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Description
The Multiple Antibiotic Resistance Regulator Family (MarR) are transcriptional regulators, many of which forms a dimer. Transcriptional regulation provides bacteria a stabilized responding system to ensure the bacteria is able to efficiently adapt to different environmental conditions. The main function of the MarR family is to create multiple antibiotic resistance from a mutated protein; this process occurs when the MarR regulates an operon. We hypothesized that different transcriptional regulator genes have interactions with each other. It is known that Salmonella pagC transcription is activated by three regulators, i.e., SlyA, MprA, and PhoP. Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) system was used to research the protein-protein interactions in SlyA, MprA, and PhoP as heterodimers and homodimers in vivo. Two fragments, T25 and T18, that lack endogenous adenylate cyclase activity, were used for construction of chimeric proteins and reconstruction of adenylate cyclase activity was tested. The significant adenylate cyclase activities has proved that SlyA is able to form homodimers. However, weak adenylate cyclase activities in this study has proved that MprA and PhoP are not likely to form homodimers, and no protein-protein interactions were detected in between SlyA, MprA and PhoP, which no heterodimers have formed in between three transcriptional regulators.
ContributorsTao, Zenan (Author) / Shi, Yixin (Thesis advisor) / Wang, Xuan (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2018
Description
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the 10th leading cause of death, worldwide. The prevalence of drug-resistant clinical isolates and the paucity of newly-approved antituberculosis drugs impedes the successful eradication of Mtb. Bacteria commonly use two-component systems (TCS) to sense their environment and genetically modulate adaptive responses. The prrAB TCS is essential in Mtb, thus representing an auspicious drug target; however, the inability to generate an Mtb ΔprrAB mutant complicates investigating how this TCS contributes to pathogenesis. Mycobacterium smegmatis, a commonly used M. tuberculosis genetic surrogate was used here. This work shows that prrAB is not essential in M. smegmatis. During ammonium stress, the ΔprrAB mutant excessively accumulates triacylglycerol lipids, a phenotype associated with M. tuberculosis dormancy and chronic infection. Additionally, triacylglycerol biosynthetic genes were induced in the ΔprrAB mutant relative to the wild-type and complementation strains during ammonium stress. Next, RNA-seq was used to define the M. smegmatis PrrAB regulon. PrrAB regulates genes participating in respiration, metabolism, redox balance, and oxidative phosphorylation. The M. smegmatis ΔprrAB mutant is compromised for growth under hypoxia, is hypersensitive to cyanide, and fails to induce high-affinity respiratory genes during hypoxia. Furthermore, PrrAB positively regulates the hypoxia-responsive dosR TCS response regulator, potentially explaining the hypoxia-mediated growth defects in the ΔprrAB mutant. Despite inducing genes encoding the F1F0 ATP synthase, the ΔprrAB mutant accumulates significantly less ATP during aerobic, exponential growth compared to the wild-type and complementation strains. Finally, the M. smegmatis ΔprrAB mutant exhibited growth impairment in media containing gluconeogenic carbon sources. M. tuberculosis mutants unable to utilize these substrates fail to establish chronic infection, suggesting that PrrAB may regulate Mtb central carbon metabolism in response to chronic infection. In conclusion, 1) prrAB is not universally essential in mycobacteria; 2) M. smegmatis PrrAB regulates genetic responsiveness to nutrient and oxygen stress; and 3) PrrAB may provide feed-forward control of the DosRS TCS and dormancy phenotypes. The data generated in these studies provide insight into the mycobacterial PrrAB TCS transcriptional regulon, PrrAB essentiality in Mtb, and how PrrAB may mediate stresses encountered by Mtb during the transition to chronic infection.
ContributorsMaarsingh, Jason (Author) / Haydel, Shelley E (Thesis advisor) / Roland, Kenneth (Committee member) / Sandrin, Todd (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2019
Description
One out of ten women has a difficult time getting or staying pregnant in the United States. Recent studies have identified aging as one of the key factors attributed to a decline in female reproductive health. Existing fertility diagnostic methods do not allow for the non-invasive monitoring of hormone levels across time. In recent years, olfactory sensing has emerged as a promising diagnostic tool for its potential for real-time, non-invasive monitoring. This technology has been proven promising in the areas of oncology, diabetes, and neurological disorders. Little work, however, has addressed the use of olfactory sensing with respect to female fertility. In this work, we perform a study on ten healthy female subjects to determine the volatile signature in biological samples across 28 days, correlating to fertility hormones. Volatile organic compounds (VOCs) present in the air above the biological sample, or headspace, were collected by solid phase microextraction (SPME), using a 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) coated fiber. Samples were analyzed, using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS). A regression model was used to identify key analytes, corresponding to the fertility hormones estrogen and progesterone. Results indicate shifts in volatile signatures in biological samples across the 28 days, relevant to hormonal changes. Further work includes evaluating metabolic changes in volatile hormone expression as an early indicator of declining fertility, so women may one day be able to monitor their reproductive health in real-time as they age.
ContributorsOng, Stephanie (Author) / Smith, Barbara (Thesis advisor) / Bean, Heather (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2018
Description
Hypoxia is a pathophysiological condition which results from lack of oxygen supply in tumors. The assessment of tumor hypoxia and its response to therapies can provide guidelines for optimization and personalization of therapeutic protocols for better treatment. Previous research has shown the difficulty in measuring hypoxia anatomically due to its heterogenous nature. This makes the study of hypoxia through various imaging modalities and mapping techniques crucial. The potential of hypoxia targeting T1 contrast agent GdDO3NI in generating hypoxia maps has been studied earlier. In this work, the similarities between hypoxia maps generated by MRI using GdDO3NI and pimonidazole based immunohistochemistry (IHC) in non-small cell lung carcinoma bearing mice have been studied. Six NCI-H1975 tumor-bearing mice were studied. All animal studies were approved by Arizona State University’s Institute of Animal Care and Use Committee (IACUC). Post co-injection of GdDO3NI and pimonidazole, T1 weighted 3D gradient echo MR images were acquired. For ex-vivo analysis of hypoxia, 30 μm thick tumor sections were obtained for each harvested tumor and were stained for pimonidazole and counter-stained with DAPI for nuclear staining. Pimonidazole (PIMO) is clinically used as a “gold standard” hypoxia marker. The key process involved stacking and iterative registration based on quality metric SSIM (Structural Similarity) Index of DAPI stained images of 5 consecutive tumor sections to produce a 3D volume stack of 150 μm thickness. Information from the 3D volume is combined to produce one final slide by averaging. The same registration transform was applied to stack the pimonidazole images which were previously thresholded to highlight hypoxic regions. The registered IHC stack was then co-registered with a single thresholded T1 weighted gradient echo MRI slice of the same location (~156 μm thick) using an elastic B-splines transform. The same transform was applied to achieve the co-registration of pimonidazole and MR percentage enhancement image. Image similarity index after the co-registration was found to be greater than 0.5 for 5 of the animals suggesting good correlation. R2 values were calculated for both hypoxic regions as well as tumor boundaries. All the tumors showed a high boundary correlation value of R2 greater than 0.8. Half of the animals showed high R2 values greater than 0.5 for hypoxic fractions. The RMSE values for the co-registration of all the animals were found to be low further suggesting better correspondence and validating the MR based hypoxia imaging.
ContributorsSahu, Sulagna (Author) / Kodibagkar, Vikram D. (Thesis advisor) / Sadleir, Rosalind (Committee member) / Smith, Barbara (Committee member) / Arizona State University (Publisher)
Created2018
Description
This work describes efforts made toward the development of a compact, quantitative fluorescence-based multiplexed detection platform for point-of-care diagnostics. This includes the development of a microfluidic delivery and actuation system for multistep detection assays. Early detection of infectious diseases requires high sensitivity dependent on the precise actuation of fluids.
Methods of fluid actuation were explored to allow delayed delivery of fluidic reagents in multistep detection lateral flow assays (LFAs). Certain hydrophobic materials such as wax were successfully implemented in the LFA with the use of precision dispensed valves. Sublimating materials such as naphthalene were also characterized along with the implementation of a heating system for precision printing of the valves.
Various techniques of blood fractionation were also investigated and this work demonstrates successful blood fractionation in an LFA. The fluid flow of reagents was also characterized and validated with the use of mathematical models and multiphysics modeling software. Lastly intuitive, user-friendly mobile and desktop applications were developed to interface the underlying Arduino software. The work advances the development of a system which successfully integrates all components of fluid separation and delivery along with highly sensitive detection and a user-friendly interface; the system will ultimately provide clinically significant diagnostics in a of point-of-care device.
Methods of fluid actuation were explored to allow delayed delivery of fluidic reagents in multistep detection lateral flow assays (LFAs). Certain hydrophobic materials such as wax were successfully implemented in the LFA with the use of precision dispensed valves. Sublimating materials such as naphthalene were also characterized along with the implementation of a heating system for precision printing of the valves.
Various techniques of blood fractionation were also investigated and this work demonstrates successful blood fractionation in an LFA. The fluid flow of reagents was also characterized and validated with the use of mathematical models and multiphysics modeling software. Lastly intuitive, user-friendly mobile and desktop applications were developed to interface the underlying Arduino software. The work advances the development of a system which successfully integrates all components of fluid separation and delivery along with highly sensitive detection and a user-friendly interface; the system will ultimately provide clinically significant diagnostics in a of point-of-care device.
ContributorsArafa, Hany M (Author) / Blain Christen, Jennifer M (Thesis advisor) / Goryll, Michael (Committee member) / Smith, Barbara (Committee member) / Arizona State University (Publisher)
Created2018
Description
Severe cases of congenital heart defect (CHD) require surgeries to fix the structural problem, in which artificial grafts are often used. Although outcome of surgeries has improved over the past decades, there remains to be patients who require re-operations due to graft-related complications and the growth of patients which results in a mismatch in size between the patient’s anatomy and the implanted graft. A graft in which cells of the patient could infiltrate, facilitating transformation of the graft to a native-like tissue, and allow the graft to grow with the patient heart would be ideal. Cardiac tissue engineering (CTE) technologies, including extracellular matrix (ECM)-based hydrogels has emerged as a promising approach for the repair of cardiac damage. However, most of the previous studies have mainly focused on treatments for ischemic heart disease and related heart failure in adults, therefore the potential of CTE for CHD treatment is underexplored. In this study, a hybrid hydrogel was developed by combining the ECM derived from cardiac tissue of pediatric CHD patients and gelatin methacrylate (GelMA). In addition, the influence of incorporating gold nanorods (GNRs) within the hybrid hydrogels was studied. The functionalities of the ECM-GelMA-GNR hydrogels as a CTE scaffold were assessed by culturing neonatal rat cardiomyocytes on the hydrogel. After 8 days of cell culture, highly organized sarcomeric alpha-actinin structures and connexin 43 expression were evident in ECM- and GNR-incorporated hydrogels compared to pristine GelMA hydrogel, indicating cell maturation and formation of cardiac tissue. The findings of this study indicate the promising potential of ECM-GelMA-GNR hybrid hydrogels as a CTE approach for CHD treatment.
As another approach to improve CHD treatment, this study sought the possibility of performing a proteomic analysis on cardiac ECM of pediatric CHD patient tissue. As the ECM play important roles in regulating cell signaling, there is an increasing interest in studying the ECM proteome and the influences caused by diseases. Proteomics on ECM is challenging due to the insoluble nature of ECM proteins which makes protein extraction and digestion difficult. In this study, as a first step to perform proteomics, optimization on sample preparation procedure was attempted.
As another approach to improve CHD treatment, this study sought the possibility of performing a proteomic analysis on cardiac ECM of pediatric CHD patient tissue. As the ECM play important roles in regulating cell signaling, there is an increasing interest in studying the ECM proteome and the influences caused by diseases. Proteomics on ECM is challenging due to the insoluble nature of ECM proteins which makes protein extraction and digestion difficult. In this study, as a first step to perform proteomics, optimization on sample preparation procedure was attempted.
ContributorsSugamura, Yuka (Author) / Nikkhah, Mehdi (Thesis advisor) / Smith, Barbara (Committee member) / Willis, Brigham (Committee member) / Arizona State University (Publisher)
Created2018
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 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
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 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
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 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
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 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