Thesis (D.P.H.)--University of Michigan, 1948. / Type-written. Bibliography: leaves 60-64.

Thesis (Ph.D.)--Kent State University, 2008. / Title from PDF t.p. (viewed Dec. 14, 2009) Advisor: Miguel E. Quinones-Mateu. Keywords: biomedical research, cellular biology, molecular biology, virology. Includes bibliographical references (p. 201-228)

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph.D.)--York University, 2008. Graduate Programme in Mathematics and Statistics. / Typescript. Includes bibliographical references (leaves 166-175). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR46017

Thesis (M.P.H.)--University of Michigan, 1947.

Thesis (M.P.H.)--University of Michigan, 1947.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 54-57).

The segmented negative-sense vRNA genome of influenza A virus is replicated via a complementary RNA (cRNA) intermediate by the viral RNA-dependent RNA polymerase (RdRP). The replicative intermediate has been thought to exist as a complementary ribonucleoprotein (cRNP) complex. Development of an RNA-based affinity-purification strategy for the isolation of viral ribonucleoprotein complexes enabled the structural and functional characterisation of the previously uncharacterised cRNP complex. The cRNP exhibits a filamentous double-helical organisation with defined termini, containing the viral RdRP at one end and a loop structure at the other end. In vitro characterisation of cRNP activity led to a proposal of a model of vRNA synthesis that relies on a trans-activating RdRP. The viral RdRP is an important host range determinant indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Affinity-purification followed by mass spectrometry was performed to identify cellular proteins that interact with the viral RdRP. 171 cellular proteins were found to co-purify with the viral RdRP, the most abundant of which were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, mitochondrial and ribosomal proteins. Among the phosphatases, three subunits of the cellular serine/threonine-protein phosphatase 6 (PP6) were identified. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and siRNA-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. Taken together, these results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP.

No description available.

Protective immunity against influenza virus infection is believed to be mediated by neutralising antibodies. Despite substantial evidence in animal models which suggests critical roles for T cells in viral clearance, the precise role of cellular immunity in human influenza immunity remains uncertain. The first aim of this project was to determine cellular immune responses in seronegative human volunteers following nasal challenge with live seasonal H3N2 or H1N1 viruses. T cell responses before and during infection were mapped. A large increase in both breadth and magnitude in influenza-specific CD4⁺ T cell responses was seen in the blood by day 7, when virus was completely cleared from nasal samples and serum antibodies were still undetectable. These acutely expanded T cells were shown to be highly activated (CD38⁺) and proliferating (Ki-67⁺). Pre-existing CD4⁺ T cells, but not CD8⁺, specific to internal proteins nucleoprotein and matrix proteins, were associated with lower virus shedding and less severe illness. These influenza-specific T cells also responded to A/California/07/2009 (H1N1) peptides, were able to kill antigen-loaded autologous B cell lines in vitro and were polyfunctional in cytokine production. The second aim was to assess the cellular immune responses to unadjuvanted, inactivated seasonal and pandemic A/California/07/2009 (H1N1) influenza vaccines. 151 healthy adult volunteers were vaccinated: modest influenza-specific T cell responses were induced, and specific responses to HA and NA peptide pools were found to be mediated by CD4⁺ T cells. Activated and proliferating cells induced during vaccination were found to be of a central memory phenotype. Lastly, I explored the link between antibody production and CD4⁺ T cells. The ability of CXCR5⁺ CD4⁺ T cells from peripheral blood to support antibody production was examined and antigen-specific CD4⁺ T cells with helper functions could be found in the peripheral blood of healthy volunteers with memory influenza-specific T cells. This thesis suggests that influenzaspecific CD4⁺ T cells have an important role in limiting the severity of influenza infection in the absence of specific antibody responses through a number of mechanisms. This work provides information on how cellular immunity can be targeted in conferring broad protection against different subtypes of influenza A viruses in the development of universal flu vaccine.