Thesis (Ph. D. in Microbiology and Immunology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

CD4 is a co-receptor for binding of T cells to antigen-presenting cells (APC) and the primary receptor for human immunodeficiency virus-I. The disulfide bond in the second extracellular domain (D2) of CD4 is reduced on the cell surface, which leads to formation of disulfide-linked homodimers. A large conformational change must take place in D2 to allow for formation of the disulfide-linked dimer. Domain swapping of D2 is the most likely candidate for the conformational change leading to formation of two disulfide-bonds between Cysl30 in one monomer and Cysl59 in the other one (Cys133 and Cysl62 in the mouse CD4). Thus, we hypothesized that the domain swapping of D2 in CD4 regulates its co-receptor function of antigenspecific T cell activation. We found that mild reduction of the extracellular part of human CD4 resulted in formation of disulfide-linked dimers. We then tested the functional significance of dimer formation for co-receptor function using the engineered Jurkat T cell system by expressing wild-type or disulfide-bond mutant mouse CD4. Eliminating the D2 disulfide bond markedly impaired CD4's coreceptor function as assessed by antigen-specific IL-2 production. Exogenous wild type thioredoxin, but not redox-inactive thioredoxin, could inhibit the CD4-mediated IL-2 production, suggesting that the redox state of D2 disulfide bond is controlled by this oxidoreductase. Furthermore, structural modeling of the complex ofthe T cell receptor and domain-swapped CD4 dimer bound to class II major histocompatibility complex and antigen supports the domain-swapped dimer as the immune co-receptor. The known involvement of D4 residues Lys318 and Gln344 in dimer formation isalso accommodated by this model. These findings imply that disulfide-linked dimeric CD4 is the preferred functional co-receptor for binding to APC. Strategies to promote dimerisation of CD4 should, therefore, enhance the immune response, while inhibiting dimer formation is predicted to be immunosuppressive.

Thesis (Ph. D. in Microbiology and Immunology)--Vanderbilt University, May 2008. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1949. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 136-145).

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 76-81). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 139-160). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 139-160). Also available online.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

An in vitro initiation of a DNA replication system was developed to study the function of Ø29 terminal protein. Cell free extracts prepared from Bacillus phage Ø29-infected cells catalyzed the formation of a complex between a 30,000 dalton protein and dAMP in the presence of MgCl₂, Ø29 DNA-protein template and α-³²P dATP. Uninfected cell extracts did not support this reaction. The molecular weight of this complex, the nature of linkage between dAMP and 30,000 dalton protein as well as nucleotide specificity for this reaction suggest that the protein moiety of this complex is the terminal protein of Ø29. Similar results were obtained with phages GA-1 and M2Y infected cell extracts. The similar requirements for complex formation and DNA replication in vitro implies that the complex formation is an initiation step in DNA replication. This notion was supported by characterizing the elongation product which formed in the presence of ddCTP. Two distinct antibodies were prepared for analyse the function of the terminal protein in Ø29 DNA replication. These antibodies react with native terminal protein as assayed by immunoprecipitation and ELISA methods. The inhibition of complex formation by these antibodies provides strong evidence that the terminal protein is involved in complex formation. The notion that complex formation is an initial step of DNA replication was demonstrated conclusively by inhibition of anti-TP on DNA replication in vitro. The results presented in this dissertation provide evidence supporting the protein-priming mode of linear Ø29 DNA replication.

Early function of a temperature-sensitive mutant of staphylophage 44A HJD was examined during the twenty-five-minute period following infection. Host cell and phage DNA were labeled with C and3H respectively. DNA was separated into linear and covalently closed circular (CCC) forms by density-gradient centrifugation. The host, S. aureus, shows no CCC DNA, and apparently carries no plasmid. Following infection with wild type phage, CCC DNA forms occur in tritiated and 1 C DNA fractions 10 to 15 min after infection. Infection with mutant at permissive temperature also demonstrates CCC DNA with both labels. Infection with mutant at nonpermissive temperature produced no CCC DNA during the first 25 min after infection. The impaired function in this mutant may be a linker protein.