Thesis--University of Florida. / Description based on print version record. Typescript. Vita. Includes bibliographical references (leaves 67-69).

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Nutrition and Food Science, 1981. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Bibliography: leaves 72-76. / by Pei-Syan Liu. / M.S.

No description available.

The major goal of this study was to achieve sustained cultivation of virulent T. pallidum in vitro. The putatuive binding of host proteins to the outer membrane (OM) of intact, virulent T. pallidum subsp. pallidum has been investigated. A major breakthrough was the development of a filtration assay, usinglow protein-binding membrane filters, for the measurement of substances bound to or incorporated into th eOM of T. pallidum. This avoided the conventional manipulations which can damage the fragile OM of T. pallidum. Using this filtration assay, studies on the binding of host serum proteins demonstrated that intact treponemes did not bind host proteins as previously reported. It also indicated that previous studies were probably performed with damaged by this research. The studies on the binding of polyclonal and monoclonal antibodies to intact and detergent treated treponemes provided evidence of the low level binding of antibody to intact treponemes which was greatly enhanced but the removal of the outer membrane with 0.1% Triton X. This research research corroborated that of others which suggests that the outer membrane of T. pallidum contains very little protein or surface exposed antigen.

Many Gram-positive and Gram-negative bacteria produce an additional thick layer of carbohydrate polymers on the cell wall surface. These capsules (capsular polysaccharides; CPS) play critical roles in interactions between bacteria and their environments (Whitfield, 2006). This is especially important in infection processes since for both Gram-negative and Gram-positive pathogens CPS is the point of first contact with the host immune system (Whitfield, 2006). However, the details of CPS biosynthesis and assembly mechanisms are still unclear. Therefore, we embarked on structural and kinetic studies of the proteins Wzc, Wza and Wzb/ Cps4B from the Wzy-dependent pathway, as well as the protein WbdD from the ATP-binding cassette (ABC) transporter dependent system. Full-length Wzc failed to crystallise due to the presence of large disordered regions and the overall difficulty of membrane protein crystallisation. A truncated version of Wzc (1-480) without the C-terminal tyrosine kinase domain was crystallised and diffracted to 15 Å in house. A previous study suggested Wza and Wzc form a functional complex (Whitfield, 2006), so Wza was also studied. Since the full-length Wza structure is available (C. Dong et al., 2006), Pulsed electron–electron double resonance spectroscopy (PELDOR) was used to study the conformational change. The PELDOR spectroscopy distance fingerprint of Wza was determined. These data also confirmed that PELDOR is a powerful tool to study large, highly symmetrical membrane proteins and can be used to study other complex membrane protein systems, such as ion channels or transporters. The crystal structure of Wzb the cognate phosphatase of Wzc was determined to 2.2 Å. Also Cps4B, which is a functional homologue of Wzb but has a completely unrelated sequence, was crystallised in two crystal forms. Form I and II Cps4B crystals diffracted to 2.8 Å and 1.9 Å resolution in house, respectively. The full-length WbdD failed to crystallise due to the presence of large disordered regions. Therefore, a shorter construct, WbdD₅₅₆ (1-556) was cloned and crystallised. The structure was determined to 2.2 Å. WbdD is a bifunctional enzyme consisting of a methyltransferase (MTase) and a kinase domain. In order to better understand the function of this protein, a variety of techniques were used, such as the ADP-Glo kinase assay, Nuclear magnetic resonance (NMR) spectroscopy, small angle X-ray scattering (SAXS) and X-ray crystallography. The various findings in the current projects provide meaningful insights towards a better understanding of the CPS biosynthesis and assembly mechanisms, which may contribute to a more intensive study identifying inhibitors and beginning to unravel the mechanism of chain length regulation.

The physiological role of the teichoic acid polymers found in Gram-positive bacterial cell walls is not known. Studies of Bacillus subtilis hybrid strains implicate a defined chromosomal region, which includes the tag-1 locus, as necessary for teichoic acid biosynthesis. A set of ten mutants carrying lesions in this region was identified from among forty-four temperature-sensitive (ts) mutants generated by nitrosoguanidine mutagenesis and bacteriophage 029 selection. This protocol gave a population enriched for ts, versus auxotrophic, mutants. For each of the ten mutants, the frequency of genetic reconstruction, or correction, of the ts phenotype indicated that it was due to change(s) in a single gene. Results of two-factor transformation crosses sorted the mutants into three complementation groups; all ten could complement tag-1. Mutants in two complementation groups were transformed to ts⁺ with cloned rodC DNA. The map order of the newly isolated ts markers was determined from the results of two factor crosses. Orientation with respect to the hisA marker was inferred from transduction experiments. The newly isolated strains were shown to be conditional rod⁻ mutants. Growth at 48°C resulted in reduced growth rates and spherically shaped cells. Additional phenotypes seen for some mutants, namely 029 phage resistance and ts spore outgrowth, appeared closely associated with the ts rod⁻ mutation. Wall phosphate content for two of the mutants, following growth at 48°C, was found to be reduced in comparison to the wild-type control. Taken together these results lend support to the argument that the tag-1 region of the chromosome, which most likely directs teichoic acid biosynthesis, is important for establishment and maintenance of the normal bacillary morphology seen for B. subtilis. The importance of other gene products to the organization of newly synthesized wall was examined using B. subtilis macrofibers. Left- and right-handed macrofibers were converted to spheroplasts and the multi-celled structures regenerated under the two sets of conditions conducive for production of the original, and inverse hand. The helix hands observed for the regenerated structures always corresponded to those expected on the basis of the parental genotype.

Thesis (Ph.D.)--University of Wollongong, School of Biological Sciences. / Typescript. Includes bibliographical references: leaf 269-331.

Autolysins are endogenous enzymes responsible for the cleavage of specific bonds in the bacterial sacculus resulting in damage to the integrity and protective properties of the cell wall. The true biological functions of these enzymes are largely unknown. However, they have been implicated in various important biological synthesis processes making their characterization important. Antibiotic susceptibility testing showed these streptococcal strains to have broad spectrum inhibitory concentrations. The major autolysins of selected streptococcal strains were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymograms). The autolysins were isolated from the specific culture supematants using 4% SDS precipitation and were shown to have apparent molecular masses ranging from 60kDa to 20kDa. Four major autolysins named A, B, C, and D from the Streptococcus milleri 77 strain were characterized. Lytic enzymes were blotted onto polyvinylidene difluoride (PVDF) membrane and N-terminally sequenced. Sequences showed between 100% and 80% similarity to that of a muramidase, glucosaminidase and a peptidase from S. mutans, S. pyogenes and S. pneumonia respectively. Biochemical characterization confirmed autolysin A to exhibit muramidase activity with both autolysin Band C exhibiting endopeptidase activity. Autolysin D showed an 80% N-terminal sequence similarity to Millericin B, a peptidoglycan hydrolase that is known to exhibit peptidase activity. Autolysis was determined using different buffers at two optimal pHs. Assaying for autolytic activity at different growth stages showed autolysis to be moderate during the lag and early exponential phases of the growth cycle. The activities of autolysins were the highest in the late exponential phase and the stationary phase of growth. Zymogram analysis showed that the Streptococcal milleri strains had moderate autolytic expression during the early and late exponential phases of the growth cycle. Control regulatory mechanisms of autolysins were determined in the presence or absence of specific charged groups, such as teichoic acids. In each case the absence of these charged groups inhibited the rate of autolysis, suggesting that the absence of teichoic acids could play a role in the regulation of the autolysins. Two-dimensional-SDS and zymographic-electrophoresis was used to determine total protein profiles for each strain. This is the first report using twodimensional zymography. Specific proteins which were either up- or down-regulated were identified. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 135-149). Also available on the Internet.

Indiana University-Purdue University Indianapolis (IUPUI) / Sepsis is a clinical syndrome due to a systemic inflammatory response to severe microbial infection. Little is known about the changes in the bone marrow (BM) and how they affect the hematopoietic response to bacterial infection. Using an animal model of severe sepsis induced by Pseudomonas aeruginosa, we have previously reported that hematopoietic stem cells (HSC) undergo a significant expansion in the BM accompanied with myeloid suppression. This bone marrow response was Toll-like Receptor 4 (TLR4)-dependent. TLR4 is activated by bacterial lipopolysaccharide (LPS) and signals through two major independent downstream molecules: TRIF and MyD88. In the present study, I found that the TLR4/TRIF and the TLR4/MyD88 pathways contribute in a distinct manner to the BM response to P. aeruginosa's LPS. TRIF plays a major role in the expansion of the HSC pool, whereas MyD88 is required for myeloid suppression. Following LPS stimulation, HSCs enter in the cell cycle, expand and exhaust when transplanted in healthy mice. Loss of TRIF rescued completely the long-term engraftment and multilineage reconstitution potential of septic HSCs, but did not affect myeloid differentiation. Conversely, MyD88 deficiency prevented completely the myeloid suppression in the myeloid progenitors, but conferred limited protective effects on the HSC function. It is of great therapeutic value to identify the downstream molecules involved in TLR4/MyD88 dependent myeloid suppression. I found miR-21, a microRNA that is involved in inflammation, was up-regulated upon LPS challenge in a MyD88-dependent manner. However, deletion of miR-21 in the BM did not rescue LPS-induced bone marrow dysfunction, demonstrating that miR-21 is not a critical regulator in these processes. Further studies are warranted to determine the precise molecular mechanisms involved in the complex pathogenesis of BM response to sepsis. Taken together, my results show for the first time that the TLR4/TRIF signaling as a key mediator of HSC damage during acute LPS exposure and that activation of the TLR4/MyD88 signaling pathway play a dominant role in myeloid suppression. These results provide novel insights into our understanding of the molecular mechanisms underlying bone marrow injury during severe sepsis and may lead to the development of new therapeutic approaches in this disease.