Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 86-98.

Thèse (Ph. D.)--Université Laval, 2009. / Bibliogr.: f. 146-173. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 156-168.

Thesis (Ph.D. in Toxicology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 141-156). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Microbiology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 142-168). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Vaccinia virus (VACV), the prototypic poxvirus, undergoes a complex life cycle, with multiple stages that are not yet fully understood. This work studied two cellular proteins which had previously been identified by siRNA screens as playing proviral roles in the replication cycle of VACV: the dual specificity mitogen-activated protein kinase kinase 3 (MKK3) and vacuolar protein sorting 52 (Vps52). MKK3 is an upstream regulator in the p38 pathway which, along with MKK6, phosphorylates and therefore activates p38. In HeLa cell cultures, siRNA depletion experiments confirmed that MKK3 supported VACV replication. MKK3 knockdown reduced production of both early and late-class VACV proteins, suggesting that it facilitates viral gene expression. However, this difference did not translate to an in vivo model, as comparison between wild type and MKK3 knockout mice infected with VACV revealed no significant differences in virus replication or overall disease. The Golgi-associated retrograde protein complex (GARP) is composed of four large heteromeric proteins: Vps51, Vps52, Vps53 and Vps54, and plays a key role in retrograde transport from endosomes to the TGN. The effects of loss of GARP function were investigated using three techniques: mouse embryonic fibroblasts (MEFs) containing the hypomorphic Vps54 “wobbler” mutation, Vps52-targetting siRNA in HeLa cells and pharmacological inhibition of retrograde transport using the drug Retro-2. GARP loss resulted in a marked reduction in VACV spread due to a reduction specifically in “double wrapped” extracellular enveloped virion (EEV) production. Investigation of the mechanism by which GARP facilitates EEV production revealed a disruption of the VACV morphogenesis pathway prior to the double wrapping event, resulting in mislocalisation and aggregation of the viral membrane protein B5 within the cytoplasm. The effects of GARP loss translated to an in vivo model, as mice infected with VACV and treated with Retro-2 exhibited reduced viral replication and overall disease. These results identify GARP as a pro-viral host complex required for EEV production, and suggest that cellular retrograde transport pathways are required for double-wrapping of VACV virions. Overall, the study illustrates both the potential pitfalls of carrying out genetic screens in a transformed cell line and the power of such studies to nevertheless identify novel features of virus biology as well as druggable targets for antiviral intervention.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

La migration des cellules endothéliales est une des étapes nécessaires à l'angiogenèse physiologique et tumorale. Le facteur pro-angiogénique VEGF (vascular endothelial growth factor) induit la réorganisation du cytosquelette d'actine en fibres de tension et la migration cellulaire en activant la kinase de stress SAPK2/p38 via le récepteur à activité tyrosine kinase VEGFR-2. Nous avons étudié les événements moléculaires suivant l'activation du récepteur du VEGF et menant à l'activation de la voie SAPK2/p38 dans les cellules endothéliales. La tyrosine 1214 en C-terminal du VEGFR-2 est un site majeur d'autophosphorylation nécessaire à l'activation de SAPK2/p38 par le VEGF dans les cellules endothéliales. Le résidu phospho-tyrosine 1214 permet le recrutement de la protéine adaptatrice Nck via son domaine SH2. De plus, l'activation de Fyn, une kinase de la famille Src, mais non c-Src même, dépend de la phosphorylation de la tyrosine 1214. L'utilisation d'un dominant négatif de Fyn empêche l'activation de SAPK2/p38, inhibe la formation de fibres de tension et abolit la migration endothéliale. Nck et Fyn s'associent en réponse au VEGF et l'activité de Fyn est nécessaire à la phosphorylation sur tyrosine de Nck et de la kinase PAK-2. La formation de fibres de tension nécessite aussi l'activation de la petite GTPase Cdc42, ce qui permet d'acheminer le signal du VEGF vers SAPK2/p38, sa cible physiologique MAPKAP K2, puis vers l'actine. Ces résultats suggèrent une séquence d'étapes moléculaires précoces suivant l'activation du VEGFR-2 et régulant la dynamique d'actine nécessaire à la migration endothéliale. 11 s'agit de la première description d'une séquence d'événements couplant l'activation d'un récepteur à activité tyrosine kinase à celle d'une kinase de stress. Par ailleurs, l'identification du rôle du site tyrosine 1214 du VEGFR-2 en fait une cible précise pour le développement d'une drogue anti-angiogénique. / Endothelial cell migration is one major step of physiological and pathological angiogenesis. The vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis and induces actin reorganization into stess fibers and cell migration via activation of SAPK2/p38 downstream of the tyrosine kinase receptor VEGFR-2. We studied the molecular events following the activation of the VEGF-receptor that lead to the activation of the SAPK2/p38 pathway in endothelial cells. The tyrosine 1214 residue in C-terminal of the VEGFR-2 is a major autophosphorylation site that is required for the activation of SAPK2/p38 by VEGF in endothelial cells. The adaptor protein Nck is recruited to the phospho-tyrosine 1214 residue via its SH2 domain. Moreover, the activation of the Src-family kinase Fyn, but not c-Src itself, depends on phospho-tyrosine 1214. A dominant negative form of Fyn inhibits SAPK2/p38 activation, stress fibers formation and endothelial cell migration. Nck and Fyn associate in response to VEGF and Fyn activity is required for the tyrosine-phosphorylation of Nck and PAK-2. The activation of the small Rho GTPase Cdc42 downstream of tyrosine 1214 is also required for the activation of SAPK2/p38, its physiological target MAPKAP K2, and stress fiber formation. Altogether, these results suggest a sequence of early molecular steps following VEGFR-2 activation by VEGF that regulate the actin dynamics required for endothelial cell migration. This is the first description of a sequence of events that couple the activation of a tyrosine kinase receptor to that of a stress-activated kinase. The identification of the role of the tyrosine 1214 residue on VEGFR-2 makes it an appealing target for the development of an anti-angiogenic therapy.