Matching Items (26)
Description
The goal of this project was to design and create a genetic construct that would allow for <br/>tumor growth to be induced in the center of the wing imaginal disc of Drosophila larvae, the <br/>R85E08 domain, using a heat shock. The resulting transgene would be combined with other <br/>transgenes in a single fly that would allow for simultaneous expression of the oncogene and, in <br/>the surrounding cells, other genes of interest. This system would help establish Drosophila as a <br/>more versatile and reliable model organism for cancer research. Furthermore, pilot studies were <br/>performed, using elements of the final proposed system, to determine if tumor growth is possible <br/>in the center of the disc, which oncogene produces the best results, and if oncogene expression <br/>induced later in development causes tumor growth. Three different candidate genes were <br/>investigated: RasV12, PvrACT, and Avli.
ContributorsSt Peter, John Daniel (Author) / Harris, Rob (Thesis director) / Varsani, Arvind (Committee member) / School of Molecular Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Mycobacterium abscessus (Mabs) is a multidrug-resistant nontuberculous mycobacterium capable of causing persistent pulmonary infection. It most prominently threatens those with cystic fibrosis (CF), a progressive and genetic disorder characterized by an immunocompromised respiratory tract. Current treatments fail to eradicate Mabs, meaning novel alternatives are greatly needed. Antimicrobial peptides (AMPs) are short sequences of amino acids that display broad-spectrum antimicrobial activity and play an important role in innate immunity. To maximize their therapeutic potential, key AMP features can be rationally combined through an iterative engineering process to create synthetic, designed AMPs (dAMPs). In this investigation, two dAMPs, RP554 and RP557, reduced Mabs ATCC 19977 viability by 99.99% and were subjected to further testing. In antimicrobial susceptibility testing with Mabs ATCC 19977, RP554 and RP557 demonstrated bactericidal activity at concentrations 16-32 μM. Complete killing of Mabs ATCC 19977 by RP554 and RP557 occurred rapidly in <24 h. RP554 and RP557 also inhibited 20 Mabs clinical isolates obtained from CF patients. Furthermore, RP554 and RP557 retained anti-Mabs activity after pre-exposure to human serum, indicating potential stability in blood. Conversely, the tested dAMPs did not kill Mabs during in vitro experiments in an artificial sputum medium. Novel antimicrobials, such as the RP554 and RP557 dAMPs, offer therapeutic potential for otherwise resistant bacterial pathogens, including Mabs, that afflict both CF and non-CF patients.
ContributorsBrandt, McKenzie (Author) / Haydel, Shelley (Thesis director) / Bean, Heather (Committee member) / Dermody, Roslyn (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2022-05
Description
Coccidioidomycosis or Valley Fever (VF) is an emerging fungal respiratory infection endemic to the southwest region of the United States, and parts of Mexico, Central and South America. Satellite cases have also been reported in Washington and Oregon. It is estimated that in Maricopa County alone, VF accounts for 10-30% of community-acquired pneumonia. Difficulty in diagnosis is largely attributed to lack of antibody reactivity to antigens used in diagnosis, especially early in disease. Serological detection of VF employs mycelial-phase culture filtrates as antigen. While culture filtrates are thought to provide the most specific diagnostic antigen, preparation includes the growth of large volume Coccidioides cultures which require employment of extensive safety precautions in a BSL3 setting. An additional concern with use of culture filtrates as an antigen source is batch variability, as expression of immunogenic proteins within each lot are variable. To address safety and batch variability concerns, this thesis proposes the use of recombinant Coccidioides proteins as a consistent and reliable antigen source. For the purpose of this study, I expressed known antigenic Coccidioides proteins in a eukaryotic, recombinant protein expression system. Recombinant endochitinase-1 (rCTS1) and recombinant heat-labile antigen (rHL-Ag) were evaluated for serologic reactivity by ELISA, using a sample set of 55 known serologically positive and 55 known negative human sera specimens, previously tested in Mayo Clinic Arizona (MCA) serologic laboratories. Evaluation by ELISA demonstrated 94.55% sensitivity and 92.72% specificity using combined rCTS1 and rHL-Ag as an antigen source, indicating promising diagnostic utility.
ContributorsRoeder, Alexa Jordan (Author) / Lake, Douglas (Thesis advisor) / Grys, Thomas (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2020
Description
Biocrusts are microbial communities that inhabit arid soil surfaces, providing essential services to dryland ecosystems. A paradoxical filamentous cyanobacterium, Microcoleus vaginatus, resides within the biocrust. While is often pioneers the colonization of bare, nutrient-poor desert soils worldwide, it cannot fix dinitrogen. In nature, M. vaginatus coexists with a unique microbial community, a “cyanosphere”, that is characterized by a high abundance of diazotrophic heterotrophs. This suggests mutualistic relationships wherein nutrients are traded between phototrophs and heterotrophs. To explore these relationships, I performed targeted, pedigreed isolation of cyanosphere members and used co-cultivation to recreate the mutualism in culture. Results showed that, in the absence of fixed nitrogen, M. vaginatus grew well when co-cultured with cyanosphere diazotrophs, but only poorly or not at all when alone or with non-cyanosphere diazotrophs. In agreement with this, the experimental provision of nitrogen to natural populations resulted in a loss of diazotrophs from the cyanosphere compared to controls, but the addition of phosphorus did not. Additionally, the convergence of M. vaginatus trichomes into large bundles held by a common sheath was elicited in culture by the addition of cyanosphere diazotrophs, pointing to a role of cyanobacterial motility responses in the development of mutualistic interactions. I then demonstrated that the tendency of M. vaginatus to stay within bundles and close to the sheath-dwelling cyanosphere was dependent on the cyanosphere population size. This effect was likely mediated by glutamate that acted as a signaling molecule rather than as a N source and impacted the gliding speed and negative chemophobic responses on the cyanobacterium. Glutamate seems to be used as a cue to spatially optimize cyanobacterium-cyanosphere mutualistic exchanges. My findings have potential practical applications in restoration ecology, which I further pursued experimentally. Co-inoculation of soil with cyanosphere diazotrophs resulted in swifter development of biocrusts over inoculation with the cyanobacterium only. Further, their addition to disturbed native soils containing traces of cyanobacteria sufficed for the formation of cohesive biocrusts without cyanobacterial inoculation. The inclusion of such “biocrust probiotics” in biocrust restoration is recommended. Overall, this body of work elucidates the hitherto unknown role of beneficial heterotrophic bacteria in the initial formation and development of biocrusts.
ContributorsNelson, Corey (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Penton, C. Ryan (Committee member) / Gile, Gillian (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2021
Description
In chronic cystic fibrosis (CF) lung infections, Pseudomonas aeruginosa (PA) evolves into bacterial communities with a complex array of genetic mutations. Mutations in lasR, encoding the LasR quorum sensing (QS) response regulator, are the most common PA mutations in chronic CF lung infections. The fitness of lasR mutants are enhanced by interactions with wild-type strains through the exchange of soluble metabolites. We are investigating whether volatile metabolites play a role in interactions between ∆lasR and wild-type strains of PA. Previous experiments in our lab demonstrated that volatile metabolites produced by wild-type PA strains inhibit swarming, a QS-regulated behavior. We hypothesize that the inhibitory volatiles are under QS-control, and therefore volatiles produced by PA14 QS mutants would not alter behaviors in wild-type PA strains. To test this hypothesis we exposed PA (strain PA14) inoculated onto swarming agar to the volatiles produced by wild-type PA14, to PA14 QS mutants, and to uninoculated agar as a negative control. Depending on which QS mutant we tested for the ability to inhibit swarming, we observed a range of swarming phenotypes, from fully inhibited to full swarming. We further hypothesize that volatile interactions between PA strains form a communication feedback loop that gives the volatile metabolomes emergent properties. To test this hypothesis, we analyzed volatile metabolites from co-cultures using two-dimensional gas chromatography. We observed novel metabolites produced in co-cultures that are not observed in mono-cultures. Investigating the volatile interactions of PA strains will provide insight into how complex PA communities interact within the CF lung.
ContributorsThallikar, Kiran (Author) / Bean, Heather (Thesis director) / Misra, Rajeev (Committee member) / Gutiérrez-Muñoz, Daniela (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2024-12
Genetic Ablation Systems of the Drosophila Imaginal Wing Disc: Building and Testing the TASER System
Description
Drosophila melanogaster, commonly known as the fruit fly, is widely used in genetic research due to its ease of genetic manipulation and well characterized genome. One of its structures, the imaginal wing disc, has played a key role in uncovering the mechanisms underlying tissue regeneration. Damage is typically induced via physical, irradiation, or genetic ablation to study these regenerative processes. Recently, genetic techniques have been developed to cause targeted damage in situ by triggering cell death through the expression of pro-apoptotic or necrotic genes. The GAL4/UAS system allows for precise, tissue-specific cell death by the induction of a heat shift, an exposure of a higher temperature for an extended period of time. However, this 1st system is unable to express genes of interest in the wing disc to determine how other factors influence the regenerative process. Therefore, another approach was created, known as DUAL (Duration and Location) control, to allow the induction of both cell death as well as gene expression in the surrounding cells. The DUAL control system is regulated with exposure to higher temperature for a shorter period, referred to as a heat shock. The limitation of this 2nd system is its inability to temporally separate the timing of cell death from gene expression. This lack of control over timing highlights the need for a newer ablation system. In this work, we chose to create a novel system, termed the Temporal Ablation with Separation of Expression Regulation (TASER) system. The TASER system uses both heat shift and heat shock to provide refined control over the timing of cell ablation and gene expression. The system was developed by crossing specific Drosophila stocks that include genetic components like GAL80ts and GAL4tp, allowing for differing temporal regulation. We confirmed that the system is functional at separating the gene expression from ablation when the heat shock precedes the heat shift. However, initiating the heat shift first led to not only ablation, but also premature gene expression due to prolonged high temperatures. Despite this drawback, the TASER system holds significant potential in providing a more precise temporal control in regeneration studies, making it a valuable tool for future research.
ContributorsRogler, Isabel (Author) / Harris, Rob (Thesis director) / Bean, Heather (Committee member) / Huijben, Silvie (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Department of Psychology (Contributor) / School of International Letters and Cultures (Contributor)
Created2024-12
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
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 etiological agent of the tuberculosis disease, is estimated to infect one-fourth of the human population and is responsible for 1.5 million deaths annually. The increased emergence of bacterial resistance to clinical interventions highlights the lack in development of novel antimicrobial therapeutics. Prototypical bacterial two-component systems (TCS) allow for sensing of extracellular stimuli and relay thereof to create a transcriptional response. The prrAB TCS is essential for viability in Mtb, presenting itself as an attractive novel drug target. In Mtb, PrrAB is involved in the adaptation to the intra-macrophage environment and recent work implicates PrrAB in the dosR-dependent hypoxia adaptation. This work defines a direct molecular and regulatory connection between Mtb PrrAB and the dosR-dependent hypoxia response. Using electrophoretic mobility shift assays combined with surface plasmon resonance, the Mtb dosR gene is established as a specific target of PrrA, corroborated by fluorescence reporter assays demonstrating a regulatory relationship. Considering the scarce understanding of prrAB essentiality in nontuberculous mycobacteria and the presence of multiple prrAB orthologs in Mycobacterium smegmatis and Mycobacterium abscessus, CRISPR interference was utilized to evaluate the essentiality of PrrAB beyond Mtb. prrAB was found to be inessential for viability in M. smegmatis yet required for in vitro growth. Conversely, M. abscessus prrAB repression led to enhanced in vitro growth. Diarylthiazole-48 (DAT-48) displayed decreased selectivity against M. abscessus but demonstrated enhanced intrinsic activity upon prrAB repression in M. abscessus. Lastly, to aid in the rapid determination of mycobacterial drug susceptibility and the detection of mycobacterial heteroresistance, the large volume scattering imaging (LVSim) platform was adapted for mycobacteria. Using LVSim, Mtb drug susceptibility was detected phenotypically within 6 hours, and clinically relevant mycobacterial heteroresistance was detected phenotypically within 10 generations. The data generated in these studies provide insight into the essential role of PrrAB in Mtb and its involvement in the dosR-dependent hypoxia adaptation, advance the understanding of mycobacterial PrrAB essentiality and PrrAB-associated mycobacterial growth dependency. These studies further establish molecular and mechanistic connection between PrrAB and DAT-48 in Mtb and M. abscessus and develop a rapid phenotypic drug susceptibility testing platform for mycobacteria.
ContributorsHaller, Yannik Alex (Author) / Haydel, Shelley E (Thesis advisor) / Bean, Heather (Committee member) / Nickerson, Cheryl (Committee member) / Plaisier, Christopher (Committee member) / Acharya, Abhinav (Committee member) / Arizona State University (Publisher)
Created2023
Description
To combat the global antimalarial resistance crisis effective resistance management strategies are needed. To do so, I need to gain a better understanding of the ecological interactions occurring within malaria infections. Despite the importance of the complex interplay among co-infecting strains, our current knowledge and empirical data of within-host diversity and malaria disease dynamics is limited. In this thesis, I explore the multifaceted dynamics of malaria infections through an ecological lens. My overall research question is: "How do ecological interactions, including niche complementarity, competition dynamics, and the cost of resistance, shape the outcomes of malaria infections, and what implications does this have on understanding and improving resistance management strategies?” In Chapter II, titled “Niche Complementarity in Malaria Infections” I demonstrate that ecological principles are observed in malarial infections by experimentally manipulating the biodiversity of rodent malaria P. chabaudi infections. I observed that some parasites experienced competitive suppression, others experienced competitive facilitation, while others were not impacted. Next, in Chapter III, titled “Determining the Differential Impact of Competition Between Genetically Distinct Plasmodium falciparum Strains” I investigate the differential effect of competition among six genetically distinct strains. The impact of competition varied between strain combinations, and both suppression and facilitation were observed, but most pairings had no competitive interactions. Lastly, in Chapter IV, titled “Assessing Fitness Costs in Malaria Parasites: A Comprehensive Review and Implications for Drug Resistance Management”, I summarize where the field currently stands and what evidence there is for the presence of a fitness cost, or lack thereof, and I highlight the current gaps in knowledge. I found that evidence from field, in vitro, and animal models are overall suggestive of the presence of a fitness cost, however, these costs were not always found. Amid the current focus on malaria eradication, it is crucial to understand the impact of biodiversity on disease severity. By incorporating an ecological approach to infectious disease systems, I can gain insights on within-host interactions and how they impact parasite fitness and transmissibility.
ContributorsSegovia, Xyonane (Author) / Huijben, Silvie (Thesis advisor) / Bean, Heather (Committee member) / Gile, Gillian (Committee member) / Hogue, Ian (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2024