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Title: SYNTHESIS AND CHARACTERIZATION OF BIOACTIVE GLASS-CERAMIC PARTICLES WITH ADVANCED ANTIBACTERIAL PROPERTIES FOR APPLICATIONS IN BONE REGENERATION
Creator: Pajares Chamorro, Natalia
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Bacterial infections are major surgical complications, which have worsened due to the continued evolution of drug-resistance. In coping with the decay of the antibiotic era, scientists eagerly search for alternative treatments. Multi-functional biomaterials capable of combating infections while triggering tissue regeneration are of great interest. For example, bioactive glasses have been regularly used to deliver drugs and regenerate tissue owed to their unique bone-bonding ability. Doping...
Show moreBacterial infections are major surgical complications, which have worsened due to the continued evolution of drug-resistance. In coping with the decay of the antibiotic era, scientists eagerly search for alternative treatments. Multi-functional biomaterials capable of combating infections while triggering tissue regeneration are of great interest. For example, bioactive glasses have been regularly used to deliver drugs and regenerate tissue owed to their unique bone-bonding ability. Doping the bioactive glass structure with broad-spectrum biocide ions such as Ag+ confers advanced antibacterial properties. The release of Ag+ is controlled by the degradation process of the glass network, maintaining the dose within a therapeutic window that is not cytotoxic to eukaryotic cells. Despite the extensive research performed on Ag-doped bioactive glasses, their regenerative properties in bone tissues have been rarely investigated. This thesis presents promising interactions between Ag-doped bioactive glass (Ag-BG) microparticles and osteoprogenitor cells, providing evidence of the ability to support bone regeneration. Ag-BG’s degradation provoked cell proliferation and cell differentiation in vitro and demonstrated healing of critical calvaria defects in mice after one month of implantation, thanks to the release of Si and Ca ions. Additionally, Ag-BG was antibacterial against Staphylococcus aureus (S. aureus), the most common cause of bone-degenerative diseases like osteomyelitis, and demonstrated low proclivity to induce resistance. The antibacterial potential originated from the degradation by-products of the structure. The mechanism of inhibition was built upon four main sources from higher to lower contribution: Ag+ release, oxidative stress, mechanical damage by nano-sized debris, and osmotic effect. In addition, Ag-BG was capable of restoring ineffective antibiotics with cell-wall-related inhibitory mechanisms by simple combinatorial therapies, rendering them effective in clearing infections. This unprecedented functionality of Ag-BG was expanded with antibiotic depots, where Ag-BG served as a carrier for an ineffective drug. Bioactive glass nanoparticles (BGNs) have been proposed to advance biological and antibacterial properties compared to their micro-sized counterparts. However, the challenges of producing BGNs with multifold metallic ions in a reproducible manner have limited their use. Here, the Stöber method was comprehensively studied to understand the effect of process variables on BGNs’ composition, structure, and morphology. The use of methanol as solvent and the early addition of metallic ion reagents before catalysis helped improved their cation incorporation within the glass network. Extended stirring was key to achieving the targeted composition and controlling the particle size. Monodispersed 10 nm Ag-doped BGNs (Ag-BGNs) were achieved. These Ag-BGNs were stronger antimicrobial weapons, providing bacterial inhibition within hours of treatment. The biological properties were not significantly advanced in the Ag-BGNs compared to Ag-BG; however, cell proliferation, differentiation, and bone re-growth were still provoked. These Ag-BGNs were used as fillers in hydrogel nanocomposites with natural matrices consisting of collagen type I or extracellular matrix. Ag-BGNs distributed homogeneously along the polymer fibrils and allowed polymerization within hours at physiological conditions. These materials hold potential for injectable devices, designing minimally invasive single-step treatment for debilitating bone infections while promoting tissue recovery.
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Title: SYNTHESIS OF 3-HYDROXYPROPIONIC ACID FROM ACETYLENECARBOXYLIC ACID
Creator: Mathes Hewage, Amaya Nethmini Sirinimal
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Throughout the past several decades, the petroleum industry has remained a dominant player in the production of fuels, chemicals, and consumer products. However, rising global temperatures and declining fossil fuel reserves demand the need for sustainable processes that can compete with the existing crude oil-based economy. Employing microbes such as E. coli for the chemical production using renewable starch-derived feedstocks such as glucose has been identified as a preferred alternative....
Show moreThroughout the past several decades, the petroleum industry has remained a dominant player in the production of fuels, chemicals, and consumer products. However, rising global temperatures and declining fossil fuel reserves demand the need for sustainable processes that can compete with the existing crude oil-based economy. Employing microbes such as E. coli for the chemical production using renewable starch-derived feedstocks such as glucose has been identified as a preferred alternative. Although lignocellulosic feedstocks have been explored as an alternative to renewable sugars, these processes are yet to be successfully implemented in an industrial setting. Despite more recent research efforts to incorporate C1 feedstocks such as methane, methanol and carbon dioxide in microbial catalysis, development of efficient bacterial metabolic pathways using these feedstocks have proven to be challenging. Herein, acetylenecarboxylic acid (ACA) is proposed as a unique feedstock for microbial catalysis. ACA can be derived from dehydrodimerization of methane to acetylene and subsequent carboxylation of acetylene. Cg10062 was previously identified as an enzyme capable of hydrating ACA to form a mixture of malonate semialdehyde (MSA) and acetaldehyde. In this study, novel variant Cg10062(E114N)) that forms exclusively MSA from ACA was discovered using rational mutagenesis. Cg10062(E114N) was coupled with NADPH-dependent dehydrogenase YdfG to develop a unique biocatalytic route to building block chemical 3-hydroxypropionic acid (3-HP). In vitro synthesis of 3-HP from ACA was demonstrated using catalytic amounts of NADP(H) where cofactor regeneration was achieved using NADP+-dependent phosphite dehydrogenase PTDH.
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Title: SYNTHETIC BIOLOGY APPROACHES ESTABLISH THE FOUNDATION FOR SUSTAINABLE PRODUCTION OF HIGH VALUE TERPENOIDS
Creator: Bibik, Jacob David
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Plants have become a promising platform for sustainable bioproduction of an array of natural products and specialty chemicals. Of particular interest are terpenes and the functionalized terpenoids, which represent the largest and most diverse class of natural products. These natural products are commonly used commercially as major constituents of flavorings and fragrances, oils, pigments, and pharmaceuticals, while having many other applications. Given the diversity and structural complexity...
Show morePlants have become a promising platform for sustainable bioproduction of an array of natural products and specialty chemicals. Of particular interest are terpenes and the functionalized terpenoids, which represent the largest and most diverse class of natural products. These natural products are commonly used commercially as major constituents of flavorings and fragrances, oils, pigments, and pharmaceuticals, while having many other applications. Given the diversity and structural complexity of many terpenoids, they are often expensive and difficult, if not impossible, to chemically synthesize. Engineering these biosynthetic pathways in plant hosts may provide a sustainable platform to access terpenoids for industrial production. While plants offer a sustainable production platform, metabolic engineering for chemical production has largely focused on microbial hosts, and further development of strategies and tools for plant engineering is needed. In my dissertation, I have taken multi-pronged approaches to further develop sustainable bioproduction of terpenoids in plants. First, I developed strategies to optimize, re-target, and compartmentalize production of squalene, a C30 triterpene, within plant cells to improve yields in plants. Re-targeting the final steps in squalene production, farnesyl diphosphate synthase (FDPS) and squalene synthase (SQS), from the cytosol to plastids enabled compartmentalization of biosynthesis away from competing cytosolic enzymes. I then anchored an optimized FDPS and SQS pair to the surface of cytosolic lipid droplets through fusions to the Nannochloropsis oceanica Lipid Droplet Surface Protein (NoLDSP), where squalene can be sequestered and stored. Scaffolding the pathway to the surface of lipid droplets increased yields to more than twice that of plastidial targeting. Re-targeting this lipid droplet scaffolding to plastids, produced similar squalene yields as the soluble, plastid targeted pathway, and ameliorated some of the negative effects on photosynthesis. Second, I worked to engineer poplar, a bioenergy crop which emits large amounts of the hemiterpene isoprene, with these pathways as a platform for bioproduction and adding value to a bioenergy pipeline. Transformants were successfully created for plastid targeted squalene production, producing up to 0.63mg/gFW of squalene. The lipid droplet scaffolding strategies appeared toxic during tissue regeneration, suggesting a need for tissue specific engineering of these pathways in future iterations. Third, I developed a pipeline to identify, characterize, and engineer bidirectional promoters (BDPs), which enable divergent expression of two genes and improve gene stacking in plant constructs. As seen above with poplar, plant engineering is often limited by construct size, diverse promoter availability, and expression regulation, and a BDP library enables a range of expression in more compact constructs. I identified 34 BDPs from Populus trichocarpa and Arabidopsis thaliana, characterized their activity via Nicotiana benthamiana transient expression, and engineered select BDPs to further alter activities. Combining these BDPs with previously developed terminator sequences provided further regulation of expression. These genetic tools provide an array of expression activities and enable greater gene stacking options while offering the potential for more fine tuning of expression for multiple genes in a metabolic pathway. The work performed in this dissertation provide strategies to improve production of terpenoids in plants, establish production hosts, and engineer larger, complex pathways.
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Title: SYSTEMIC DELIVERY OF INSECTICIDES IN BLUEBERRIES FOR CONTROL OF BLUEBERRY STEM GALL WASP, HEMADAS NUBILIPENNIS
Creator: Bosch, Amber Kay
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Blueberry stem gall wasp (Hemadas nubilipennis) is a pest of highbush blueberry and can pose a challenge to control with foliar sprays due to adult activity during bloom and larval development within the plant tissues. In this thesis, systemic delivery of insecticides in blueberry bushes was evaluated using three application methods on potted bushes, in blueberry shoot bioassays, and on a commercial blueberry farm. Each study aimed to evaluate impact on gall and gall wasp development, along...
Show moreBlueberry stem gall wasp (Hemadas nubilipennis) is a pest of highbush blueberry and can pose a challenge to control with foliar sprays due to adult activity during bloom and larval development within the plant tissues. In this thesis, systemic delivery of insecticides in blueberry bushes was evaluated using three application methods on potted bushes, in blueberry shoot bioassays, and on a commercial blueberry farm. Each study aimed to evaluate impact on gall and gall wasp development, along with active ingredient residue delivery to plant tissues. I hypothesized that if the insecticide moved systemically within the blueberry vascular system to areas where the blueberry stem gall wasp larvae are developing, then the insecticide will kill the larvae leading to a reduction in gall formation and number of surviving adults per gall. In the potted bush study, applications were made by crown injection, soil drench and foliar sprays. Imidacloprid, flupyradifurone, and spirotetramat were recovered in shoot and leaf tissues, however there was no evidence of inhibited gall or gall wasp development. In the shoot bioassays, imidacloprid and spirotetramat were found to have the greatest potential for control of blueberry stem gall wasp. Active ingredient recovery in bioassay gall tissue revealed the concentration needed to get moribund/ lethal larval response. The on-farm study indicated there was successful movement of imidacloprid and flupyradifurone using chemigation, however, the impact on blueberry stem gall wasp was not great enough to provide control.
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