Thesis (M.S.)--University of Wisconsin--Madison. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 88-95).

Thesis advisor: Christina Faherty / Shigella, a Gram-negative bacterial pathogen, induces inflammation and diarrhea by invading the colonic epithelium. Annually, millions of Shigella infections occur globally, mainly in malnourished children. Despite extensive research, no effective vaccine exists. This work explores the mechanisms of Shigella proliferation before colonic infection, where an adverse environment is encountered, including bile salts exposure. One means of bile salts evasion is possibly lipopolysaccharide (LPS) modification. LPS—O-antigen, the polysaccharide core, and the lipid A—is a crucial outer membrane component for virulence. Transposon mutant analysis suggested a role of LPS in bile salts resistance; thus, the goal of this study was to define Shigella LPS modifications following bile salts exposure. LPS mutants were investigated to distinguish crucial components of the LPS structure for bile salts resistance. Mutants were analyzed relative to wild type for growth in bile salts and biofilm formation. The LPS from all strains was purified and analyzed by polyacrylamide gel electrophoresis. Stained gels show modifications in the Oag, lipid A, and core components. Key bands were sent for mass spectrophotometry sequencing. Results indicate that the O-antigen regulates Shigella bile salts resistance, as the complete O-antigen deletion mutant and partial deletion mutants exhibited slow growth in bile salts and failed to form a biofilm in the presence of bile salts. This work highlights the importance of bile salts exposure for Shigella in future targeted antibodies against the pathogen. / Thesis (BS) — Boston College, 2019. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 98-116). Also available in electronic version. Access restricted to campus users.

Photochromism is a phenomenon where two isomers with markedly different absorption spectra are interconverted by a reversible photochemical reaction. The photochromism of 1’,3’,3’-trimethyl-6-nitrospiro[2H-1]-benzopyran-2,2’-indoline (NSP) and 1,2-bis(2,4-dimethyl-5-phenyl-3-thienyl)-3,3,4,4,5,5-hexafluoro-1-cyclopentene) (DAE) was studied in aqueous solutions containing NaCl and the bile salts sodium cholate (NaC), sodium deoxycholate (NaDC) or sodium taurocholate (NaTC). Bile salts are amphiphilic compounds that aggregate in water. These aggregates can solubilize hydrophobic organic compounds in water and affect the reactivity of the bound compounds. NSP and DAE are photochromic compounds that can be switched between a colored and a colorless isomer. The colored isomer of DAE can only be transformed into the colorless form by irradiation of light, while the colored isomer of NSP is also converted into the colorless form by a dark reaction. The dark reaction rate constant of NSP increases at high concentrations of bile salt and NaCl. The bile salt structure also affects the dark reaction rate constant, which is smaller in NaTC and approximately the same in NaC and NaDC. The activation energy for the reaction in all conditions studied is similar to the value reported for polar organic solvents. A method that employs HPLC was developed to determine the molar absorptivity coefficients of photochromic compounds. The values obtained were important to determine the quantum yields for photocoloration (ΦAB) and photodecoloration (ΦBA). Quantum yield values were determined by a photokinetic method that employs irradiation at a single wavelength and numerical analysis. The values of ΦAB and ΦBA for DAE in bile salts are the same as the values in cyclohexane. For NSP, ΦAB is dependent on the structure of the bile salt and increases in the order NaTC < NaC < NaDC. / Graduate

Photochromism is a phenomenon where two isomers with markedly different absorption spectra are interconverted by a reversible photochemical reaction. The photochromism of 1,3,3-trimethyl-6-nitrospiro[2H-1]-benzopyran-2,2-indoline (NSP) and 1,2-bis(2,4-dimethyl-5-phenyl-3-thienyl)-3,3,4,4,5,5-hexafluoro-1-cyclopentene) (DAE) was studied in aqueous solutions containing NaCl and the bile salts sodium cholate (NaC), sodium deoxycholate (NaDC) or sodium taurocholate (NaTC). Bile salts are amphiphilic compounds that aggregate in water. These aggregates can solubilize hydrophobic organic compounds in water and affect the reactivity of the bound compounds. NSP and DAE are photochromic compounds that can be switched between a colored and a colorless isomer. The colored isomer of DAE can only be transformed into the colorless form by irradiation of light, while the colored isomer of NSP is also converted into the colorless form by a dark reaction. The dark reaction rate constant of NSP increases at high concentrations of bile salt and NaCl. The bile salt structure also affects the dark reaction rate constant, which is smaller in NaTC and approximately the same in NaC and NaDC. The activation energy for the reaction in all conditions studied is similar to the value reported for polar organic solvents. A method that employs HPLC was developed to determine the molar absorptivity coefficients of photochromic compounds. The values obtained were important to determine the quantum yields for photocoloration (AB) and photodecoloration (BA). Quantum yield values were determined by a photokinetic method that employs irradiation at a single wavelength and numerical analysis. The values of AB and BA for DAE in bile salts are the same as the values in cyclohexane. For NSP, AB is dependent on the structure of the bile salt and increases in the order NaTC < NaC < NaDC. / Graduate / 2015-03-31

Three different types (A, B, and C) of bile salt hydrolase from different Bifidobacterium strains revealed during the purification study showed the type-specific characteristics in their electrophoretic migration and elution profiles from anion exchange and hydrophobic interaction chromatographic columns. The subunit molecular mass estimated by SDS-PAGE was around 35 kDa and the native molecular mass in all types of BSH was estimated to be between 130 and 150 kDa by gel filtration chromatography, indicating that all BSH enzymes have tetrameric structure. From the isoelectric focusing, pI value of 4.45 was obtained with type B, but type A and C BSHs showed similar pI values of around 4.65. While the N-terminal amino acid sequences of types A and B were highly homologous (19/20), six out of twenty amino acid residues were different in the N-terminal sequences of types A and C. / As the type A bsh gene was cloned from a strain of B. longum and the nucleotide sequence became available from the GenBank, our study has focused on the cloning and characterization of the type B and C bsh genes from Bifidobacterium strains. / The type B bsh gene was cloned from B. bifidum ATCC11863 and the DNA flanking the bsh gene was sequenced. The 951 by-long bsh gene encoded a 316-amino-acid protein with a molecular mass of 35 kDa and a pI of 4.48. For the first time in the genus Bifidobacterium, the transcriptional start point of the bsh gene was identified by primer extension analysis. Furthermore, Northern blot analysis revealed that B. bifidum bsh was transcribed as a monocistronic unit, contrary to that of B. longum bsh. Despite a high level of sequence similarity among the bsh genes, a BSH type-specific primer set based on the variable regions of bsh genes was designed in order to differentiate B. bifidum strains from the other species of Bifidobacterium commonly detected in the human gut. / The type C bsh gene was cloned from a bile salt tolerant strain of Bifidobacterium and the DNA flanking the bsh gene was further identified by a thermal asymmetric interlaced PCR (TAIL-PCR) technique. The 945 by-long bsh gene encoded a 314-amino-acid protein with a molecular mass of 35 kDa and a pI of 4.71. A predicted BSH promoter (Pbsh) sequence was experimentally verified and the transcriptional start point of the type C bsh gene was determined by primer extension analysis. An operonic structure including the type C bsh gene and two more ORFs, which were found within a complete set of a promoter and a transcription terminator, was identified in this study for the first time in the genus Bifidobacterium, and the polycistronic bsh transcript was revealed by RT-PCR and Northern blot analysis.

In order to gain an understanding of the interaction of probe molecules with NaCh aggregates, steady-state and time-resolved photophysical methods were used. By employing several probe molecules, an understanding of how the structure of the probe affected binding to the two distinct sites in the aggregates was investigated. The first half of the thesis examined the reactivity of benzophenone (Bp) and 4,4-dimethylbenzophenone (DMBp) with NaCh aggregates by studying the kinetics of both the ketone triplet excited states and the ketone ketyl radicals. There were three species of triplet excited states observed in the presence of primary aggregates. One of the ketone triplet excited state species was located within the primary aggregate, and it reacted to form ketyl radicals. Other triplet states included in the primary aggregate were found to be long-lived, while a third species of triplet states was present in the aqueous phase. At higher bile salt concentrations, and in the presence of secondary aggregates, a layer of complexity was added. The binding dynamics for the triplet excited state with the secondary binding sites were much faster than those observed for the primary binding site. Hydrogen abstraction did not compete with other deactivation pathways in the presence of secondary aggregates, and thus only self-quenching and exit of the excited state probe from the secondary site were observed. Ketyl radical recombination took place in water and in the secondary sites. The second half of the research focused on the study a series of naphthalene (Np) derivatives in order to look at the effects of shape and hydrophobicity of probe molecules on the interactions between these probes and the host NaCh aggregates. 1-Ethylnaphthalene, 2-ethylnaphthalene, 1-acetonaphthone, 2-acetonaphthone, 1-naphthyl-1-ethanol and 2-naphthyl-1-ethanol were studied. 1-Ethylnaphthalene and 2-ethylnaphthalene were Ethylnaphthalene, 2-ethylnaphthalene, 1-acetonaphihone, 2-acetonaphthone, 1-naphthyl- 1-ethanol and 2-naphthyl-1-ethanol were studied. I-Ethylnaphthalene and 2- ethylnaphthalene were contained within the primary binding site, while 1-naphthyl-1-ethanol, 2-naphthyl-1-ethanol, 1-acetonaphthone and 2-acetonaphthone were contained within the secondary binding site. The effect of the position of the substituent was only noticed when the probe molecules formed weak interactions with the outside of the primary aggregate, and not when the probe was complexed to one of the binding sites present in the NaCh system. The naphthalene probe molecules were also used to study the effect of ionic strength on NaCh aggregate formation. It was found that primary aggregation occurred at lower NaCh concentration as the ionic strength was increased. No effect of ionic strength was observed on the formation of secondary aggregates. All the findings in this study are consistent with an aggregation model in which two distinct binding sites are present. The shape of the probe as well as its hydrophobicity are critical to its interaction with the NaCh aggregates. From these dynamic studies it was found that only a small number of NaCh monomers (6-13) are needed to define both the primary and secondary binding sites. / Graduate

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Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis. The infectious process depends upon survival in high bile salt conditions encountered throughout the gastrointestinal tract, including the gallbladder. However, it is not clear how bile salt resistance mechanisms are induced, especially under physiologically relevant conditions. This study sought to determine how L. monocytogenes responds to bile salts under anaerobic conditions. The study found resistance to be strain specific and not dependent upon virulence. Changes in the expressed proteome were analyzed using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. A general response among virulent and avirulent strains found significant alterations in intensity of cell wall associated proteins, DNA repair proteins, protein folding chaperones and oxidative response proteins. Strain viability was correlated with an initial osmotic stress response followed by strain specific proteins associated with biofilm formation in EGDe and a transmembrane efflux pump in F2365.