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Salmonella enterica is a gastrointestinal (GI) pathogen that can cause systemic diseases. It invades the host through the GI tract and can induce powerful immune responses in addition to disease. Thus, it is considered as a promising candidate to use as oral live vaccine vectors. Scientists have been making great efforts to get a properly attenuated Salmonella vaccine strain for a long time, but could not achieve a balance between attenuation and immunogenicity. So the regulated delayed attenuation/lysis Salmonella vaccine vectors were proposed as a design to seek this balance. The research work is progressing steadily, but more improvements need to be made. As one of the possible improvements, the cyclic adenosine monophosphate (cAMP) -independent cAMP receptor protein (Crp*) is expected to protect the Crp-dependent crucial regulator, araC PBAD, in these vaccine designs from interference by glucose, which decreases synthesis of cAMP, and enhance the colonizing ability by and immunogenicity of the vaccine strains. In this study, the cAMP-independent crp gene mutation, crp-70, with or without araC PBAD promoter cassette, was introduced into existing Salmonella vaccine strains. Then the plasmid stability, growth rate, resistance to catabolite repression, colonizing ability, immunogenicity and protection to challenge of these new strains were compared with wild-type crp or araC PBAD crp strains using western blots, enzyme-linked immunosorbent assays (ELISA) and animal studies, so as to evaluate the effects of the crp-70 mutation on the vaccine strains. The performances of the crp-70 strains in some aspects were closed to or even exceeded the crp+ strains, but generally they did not exhibit the expected advantages compared to their wild-type parents. Crp-70 rescued the expression of araC PBAD fur from catabolite repression. The strain harboring araC PBAD crp-70 was severely affected by its slow growth, and its colonizing ability and immunogenicity was much weaker than the other strains. The Pcrp crp-70 strain showed relatively good ability in colonization and immune stimulation. Both the araC PBAD crp-70 and the Pcrp crp-70 strains could provide certain levels of protection against the challenge with virulent pneumococci, which were a little lower than for the crp+ strains.
ContributorsShao, Shihuan (Author) / Curtiss, Roy (Thesis advisor) / Arizona State University (Publisher)
Created2012
Description
It is important to consider factors that contribute to successful fertilization and the development of viable offspring. Better understanding the factors that contribute to infertility can be used to assist in the development of viable offspring, especially for human beings looking to successfully reproduce. Identifying paternal effect genes, genes that come from the father, introduces more targets that can be manipulated to produce specific reproductive effects. Use of Drosophila melanogaster as a model to study reproduction has increased, in part, due to the use of the GAL4 system. In this system, the GAL4 gene encodes an 881 amino acid protein that binds to the 4-site Upstream Activating Sequence (UAS) to induce transcription of the gene of interest. These sequences constitute the two components of the system: the driver (GAL4) and the responder (gene of interest) \u2014 each of which is maintained as a separate parental line. Effects of the GAL4 driver line "driving" transcription of the responder can be assessed by examining the offspring. One of the more common uses of the GAL4 system involves analyzing phenotypic effects of reducing or eliminating expression of a target gene through the induction of RNAi transcription, which often results in toxicity, lethality, or reduced viability. Utilizing these principles, we strove to demonstrate the effect of knocking down the expression of testis-specific sperm-leucyl-aminopeptidases gene CG13340 on progeny by inducing expression of RNAi with two distinct GAL4 driver lines - one with a nonspecific actin-binding activation sequence and the other with a testis-specific activation sequence. Comparison of both GAL4 driver lines to crosses using N01 wild type ("wt") flies verify that inducing RNAi transcription using the GAL4 system results in reduction of proper offspring development. Further studies using D. melanogaster and the GAL4 system can improve knowledge of factors contributing to male fertility and also be applied to better understand mammalian, specifically human, fertility.
ContributorsEvans, Donna Marie (Author) / Karr, Timothy L. (Thesis director) / Roland, Kenneth (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Department of English (Contributor)
Created2014-05
DescriptionA novel and unconventional approach for delivering a eukaryotic apoptosis factor, TNF-related apoptosis-inducing ligand (TRAIL), to cancer cells within and around necrotizing tumors by utilizing a S. Typhimurium purine requiring auxotroph as a biological vector to develop two anticancer therapies with multiple modality and broad economic feasibility.
ContributorsKoons, Andrew (Author) / Curtiss, Roy (Thesis director) / Lake, Douglas (Committee member) / Janthakahalli, Nagaraj Vinay (Committee member) / Barrett, The Honors College (Contributor)
Created2013-12
Description
Five immunocompetent C57BL/6-cBrd/cBrd/Cr (albino C57BL/6) mice were injected with GL261-luc2 cells, a cell line sharing characteristics of human glioblastoma multiforme (GBM). The mice were imaged using magnetic resonance (MR) at five separate time points to characterize growth and development of the tumor. After 25 days, the final tumor volumes of the mice varied from 12 mm3 to 62 mm3, even though mice were inoculated from the same tumor cell line under carefully controlled conditions. We generated hypotheses to explore large variances in final tumor size and tested them with our simple reaction-diffusion model in both a 3-dimensional (3D) finite difference method and a 2-dimensional (2D) level set method. The parameters obtained from a best-fit procedure, designed to yield simulated tumors as close as possible to the observed ones, vary by an order of magnitude between the three mice analyzed in detail. These differences may reflect morphological and biological variability in tumor growth, as well as errors in the mathematical model, perhaps from an oversimplification of the tumor dynamics or nonidentifiability of parameters. Our results generate parameters that match other experimental in vitro and in vivo measurements. Additionally, we calculate wave speed, which matches with other rat and human measurements.
ContributorsRutter, Erica (Author) / Stepien, Tracy (Author) / Anderies, Barrett (Author) / Plasencia, Jonathan (Author) / Woolf, Eric C. (Author) / Scheck, Adrienne C. (Author) / Turner, Gregory H. (Author) / Liu, Qingwei (Author) / Frakes, David (Author) / Kodibagkar, Vikram (Author) / Kuang, Yang (Author) / Preul, Mark C. (Author) / Kostelich, Eric (Author) / College of Liberal Arts and Sciences (Contributor)
Created2017-05-31
Description
Transorbital surgery has gained recent notoriety due to its incorporation into endoscopic skull base surgery. The body of published literature on the field is cadaveric and observation. The pre-clinical studies are focused on the use of the endoscope only. Furthermore the methodology utilised in the published literature is inconsistent and does not embody the optimal principles of scientific experimentation. This body of work evaluates a minimally invasive novel surgical corridor - the transorbital approach - its validity in neurosurgical practice, as well as both qualitatively and quantitatively assessing available technological advances in a robust experimental fashion. While the endoscope is an established means of visualisation used in clinical transorbital surgery, the microscope has never been assessed with respect to the transorbital approach. This question is investigated here and the anatomical and surgical benefits and limitations of microscopic visualisation demonstrated. The comparative studies provide increased knowledge on specifics pertinent to neurosurgeons and other skull base specialists when planning pre-operatively, such as pathology location, involved anatomical structures, instrument maneuvrability and the advantages and disadvantages of the distinct visualisation technologies. This is all with the intention of selecting the most suitable surgical approach and technology, specific to the patient, pathology and anatomy, so as to perform the best surgical procedure. The research findings illustrated in this body of work are diverse, reproducible and applicable. The transorbital surgical corridor has substantive potential for access to the anterior cranial fossa and specific surgical target structures. The neuroquantitative metrics investigated confirm the utility and benefits specific to the respective visualisation technologies i.e. the endoscope and microscope. The most appropriate setting wherein the approach should be used is also discussed. The transorbital corridor has impressive potential, can utilise all available technological advances, promotes multi-disciplinary co-operation and learning amongst clinicians and ultimately, is a means of improving operative patient care.
ContributorsHoulihan, Lena Mary (Author) / Preul, Mark C. (Thesis advisor) / Vernon, Brent (Thesis advisor) / O' Sullivan, Michael G.J. (Committee member) / Lawton, Michael T. (Committee member) / Santarelli, Griffin (Committee member) / Smith, Brian (Committee member) / Arizona State University (Publisher)
Created2021
Description
A description of numerical and analytical work pertaining to models that describe the growth and progression of glioblastoma multiforme (GBM), an aggressive form of primary brain cancer. Two reaction-diffusion models are used: the Fisher-Kolmogorov-Petrovsky-Piskunov equation and a 2-population model that divides the tumor into actively proliferating and quiescent (or necrotic) cells. The numerical portion of this work (chapter 2) focuses on simulating GBM expansion in patients undergoing treatment for recurrence of tumor following initial surgery. The models are simulated on 3-dimensional brain geometries derived from magnetic resonance imaging (MRI) scans provided by the Barrow Neurological Institute. The study consists of 17 clinical time intervals across 10 patients that have been followed in detail, each of whom shows significant progression of tumor over a period of 1 to 3 months on sequential follow up scans. A Taguchi sampling design is implemented to estimate the variability of the predicted tumors to using 144 different choices of model parameters. In 9 cases, model parameters can be identified such that the simulated tumor contains at least 40 percent of the volume of the observed tumor. In the analytical portion of the paper (chapters 3 and 4), a positively invariant region for our 2-population model is identified. Then, a rigorous derivation of the critical patch size associated with the model is performed. The critical patch (KISS) size is the minimum habitat size needed for a population to survive in a region. Habitats larger than the critical patch size allow a population to persist, while smaller habitats lead to extinction. The critical patch size of the 2-population model is consistent with that of the Fisher-Kolmogorov-Petrovsky-Piskunov equation, one of the first reaction-diffusion models proposed for GBM. The critical patch size may indicate that GBM tumors have a minimum size depending on the location in the brain. A theoretical relationship between the size of a GBM tumor at steady-state and its maximum cell density is also derived, which has potential applications for patient-specific parameter estimation based on magnetic resonance imaging data.
ContributorsHarris, Duane C. (Author) / Kuang, Yang (Thesis advisor) / Kostelich, Eric J. (Thesis advisor) / Preul, Mark C. (Committee member) / Crook, Sharon (Committee member) / Gardner, Carl (Committee member) / Arizona State University (Publisher)
Created2023
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
The emergence of invasive non-Typhoidal Salmonella (iNTS) infections belonging to sequence type (ST) 313 are associated with severe bacteremia and high mortality in sub-Saharan Africa. Distinct features of ST313 strains include resistance to multiple antibiotics, extensive genomic degradation, and atypical clinical diagnosis including bloodstream infections, respiratory symptoms, and fever. Herein, I report the use of dynamic bioreactor technology to profile the impact of physiological fluid shear levels on the pathogenesis-related responses of ST313 pathovar, 5579. I show that culture of 5579 under these conditions induces profoundly different pathogenesis-related phenotypes than those normally observed when cultures are grown conventionally. Surprisingly, in response to physiological fluid shear, 5579 exhibited positive swimming motility, which was unexpected, since this strain was initially thought to be non-motile. Moreover, fluid shear altered the resistance of 5579 to acid, oxidative and bile stress, as well as its ability to colonize human colonic epithelial cells. This work leverages from and advances studies over the past 16 years in the Nickerson lab, which are at the forefront of bacterial mechanosensation and further demonstrates that bacterial pathogens are “hardwired” to respond to the force of fluid shear in ways that are not observed during conventional culture, and stresses the importance of mimicking the dynamic physical force microenvironment when studying host-pathogen interactions. The results from this study lay the foundation for future work to determine the underlying mechanisms operative in 5579 that are responsible for these phenotypic observations.
ContributorsCastro, Christian (Author) / Nickerson, Cheryl A. (Thesis advisor) / Ott, C. Mark (Committee member) / Roland, Kenneth (Committee member) / Barrila, Jennifer (Committee member) / Arizona State University (Publisher)
Created2016
Description
Invasive salmonellosis caused by Salmonella enterica serovar Typhimurium ST313 is a major health crisis in sub-Saharan Africa, with multidrug resistance and atypical clinical presentation challenging current treatment regimens and resulting in high mortality. Moreover, the increased risk of spreading ST313 pathovars worldwide is of major concern, given global public transportation networks and increased populations of immunocompromised individuals (as a result of HIV infection, drug use, cancer therapy, aging, etc). While it is unclear as to how Salmonella ST313 strains cause invasive disease in humans, it is intriguing that the genomic profile of some of these pathovars indicates key differences between classic Typhimurium (broad host range), but similarities to human-specific typhoidal Salmonella Typhi and Paratyphi. In an effort to advance fundamental understanding of the pathogenesis mechanisms of ST313 in humans, I report characterization of the molecular genetic, phenotypic and virulence profiles of D23580 (a representative ST313 strain). Preliminary studies to characterize D23580 virulence, baseline stress responses, and biochemical profiles, and in vitro infection profiles in human surrogate 3-D tissue culture models were done using conventional bacterial culture conditions; while subsequent studies integrated a range of incrementally increasing fluid shear levels relevant to those naturally encountered by D23580 in the infected host to understand the impact of biomechanical forces in altering these characteristics. In response to culture of D23580 under these conditions, distinct differences in transcriptional biosignatures, pathogenesis-related stress responses, in vitro infection profiles and in vivo virulence in mice were observed as compared to those of classic Salmonella pathovars tested.
Collectively, this work represents the first characterization of in vivo virulence and in vitro pathogenesis properties of D23580, the latter using advanced human surrogate models that mimic key aspects of the parental tissue. Results from these studies highlight the importance of studying infectious diseases using an integrated approach that combines actions of biological and physical networks that mimic the host-pathogen microenvironment and regulate pathogen responses.
Collectively, this work represents the first characterization of in vivo virulence and in vitro pathogenesis properties of D23580, the latter using advanced human surrogate models that mimic key aspects of the parental tissue. Results from these studies highlight the importance of studying infectious diseases using an integrated approach that combines actions of biological and physical networks that mimic the host-pathogen microenvironment and regulate pathogen responses.
ContributorsYang, Jiseon (Author) / Nickerson, Cheryl A. (Thesis advisor) / Chang, Yung (Committee member) / Stout, Valerie (Committee member) / Ott, C Mark (Committee member) / Roland, Kenneth (Committee member) / Barrila, Jennifer (Committee member) / Arizona State University (Publisher)
Created2015
Description
Energy use within urban building stocks is continuing to increase globally as populations expand and access to electricity improves. This projected increase in demand could require deployment of new generation capacity, but there is potential to offset some of this demand through modification of the buildings themselves. Building stocks are quasi-permanent infrastructures which have enduring influence on urban energy consumption, and research is needed to understand: 1) how development patterns constrain energy use decisions and 2) how cities can achieve energy and environmental goals given the constraints of the stock. This requires a thorough evaluation of both the growth of the stock and as well as the spatial distribution of use throughout the city. In this dissertation, a case study in Los Angeles County, California (LAC) is used to quantify urban growth, forecast future energy use under climate change, and to make recommendations for mitigating energy consumption increases. A reproducible methodological framework is included for application to other urban areas.
In LAC, residential electricity demand could increase as much as 55-68% between 2020 and 2060, and building technology lock-in has constricted the options for mitigating energy demand, as major changes to the building stock itself are not possible, as only a small portion of the stock is turned over every year. Aggressive and timely efficiency upgrades to residential appliances and building thermal shells can significantly offset the projected increases, potentially avoiding installation of new generation capacity, but regulations on new construction will likely be ineffectual due to the long residence time of the stock (60+ years and increasing). These findings can be extrapolated to other U.S. cities where the majority of urban expansion has already occurred, such as the older cities on the eastern coast. U.S. population is projected to increase 40% by 2060, with growth occurring in the warmer southern and western regions. In these growing cities, improving new construction buildings can help offset electricity demand increases before the city reaches the lock-in phase.
In LAC, residential electricity demand could increase as much as 55-68% between 2020 and 2060, and building technology lock-in has constricted the options for mitigating energy demand, as major changes to the building stock itself are not possible, as only a small portion of the stock is turned over every year. Aggressive and timely efficiency upgrades to residential appliances and building thermal shells can significantly offset the projected increases, potentially avoiding installation of new generation capacity, but regulations on new construction will likely be ineffectual due to the long residence time of the stock (60+ years and increasing). These findings can be extrapolated to other U.S. cities where the majority of urban expansion has already occurred, such as the older cities on the eastern coast. U.S. population is projected to increase 40% by 2060, with growth occurring in the warmer southern and western regions. In these growing cities, improving new construction buildings can help offset electricity demand increases before the city reaches the lock-in phase.
ContributorsReyna, Janet Lorel (Author) / Chester, Mikhail V (Thesis advisor) / Gurney, Kevin (Committee member) / Reddy, T. Agami (Committee member) / Rey, Sergio (Committee member) / Arizona State University (Publisher)
Created2016