Matching Items (46)
Description
Glioblastoma multiforme is the most common and aggressive primary malignant brain tumor in adults, exhibiting a median survival of only 15 months after diagnosis. A significant challenge in treating GBM is the ability of glioma cells to invade normal brain tissue, escape surgical resection, and resist radiotherapy and chemotherapy. We have previously demonstrated that the TWEAK-Fn14 signaling axis plays an important role in glioma cell invasion and discovered a small molecule, L524-0366, that specifically disrupts the TWEAK-Fn14 interaction. However, low affinity limits L524-0366’s clinical feasibility. By utilizing structure-activity relationship analyses of L524-0366, we identified additional small molecules that may inhibit TWEAK-Fn14 signaling. Here, we identify five additional novel Fn14 signaling inhibitors that specifically inhibited TWEAK-Fn14 NF-κB-dependent signaling and suppressed TWEAK-induced glioma cell migration. Furthermore, we demonstrate that two molecules exhibit improved affinity for Fn14, two molecules showed binding to the TWEAK ligand but not Fn14, and one showed no binding to either TWEAK or Fn14. These molecules will be further tested for in vitro and in vivo functionality, and serve as foundations for additional medicinal chemistry for drug modifications.
ContributorsMillard, Nghia Patrick (Author) / Misra, Rajeev (Thesis director) / Chang, Yung (Committee member) / Tran, Nhan (Committee member) / School of Life Sciences (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Translating research has been a goal of the Department of Health and Human Services since 1999. Through two years of iteration and interview with our community members, we have collected insights into the barriers to accomplishing this goal. Liberating Science is a think-tank of researchers and scientists who seek to create a more transparent process to accelerate innovation starting with behavioral health research.
ContributorsRaghani, Pooja Sioux (Author) / Hekler, Eric (Thesis director) / Buman, Matthew (Committee member) / Pruthi, Virgilia Kaur (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Biomedical Informatics Program (Contributor)
Created2014-05
Description
Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E) protein as an immunogen to be vectored by a measles virus (MV) vaccine. However the domain III (DIII) of the dengue 2 E protein is too small to be immunogenic by itself. In order for it to be displayed on a larger particle, it was inserted into the amino terminus of small hepatitis B surface antigen (HBsAg, S) coding sequence. To generate the recombinant MV vector and verify the efficiency of this concept, a reverse genetics system was used where the MV vectors express one or two additional transcription units to direct the assembly of hybrid HBsAg particles. Two types of recombinant measles virus were produced: pB(+)MVvac2(DIII-S,S)P and pB(+)MVvac2(DIII-S)N. Virus recovered from pB(+)MVvac2(DIII-S,S)P was viable. An ELISA assay was performed to demonstrate the expression and secretion of HBsAg. Supernatant from MVvac2(DIII-S,S)P infected cells confirmed that hybrid HBsAg-domain III particles with a density similar to traditional HBsAg particles were released. Characteristics of the subviral particle have been analyzed for the successful incorporation of domain III. The replication fitness of the recombinant MV was evaluated using multi-step growth kinetics and showed reduced replication fitness when compared to the parental strain MVvac2. This demonstrates that viral replication is hindered by the addition of the two inserts into MV genome. Further analysis of MVvac2(DIII-S)N is needed to justify immune response studies in a small animal model using both of the generated recombinant vectors.
ContributorsHarahap, Indira Saridewi (Author) / Reyes del Valle, Jorge (Thesis director) / Hogue, Brenda (Committee member) / Misra, Rajeev (Committee member) / Barrett, The Honors College (Contributor) / T. Denny Sanford School of Social and Family Dynamics (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Methane (CH4) is very important in the environment as it is a greenhouse gas and important for the degradation of organic matter. During the last 200 years the atmospheric concentration of CH4 has tripled. Methanogens are methane-producing microbes from the Archaea domain that complete the final step in breaking down organic matter to generate methane through a process called methanogenesis. They contribute to about 74% of the CH4 present on the Earth's atmosphere, producing 1 billion tons of methane annually. The purpose of this work is to generate a preliminary metabolic reconstruction model of two methanogens: Methanoregula boonei 6A8 and Methanosphaerula palustris E1-9c. M. boonei and M. palustris are part of the Methanomicrobiales order and perform hydrogenotrophic methanogenesis, which means that they reduce CO2 to CH4 by using H2 as their major electron donor. Metabolic models are frameworks for understanding a cell as a system and they provide the means to assess the changes in gene regulation in response in various environmental and physiological constraints. The Pathway-Tools software v16 was used to generate these draft models. The models were manually curated using literature searches, the KEGG database and homology methods with the Methanosarcina acetivorans strain, the closest methanogen strain with a nearly complete metabolic reconstruction. These preliminary models attempt to complete the pathways required for amino acid biosynthesis, methanogenesis, and major cofactors related to methanogenesis. The M. boonei reconstruction currently includes 99 pathways and has 82% of its reactions completed, while the M. palustris reconstruction includes 102 pathways and has 89% of its reactions completed.
ContributorsMahendra, Divya (Author) / Cadillo-Quiroz, Hinsby (Thesis director) / Wang, Xuan (Committee member) / Stout, Valerie (Committee member) / Barrett, The Honors College (Contributor) / Computing and Informatics Program (Contributor) / School of Life Sciences (Contributor) / Biomedical Informatics Program (Contributor)
Created2014-05
Description
This study focused on the connection between the EnvZ/OmpR two-component regulatory system and the iron homeostasis system in Escherichia coli, specifically how a mutant form of EnvZ11/OmpR is able to reduce the expression of fepA::lacZ, a reporter gene fusion in E. coli. FepA is one of several outer membrane siderophore receptors that allow extracellular siderophores bound to iron to enter the cells to power various biological processes. Previous studies have shown that in E. coli cells that expressed a mutant allele of envZ, called envZ11, which led to altered expression of various iron genes including down regulation of fepA::lacZ. The wild type EnvZ/OmpR system is not considered to regulate iron genes, but because these envz11 strains had downregulated fepA::lacZ, this study was undertaken to understand the connection and mechanisms of this downregulation. A large number of Lac+ revertants were obtained from the B32-2483 strain (envz11 and fepA::lacZ) and 7 Lac+ revertants that had reversion mutations not directly correcting the envZ11 allele were further characterized. With P1 phage transduction genetic mapping that involved moving a kanamycin resistance marker linked to fepA::lacZ, two Lac+ revertants were found to have their reversion mutations in the fepA promoter region, while the other five revertants had their mutations mapping outside the fepA region. These two revertants underwent DNA sequencing and found to carry two different single base pair mutations in two different locations of the fepA promoter region. Each one is in the Fur repressor binding region, but one also may have affected the Shine-Dalgarno region involved in translation initiation. All 7 reveratants underwent beta-galactosidase assays to measure fepA::lacZ expression. The two revertants that had mutations in the fepA promoter region had significantly increased fepA activity, with the revertant with the Shine-Dalgarno mutation having the most elevated fepA expression. The other 5 revertants that did not map in the fepA region had fepA expression elevated to the same level as that found in the wild type EnvZ/OmpR background. The data suggest that the negative effect of envZ11 can be overcome by multiple mechanisms, including directly correcting the envZ11 allele or changing the fepA promoter region.
ContributorsKalinkin, Victor Arkady (Co-author) / Misra, Rajeev (Co-author, Thesis director) / Mason, Hugh (Committee member) / Foy, Joseph (Committee member) / Biomedical Informatics Program (Contributor) / School of Life Sciences (Contributor) / W. P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Little is known about the diversity and role of bacteriophages in carbon (C) rich ecosystems such as peatlands in tropical and temperate regions. In fact, there is no currently published assessment of phage abundance on diversity in a key tropical ecosystem such as Amazon peatlands. To better understand phage assemblages in terrestrial ecosystems and how bacteriophages influence organic C cycling to final products like CO2 and CH4, phage communities and phage-like particles were recovered, quantified, and viable phage particles were enriched from pore water from contrasting Amazon peatlands. Here we present the first results on assessing Amazon bacteriophages on native heterotrophic bacteria. Several steps to test for methodological suitability were taken. First, the efficiency of iron flocculation method was determined using fluorescent microscopy counts of phage TLS, a TolC-specific and LPS-specific bacteriophage, and Escherichia coli host pre- and post-extraction method. One-hundred percent efficiency and 0.15% infectivity was evidenced. Infectivity effects were determined by calculating plaque forming units pre and post extraction method. After testing these methods, fieldwork in the Amazon peatlands ensued, where phages were enriched from pore water samples. Phages were extracted and concentrated by in tandem filtering rounds to remove organic matter and bacteria, and then iron flocculation to bind the phages and allow for precipitation onto a filter. Phage concentrates were then used for overall counts, with fluorescent microscopy, as well as phage isolation attempts. Phage isolations were performed by first testing for lysis of host cells in liquid media using OD600 absorbance of cultures with and without phage concentrate as well as attempts with the cross-streaking methods. Forty-five heterotrophic bacterial isolates obtained from the same Amazon peatland were challenged with phage concentrates. Once a putative host was found, steps were taken to further propagate and isolate the phage. Several putative phages were enriched from Amazon peatland pore water and require further characterization. TEM imaging was taken of two phages isolated from two plaques. Genomes of selected phages will be sequenced for identification. These results provide the groundwork for further characterizing the role bacteriophage play in C cycling and greenhouse gas production from Amazon peatland soils.
ContributorsSpring, Jessica Lynette (Author) / Cadillo-Quiroz, Hinsby (Thesis director) / Haydel, Shelley (Committee member) / Misra, Rajeev (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
The spread of antibiotic resistant bacteria is currently a pressing global health concern, especially considering the prevalence of multi-drug resistance. Efflux pumps, bacterial machinery involved in various active transport functions, are capable of removing a broad range of antibiotics from the periplasmic space and the outer leaflet of the inner membrane, frequently conferring multi-drug resistance. Many aspects of efflux machinery’s structure, functions, and inter-protein interactions are still not fully understood; further characterization of these components of efflux will provide a strong foundation for combating this resistance mechanism. In this project, I further characterize the channel protein TolC as a part of the AcrAB-TolC efflux pump complex in Escherichia coli by first determining the specificity of compensatory mutations in TolC against defective AcrA and AcrB, and then identifying TolC residues that might influence TolC aperture dynamics or stability when altered. Specificity of compensatory mutations was determined using an array of TolC mutants, previously generated from defective AcrA or AcrB, against a different mutant AcrB protein; these new mutant combinations were then analyzed by real-time efflux and antibiotic susceptibility assays. A vancomycin susceptible TolC mutant—a phenotype that has been associated with constitutively open TolC channels—was then used to generate vancomycin-resistant revertants which were evaluated with DNA sequencing, protein quantification by Western blots, and real-time efflux assays to identify residues important for TolC aperture dynamics and protein stability and complex activity. Mutations identified in revertant strains corresponded to residues located in the lower half of the periplasmic domain of TolC; generally, these revertants had poorer efflux than wild-type TolC in the mutant AcrB background, and all revertants had poorer efflux activity than the parental mutant strain.
ContributorsMcFeely, Megan Elizabeth (Author) / Misra, Rajeev (Thesis director) / Haydel, Shelley (Committee member) / Stout, Valerie (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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 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.
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
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 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
Description
The study of bacterial resistance to antimicrobial peptides (AMPs) is a significant area of interest as these peptides have the potential to be developed into alternative drug therapies to combat microbial pathogens. AMPs represent a class of host-mediated factors that function to prevent microbial infection of their host and serve as a first line of defense. To date, over 1,000 AMPs of various natures have been predicted or experimentally characterized. Their potent bactericidal activities and broad-based target repertoire make them a promising next-generation pharmaceutical therapy to combat bacterial pathogens. It is important to understand the molecular mechanisms, both genetic and physiological, that bacteria employ to circumvent the bactericidal activities of AMPs. These understandings will allow researchers to overcome challenges posed with the development of new drug therapies; as well as identify, at a fundamental level, how bacteria are able to adapt and survive within varied host environments. Here, results are presented from the first reported large scale, systematic screen in which the Keio collection of ~4,000 Escherichia coli deletion mutants were challenged against physiologically significant AMPs to identify genes required for resistance. Less than 3% of the total number of genes on the E. coli chromosome was determined to contribute to bacterial resistance to at least one AMP analyzed in the screen. Further, the screen implicated a single cellular component (enterobacterial common antigen, ECA) and a single transporter system (twin-arginine transporter, Tat) as being required for resistance to each AMP class. Using antimicrobial resistance as a tool to identify novel genetic mechanisms, subsequent analyses were able to identify a two-component system, CpxR/CpxA, as a global regulator in bacterial resistance to AMPs. Multiple previously characterized CpxR/A members, as well as members found in this study, were identified in the screen. Notably, CpxR/A was found to transcriptionally regulate the gene cluster responsible for the biosynthesis of the ECA. Thus, a novel genetic mechanism was uncovered that directly correlates with a physiologically significant cellular component that appears to globally contribute to bacterial resistance to AMPs.
ContributorsWeatherspoon-Griffin, Natasha (Author) / Shi, Yixin (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Misra, Rajeev (Committee member) / Nickerson, Cheryl (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2013