Matching Items (20)
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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

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
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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

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
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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

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
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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

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
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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

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
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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

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
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Description
Though for most of the twentieth century, dogma held that the adult brain was post-mitotic, it is now known that adult neurogenesis is widespread among vertebrates, from fish, amphibians, reptiles and birds to mammals including humans. Seasonal changes in adult neurogenesis are well characterized in the song control system of

Though for most of the twentieth century, dogma held that the adult brain was post-mitotic, it is now known that adult neurogenesis is widespread among vertebrates, from fish, amphibians, reptiles and birds to mammals including humans. Seasonal changes in adult neurogenesis are well characterized in the song control system of song birds, and have been found in seasonally breeding mammals as well. In contrast to more derived vertebrates, such as mammals, where adult neurogenesis is restricted primarily to the olfactory bulb and the dentate gyrus of the hippocampus, neurogenesis is widespread along the ventricles of adult amphibians. I hypothesized that seasonal changes in adult amphibian brain cell proliferation and survival are a potential regulator of reproductive neuroendocrine function. Adult, male American bullfrogs (Rana catesbeiana; aka Lithobates catesbeianus), were maintained in captivity for up to a year under season-appropriate photoperiod. Analysis of hormone levels indicated seasonal changes in plasma testosterone concentration consistent with field studies. Using the thymidine analogue 5-bromo-2-deoxyuridine (BrdU) as a marker for newly generated cells, two differentially regulated aspects of brain cell neogenesis were tracked; that is, proliferation and survival. Seasonal differences were found in BrdU labeling in several brain areas, including the olfactory bulb, medial pallium, nucleus accumbens and the infundibular hypothalamus. Clear seasonal differences were also found in the pars distalis region of the pituitary gland, an important component of neuroendocrine pathways. BrdU labeling was also examined in relation to two neuropeptides important for amphibian reproduction: arginine vasotocin and gonadotropin releasing hormone. No cells co-localized with BrdU and either neuropeptide, but new born cells were found in close proximity to neuropeptide-containing neurons. These data suggest that seasonal differences in brain and pituitary gland cell neogenesis are a potential neuroendocrine regulatory mechanism.
ContributorsMumaw, Luke (Author) / Orchinik, Miles (Thesis advisor) / Deviche, Pierre (Committee member) / Chandler, Douglas (Committee member) / Arizona State University (Publisher)
Created2012
Description
Skeletal muscle (SM) mitochondria generate the majority of adenosine triphosphate (ATP) in SM, and help regulate whole-body energy expenditure. Obesity is associated with alterations in SM mitochondria, which are unique with respect to their arrangement within cells; some mitochondria are located directly beneath the sarcolemma (i.e., subsarcolemmal (SS) mitochondria), while

Skeletal muscle (SM) mitochondria generate the majority of adenosine triphosphate (ATP) in SM, and help regulate whole-body energy expenditure. Obesity is associated with alterations in SM mitochondria, which are unique with respect to their arrangement within cells; some mitochondria are located directly beneath the sarcolemma (i.e., subsarcolemmal (SS) mitochondria), while other are nested between the myofibrils (i.e., intermyofibrillar (IMF) mitochondria). Functional and proteome differences specific to SS versus IMF mitochondria in obese individuals may contribute to reduced capacity for muscle ATP production seen in obesity. The overall goals of this work were to (1) isolate functional muscle SS and IMF mitochondria from lean and obese individuals, (2) assess enzyme activities associated with the electron transport chain and ATP production, (3) determine if elevated plasma amino acids enhance SS and IMF mitochondrial respiration and ATP production rates in SM of obese humans, and (4) determine differences in mitochondrial proteome regulating energy metabolism and key biological processes associated with SS and IMF mitochondria between lean and obese humans.

Polarography was used to determine functional differences in isolated SS and IMF mitochondria between lean (37 ± 3 yrs; n = 10) and obese (35 ± 3 yrs; n = 11) subjects during either saline (control) or amino acid (AA) infusions. AA infusion increased ADP-stimulated respiration (i.e., coupled respiration), non-ADP stimulated respiration (i.e., uncoupled respiration), and ATP production rates in SS, but not IMF mitochondria in lean (n = 10; P < 0.05). Neither infusion increased any of the above parameters in muscle SS or IMF mitochondria of the obese subjects.

Using label free quantitative mass spectrometry, we determined differences in proteomes of SM SS and IMF mitochondria between lean (33 ± 3 yrs; n = 16) and obese (32 ± 3 yrs; n = 17) subjects. Differentially-expressed mitochondrial proteins in SS versus IMF mitochondria of obese subjects were associated with biological processes that regulate: electron transport chain (P<0.0001), citric acid cycle (P<0.0001), oxidative phosphorylation (P<0.001), branched-chain amino acid degradation, (P<0.0001), and fatty acid degradation (P<0.001). Overall, these findings show that obesity is associated with redistribution of key biological processes within the mitochondrial reticulum responsible for regulating energy metabolism in human skeletal muscle.
ContributorsKras, Katon Anthony (Author) / Katsanos, Christos (Thesis advisor) / Chandler, Douglas (Committee member) / Dinu, Valentin (Committee member) / Mor, Tsafrir S. (Committee member) / Arizona State University (Publisher)
Created2017
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Description
Cell-free protein synthesis (CFPS) is becoming an increasingly popular method of in vitro protein expression for biotechnology applications. However, there is still no comprehensive resource that outlines the most effective lysate and template combinations for efficient eukaryotic CFPS. To address this issue, expression vectors were constructed and assayed in order

Cell-free protein synthesis (CFPS) is becoming an increasingly popular method of in vitro protein expression for biotechnology applications. However, there is still no comprehensive resource that outlines the most effective lysate and template combinations for efficient eukaryotic CFPS. To address this issue, expression vectors were constructed and assayed in order to determine their activity within three commercial eukaryotic CFPS systems: Wheat Germ Extract (WGE), Rabbit Reticulocyte Lysate (RRL), and HeLa Cell Lysate (HCL). Previously in the Chaput lab, a luciferase reporter vector was expressed in each lysate system, testing different template variables impacting protein expression, including the 5' UTR sequence, presence of poly(A) tail, and DNA type. It was found that plasmid DNA templates generally yielded ~500-fold greater amount of protein than linear DNA templates and the inclusion of a poly(A) tail did not significantly increase protein expression in the plasmid systems. Additionally, the incorporation of a viral translation enhancing sequence into the 5' UTR increased translation in a lysate-specific manner. The HCL system had a strong preference for the EMCV sequence, WGE had a preference for the sequences from AMV and TMV, and RRL showed no specific preference. Overall, the HCL-EMCV system generated the greatest amount of protein per volume, producing 10-fold more protein than the second best template-lysate combination tested. Here, four human genes fused with a c-Myc tag were expressed in each lysate using the EMCV 5' UTR sequence in order to test the generality of the previous results. Protein synthesis was assayed using a luciferase construct with a c-Myc tag to recapitulate the previous luminometer data and western blotting of the human proteins. These analyses showed the same EMCV expression trends across all systems, with the HCL system synthesizing the greatest amount of each protein. In the future, when choosing commercial eukaryotic CFPS systems for gene expression, these template variables should be considered when performing cost analysis for cell-free protein production.
ContributorsHartsough, Emily Mae (Author) / Chaput, John (Thesis director) / Chandler, Douglas (Committee member) / Larsen, Andrew (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Arts, Media and Engineering (Contributor)
Created2015-05
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Description
Birds have unusually high plasma glucose concentrations compared to mammals of similar size despite their high metabolic rate. While birds use lipids as their main source of energy, it is still unclear how and why they maintain high plasma glucose concentrations. To investigate a potential underlying mechanism, this study looks

Birds have unusually high plasma glucose concentrations compared to mammals of similar size despite their high metabolic rate. While birds use lipids as their main source of energy, it is still unclear how and why they maintain high plasma glucose concentrations. To investigate a potential underlying mechanism, this study looks at the role of lipolysis in glucose homeostasis. The purpose of this study is to examine the effects of decreased glycerol availability (through inhibition of lipolysis) on plasma glucose concentrations in mourning doves. The hypothesis is that decreased availability of glycerol will result in decreased production of glucose through gluconeogenesis leading to reduced plasma glucose concentrations. In the morning of each experiment, mourning doves were collected at the Arizona State University Tempe campus, and randomized into either a control group (0.9% saline) or experimental group (acipimox, 50mg/kg BM). Blood samples were collected prior to treatment, and at 1, 2, and 3 hours post-treatment. At 3 hours, doves were euthanized, and tissue samples were collected for analysis. Acipimox treatment resulted in significant increases in blood glucose concentrations at 1 and 2 hours post- treatment as well as renal triglyceride concentrations at 3 hours post-treatment. Change in plasma free glycerol between 0h and 3h followed an increasing trend for the acipimox treated animals, and a decreasing trend in the saline treated animals. These results do not support the hypothesis that inhibition of lipolysis should decrease blood glycerol and blood glucose levels. Rather, the effects of acipimox in glucose homeostasis appear to differ significantly between birds and mammals suggesting differing mechanisms for glucose homeostasis.
ContributorsKouteib, Soukaina (Author) / Sweazea, Karen (Thesis director) / Deviche, Pierre (Committee member) / Chandler, Douglas (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05