mutations in the Human Cardiac Ca2+-Regulatory Proteins Affect the Function of the Thin Filament: Lessons for Inherited CardiomyopathiesUnknown Date (has links)
Familial hypertrophic cardiomyopathy (FHC) is the leading cause of sudden cardiac death in both preadolescents and adolescents. The hallmark of the disorder is myocardial hypertrophy of the left ventricle, which results in an obstruction of blood flow through the left ventricular outflow tract. FHC has been associated with well over 100 mutations, primarily in proteins of the contractile apparatus of the heart. The molecular mechanisms involved in the pathogenesis of FHC are not well understood. For this study, in vitro motility assays (IVM) were conducted to assess the relationship between structure and function of cardiac thin filaments and to elucidate the molecular basis for sequelae of FHC caused by point mutations in the human cardiac regulatory proteins. Our research aims to contribute to the study of FHC by the accomplishment of the following innovations: 1) Development of a simple strategy to express recombinant human cardiac regulatory proteins in E. coli for molecular assays of human cardiac contractility. 2) Fabrication of a thermo-electric controller that allows rapid and reversible characterization over a broad temperature range of the effects of FHC mutations in troponin and tropomyosin using IVM assays. My research yielded the following novel physiological findings 1) Ca2+-sensitivity of human cardiac thin filament sliding is affected by some of the FHC mutations in the cardiac regulatory proteins, but not by changes in myosin isoform, indicating that Ca2+-sensitivity does not depend upon the kinetics of cross-bridge cycling. This finding implies that the hypertrophic response is communicated through different pathways depending whether the mutation is in troponin, tropomyosin, or myosin 2) Troponin and tropomyosin affect the temperature sensitivity as well as the maximum speed of unloaded filament sliding by reducing the myosin cross-bridge duty ratio. Our results suggest that the duty ratio also might be affected by clinically relevant mutations in troponin and tropomyosin. Finally, functional characterization of troponin, bearing FHC mutations in subunit I, subunit T, or both subunits, predicts structural relationships of the thin filament. / A Dissertation submitted to the Program in Molecular Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2006. / October 25, 2006. / Troponin, Calciun Sensitivity, Temperature, Familial Hypertrophic Cardiomyopathy, Thermal Stability, Cross-Bridge Cycle, Tropomyosin / Includes bibliographical references. / P. Bryant Chase, Professor Directing Dissertation; Peng Xiong, Outside Committee Member; Timothy S. Moerland, Committee Member; Peter G. Fajer, Committee Member; Thomas C. S. Keller, III, Committee Member.
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Predictive Sampling of Protein Conformational ChangesUnknown Date (has links)
In aqueous solution, solute conformational transitions are governed by intimate interplays of the fluctuations of
solute–solute, solute–water, and water–water interactions. To more effectively sample conformational transitions in aqueous solution, we
devised a predictive sampling method: the generalized orthogonal space tempering (gOST) algorithm. Specifically, in the Hamiltonian
perturbation part, a solvent-accessible-surface-area-dependent term is introduced to implicitly perturb near-solute water–water
fluctuations; more importantly in the orthogonal space response part, the generalized force order parameter is generalized as a
two-dimension order parameter set, in which essential solute–solvent and solute–solute components are separately treated. The gOST
algorithm is evaluated through a molecular dynamics simulation study on the explicitly solvated deca-alanine peptide. On the basis of a
fully automated sampling protocol, the gOST simulation enabled repetitive folding and unfolding of the solvated peptide within a single
continuous trajectory and allowed for detailed constructions of deca-alanine folding/unfolding free energy surfaces. In addition, by
employing the gOST method we enabled efficient molecular dynamics simulation of repetitive breaking and reforming of salt bridge
structures within a minimalist salt-bridge model, the Asp-Arg dipeptide and thereby were able to map its detailed free energy landscape in
aqueous solution. Our results reveal the critical role of local solvent structures in modulating salt-bridge partner interactions and
imply the importance of water fluctuations on conformational dynamics that involves solvent accessible salt bridge formations. Based on
the gOST method, we have developed a solvation force orthogonal space tempering (SFOST) algorithm, in which several major changes were
made from the original gOST method. Due to compensating fluctuations of essential solute-solvent and solute-solute interactions, only
essential solute-solvent interactions are perturbed in the SFOST algorithm. Importantly, the above treatment enabled us to incorporate a
high order orthogonal space sampling strategy. Specifically, to enlarge fluctuations of essential solute-solvent interactions, a third
order treatment was introduced to accelerate the coupled responses caused by fluctuations of essential solute-solvent interactions, which
come from synchronous fluctuations of essential solute-solute interactions and solvent-solvent interactions. The SFOST algorithm was
evaluated through a molecular dynamics simulation study on the explicitly solvated deca-alanine peptide. More importantly, the SFOST
simulation explicitly revealed the compensating fluctuations between the essential solute-solvent interactions and the solvent-solvent
interactions, suggesting that solvent cooperative fluctuations intimately interplay with deca-alanine conformational transitions. In
addition, the SFOST algorithm was also employed to study ion conduction through gramicidin A (gA). By enlarging fluctuations of the
ion-environment interactions, the SFOST simulation enabled several round trips of ion permeation through the channel and allowed detailed
construction of free energy surfaces along the conduction. The calculated observables agree very well with experiment. We also found that
fluctuations of channel orientations play an essential role in ion conduction. Furthermore, by employing the SFOST algorithm we enabled
predictive sampling of the conformational ensemble of the p53 transcriptional activation domain 1 (TAD1). Strikingly, a helical structure
resembling the MDM2-bound form was found in our SFOST simulation, indicating the pre-existing nature of the structure. Detailed studies of
free energy surfaces revealed that the most popular state is not a fully disordered form but a partially helical state. Upon binding to
MDM2, the hydrophobic interactions at the interface shift the conformational equilibrium to favor the total helical structure. In addition
to the predictive sampling methods, we developed a Gaussian kernel Monte Carlo (GKMC) method to smoothly approximate multidimensional free
energy surfaces of biomolecular processes. By taking a discrete probability distribution of sampled collective variables as an input, a
biased Monte Carlo simulation is performed to efficiently resample the distribution in the collective variable space, leading to a smooth
analytical estimate of the free energy surface. The GKMC method is evaluated by resampling data of a generalized orthogonal space
tempering simulation of deca-alanine peptide, aiming to construct smooth one-dimensional and two-dimensional free energy surfaces along
certain collective variables. As demonstrated in these model studies, the GKMC method can robustly construct smooth multidimensional free
energy surfaces with super resolutions, which preserve probability distributions of target molecular processes. Constructing smooth free
energy surfaces plays a vital role in interpreting simulation data to understand molecular processes of interest. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of
the Doctor of Philosophy. / Fall Semester 2016. / November 22, 2016. / Orthogonal Space Tempering, Predictive Sampling, Protein Dynamics, Solvation Force / Includes bibliographical references. / Wei Yang, Professor Directing Dissertation; Kenneth A. Taylor, University Representative; Oliver
Steinbock, Committee Member; Hong Li, Committee Member; Timothy A. Cross, Committee Member.
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Self-Organization of Complex Polycrystalline Silica-Carbonate BiomorphsUnknown Date (has links)
A key challenge for modern chemistry is the production of mesoscopic complexity and hierarchical order to ultimately bridge the
gap between the molecular world and functional microdevices. As proof of concept, nature shows unambiguously that this approach can be
rewarding. In particular, natural biominerals such as nacre, bone, and tooth enamel, consist of ordinary nanoscale components, yet
assemble complex polycrystalline materials that are clearly superior to their synthetic counterparts. In this context, an exciting model
system for biomimetic crystallization is the assembly of biomorphs by the co-precipitation of silica and metal carbonates. Despite being
formed by purely inorganic processes, these structures show "life-like" morphologies such as twisted helices and cardioid leaves. At the
nanoscale, silica-carbonate biomorphs consist of crystalline nanorods that assemble hierarchical architectures reminiscent of natural
biominerals. In this research work, we improve the level of control over the growth process and quantitatively characterize the biomorph
structures beyond simple qualitative observations. We report the synthesis of silica-carbonate biomorphs in single-phase, gradient-free
solutions that differ markedly from the typical solution-gas or gel-solution setups. Our experimental approach reveals novel biomorph
structural motifs, reduces transients in the chemical conditions, and expands the upper pH limit for biomorph formation to over 12 where
silica is essentially soluble. Moreover, the single-phase approach significantly increases the duration of growth to assemble biomorph
networks that extend to several millimeters. These unusually long biomorphs allow the first quantitative measurements of mesoscopic
parameters such as the helix wavelength, period, width, and linear as well as tangential growth velocities. We find that the latter
quantities are system-specific and tightly conserved during many hours of growth. We also systematically characterize the biomorph sheets
and report the existence of an additional level of self-organization that creates oscillatory height variations along the sheet surface.
These topographic features take the form of either concentric rings or disordered, patchy patterns with a wavelength of approximately 6.5
μm that shows no pronounced dependence on the reactant concentrations. These undulations are accompanied by a systematic out-of-plane
displacement of the nanorods. Our results are discussed in the context of an earlier hypothesis that predicts pH oscillations near the
crystallization front. We further investigate the effect of inorganic dopants that influence the morphological, compositional, and
crystallographic properties of biomorphs. In the case of Pb²⁺ and Ag⁺ ions, biomorph growth is disrupted by the formation of competing
precipitates. Similarly, the addition of Ca²⁺, Mg²⁺, and Zn²⁺ induces the rapid crystallization of witherite or amorphous silica-carbonate
aggregates at enhanced growth rates. By comparison, the addition of strontium ions results in the assembly of classic biomorphs such as
cardioid sheets and helices. Another aspect of the project lies at the overlap between geochemistry, paleontology, and astrobiology. To
date, these fascinating biomorph microstructures have only been synthesized using model laboratory solutions. We report that mineral
self-assembly can be also obtained from natural alkaline silica-rich water deriving from serpentinization. Specifically, we obtain water
samples from the Ney springs in California and demonstrate the self-assembly of nanocrystalline biomorphs of barium carbonate and silica,
as well as the formation mesocrystals and crystal aggregates of calcium carbonate with complex biomimetic textures. Our results suggest
that silica-induced mineral self-assembly could have been a common phenomenon in alkaline environments of the early Earth and Earth-like
planets. Moreover, the structural complexity obtained from these simple crystallization reactions in the natural Ney water further blurs
the boundaries between geochemical and biological microscale morphologies that not too long ago were perceived as sharp and
well-defined. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of
the Doctor of Philosophy. / Fall Semester 2016. / November 17, 2016. / Biomimetics, Biomorph, Crystallization, Hierarchical, Self-organization, Silica / Includes bibliographical references. / Oliver Steinbock, Professor Directing Dissertation; Richard Bertram, University Representative;
Ken L. Knappenberger, Committee Member; Hedi Mattoussi, Committee Member.
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Understanding the "Microwave" in Microwave ChemistryUnknown Date (has links)
Microwave chemistry has long been a subject of interest in both the organic and inorganic synthesis communities. Microwave heating has
the potential to become a powerful force for green synthesis in industry as it uses much less power to accomplish the same goals, but a lack
of understanding in how to translate traditional convective reactions into microwave reactions is hampering this progress. In this manuscript
an overview of microwave physics and mathematics is given first. Then the role of microwave source and choice of microwave reaction vessel,
along with precursor and solvent choice in the design of a microwave chemical reaction is explored. Next, synthesis of nickel and gold
nanoparticles—chosen because of their ubiquitous nature in the literature—in a microwave is explored, and the kinetics examined.
Additionally, the role of size dependent properties of the nanoparticles, as well as the role of the oxide layer on the nanoparticle, are
explored in relationship to how the reaction heats in a standard laboratory microwave. Lastly, the role of power and frequency of the
microwave radiation in the synthesis of nickel nanoparticles is examined, and relationships between the kinetics of the synthesis and the
applied power and frequency of the microwave is extracted. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the
requirements for the degree of Doctor of Philosophy. / Fall Semester 2017. / November 15, 2017. / microwave, nanoparticle, physical chemistry / Includes bibliographical references. / Geoffrey F. Strouse, Professor Directing Dissertation; Stephen Hill, University Representative; Albert
E. Stiegman, Committee Member; Michael Shatruk, Committee Member.
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Topics in theoretical chemistry : a CNDOBW calculation on a Friedel-Crafts intermediate, and Riccati equation solutions in mathematical physicsPulfer, James Douglas. January 1975 (has links)
No description available.
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Equilibria existing in the three-component system, calcium oxide - sulphur dioxide - water, over the temperature range 250-1300C.Beazley, Warren Benson. January 1937 (has links)
No description available.
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Free radicals in organic decomposition reactions. --.Alexander, Wendal Arthur January 1938 (has links)
No description available.
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Reducing the computational cost of Ab Initio methodsMintz, Benjamin. Wilson, Angela K., January 2008 (has links)
Thesis (Ph. D.)--University of North Texas, August, 2008. / Title from title page display. Includes bibliographical references.
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