Thesis (Ph.D.)--Ohio University, August, 2005. / Includes bibliographical references (leaves 130-156)

Theses (M.S.)--Marshall University, 2006. / Title from document title page. Includes abstract. Document formatted into pages: contains viii, 87 p. including illustrations. Bibliography: p. 78-86.

Thesis (Ph. D.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: Ed Schmidt. Includes bibliographical references (leaves 88-107).

Thesis (Ph. D.)--Florida State University, 2005. / Advisor: Timothy S. Moerland, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed May 11, 2006). Document formatted into pages; contains viii, 76 pages. Includes bibliographical references.

A novel x-ray anode for electron spectroscopy is investigated for application in the surface analysis field, monochromatic Ag Lalpha (hnu =2984. 3eV), its energy being capable of exciting 1s electrons up to chlorine in the periodic table. Resolution available with this source is satisfactory, with a limitation of approximately 1. 3eV. An increase in sensitivity is achieved for those peaks in the range 1500-3000BE, whilst there is no serious reduction within the conventional XPS energy range. The agreement between experimental sensitivity factors and theoretical cross-section values is good, allowing the transmission function for the VG ESCA3 Mkll spectrometer to be confirmed constant from 0-3000eV. A comprehensive investigation of the LEG51 electron flood gun preceded its successful application for the charge neutralisation of insulating materials. This allowed the application of this source to such materials as chlorides, pertinent to the breakdown in passivity on stainless steels, and silicon compounds, involving thermal oxides on silicon of interest to the microchip industry, zeolites for catalysis in the petrochemical industry and siloxane copolymers for the opthalmic industry. The ability to excite the 1s orbital, together with the 2p and KLL Auger lines, affords calculation of Auger parameters and extra-atomic relaxation energies for the accurate description of the chemical environment of a particular chemical species. A method for internal energy referencing is investigated, involving the vaccum-deposition of Au, Cu and Pt metals. This allows the measurement of photoelectron binding energies to an accuracy of +/-0.1-0.2eV for insulating materials, when referenced to the vacuum level.

No description available.

No description available.

Shope fibroma virus (SFV) N1R is a member of a family of poxvirus proteins that is associated with virulence and largely defined by the presence of a C-terminal RING finger motif and localization to virus factories within the cytoplasm of infected cells. Altered proteins, with deletions and site-specific mutations, were transiently expressed in vaccinia virus infected cells to discern regions of the protein that are required for localization. Deletion mutagenesis implicated a requirement of a small central region of the RING for localization, but the RING motif alone was not sufficient. A chimeric protein, however, in which the RING motif of the herpes simplex virus-1 ICP0 protein replaced the SFV N1R RING motif did localize to virus factories, indicating that the specificity for factory localization resided outside the RING motif of N1R. Critical evaluation of an alignment of poxviral N1R homologs identified a short, highly conserved N-terminal sequence 24-YINIT-28. When this sequence was deleted from N1R localization was abolished. Recombinant N1R protein isolated from vaccinia virus (VV) infected cells bound to calf-thymus DNA cellulose. Elution from this matrix required 0.5–0.75M NaCl, suggesting N1R localizes to the factory through an inherent DNA binding activity. Structural prediction analysis inferred that the conserved N-terminal region required for N1Rs factory localization forms a short β strand and subsequent alignment analysis with β sheet DNA binding proteins uncovered significant homology with the ribbon-helix-helix motif family which utilize a short β sheet for specific DNA interaction. Characterization of the factory localization of five N1R mutants, each having a single potential β strand residue replaced with Ala revealed that Asn 26 was the most important residue for factory localization. In contrast to N1R, which strongly binds DNA and rapidly sediments with the virus factories, SFV-N1RAsn26ΔAla mutant protein was found in the soluble fraction of infected cell lysates and failed to bind DNA cellulose. These results indicate that the N1R RING finger motif may not be central to DNA interactions and that N1R β strand residues particularly Asn 26 are involved in DNA binding and targeting N1R to the virus factories. Overexpression of N1R in vaccinia virus (VV) infected cells was found to inhibit virus induced apoptosis. To clarify the role of N1R protein with respect to apoptosis and to examine whether the related ectromelia virus virulence factor p28 (EVp28) might also play a role in apoptosis protection, a p28-mutant EV virus and the VV-N1R virus were tested for their ability to interfere with apoptosis induced by different signals. VV and EV infection were found to protect cells from Ultra Violet (UV) light, Tumor necrosis factor alpha (TNFα) and anti-Fas induced apoptosis. Expression of SFV N1R and EVp28 however, only protected infected HeLa cells from apoptosis induced by UV light, and did not protect from apoptosis induced by TNFα or anti-Fas antibody. Immunoblot analysis indicated EVp28 blocks processing of procaspase-3 suggesting EVp28 acts upstream of this protease in response to UV induced apoptotic signals. The requirement of EVp28 to promote replication and virulence in vivo may be related to apoptosis suppression because the number of progeny virus harvested from p28-mutant EV virus infected cells compared to wild type EV was similar following mock UV induced apoptosis, but significantly reduced following apoptosis induction by UV. / Graduate

La tuberculose est la première cause de mortalité due à une infection par une bactérie : Mycobacterium tuberculosis. Récemment, une adhésine a été caractérisée à sa surface, son nom est HBHA (Heparin Binding Haemagglutinin). Il y a 10 ans, elle a été identifiée chez M. tuberculosis comme une adhésine majeure impliquée dans la reconnaissance des cellules épithéliales par le bacille tuberculeux via les polysaccharides sulfatés. Cette interaction aboutit à l’internalisation de la bactérie qui peut alors franchir la barrière épithéliale pulmonaire. HBHA est une protéine de 198 résidus organisés en quatre domaines : un domaine hydrophobe (résidus 12 à 36), un domaine coiled-coil (résidus 36 à 111), un domaine de liaison (111-160) et un domaine riche en lysine (ou Heparin Binding Domain, HBD) (161-198). Le HBD constitue le site autonome d’interaction entre HBHA et les polysaccharides sulfatés (ou GAGs). Il est essentiellement composé d’alanines, de prolines et de lysines arrangées en six motifs répétés. Ce domaine est mono ou di-méthylé sur les lysines. Nos études de SAXS, DLS et de CD ont mis en évidence le caractère fortement désordonné de HBHA, suggérant que celle-ci est une PID (Protéine Intrinsèquement Désordonnée). Nous avons donc entrepris d’étudier la structure de cette protéine par deux approches. Une première approche nous a permis de caractériser sa structure quaternaire en solution et dans la mycobactérie par des études de crosslinking, de gel filtration et de SAXS. Celles-ci ont montré que le degré d’oligomérisation de HBHA est plus important dans la mycobactérie qu’en solution. Nous avons également caractérisé le mécanisme d’interaction entre HBHA et les polysaccharides sulfatés par des études de RMN et de CD. Ces études ont montré que l’interaction avec les GAGs n’est pas homogène sur le domaine HBD. Celle-ci induit un réarrangement structural différent pour chaque partie du domaine. On a observé la stabilisation d’une structure plus étendue dans la première moitié du HBD et une stabilisation d’hélice alpha dans la deuxième partie. Cette étude nous a permis de mieux comprendre la spécificité de cette interaction, et nous a donné des pistes sur le rôle de cette protéine dans le tropisme bactérien. / Tuberculosis is a leading cause of mortality due to an infectious agent worldwide: Mycobacterium tuberculosis. One of the most recently characterized mycobacterial cell wall protein is the Heparin binding Haemagglutinin (HBHA). Roughly 10 years ago, it was identified in M. tuberculosis as one of the major adhesin involved in binding of mycobacteria to the epithelial cells by interaction with sulphated polysaccharides. This interaction induced internalization of M. tuberculosis in pulmonary epithelial cells leading to serious forms of disease. HBHA have 198 residues organized in four domains: a hydrophobic domain (residue 12 to 36), a coiled-coil domain (36 to 111), a linker domain (111-160) and a poly-lysine rich domain (161 to 198) (or Heparin Binding Domain, HBD) which forms the independent interaction domain between HBHA and sulphated polysaccharides. The approximately 40 amino acid residues long C-terminal domain is essentially composed of alanines, prolines and lysines, arranged in repeated motifs. This domain is mono or di methylated on the lysines in Mycobacteria, this post-traductional modification does not occur when expressing HBHA in Escherichia coli. Circular dichroïsm, DLS and SAXS studies indicated that HBHA is an IPD (Intrinsically Disordered Protein). This feature is not compatible with crystallography. Two approaches have been used in our study. In the first one, we studied the quaternary structure of HBHA by crosslink, SAXS and gel-filtration. All data showed that the oligomerization state of HBHA in mycobacteria is larger than after purification in E. coli. In the second part, we studied the binding mechanism between the HBD of HBHA and GAGs. This study was carried out by circular dichroïsm and NMR. Our data showed that the GAGs binding is not homogeneous, two specific rearrangements of the domain have been observed; GAGs binding induced a more extended structure in the first part of the HBD and induce an alpha helix stabilization in the second part. These data helped us to understand the specificity of this interaction and maybe the role of this interaction in the bacterial tropism in the host.

A diafiltration technique was used to study drug-protein interactions. Fraction V human serum albumin and plasma and two drugs (phenylbutazone and bishydroxycoumarin) with a high affinity for these substances were used in this investigation. Preliminary experiments were carried out to check for release of foreign substances and for binding of drug to the Amicon diafiltration apparatus. A binding experiment, in the absence of drug, revealed release of a protein-like, ultraviolet absorbing substance from Fraction V human serum albumin. The most suitable method of purification for albumin was by diafiltration with Tris buffer. Binding curves for bishydroxycoumarin - human serum albumin, phenylbutazone - human serum albumin, and bishydroxycoumarin - plasma interactions were obtained. The r and r/Df [subscript omitted] values were calculated and binding parameters estimated by both graphical extrapolation and by a computer non-linear least squares fit analysis. Binding curves were not independent of human serum albumin concentration, but the cause of this effect was not fully resolved. Results showed the diafiltration technique can yield precise data, can be used over a wide macromolecule concentration range and produces a binding curve, from one experiment, over a wide range of molar binding ratios. Use of the Amicon diafiltration apparatus in desorption (washout) experiments and equilibrium or direct experiments was also investigated. Attempts were made to obtain binding data by centrifugation (ultrafiltration) and by a gel filtration technique (Sephadex G-25 batch method). These methods yielded unsatisfactory results which could not be compared with those obtained by diafiltration. This abstract represents the true contents of the thesis submitted. / Pharmaceutical Sciences, Faculty of / Graduate