Matching Items (27)
Description
Synechocystis sp PCC 6803 is a photosynthetic cyanobacterium that can be easily transformed to produce molecules of interest; this has increased Synechocystis’ popularity as a clean energy platform. Synechocystis has been shown to produce and excrete molecules such as fatty acids, isoprene, etc. after appropriate genetic modification. Challenges faced for large–scale growth of modified Synechocystis include abiotic stress, microbial contamination and high processing costs of product and cell material. Research reported in this dissertation contributes to solutions to these challenges. First, abiotic stress was addressed by overexpression of the heat shock protein ClpB1. In contrast to the wild type, the ClpB1 overexpression mutant (Slr1641+) tolerated rapid temperature changes, but no difference was found between the strains when temperature shifts were slower. Combination of ClpB1 overexpression with DnaK2 overexpression (Slr1641+/Sll0170+) further increased thermotolerance. Next, we used a Synechocystis strain that carries an introduced isoprene synthase gene (IspS+) and that therefore produces isoprene. We attempted to increase isoprene yields by overexpression of key enzymes in the methyl erythritol phosphate (MEP) pathway that leads to synthesis of the isoprene precursor. Isoprene production was not increased greatly by MEP pathway induction, likely because of limitations in the affinity of the isoprene synthase for the substrate. Finally, two extraction principles, two–phase liquid extraction (e.g., with an organic and aqueous phase) and solid–liquid extraction (e.g., with a resin) were tested. Two–phase liquid extraction is suitable for separating isoprene but not fatty acids from the culture medium. Fatty acid removal required acidification or surfactant addition, which affected biocompatibility. Therefore, improvements of both the organism and product–harvesting methods can contribute to enhancing the potential of cyanobacteria as solar–powered biocatalysts for the production of petroleum substitutes.
ContributorsGonzalez Esquer, Cesar Raul (Author) / Vermaas, Willem (Thesis advisor) / Chandler, Douglas (Committee member) / Bingham, Scott (Committee member) / Nielsen, David (Committee member) / Arizona State University (Publisher)
Created2013
Description
In somatic cells, the mitotic spindle apparatus is centrosomal and several isoforms of Protein Kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is unclear. Other protein kinases such as, Glycogen Synthase Kinase 3â (GSK3â) also have been shown to be associated with the mitotic spindle. In the study in chapter 2, we show the enrichment of active (phosphorylated) PKCæ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases, PKC and GSK3â are associated with the mitotic spindle, first, the co-localization and close molecular proximity of PKC isoforms with GSK3â was studied in metaphase cells. Second, the involvement of inactive GSK3â in maintaining an intact mitotic spindle was shown. Third, this study showed that addition of a phospho-PKCæ specific inhibitor to cells can disrupt the mitotic spindle microtubules. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCæ acting through GSK3â. The MAPK pathway has been implicated in various functions related to cell cycle regulation. MAPKK (MEK) is part of this pathway and the extracellular regulated kinase (ERK) is its known downstream target. GSK3â and PKCæ also have been implicated in cell cycle regulation. In the study in chapter 3, we tested the effects of inhibiting MEK on the activities of ERK, GSK3â, PKCæ, and á-tubulin. Results from this study indicate that inhibition of MEK did not inhibit GSK3â and PKCæ enrichment at the centrosomes. However, the mitotic spindle showed a reduction in the pixel intensity of microtubules and also a reduction in the number of cells in each of the M-phase stages. A peptide activation inhibitor of ERK was also used. Our results indicated a decrease in mitotic spindle microtubules and an absence of cells in most of the M-phase stages. GSK3â and PKCæ enrichment were however not inhibited at the centrosomes. Taken together, the kinases GSK3â and PKCæ may not function as a part of the MAPK pathway to regulate the mitotic spindle.
ContributorsChakravadhanula, Madhavi (Author) / Capco, David G. (Thesis advisor) / Chandler, Douglas (Committee member) / Clark-Curtiss, Josephine (Committee member) / Newfeld, Stuart (Committee member) / Arizona State University (Publisher)
Created2012
Description
The F1Fo ATP synthase is required for energy conversion in almost all living organisms. The F1 complex is a molecular motor that uses ATP hydrolysis to drive rotation of the γ–subunit. It has not been previously possible to resolve the speed and position of the γ–subunit of the F1–ATPase as it rotates during a power stroke. The single molecule experiments presented here measured light scattered from 45X91 nm gold nanorods attached to the γ–subunit that provide an unprecedented 5 μs resolution of rotational position as a function of time. The product of velocity and drag, which were both measured directly, resulted in an average torque of 63±8 pN nm for the Escherichia coli F1-ATPase that was determined to be independent of the load. The rotational velocity had an initial (I) acceleration phase 15° from the end of the catalytic dwell, a slow (S) acceleration phase during ATP binding/ADP release (15°–60°), and a fast (F) acceleration phase (60°–90°) containing an interim deceleration (ID) phase (75°–82°). High ADP concentrations decreased the velocity of the S phase proportional to 'ADP-release' dwells, and the F phase proportional to the free energy derived from the [ADP][Pi]/[ATP] chemical equilibrium. The decreased affinity for ITP increased ITP-binding dwells by 10%, but decreased velocity by 40% during the S phase. This is the first direct evidence that nucleotide binding contributes to F1–ATPase torque. Mutations that affect specific phases of rotation were identified, some in regions of F1 previously considered not to contribute to rotation. Mutations βD372V and γK9I increased the F phase velocity, and γK9I increased the depth of the ID phase. The conversion between S and F phases was specifically affected by γQ269L. While βT273D, βD305E, and αR283Q decreased the velocity of all phases, decreases in velocity due to βD302T, γR268L and γT82A were confined to the I and S phases. The correlations between the structural locations of these mutations and the phases of rotation they affect provide new insight into the molecular basis for F1–ATPase γ-subunit rotation.
ContributorsMartin, James (Author) / Frasch, Wayne D (Thesis advisor) / Chandler, Douglas (Committee member) / Gaxiola, Roberto (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2012
Description
Malaria is a vector-borne parasitic disease affecting tropical and subtropical regions. Regardless control efforts, malaria incidence is still incredible high with 219 million clinical cases and an estimated 660,000 related deaths (WHO, 2012). In this project, different population genetic approaches were explored to characterize parasite populations. The goal was to create a framework that considered temporal and spatial changes of Plasmodium populations in malaria surveillance. This is critical in a vector borne disease in areas of low transmission where there is not accurate information of when and where a patient was infected. In this study, fragment analysis data and single nucleotide polymorphism (SNPs) from South American samples were used to characterize Plasmodium population structure, patterns of migration and gene flow, and discuss approaches to differentiate reinfection vs. recrudescence cases in clinical trials. A Bayesian approach was also applied to analyze the Plasmodium population history by inferring genealogies using microsatellites data. Specifically, fluctuations in the parasite population and the age of different parasite lineages were evaluated through time in order to relate them with the malaria control plan in force. These studies are important to understand the turnover or persistence of "clones" circulating in a specific area through time and consider them in drug efficacy studies. Moreover, this methodology is useful for assessing changes in malaria transmission and for more efficiently manage resources to deploy control measures in locations that act as parasite "sources" for other regions. Overall, these results stress the importance of monitoring malaria demographic changes when assessing the success of elimination programs in areas of low transmission.
ContributorsChenet, Stella M (Author) / Escalante, Ananias A (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Rosenberg, Michael (Committee member) / Taylor, Jesse E (Committee member) / Arizona State University (Publisher)
Created2014
Description
Proper cell growth and differentiation requires the integration of multiple signaling pathways that are maintained by various post-translational modifications. Many proteins in signal transduction pathways are conserved between humans and model organisms. My dissertation characterizes four previously unknown manners of regulation in the Drosophila Decapentaplegic (Dpp) pathway, a pathway within TGF-beta family. First, I present data that the Dpp signal transducer, Mothers Against Dpp (Mad), is phosphorylated by Zeste-white 3 (Zw3), a kinase involved in the Wingless pathway. This phosphorylation event occurs independently of canonical phosphorylation of Mad by the Dpp receptor. Using ectopic expression of different alleles of Mad, I show that Zw3 phosphorylation of Mad occurs during the cell cycle in pro-neuronal cells and the loss of phosphorylation of Mad by Zw3 results in ectopic neuronal cells. Thus, Mad phosphorylation by Zw3 is necessary for cell cycle control in pro-neuronal cells. Second, I have shown that the regulator dSno, which has previously been shown to be a TGF-beta antagonist and agonist, is also a Wingless pathway antagonist. Loss of function flip-out clones and ectopic expression of dSno both resulted in changes of Wingless signaling. Further analysis revealed that dSno acts at or below the level of Armadillo (Arm) to inhibit target gene expression. Third, I have demonstrated that the protein Bonus, which is known to be involved in chromatin modification, is required in dorsal-ventral patterning. Further experiments discovered that the chromatin modifier is not only a necessary Dpp agonist, but it is also necessary for nuclear localization of Dorsal during Toll signaling. Last, I showed that longitudinal lacking-like (lola-like) is also required in dorsal-ventral patterning. The loss of maternally expressed lola-like prevents dpp transcription. This shows that lola-like is integral in the Dpp pathway. The study of these four proteins integrates different signaling pathways, demonstrating that the process of development is a web of connections rather than a linear pathway.
ContributorsQuijano, Janine C (Author) / Newfeld, Stuart J (Thesis advisor) / Goldstein, Elliott (Committee member) / Chandler, Douglas (Committee member) / Capco, David (Committee member) / Arizona State University (Publisher)
Created2014
Description
Engineered nanoparticles (NP; 10-9 m) have found use in a variety of consumer goods and medical devices because of the unique changes in material properties that occur when synthesized on the nanoscale. Although many definitions for nanoparticle exist, from the perspective of size, nanoparticle is defined as particles with diameters less than 100 nm in any external dimension. Examples of their use include titanium dioxide added as a pigment in products intended to be ingested by humans, silicon dioxide NPs are used in foods as an anticaking agent, and gold or iron oxide NPs can be used as vectors for drug delivery or contrast agents for specialized medical imaging. Although the intended use of these NPs is often to improve human health, it has come to the attention of investigators that NPs can have unintended or even detrimental effects on the organism. This work describes one such unintended effect of NP exposure from the perspective of exposure via the oral route. First, this Dissertation will explain an event referred to as brush border disruption that occurred after nanoparticles interacted with an in vitro model of the human intestinal epithelium. Second, this Dissertation will identify and characterize several consumer goods that were shown to contain titanium dioxide that are intended to be ingested. Third, this Dissertation shows that sedimentation due to gravity does not artifactually result in disruption of brush borders as a consequence of exposure to food grade titanium dioxide in vitro. Finally, this Dissertation will demonstrate that iron oxide nanoparticles elicited similar effects after exposure to an in vitro brush border expressing model of the human placenta. Together, these data suggest that brush border disruption is not an artifact of the material/cell culture model, but instead represents a bona fide biological response as a result of exposure to nanomaterial.
ContributorsFaust, James J (Author) / Capco, David G. (Thesis advisor) / Ugarova, Tatiana (Committee member) / Chandler, Douglas (Committee member) / Baluch, Page (Committee member) / Herman, Richard (Committee member) / Arizona State University (Publisher)
Created2014
Description
The viscous lung mucus of cystic fibrosis (CF) patients is characterized by oxygen gradients, which creates a unique niche for bacterial growth. Pseudomonas aeruginosa and Staphylococcus aureus, two predominant microorganisms chronically infecting the airways of CF patients, typically localize in hypoxic regions of the mucus. While interspecies interactions between P. aeruginosa and S. aureus have been reported, little is known about the role of low oxygen in regulating these interactions. Studying interspecies interactions in CF lung disease is important as evidence suggests that microbial community composition governs disease progression. In this study, P. aeruginosa lab strain PAO1 and two primary clinical isolates from hypoxic tissues were cultured alone, or in combination, with methicillin resistant S. aureus (MRSA) strain N315 under hypoxic or normoxic conditions. Herein, it is shown for the first time that low oxygen conditions relevant to the CF lung affect the competitive behavior between P. aeruginosa and S. aureus. Specifically, S. aureus was able to better survive competition in hypoxic versus normoxic conditions. Competition data from different oxygen concentrations were consistent using PAO1 and clinical isolates even though differences in the level of competition were observed. PAO1 strains carrying mutations in virulence factors known to contribute to S. aureus competition (pyocyanin/phzS, elastase/lasA and lasI quorum sensing/lasI) were used to determine which genes play a role in the differential growth inhibition. The lasA and lasI mutants competed less effectively with S. aureus regardless of the oxygen level present in the culture compared to the isogenic wild type strain. These results are consistent with previous findings that elastase and lasI quorum sensing play a role in competitive behavior of P. aeruginosa and S. aureus. Interestingly, the phzS mutant competed less effectively in hypoxic conditions suggesting that pyocyanin may be important in microaerophilic conditions. This study demonstrates that oxygen plays a role in competition between P. aeruginosa and S. aureus and contributes to understanding CF environmental factors that may regulate microbial community dynamics important for disease progression with potential for development of therapeutic avenues.
ContributorsLedesma Barrera, Maria Alexandra (Author) / Nickerson, Cheryl A. (Thesis advisor) / Reyes del Valle, Jorge (Committee member) / Clark-Curtiss, Josephine (Committee member) / Stout, Valerie (Committee member) / Ott, C M (Committee member) / Arizona State University (Publisher)
Created2014
Description
The Philadelphia chromosome in humans, is on oncogenic translocation between chromosomes 9 and 22 that gives rise to the fusion protein BCR-Abl. This protein is constitutively active resulting in rapid and uncontrolled cell growth in affected cells. The BCR-Abl protein is the hallmark feature of chronic myeloid leukemia (CML) and is seen in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cases. Currently, the first line of treatment is the Abl specific inhibitor Imatinib. Some patients will, however, develop resistance to Imatinib. Research has shown how transformation of progenitor B cells with v-Abl, an oncogene expressed by the Abelson murine leukemia virus, causes rapid proliferation, prevents further differentiation and produces a potentially malignant transformation. We have used progenitor B cells transformed with a temperature-sensitive form of the v-Abl protein that allows us to inactivate or re-activate v-Abl by shifting the incubation temperature. We are trying to use this line as a model to study both the progression from pre-malignancy to malignancy in CML and Imatinib resistance in Ph+ ALL and CML. These progenitor B cells, once v-Abl is reactivated, in most cases, will not return to their natural cell cycle. In this they resemble Ph+ ALL and CML under Imatinib treatment. With some manipulation these cells can break this prolonged G1 arrested phenotype and become a malignant cell line and resistant to Imatinib treatment. Cellular senescence can be a complicated process requiring inter-play between a variety of players. It serves as an alternate option to apoptosis, in that the cell loses proliferative potential, but does not die. Treatment with some cancer therapeutics will induce senescence in some cancers. Such is the case with Imatinib treatment of CML and Ph+ ALL. By using the S9 cell line we have been able to explore the possible routes for breaking of prolonged G1 arrest in these Ph+ leukemias. We inhibited the DNA damage sensor protein ataxia telangiectasia mutated (ATM) and found that prolonged G1 arrest in our S9 cells was broken. While previous research has suggested that the DNA damage sensor protein ataxia-telangiectasia mutated (ATM) has little impact in CML, our research indicates that ATM may play a role in either senescence induction or release.
ContributorsDixon, Sarah E (Author) / Chang, Yung (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Touchman, Jeffrey (Committee member) / Arizona State University (Publisher)
Created2011
Description
Teleosts have the most primitive adaptive immune system. However, in terms of functionality the teleost immune system is similar to birds and mammals. On the other hand, enteric bacterial pathogens of mammals and birds present conserved regulatory mechanisms that control virulence factors. In this context, deletion of conserved genes that control virulence factors have been successfully used as measure to construct live attenuated bacterial vaccines for mammals and birds. Here, I hypothesize that evolutionary conserved genes, which control virulence factors or are essential for bacterial physiology in Enterobacteriaceae, could be used as universal tools to design live attenuated recombinant bacterial vaccines from fish to mammals. The evolutionary conserved genes that control virulence factors, crp and fur, and the essential gene for the synthesis of the cell wall, asd, were studied in Edwardsiella ictaluri to develop a live recombinant vaccine for fish host. The genus Edwardsiella is one of the most ancient represent of the Enterobacteriaceae family. E. ictaluri, a host restricted pathogen of catfish (Ictalurus punctatus), is the causative agent of the enteric septicemia and one of the most important pathogens of this fish aquaculture. Although, crp and fur control different virulence factors in Edwardsiella, in comparison to other enterics, individual deletion of these genes triggered protective immune response at the systemic and mucosal level of the fish. Deletion of asdA gene allowed the creation of a balanced-lethal system to syntheses heterologous antigens. I concluded that crp, fur and asd could be universally used to develop live attenuate recombinant Enterobacteriaceae base vaccines for different hosts.
ContributorsSantander Morales, Javier Alonso (Author) / Curtiss, Roy Iii (Thesis advisor) / Chandler, Douglas (Committee member) / Chang, Yung (Committee member) / Shi, Yixin (Committee member) / Arizona State University (Publisher)
Created2012
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