Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2013)

Studies of cell walls have long contributed to understanding plant function. Relevance to the production of biofuels from biomass has recently added urgency to the study of plant cell walls. Arabinogalactan proteins (AGPs) are highly glycosylated glycoproteins at the plant plasma membrane and cell wall. Consisting of a core polypeptide surrounded by glycans, AGPs contain abundant galactosyl and arabinosyl residues and often some glucuronosyl, rhamnosyl, or other residues. Previous study revealed that AGPs in the moss Physcomitrella patens contain unusual 3-O-methyl-rhamnosyl residues (3-O-Me-Rha) in amounts as high as 15 mole percent. The goals of this dissertation were to identify and evaluate Physcomitrella genes that are candidates to encode the rhamnosyl 3-O-methyltransferase that forms 3-O-Me-Rha. A Mycobacteria gene encoding a rhamnosyl 3-O-methyltransferase and an Arabidopsis gene encoding a glucuronosyl 4-O-methyltransferase were used as queries in bioinformatics searches of the Physcomitrella genome. Eleven candidate genes were identified and, through homologous recombination in Physcomitrella, targeted knockouts of these genes were generated. Based on reductions in 3-O-Me-Rha/Rha content ratio in AGPs of the knockouts, two genes,KO1 and KO9, were selected for further study. A third gene, KO11, was selected for further study because it shares a domain with the Arabidopsis glucuronosyl 4-O-methyltransferase. Further study of KO1 led to revision of its gene model. A phylogenic tree suggested that the revised KO1 protein contains a LpxB domain, which is characteristic of bacterial lipid A synthesis. The Physcomitrella ko1 knockout exhibited phenotypic effects including abnormal growth in protonema and rhizoids, reduced 3-O-Me-Rha/Rha in AGPs, and reduced lignin-like content in cell walls. Heterologous expression of KO1 did not produce detectable 3-O-Me-Rha in tobacco AGPs. Further bioinformatics study of KO9 showed it to be an ortholog of caffeoyl-CoA O-methyltransferase of lignin biosynthesis. The Physcomitrella ko9 knockout did not exhibit altered lignin-like content in its cell walls. Heterologous expression of KO9 in tobacco did not produce detectable 3-O-Me-Rha in tobacco AGPs. The Physcomitrella ko11 knockout exhibited no effect on 3-O-Me-Rha/Rha in AGPs, and no 3-O-Me-Rha was detected in AGPs from transgenic KO11 tobacco. It remains uncertain whether any of the candidate genes encodes rhamnosyl 3-O-methyltransferase in Physcomitrella.

    Cover page: Functional Analysis of Candidate Genes to Encode Rhamnosyl 3-O-Methyltransferase in the Moss Physcomitrella patens
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2016)
      Cover page: Unexpected nicotine in Do-it-Yourself electronic cigarette flavourings
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2004)

      Background

      It is known that "second-hand" cigarette smoke leads to abnormal tissue repair and remodelling but the cellular mechanisms involved in these adverse effects are not well understood. Fibroblasts play a major role in repair and remodelling. They orchestrate these processes by proliferating, migrating, and secreting proteins such as, cytokines, growth factors and extracellular matrix molecules. Therefore, we focus our studies on the effects of "second-hand" cigarette smoke on the structure and function of these cells.

      Results

      We used sidestream whole (SSW) smoke, a major component of "second-hand" smoke, primary embryonic fibroblasts, cells that behave very much like wound fibroblasts, and a variety of cellular and molecular approaches. We show that doses of smoke similar to those found in tissues cause cytoskeletal changes in the fibroblasts that may lead to a decrease in cell migration. In addition, we also show that these levels of cigarette smoke stimulate an increase in cell survival that is reflected in an increase and/or activation of stress/survival proteins such as cIL-8, grp78, PKB/Akt, p53, and p21. We further show that SSW affects the endomembrane system and that this effect is also accomplished by nicotine alone.

      Conclusions

      Taken together, our results suggest that: (i) SSW may delay wound repair because of the inability of the fibroblasts to migrate into the wounded area, leading to an accumulation of these cells at the edge of the wound, thus preventing the formation of the healing tissue; (ii) the increase in cell survival coupled to the decrease in cell migration can lead to a build-up of connective tissue, thereby causing fibrosis and excess scarring.
        Cover page: Effects of "second-hand" smoke on structure and function of fibroblasts, cells that are critical for tissue repair and remodeling
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2014)
        By 2025, more than 500 M people worldwide will suffer from diabetes; 125 M will develop foot ulcer(s) and 20 M will undergo an amputation, creating a major health problem. Understanding how these wounds become chronic will provide insights to reverse chronicity. We hypothesized that oxidative stress (OS) in wounds is a critical component for generation of chronicity. We used the db/db mouse model of impaired healing and inhibited, at time of injury, two major antioxidant enzymes, catalase and glutathione peroxidase, creating high OS in the wounds. This was necessary and sufficient to trigger wounds to become chronic. The wounds initially contained a polymicrobial community that with time selected for specific biofilm-forming bacteria. To reverse chronicity we treated the wounds with the antioxidants α-tocopherol and N-acetylcysteine and found that OS was highly reduced, biofilms had increased sensitivity to antibiotics, and granulation tissue was formed with proper collagen deposition and remodeling. We show for the first time generation of chronic wounds in which biofilm develops spontaneously, illustrating importance of early and continued redox imbalance coupled with the presence of biofilm in development of wound chronicity. This model will help decipher additional mechanisms and potentially better diagnosis of chronicity and treatment of human chronic wounds.
          Cover page: Generating and reversing chronic wounds in diabetic mice by manipulating wound redox parameters.
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2014)
          Chronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼$25B/year in the US alone. We previously discovered that a genetically modified mouse model displays impaired healing similar to problematic wounds in humans and that sometimes the wounds become chronic. Here we show how and why these impaired wounds become chronic, describe a way whereby we can drive impaired wounds to chronicity at will and propose that the same processes are involved in chronic wound development in humans. We hypothesize that exacerbated levels of oxidative stress are critical for initiation of chronicity. We show that, very early after injury, wounds with impaired healing contain elevated levels of reactive oxygen and nitrogen species and, much like in humans, these levels increase with age. Moreover, the activity of anti-oxidant enzymes is not elevated, leading to buildup of oxidative stress in the wound environment. To induce chronicity, we exacerbated the redox imbalance by further inhibiting the antioxidant enzymes and by infecting the wounds with biofilm-forming bacteria isolated from the chronic wounds that developed naturally in these mice. These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks. These findings are highly significant because they show for the first time that chronic wounds can be generated in an animal model effectively and consistently. The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue. Furthermore, the model can lead to the understanding of other fundamental mechanisms of chronic wound development that can potentially lead to novel therapies.
            Cover page: A Novel Model of Chronic Wounds: Importance of Redox Imbalance and Biofilm-Forming Bacteria for Establishment of Chronicity
            Top