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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 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
Glioblastoma (GBM) is the most common primary brain tumor with an incidence of approximately 11,000 Americans. Despite decades of research, average survival for GBM patients is a modest 15 months. Increasing the extent of GBM resection increases patient survival. However, extending neurosurgical margins also threatens the removal of eloquent brain. For this reason, the infiltrative nature of GBM is an obstacle to its complete resection. We hypothesize that targeting genes and proteins that regulate GBM motility, and developing techniques that safely enhance extent of surgical resection, will improve GBM patient survival by decreasing infiltration into eloquent brain regions and enhancing tumor cytoreduction during surgery. Chapter 2 of this dissertation describes a gene and protein we identified; aquaporin-1 (aqp1) that enhances infiltration of GBM. In chapter 3, we describe a method for enhancing the diagnostic yield of GBM patient biopsies which will assist in identifying future molecular targets for GBM therapies. In chapter 4 we develop an intraoperative optical imaging technique that will assist identifying GBM and its infiltrative margins during surgical resection. The topic of this dissertation aims to target glioblastoma infiltration from molecular and cellular biology and neurosurgical disciplines. In the introduction we; 1. Provide a background of GBM and current therapies. 2. Discuss a protein we found that decreases GBM survival. 3. Describe an imaging modality we utilized for improving the quality of accrued patient GBM samples. 4. We provide an overview of intraoperative contrast agents available for neurosurgical resection of GBM, and discuss a new agent we studied for intraoperative visualization of GBM.
ContributorsGeorges, Joseph F (Author) / Feuerstein, Burt G (Thesis advisor) / Smith, Brian H. (Thesis advisor) / Van Keuren-Jensen, Kendall (Committee member) / Deviche, Pierre (Committee member) / Bennett, Kevin (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
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
Reproduction is energetically costly and seasonal breeding has evolved to capitalize on predictable increases in food availability. The synchronization of breeding with periods of peak food availability is especially important for small birds, most of which do not store an extensive amount of energy. The annual change in photoperiod is the primary environmental cue regulating reproductive development, but must be integrated with supplementary cues relating to local energetic conditions. Photoperiodic regulation of the reproductive neuroendocrine system is well described in seasonally breeding birds, but the mechanisms that these animals use to integrate supplementary cues remain unclear. I hypothesized that (a) environmental cues that negatively affect energy balance inhibit reproductive development by acting at multiple levels along the reproductive endocrine axis including the hypothalamus (b) that the availability of metabolic fuels conveys alterations in energy balance to the reproductive system. I investigated these hypotheses in male house finches, Haemorhous mexicanus, caught in the wild and brought into captivity. I first experimentally reduced body condition through food restriction and found that gonadal development and function are inhibited and these changes are associated with changes in hypothalamic gonadotropin-releasing hormone (GnRH). I then investigated this neuroendocrine integration and found that finches maintain reproductive flexibility through modifying the release of accumulated GnRH stores in response to energetic conditions. Lastly, I investigated the role of metabolic fuels in coordinating reproductive responses under two different models of negative energy balance, decreased energy intake (food restriction) and increased energy expenditure (high temperatures). Exposure to high temperatures lowered body condition and reduced food intake. Reproductive development was inhibited under both energy challenges, and occurred with decreased gonadal gene expression of enzymes involved in steroid synthesis. Minor changes in fuel utilization occurred under food restriction but not high temperatures. My results support the hypothesis that negative energy balance inhibits reproductive development through multilevel effects on the hypothalamus and gonads. These studies are among the first to demonstrate a negative effect of high temperatures on reproductive development in a wild bird. Overall, the above findings provide important foundations for investigations into adaptive responses of breeding in energetically variable environments.
ContributorsValle, Shelley (Author) / Deviche, Pierre (Thesis advisor) / McGraw, Kevin (Committee member) / Orchinik, Miles (Committee member) / Propper, Catherine (Committee member) / Sweazea, Karen (Committee member) / Arizona State University (Publisher)
Created2018