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- Genre: Masters Thesis
Description
The alkali activation of aluminosilicate materials as binder systems derived from industrial byproducts have been extensively studied due to the advantages they offer in terms enhanced material properties, while increasing sustainability by the reuse of industrial waste and byproducts and reducing the adverse impacts of OPC production. Fly ash and ground granulated blast furnace slag are commonly used for their content of soluble silica and aluminate species that can undergo dissolution, polymerization with the alkali, condensation on particle surfaces and solidification. The following topics are the focus of this thesis: (i) the use of microwave assisted thermal processing, in addition to heat-curing as a means of alkali activation and (ii) the relative effects of alkali cations (K or Na) in the activator (powder activators) on the mechanical properties and chemical structure of these systems. Unsuitable curing conditions instigate carbonation, which in turn lowers the pH of the system causing significant reductions in the rate of fly ash activation and mechanical strength development. This study explores the effects of sealing the samples during the curing process, which effectively traps the free water in the system, and allows for increased aluminosilicate activation. The use of microwave-curing in lieu of thermal-curing is also studied in order to reduce energy consumption and for its ability to provide fast volumetric heating. Potassium-based powder activators dry blended into the slag binder system is shown to be effective in obtaining very high compressive strengths under moist curing conditions (greater than 70 MPa), whereas sodium-based powder activation is much weaker (around 25 MPa). Compressive strength decreases when fly ash is introduced into the system. Isothermal calorimetry is used to evaluate the early hydration process, and to understand the reaction kinetics of the alkali powder activated systems. A qualitative evidence of the alkali-hydroxide concentration of the paste pore solution through the use of electrical conductivity measurements is also presented, with the results indicating the ion concentration of alkali is more prevalent in the pore solution of potassium-based systems. The use of advanced spectroscopic and thermal analysis techniques to distinguish the influence of studied parameters is also discussed.
ContributorsChowdhury, Ussala (Author) / Neithalath, Narayanan (Thesis advisor) / Rajan, Subramanium D. (Committee member) / Mobasher, Barzin (Committee member) / Arizona State University (Publisher)
Created2013
Description
Reactive oxygen species (ROS) including superoxide, hydrogen peroxide, and hydroxyl radicals occur naturally as a byproduct of aerobic respiration. To mitigate damages caused by ROS, Escherichia coli employs defenses including two cytosolic superoxide dismutases (SODs), which convert superoxide to hydrogen peroxide. Deletion of both sodA and sodB, the genes coding for the cytosolic SOD enzymes, results in a strain that is unable to grow on minimal medium without amino acid supplementation. Additionally, deletion of both cytosolic SOD enzymes in a background containing the relA1 allele, an inactive version of the relA gene that contributes to activation of stringent response by amino acid starvation, results in a strain that is unable to grow aerobically, even on rich medium. These observations point to a relationship between the stringent response and oxidative stress. To gain insight into this relationship, suppressors were isolated by growing the ∆sodAB relA1 cells aerobically on rich medium, and seven suppressors were further examined to characterize distinct colony sizes and temperature sensitivity phenotypes. In three of these suppressor-containing strains, the relA1 allele was successfully replaced by the wild type relA allele to allow further study in aerobic conditions. None of those three suppressors were found to increase tolerance to exogenous superoxides produced by paraquat, which shows that these mutations only overcome the superoxide buildup that naturally occurs from deletion of SODs. Because each of these suppressors had unique phenotypes, it is likely that they confer tolerance to SOD-dependent superoxide buildup by different mechanisms. Two of these three suppressors have been sent for whole-genome sequencing to identify the location of the suppressor mutation and determine the mechanism by which they confer superoxide tolerance.
ContributorsFlake, Melissa (Author) / Misra, Rajeev (Thesis advisor) / Shah, Dhara (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2024
Description
As the utilization of tyrosine is needed by both eukaryotes and prokaryotes, this versatile amino acid contributes towards a variety of operations including protein synthesis, pigment production, and host or habitat impacting metabolite creation. While there are numerous pathways which involve the degradation of tyrosine to create different products, the one that is central in this thesis is a pathway with homogentisate as an intermediate. This pathway brings an interest due to its association with metabolic disorders like Tyrosinemia (I, II, or III), and its impact within an agricultural environment. In other words, for humans and plant microbiomes to maintain their optimal metabolic homeostasis, tyrosine is required to participate in numerous demands. This necessity can ultimately create competition between organisms present in microbial communities, as there are a multitude of species that can metabolize tyrosine for the creation of diverse products. In this work, a primary objective is to characterize the breakdown of tyrosine within a competitive environment where there are multiple available pathways. There are many factors that could influence the catabolism of tyrosine like catalytic efficiency of enzymes, availability of breakdown routes, and pathway regulations. Here, the start will be creating a proof of concept developed by studying the competition for tyrosine utilization by environmental microbial enzymes; 4-hydroxyphenylpyruvate dioxygenase from Streptomyces avermitilis, 4-hydroxymandelate synthase from Amycolatopsis orientalis, and tyrosine ammonia lyase from Flavobacterium johnsoniae. Through phenotypic assays and by quantifying secreted metabolites, rerouting of this pathway is observed. This insight towards the ability of diverting the homogentisate pathway was then utilized for the analysis of contest between human enzyme, 4-hydroxyphenylpyruvate dioxygenase, and gut microbial enzyme, tyrosine ammonia lyase from Bacteroides ovatus. Within both aims it is seen that due to successful diversion of the pathway, there is a reduction in tyrosine with the formation of more favorable products. The strategy of redirecting this tyrosine catabolism pathway will provide baseline knowledge for future efforts to contribute towards alternative methods of intervention to alleviate the burdens from tyrosine metabolic dysfunction and disorders.
ContributorsMitchem, Madison (Author) / Shah, Dhara D (Thesis advisor) / Misra, Rajeev (Thesis advisor) / Krajmalnik-Brown, Rosa (Committee member) / Arizona State University (Publisher)
Created2024
Description
Directed evolution using genetically diverse libraries is integral to advancing research in industrial microbial production and protein functionality enhancement. This process typically involves a step of sequence diversification and subsequent selection/screening steps for improved variants. While CRISPR-Cas9 systems are known to offer efficient and targeted modification of genes in vivo, concerns arise regarding off-target effects and the emergence of escaper cells evading Cas9 cleavage. This study investigated a strategy to leverage CRISPR-Cas9 counter-selection in Escherichia coli for targeted chromosomal mutagenesis. By designing gRNAs to target a desired region, the spontaneous mutations occurring at the targeted region will potentially disrupt Cas9 binding and thus allow the cell to avoid death caused by Cas9-induced double-stranded DNA breaks. This population of ‘escaper’ cells surviving the counter-selection will have mutations in the gRNA-targeting region at a higher frequency than their non-escaper counterparts. To optimize this counter-selection method, the design for the CRISPR-Cas9 expression system was improved, Cas9 variants with varied fidelities and activities were investigated, and the strategy of using truncated gRNAs for enhanced mutation selectivity was explored. Using the E. coli rpoB gene as a target for editing, the rifampicin-resistant mutation (caused by mutations in rpoB) frequency was increased by more than five orders of magnitude compared to the control E. coli strain without CRISPR targeting. Nanopore DNA sequencing of the mutants’ rpoB region confirmed the promising targeting efficacy of this approach. This study demonstrates a streamlined method for targeted genetic diversification in vivo, facilitating efficient protein engineering in bacterial systems.
ContributorsRick, Rachel Nicole (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Misra, Rajeev (Committee member) / Arizona State University (Publisher)
Created2024
Description
Emergence of multidrug resistant (MDR) bacteria is a major concern to global health. One of the major MDR mechanisms bacteria employ is efflux pumps for the expulsion of drugs from the cell. In Escherichia coli, AcrAB-TolC proteins constitute the major chromosomally-encoded drug efflux system. AcrB, a trimeric membrane protein is well-known for its substrate promiscuity. It has the ability to efflux a broad spectrum of substrates alongside compounds such as dyes, detergent, bile salts and metabolites. Newly identified AcrB residues were shown to be functionally relevant in the drug binding and translocation pathway using a positive genetic selection strategy. These residues—Y49, V127, D153, G288, F453, and L486—were identified as the sites of suppressors of an alteration, F610A, that confers a drug hypersensitivity phenotype. Using site-directed mutagenesis (SDM) along with the real-time efflux and the classical minimum inhibitory concentration (MIC) assays, I was able to characterize the mechanism of suppression.
Three approaches were used for the characterization of these suppressors. The first approach focused on side chain specificity. The results showed that certain suppressor sites prefer a particular side chain property, such as size, to overcome the F610A defect. The second approach focused on the effects of efflux pump inhibitors. The results showed that though the suppressor residues were able to overcome the intrinsic defect of F610A, they were unable to overcome the extrinsic defect caused by the efflux pump inhibitors. This showed that the mechanism by which F610A imposes its effect on AcrB function is different than that of the efflux pump inhibitors. The final approach was to determine whether suppressors mapping in the periplasmic and trans-membrane domains act by the same or different mechanisms. The results showed both overlapping and distinct mechanisms of suppression.
To conclude, these approaches have provided a deeper understanding of the mechanisms by which novel suppressor residues of AcrB overcome the functional defect of the drug binding domain alteration, F610A.
Three approaches were used for the characterization of these suppressors. The first approach focused on side chain specificity. The results showed that certain suppressor sites prefer a particular side chain property, such as size, to overcome the F610A defect. The second approach focused on the effects of efflux pump inhibitors. The results showed that though the suppressor residues were able to overcome the intrinsic defect of F610A, they were unable to overcome the extrinsic defect caused by the efflux pump inhibitors. This showed that the mechanism by which F610A imposes its effect on AcrB function is different than that of the efflux pump inhibitors. The final approach was to determine whether suppressors mapping in the periplasmic and trans-membrane domains act by the same or different mechanisms. The results showed both overlapping and distinct mechanisms of suppression.
To conclude, these approaches have provided a deeper understanding of the mechanisms by which novel suppressor residues of AcrB overcome the functional defect of the drug binding domain alteration, F610A.
ContributorsBlake, Mellecha (Author) / Misra, Rajeev (Thesis advisor) / Stout, Valerie (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016