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Description
V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2)

V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy.
ContributorsWang, Guannan (Author) / Chang, Yung (Thesis advisor) / Levitus, Marcia (Committee member) / Misra, Rajeev (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2012
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Description
Intrinsic antibiotic resistance is of growing concern in modern medical treatment. The primary action of multidrug resistant strains is through over-expression of active transporters which recognize a broad range of antibiotics. In Escherichia coli, the TolC-AcrAB complex has become a model system to understand antibiotic efflux. While the structures of

Intrinsic antibiotic resistance is of growing concern in modern medical treatment. The primary action of multidrug resistant strains is through over-expression of active transporters which recognize a broad range of antibiotics. In Escherichia coli, the TolC-AcrAB complex has become a model system to understand antibiotic efflux. While the structures of these three proteins (and many of their homologs) are known, the exact mechanisms of interaction are still poorly understood. By mutational analysis of the TolC turn 1 residues, a drug hypersensitive mutant has been identified which is defective in functional interactions with AcrA and AcrB. Antibiotic resistant revertants carry alterations in both TolC and AcrA act by stabilizing functional complex assembly and opening of the TolC aperture, as monitored by stability of a labile TolC mutant and sensitivity to vancomycin, respectively. Alterations in the AcrB periplasmic hairpin loops lead to a similar antibiotic hypersensitivity phenotype and destabilized complex assembly. Likewise, alterations in TolC which constitutively open the aperture suppress this antibiotic sensitivity. Suppressor alterations in AcrA and AcrB partially restore antibiotic resistance by mediating stability of the complex. The AcrA suppressor alterations isolated in these studies map to the three crystallized domains and it is concluded they alter the AcrA conformation such that it is permanently fixed in an active state, which wild type only transiently goes through when activated by AcrB. Through this genetic evidence, a direct interaction between TolC and AcrB which is stabilized by AcrA has been proposed. In addition to stabilizing the interactions between TolC and AcrB, AcrA is also responsible for triggering opening of the TolC aperture by mediating energy flow from AcrB to TolC. By permanently altering the conformation of AcrA, suppressor mutants allow defective TolC or AcrB mutants to regain functional interactions lost by the initial mutations. The data provide the genetic proof for direct interaction between AcrB and that AcrA mediated opening of TolC requires AcrB as a scaffold.
ContributorsWeeks, Jon William (Author) / Misra, Rajeev (Thesis advisor) / Stout, Valerie (Committee member) / Shi, Yixin (Committee member) / Clark-Curtiss, Josephine (Committee member) / Arizona State University (Publisher)
Created2012
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Description
The discovery and development of novel antibacterial agents is essential to address the rising health concern over antibiotic resistant bacteria. This research investigated the antibacterial activity of a natural clay deposit near Crater Lake, Oregon, that is effective at killing antibiotic resistant human pathogens. The primary rock types in the

The discovery and development of novel antibacterial agents is essential to address the rising health concern over antibiotic resistant bacteria. This research investigated the antibacterial activity of a natural clay deposit near Crater Lake, Oregon, that is effective at killing antibiotic resistant human pathogens. The primary rock types in the deposit are andesitic pyroclastic materials, which have been hydrothermally altered into argillic clay zones. High-sulfidation (acidic) alteration produced clay zones with elevated pyrite (18%), illite-smectite (I-S) (70% illite), elemental sulfur, kaolinite and carbonates. Low-sulfidation alteration at neutral pH generated clay zones with lower pyrite concentrations pyrite (4-6%), the mixed-layered I-S clay rectorite (R1, I-S) and quartz.

Antibacterial susceptibility testing reveals that hydrated clays containing pyrite and I-S are effective at killing (100%) of the model pathogens tested (E. coli and S. epidermidis) when pH (< 4.2) and Eh (> 450 mV) promote pyrite oxidation and mineral dissolution, releasing > 1 mM concentrations of Fe2+, Fe3+ and Al3+. However, certain oxidized clay zones containing no pyrite still inhibited bacterial growth. These clays buffered solutions to low pH (< 4.7) and oxidizing Eh (> 400 mV) conditions, releasing lower amounts (< 1 mM) of Fe and Al. The presence of carbonate in the clays eliminated antibacterial activity due to increases in pH, which lower pyrite oxidation and mineral dissolution rates.

The antibacterial mechanism of these natural clays was explored using metal toxicity and genetic assays, along with advanced bioimaging techniques. Antibacterial clays provide a continuous reservoir of Fe2+, Fe3+ and Al3+ that synergistically attack pathogens while generating hydrogen peroxide (H2O¬2). Results show that dissolved Fe2+ and Al3+ are adsorbed to bacterial envelopes, causing protein misfolding and oxidation in the outer membrane. Only Fe2+ is taken up by the cells, generating oxidative stress that damages DNA and proteins. Excess Fe2+ oxidizes inside the cell and precipitates Fe3+-oxides, marking the sites of hydroxyl radical (•OH) generation. Recognition of this novel geochemical antibacterial process should inform designs of new mineral based antibacterial agents and could provide a new economic industry for such clays.
ContributorsMorrison, Keith D (Author) / Williams, Lynda B (Thesis advisor) / Williams, Stanley N (Thesis advisor) / Misra, Rajeev (Committee member) / Shock, Everett (Committee member) / Anbar, Ariel (Committee member) / Arizona State University (Publisher)
Created2015
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Description
Lignocellulosic biomass represents a renewable domestic feedstock that can support large-scale biochemical production processes for fuels and specialty chemicals. However, cost-effective conversion of lignocellulosic sugars into valuable chemicals by microorganisms still remains a challenge. Biomass recalcitrance to saccharification, microbial substrate utilization, bioproduct titer toxicity, and toxic chemicals associated with chemical

Lignocellulosic biomass represents a renewable domestic feedstock that can support large-scale biochemical production processes for fuels and specialty chemicals. However, cost-effective conversion of lignocellulosic sugars into valuable chemicals by microorganisms still remains a challenge. Biomass recalcitrance to saccharification, microbial substrate utilization, bioproduct titer toxicity, and toxic chemicals associated with chemical pretreatments are at the center of the bottlenecks limiting further commercialization of lignocellulose conversion. Genetic and metabolic engineering has allowed researchers to manipulate microorganisms to overcome some of these challenges, but new innovative approaches are needed to make the process more commercially viable. Transport proteins represent an underexplored target in genetic engineering that can potentially help to control the input of lignocellulosic substrate and output of products/toxins in microbial biocatalysts. In this work, I characterize and explore the use of transport systems to increase substrate utilization, conserve energy, increase tolerance, and enhance biocatalyst performance.
ContributorsKurgan, Gavin (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Misra, Rajeev (Committee member) / Nannenga, Brent (Committee member) / Arizona State University (Publisher)
Created2018
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Description
The purpose of this study was to observe the effectiveness of the phenylalanyl arginine β-naphthylamide dihydrochloride inhibitor and Tween 20 when combined with an antibiotic against Escherichia. coli. As antibiotic resistance becomes more and more prevalent it is necessary to think outside the box and do more than just increase

The purpose of this study was to observe the effectiveness of the phenylalanyl arginine β-naphthylamide dihydrochloride inhibitor and Tween 20 when combined with an antibiotic against Escherichia. coli. As antibiotic resistance becomes more and more prevalent it is necessary to think outside the box and do more than just increase the dosage of currently prescribed antibiotics. This study attempted to combat two forms of antibiotic resistance. The first is the AcrAB efflux pump which is able to pump antibiotics out of the cell. The second is the biofilms that E. coli can form. By using an inhibitor, the pump should be unable to rid itself of an antibiotic. On the other hand, using Tween allows for biofilm formation to either be disrupted or for the biofilm to be dissolved. By combining these two chemicals with an antibiotic that the efflux pump is known to expel, low concentrations of each chemical should result in an equivalent or greater effect on bacteria compared to any one chemical in higher concentrations. To test this hypothesis a 96 well plate BEC screen test was performed. A range of antibiotics were used at various concentrations and with varying concentrations of both Tween and the inhibitor to find a starting point. Following this, Erythromycin and Ciprofloxacin were picked as the best candidates and the optimum range of the antibiotic, Tween, and inhibitor were established. Finally, all three chemicals were combined to observe the effects they had together as opposed to individually or paired together. From the results of this experiment several conclusions were made. First, the inhibitor did in fact increase the effectiveness of the antibiotic as less antibiotic was needed if the inhibitor was present. Second, Tween showed an ability to prevent recovery in the MBEC reading, showing that it has the ability to disrupt or dissolve biofilms. However, Tween also showed a noticeable decrease in effectiveness in the overall treatment. This negative interaction was unable to be compensated for when using the inhibitor and so the hypothesis was proven false as combining the three chemicals led to a less effective treatment method.
ContributorsPetrovich Flynn, Chandler James (Author) / Misra, Rajeev (Thesis director) / Bean, Heather (Committee member) / Perkins, Kim (Committee member) / Mechanical and Aerospace Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
An in-depth analysis on the effects vortex generators cause to the boundary layer separation that occurs when an internal flow passes through a diffuser is presented. By understanding the effects vortex generators demonstrate on the boundary layer, they can be utilized to improve the performance and efficiencies of diffusers and

An in-depth analysis on the effects vortex generators cause to the boundary layer separation that occurs when an internal flow passes through a diffuser is presented. By understanding the effects vortex generators demonstrate on the boundary layer, they can be utilized to improve the performance and efficiencies of diffusers and other internal flow applications. An experiment was constructed to acquire physical data that could assess the change in performance of the diffusers once vortex generators were applied. The experiment consisted of pushing air through rectangular diffusers with half angles of 10, 20, and 30 degrees. A velocity distribution model was created for each diffuser without the application of vortex generators before modeling the velocity distribution with the application of vortex generators. This allowed the two results to be directly compared to one another and the improvements to be quantified. This was completed by using the velocity distribution model to find the partial mass flow rate through the outer portion of the diffuser's cross-sectional area. The analysis concluded that the vortex generators noticeably increased the performance of the diffusers. This was best seen in the performance of the 30-degree diffuser. Initially the diffuser experienced airflow velocities near zero towards the edges. This led to 0.18% of the mass flow rate occurring in the outer one-fourth portion of the cross-sectional area. With the application of vortex generators, this percentage increased to 5.7%. The 20-degree diffuser improved from 2.5% to 7.9% of the total mass flow rate in the outer portion and the 10-degree diffuser improved from 11.9% to 19.2%. These results demonstrate an increase in performance by the addition of vortex generators while allowing the possibility for further investigation on improvement through the design and configuration of these vortex generators.
ContributorsSanchez, Zachary Daniel (Author) / Takahashi, Timothy (Thesis director) / Herrmann, Marcus (Committee member) / Mechanical and Aerospace Engineering Program (Contributor) / W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
The objective of this project was to design an electrically driven centrifugal pump for the Daedalus Astronautics @ASU hybrid rocket engine (HRE). The pump design was purposefully simplified due to time, fabrication, calculation, and capability constraints, which resulted in a lower fidelity design, with the option to be improved later.

The objective of this project was to design an electrically driven centrifugal pump for the Daedalus Astronautics @ASU hybrid rocket engine (HRE). The pump design was purposefully simplified due to time, fabrication, calculation, and capability constraints, which resulted in a lower fidelity design, with the option to be improved later. The impeller, shroud, volute, shaft, motor, and ESC were the main focuses of the pump assembly, but the seals, bearings, lubrication methods, and flow path connections were considered as elements which would require future attention. The resulting pump design is intended to be used on the Daedalus Astronautics HRE test cart for design verification. In the future, trade studies and more detailed analyses should and will be performed before this pump is integrated into the Daedalus Astronautics flight-ready HRE.
ContributorsShillingburg, Ryan Carl (Author) / White, Daniel (Thesis director) / Brunacini, Lauren (Committee member) / Mechanical and Aerospace Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
Human habitation of other planets requires both cost-effective transportation and low time-of-flight for human passengers and critical supplies. The current methods for interplanetary orbital transfers, such as the Hohmann transfer, require either expensive, high fuel maneuvers or extended space travel. However, by utilizing the high velocities of a super-geosynchronous space

Human habitation of other planets requires both cost-effective transportation and low time-of-flight for human passengers and critical supplies. The current methods for interplanetary orbital transfers, such as the Hohmann transfer, require either expensive, high fuel maneuvers or extended space travel. However, by utilizing the high velocities of a super-geosynchronous space elevator, spacecraft released from an apex anchor could achieve interplanetary transfers with minimal Delta V fuel and time of flight requirements. By using Lambert’s Problem and Free Release propagation to determine the minimal fuel transfer from a terrestrial space elevator to Mars under a variety of initial conditions and time-of-flight constraints, this paper demonstrates that the use of a space elevator release can address both needs by dramatically reducing the time-of-flight and the fuel budget.
ContributorsTorla, James (Author) / Peet, Matthew (Thesis director) / Swan, Peter (Committee member) / Mechanical and Aerospace Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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Description
This thesis evaluates the viability of an original design for a cost-effective wheel-mounted dynamometer for road vehicles. The goal is to show whether or not a device that generates torque and horsepower curves by processing accelerometer data collected at the edge of a wheel can yield results that are comparable

This thesis evaluates the viability of an original design for a cost-effective wheel-mounted dynamometer for road vehicles. The goal is to show whether or not a device that generates torque and horsepower curves by processing accelerometer data collected at the edge of a wheel can yield results that are comparable to results obtained using a conventional chassis dynamometer. Torque curves were generated via the experimental method under a variety of circumstances and also obtained professionally by a precision engine testing company. Metrics were created to measure the precision of the experimental device's ability to consistently generate torque curves and also to compare the similarity of these curves to the professionally obtained torque curves. The results revealed that although the test device does not quite provide the same level of precision as the professional chassis dynamometer, it does create torque curves that closely resemble the chassis dynamometer torque curves and exhibit a consistency between trials comparable to the professional results, even on rough road surfaces. The results suggest that the test device provides enough accuracy and precision to satisfy the needs of most consumers interested in measuring their vehicle's engine performance but probably lacks the level of accuracy and precision needed to appeal to professionals.
ContributorsKing, Michael (Author) / Ren, Yi (Thesis director) / Spanias, Andreas (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / Mechanical and Aerospace Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
This research project will test the structural properties of a 3D printed origami inspired structure and compare them with a standard honeycomb structure. The models have equal face areas, model heights, and overall volume but wall thicknesses will be different. Stress-deformation curves were developed from static loading testing. The area

This research project will test the structural properties of a 3D printed origami inspired structure and compare them with a standard honeycomb structure. The models have equal face areas, model heights, and overall volume but wall thicknesses will be different. Stress-deformation curves were developed from static loading testing. The area under these curves was used to calculate the toughness of the structures. These curves were analyzed to see which structures take more load and which deform more before fracture. Furthermore, graphs of the Stress-Strain plots were produced. Using 3-D printed parts in tough resin printed with a Stereolithography (SLA) printer, the origami inspired structure withstood a larger load, produced a larger toughness and deformed more before failure than the equivalent honeycomb structure.
ContributorsMcGregor, Alexander (Author) / Jiang, Hanqing (Thesis director) / Kingsbury, Dallas (Committee member) / Mechanical and Aerospace Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05