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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
11

Structural and functional characterization of a hybrid benzoate degradation pathway

Bains, Jasleen 25 October 2011 (has links)
Aromatic compounds comprise approximately one quarter of the Earth's biomass and thus play a critical role in the biogeochemical carbon cycle. These compounds are degraded almost exclusively by specialized microbial enzymes that are part of complex metabolic pathways. Detailed characterization of these enzymes is both a gateway to understanding a biological process fundamental to nature and a platform for bioengineering applications in bioremediation. Recently, a novel pathway was shown to metabolize two key aromatic intermediates: Benzoate and Benzoyl-Coenzyme A. Designated as the box pathway (benzoate oxidation), this metabolic conduit incorporates in succession; CoA-ligation, oxygenation, ring cleavage and neutralization of the aldehydic ring cleavage product, catalyzed by a Benzoate Coenzyme A Ligase (BCL), BoxAB, BoxC and an Aldehyde Dehydrogenase (ALDH) respectively. Collectively, these steps define the initial and unique segment of the box pathway. The objective of the research described here was to establish a molecular blueprint of the substrate binding pocket of the initial BCL and elucidate mechanistic details for both BoxC and ALDH enzymes from Burkholderia xenovorans LB400 through in-depth structural and functional characterizations. An intriguing feature of the box pathway in LB400 is a paralogous genetic organization. Functional studies on the BCL paralogs (BCLM and BCLC) show that BCLM is more active towards benzoate than BCLC. Structural analysis of the 1.84 Å resolution co- crystal structure of BCLM with benzoate reveals that the substrate binding pocket is closely contoured to bind benzoate, leaving little room to accommodate substituted benzoates, especially in the para position owing to a histidine (H339) residue that renders the pocket particularly shallow. Overall, while corroborative, the structural data provides a molecular rationale to our functional data where both the BCLs were seen to be highly specific for benzoate. Structural analysis of the 1.5 Å resolution crystal structure of the novel ring cleaving BoxC reveals an intriguing structural demarcation consistent with the primary sequence based divergence of BoxC within the crotonase superfamily. A highly divergent region in the C-terminus likely serves as a structural scaffold for the conserved N-terminus that harbors the active site. Isothermal titration calorimetry and molecular docking simulations contribute to a detailed view of the active site resulting in a compelling mechanistic model involving a pair of conserved glutamates (E146 and E168) and a novel cysteine (C111). Lastly, the 1.6 Å resolution co-crystal structure of ALDHC with NADPH and PEG allows identification of residues that are involved in rendering ALDHC selective for NADP+ and linear, medium to long chain aldehydes, as observed in our initial kinetic analyses. Functional and structural characterization of strategic ALDHC mutants enables us to propose a detailed reaction mechanism which involves the essential roles for C296 as the nucleophile, E257 as the general base and a proton relay network anchored by E496 and supported by E167 and K168. Overall, this research provides a molecular blueprint for three key box enzymes, thereby enhancing our understanding of central aromatic metabolism. / Graduate
12

Recent Advances in Developing Molecular Biotechnology Tools for Metabolic Engineering and Recombinant Protein Purification

Stimple, Samuel Douglas 25 May 2018 (has links)
No description available.
13

The art of maintaining a successful marriage in the Seventh-Day Adventist Church

Tembo, Lysant Molly Langwell 08 1900 (has links)
Making a successful Christian marriage is a major challenge that faces the Seventh Day Adventist church (SDA Church) of Malawi. The colonial government of Nyasaland (Malawi) created weak marriages, promoting high divorce rates by its own practices during its era, which have remained to this day. The failure of secular marriages endangers the success of SDA Christian marriages. Little has been done by the Church to educate its members concerning successful Christian marriage. This study focuses on educating the church to deal with the problems that cause marriage failure in the SDA Church. The Malawi government is another tool that the church could use to address marriage failure. I have used the Bible, and scientific research methods to suggest workable solutions for Christian marriage. / Practical Theology / M.Th. (Practical Theology)
14

Gene therapy tools: oligonucleotides and peptides

Eriksson, Jonas January 2016 (has links)
Genetic mutations can cause a wide range of diseases, e.g. cancer. Gene therapy has the potential to alleviate or even cure these diseases. One of the many gene therapies developed so far is RNA-cleaving deoxyribozymes, short DNA oligonucleotides that specifically bind to and cleave RNA. Since the development of these synthetic catalytic oligonucleotides, the main way of determining their cleavage kinetics has been through the use of a laborious and error prone gel assay to quantify substrate and product at different time-points. We have developed two new methods for this purpose. The first one includes a fluorescent intercalating dye, PicoGreen, which has an increased fluorescence upon binding double-stranded oligonucleotides; during the course of the reaction the fluorescence intensity will decrease as the RNA is cleaved and dissociates from the deoxyribozyme. A second method was developed based on the common denominator of all nucleases, each cleavage event exposes a single phosphate of the oligonucleotide phosphate backbone; the exposed phosphate can simultaneously be released by a phosphatase and directly quantified by a fluorescent phosphate sensor. This method allows for multiple turnover kinetics of diverse types of nucleases, including deoxyribozymes and protein nucleases. The main challenge of gene therapy is often the delivery into the cell. To bypass cellular defenses researchers have used a vast number of methods; one of these are cell-penetrating peptides which can be either covalently coupled to or non-covalently complexed with a cargo to deliver it into a cell. To further evolve cell-penetrating peptides and understand how they work we developed an assay to be able to quickly screen different conditions in a high-throughput manner. A luciferase up- and downregulation experiment was used together with a reduction of the experimental time by 1 day, upscaling from 24- to 96-well plates and the cost was reduced by 95% compared to commercially available assays. In the last paper we evaluated if cell-penetrating peptides could be used to improve the uptake of an LNA oligonucleotide mimic of GRN163L, a telomerase-inhibiting oligonucleotide. The combination of cell-penetrating peptides and our mimic oligonucleotide lead to an IC50 more than 20 times lower than that of GRN163L.
15

The art of maintaining a successful marriage in the Seventh-Day Adventist Church

Tembo, Lysant Molly Langwell 08 1900 (has links)
Making a successful Christian marriage is a major challenge that faces the Seventh Day Adventist church (SDA Church) of Malawi. The colonial government of Nyasaland (Malawi) created weak marriages, promoting high divorce rates by its own practices during its era, which have remained to this day. The failure of secular marriages endangers the success of SDA Christian marriages. Little has been done by the Church to educate its members concerning successful Christian marriage. This study focuses on educating the church to deal with the problems that cause marriage failure in the SDA Church. The Malawi government is another tool that the church could use to address marriage failure. I have used the Bible, and scientific research methods to suggest workable solutions for Christian marriage. / Philosophy, Practical and Systematic Theology / M.Th. (Practical Theology)
16

A Mechanistic Investigation of the Photochemical and Thermal Activation of 2,2- and 2,3-Diaryl- and 2,2,3-Triaryl-2,3-dihydro-phenanthro[9,10-b]-1,4-dioxins, a New Class of 1,4-Dioxene Based DNA Cleaving Agents

CARLE, AXEL BJORN 21 June 2002 (has links)
No description available.
17

Studies of conformational changes and dynamics accompanying substrate recognition, allostery and catalysis in bacteriophage lambda integrase

Subramaniam, Srisunder 19 April 2005 (has links)
No description available.
18

Brownian Motion, Cleaving, Healing and Interdiffusioninduced Nanopores and Defect Clusters in Ni1-xO-Co1-xO-ZrO2 System

Li, Ming-yen 12 July 2005 (has links)
Abstract This research is designed to investigate the occurrence of interdiffusion-induced mesopores, Brownian motion, cleaving and healing and defect clusters in three binary composites, i.e. Ni1-xO/Co1-xO, Ni1-xO/ZrO2 and Co1-xO/ZrO2 of the Ni1-xO-Co1-xO-ZrO2 system. Firstly, the (NimCo1-m)1-£_O/Ni-doped Co3-dO4 composites prepared by reactive sintering Ni1-xO and Co1-xO powders (1:2 molar ratio, denoted as N1C2) at 1000oC with or without further annealing at 720oC in air were studied by X-ray diffraction and electron microscopy to clarify the formation mechanism of mesoporous spinel precipitates. Submicron-sized inter- and intragranular pores, due to incomplete sintering and grain boundary detachment, prevails in (Ni0.33Co0.67)1-£_O protoxide with rock salt structure; whereas nanosize pores due to Kirkendall effect were restricted to the spinel precipitates having Ni component progressively expelled upon annealing. A rapid net vacancy flux and a tensile misfit stress perpendicular to the protoxide/spinel interface caused the formation of elongated and aligned {100}-faceted mesopores in the spinel precipitates with a relatively low equilibrium vacancy concentration. Aligned mesopores in diffusion zone of nonstoichiometric metal oxides have potential applications on thermal barrier bond coating and mass-transport limited heterogeneous catalysis. Also, this thesis deals with the reorientation and shape change of low-crystal-symmetry (non-cubic) ZrO2 within the high-crystal-symmetry grains of Co1-xO/Ni1-xO cubic rock salt-type structure. ZrO2/Co1-xO composites 1:99 and ZrO2/Ni1-xO composites 1:9 in molar ratio were sintered and then annealed at 1650oC for 24 and 100 h in air to induce reorientation of the embedded particles. Transmission electron microscopic observations in both systems indicated that the submicron tetragonal/monoclinic (t/m) ZrO2 particles fell into three topotaxial relationships with respect to the host Co1-xO/Ni1-xO grain: (1) parallel topotaxy, (2) ¡§eutectic¡¨ topotaxy i.e. [100]Z//[111]C,N, [010]Z//[0 1]C,N and (3) ¡§occasional¡¨ topotaxy [100]Z//[111]C,N, [01 ]Z//[0 1]C,N. The parallel topotaxy has a beneficial low energy for the family of {100}Z/C,N and {111}Z/C,N interfaces. The change from the occasional topotaxy to an energetically more favorable eutectic topotaxy was likely achieved by a rotation of the ZrO2 particles over a specific (100)Z/(111)C,N interface. Brownian-type rotation is probable for the embedded t-ZrO2 particles in terms of anchorage release at the interphase interface with the Co1-xO/Ni1-xO host. Detachment or bypassing of rock salt type grain boundaries could also cause orientation as well as shape changes of intergranular ZrO2 particles. Zirconia-polymorphism-induced cleaving and spontaneous healing by precipitation was studied in Co1-xO polycrystals containing a dispersion of ZrO2 particles. Conventional, analytical, and high-resolution transmission electron microscopy indicated that the Co1-xO matrix cleaves parallel to {100} and {110} planes and heals itself by co-precipitation of parallel-topotaxial ZrO2/Co3-£_O4 particles upon cooling. Due to size effect and matrix constraint, nanometer-size ZrO2 precipitates at cleavages were able to retain tetragonality upon further cooling to room temperature. Paracrystalline array of defect cluster was shown to form in Zr-doped Ni1-xO and Co1-xO polycrystals while prepared by sintering at relative high temperature, i.e., 1650oC to increase the defect concentration. Paracrystalline array of defect clusters in Co3-£_O4 spinel structure also occurred when doped with Zr4+ at high temperature or cooled below 900oC to activate oxy-precipitation of Co3-dO4 at dislocations. transmission electron microscopic observations indicated the spinel precipitate and its paracrystal predominantly formed at the ZrO2/Co1-xO interface and the cleavages/dislocations of the Co1-xO host. Defect chemistry consideration suggests the paracrystal is due to the assembly of charge- and volume-compensating defects of the 4:1 type with four octahedral vacant sites surrounding one Co3+-filled tetrahedral interstitial site. The spacing of paracrystalline distribution is 3.3, 2.9 and 4.9 times the lattice parameter for Zr-doped Ni1-xO, Zr-doped Co1-xO and Zr-doped Co3-dO4. This spacing between defect clusters is about 0.98 times that of the previously studied undoped Co3-dO4. There is much larger (3.4 times difference) paracrystalline spacing for Zr-doped Co3-£_O4 than its parent phase of Zr-doped Co1-xO.
19

Split Intein Applications for Downstream Purification and Protein Conjugation

Galiardi, Jackelyn 05 October 2021 (has links)
No description available.

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