Thesis (Ph.D.)--Texas Christian University, 2007. / Title from dissertation title page (viewed Sept. 11, 2007). Includes abstract. Includes bibliographical references.

Francisella tularensis and Francisella novicida are closely related facultative intracellular pathogens capable of survival and growth within macrophages. In this work we present evidence to show that F. tularensis uses phase variation to alter lipopolysaccharide (LPS) antigenicity, macrophage nitric oxide (NO) production, and microbial intramacrophage growth. The LPS and lipid A of F. tularensis LVS fail to stimulate production of significant levels of nitric oxide by rat macrophage monolayers. However, spontaneous variants of F. tularensis expressing an antigenically distinct LPS induce rat macrophages to produce increased levels of NO, thereby suppressing intracellular growth. This new form of LPS produced by F. tularensis is also the predominant form of LPS found normally in F. novicida. Rat macrophages infected with F. novicida produce high levels of NO and exhibit suppression of intracellular growth. LPS and lipid A isolated from F. novicida and variants of F. tularensis stimulate increased levels of NO production. In addition, a reverse phase shift can occur which returns the LPS of the F. tularensis variants to the original antigenic form, resulting in reduced macrophage NO production and restoration of intracellular growth. These results suggest that F. tularensis can modulate macrophage NO production through phase variation of its LPS. It was of interest to initiate a study that would ultimately characterize the molecular mechanism of LPS phase variation in Francisella tularensis . To this end, we used shuttle mutagenesis to create a mutant library of F. novicida. We mutagenized a size- restricted plasmid library of F. novicida with the erythromycin- resistant transposon TnMax2. Putative F. novicida LPS mutants created by shuttle mutagenesis were screened visually for aberrant colony phenotypes on agar plates. Of 10464 mutants screened, 5 unique F. novicida LPS mutants were isolated which exhibit three distinct LPS phenotypes as determined by Western immunoblot. A single mutant from each of the three phenotypic groups was further characterized with respect to DNA sequence analysis, intramacrophage growth, and sensitivity to detergent and serum complement. Furthermore, these three loci were shown to hybridize with a corresponding locus in F. tularensis LVS. However, there was no difference in the restriction pattern of the hybridizing bands between LVS and its LPS phase variants, thus indicating that no major genetic rearrangements or insertion/deletion of a large mobile genetic element occurs in these genes during the phase variation process of F. tularensis. The F. novicida valAB locus has previously been cloned, sequenced, and shown to be functionally homologous to the E. coli genes msbA/lpxK. In order to investigate the hypothesis that valAB is involved in transport of LPS to the cell surface, an E. coli strain harboring an NTG-mutagenized temperature sensitive (t.s.) allele of valAB, a nonfunctional copy of msbA/lpxK, and an IPTG-inducible copy of the gene encoding the Chlamydia trachomatis genus-specific LPS epitope (gseA) was constructed. In this study, DNA sequencing was used to locate the temperature sensitive mutations in the valAB locus. Two C to T transitions were found in the valA coding region which result in a S to F change at amino acid 543 and a T to I change at amino acid 458. The ability of E. coli cells harboring this t.s. copy of valAB to transport the Chlamydia LPS epitope across the inner membrane at the permissive and non-permissive temperatures was determined using sucrose density gradient centrifugation and ELISA. It was determined that there was increased association of the LPS epitope with the inner membrane at the non-permissive temperature, thus suggesting that ValA is required for transport of an LPS precursor across the inner membrane. / Graduate

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Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains x, 130 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 120-130).

Thesis (M.S.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains ix, 75 p. : ill. (some col.) Vita. Includes abstract. Includes bibliographical reference.

Thesis (Ph. D.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains ix, 132 p. : ill. (some col.) Vita. Includes abstract. Includes bibliographical references.

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of Gram-negative bacteria and plays a fundamental role in protecting the bacteria from harsh environments and toxic compounds. The LPS transport system is responsible for transporting LPS from the periplasmic side of the inner membrane (IM) to the OM, in a process involving seven LptA-LptG proteins. The current model for lipopolysaccharide transport (Lpt) suggests that LPS is initially extracted by a four-protein complex, LptBCFG, from the inner membrane to the periplasm, where LptA mediates further transport to the OM. Another two protein complex, LptD/E, catalyses the assembly of LPS at the OM cell surface. However, the details of this transport mechanism have remained unknown, mainly due to a lack of structural information. In chapter 1 and 2 of this thesis, I report materials and methods for all LptD/E, and Schmallenberg virus (SBV) nucleoprotein (NP) experiments and the theories and software that were used in determining structures of LptD/E, SBV NP and the SBV NP/RNA complex. In chapter 3 of this thesis, I report the first crystal structure of the outer membrane protein LptD/E complex. LptD forms a 26-strand ß-barrel in a closed form and LptE is a roll-like structure located inside LptD to form “barrel and plug” architecture. Through structural analysis, function assay and molecular dynamics simulation, we proposed a mechanism in which the hydrophilic head of LPS molecule, including the oligosaccharide core and the O antigen, directly penetrates through the hydrophilic ß- barrel whilst the hydrophobic lipid A tail is inserted into an intramembrane hole, with a lateral opening between strand ß1 and ß26 of the LptD. LptE may assist this process. In chapter 4, I report the crystal structure of the SBV NP in two conformations: tetrameric when the protein was purified under native conditions, and trimeric when denatured and refolded during purification. The SBV NP has a novel fold and we have also identified that the N-terminal arm is crucial for RNA binding, and the N- and the C-terminal arm is essential for RNA multimerisation with adjacent protomers and for viral RNA encapsidation. Chapter 5 describes the crystal structure of SBV NP in complex with a 42 nucleotide long RNA (polyU). This ribonucleoprotein (RNP) complex was crystallized as a ring-like tetramer with each protomer bound to 11 ribonucleotides. Eight of these nucleotides are bound in a positively charged cleft between N- and C- terminal domains and three are bound in the N-terminal arm. I also compared the structure to that of other NPs from negative-sense RNA viruses, and found that SBV NP sequesters RNA using a different mechanism. Furthermore, the structure suggests that when RNA binds the protein, there are conformational changes in the RNA-binding cleft, and in the N- and C-terminal arms. Thus our results reveal a novel mechanism of RNA encapsidation by orthobunyaviruses NP.

M.Sc. (Biochemistry) / During microbial invasion, a variety of defense responses are induced in host plants. In order for host plants to combat potential diseases induced by microbes, they must be equipped with pattern recognition receptors (PRRs) localized at the cell surface, since such receptors enable the perception of conserved microbial epitopes termed microbe/pathogen-associated molecular patterns (M/PAMPs), thereby resulting in the activation of plant innate immunity via M/PAMP-triggered immunity (P/MTI). Lipopolysaccharide (LPS) is the major component of the outer leaflet of the external membrane of Gram-negative bacteria. This thermo-stable lipoglycan is exposed towards the external environment and plays an important role in bacterial adaptation to external surroundings. LPS is recognized as a major M/PAMP in plants, and thus potentiates or elicits defense-related responses such as the production of antimicrobial compounds and the expression of immune response genes. One of the most widely investigated effects of LPS on plants is its ability to prevent and/or suppress the hypersensitive response (HR) induced by an array of bacteria. The HR is a programmed cell death response which ends in a local necrosis of plant tissue, thereby resulting in a reduced number of viable bacteria that can further promote disease progression in the host.

Orientador: Rogério de Castilho Jacinto / Coorientador: Frederico Canato Martinho / Banca: Eloi Dezan Junior / Banca: Giselle Prisiclla Cruz Abi Rached / Resumo: O objetivo deste estudo clínico foi avaliar a eficácia de dois sistemas rotatórios: HyFlex CM e ProTaper Next na remoção de bactérias cultiváveis e endotoxinas de canais radiculares infectados. Vinte e quatro canais radiculares de molares e pré-molares com necrose pulpar e lesão periapical foram selecionados e divididos aleatoriamente em 2 grupos: HyFlex CM (n = 12); e ProTaper Next (n = 12). As amostras foram coletadas antes e após o preparo biomecânico e inoculadas em frascos específicos. A irrigação foi realizada com hipoclorito de sódio a 2,5%. Um teste turbidimétrico LAL (Pyrogent 5000 - Lonza, Walkersville, MD, EUA) foi utilizado para quantificar endotoxinas. Cultura microbiológica foi utilizada para determinar a contagem de unidades formadoras de colônias bacterianas (UFC/mL). Os dados coletados foram analisados estatisticamente usando SigmaPlot 12.0 para Windows (Systat Software Inc., San Jose, CA). Foi realizado o teste estatístico de Two-Way ANOVA e o nível de significância foi de 5%. Nas coletas antes do preparo biomecânico, bactérias cultiváveis e endotoxinas foram evidenciadas em 100% das amostras. A análise de cultura revelou que não houve uma diferença estatisticamente significativa na redução bacteriana entre os dois sistemas de instrumentação. As endotoxinas estavam presentes em 100% dos canais após a instrumentação e não houve diferença estatística entre os dois sistemas na redução de endotoxinas. Assim, concluímos que ambos os sistemas de instrumentação for... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this clinical study was to compare the effectiveness of two rotary systems: HyFlex CM and ProTaper Next on the removal of cultivable bacteria and endotoxins from primarily infected root canals. Twenty-four root canals of molars and premolars with pulp necrosis were selected and randomly divided into 2 groups: HyFlex CM (n = 12); and ProTaper Next (n = 12). Samples were collected before and after the biomechanical preparation and inoculated in specific flasks. Irrigation was performed using 2.5% sodium hypochlorite. A kinetic turbidimetric lysate assay of limulus amoebocytes was used to quantify endotoxins. Microbiological culture was used to determine the count of bacterial colony forming units (CFU/mL). Data collected were statistically analyzed using SigmaPlot 12.0 for Windows (Systat Software Inc, San Jose, CA). The Two-Way ANOVA statistical test was performed and the level of significance was 5%. In the samples before the biomechanical preparation, cultivable bacteria and endotoxins were evidenced in 100% of the cases. The culture analysis revealed that there was no statistically significant difference in the bacterial reduction between the two instrumentation systems. Endotoxins were present in 100% of the canals after instrumentation, and there was no statistical difference between the two systems in endotoxin reduction. Thus, it was concluded that both instrumentation systems were effective in reducing root canal bacteria and endotoxins with primary endodont... (Complete abstract click electronic access below) / Mestre