Bovine papillomavirus type 1 (BPV1) induces fibropapillomas in cattle, and has long served as a useful model virus to study the molecular biology of the papillomaviruses. The cellular transforming activity of BPV1 maps to its E5, E6 and E7 genes. While the cellular transformation function of BPV1 E5 is well elucidated, the biological functions of the BPV1 E6 and E7 oncoproteins are still largely unknown. To further our understanding of the cellular functions of BPV1 proteins, I performed an unbiased mass spectrometry-based proteomic analysis to identify their cellular interacting partners. I subsequently focused on characterizing the interactions of the BPV1 E6 and E7 proteins with some of their cellular interactors. I discovered Mastermind-like 1 (MAML1) and the other components of the Notch transcription complex as novel cellular interacting partners of BPV1 E6. A parallel proteomic screen performed in the laboratory using the HPV E6 proteins as baits identified MAML1 and the Notch transcription complex as interactors of the E6 proteins from beta-genus HPVs, but not those from the alpha-genus HPVs. Further investigation revealed that the beta-genus HPV E6 proteins repress the basal expression and transcriptional activation of endogenous Notch target genes in keratinocytes. For the BPV1 E7 protein, I confirmed a previously reported interaction with UBR4, and showed that this interaction is dependent on the UBR box of UBR4 and the N- terminal of E7. Since little is known about the biological function of UBR4, I performed a proteomic screen to identify its interactors. I identified the E2 ubiquitin-conjugating enzyme UBE2A as an interactor of UBR4, and showed that UBR4 is able to discharge ubiquitin from UBE2A in an in vitro discharge assay. Together with the UBR4 auto-ubiquitylation observed in an in vitro ubiquitylation assay, these results suggest that UBR4 can function as an E3 ubiquitin ligase. In summary, these studies lay the groundwork for further systems-level studies of the biological functions of papillomavirus proteins, identify the Notch signaling pathway as a novel target of cutaneous papillomaviruses such as BPV1 and beta-genus HPVs, and provide evidence that the N-recognin UBR4 can act as an E3 ubiquitin ligase.

The generation of the cytotoxic CD8 T cell response is dependent on the functional outcomes imposed by the intrathymic constraints of differentiation and self-tolerance. Although thymic function can be partly replicated in vitro using OP9-DL1 cell cultures to yield CD8 αβ T cell receptor (TCR)-bearing cells from hematopoietic progenitor cells, a comprehensive and functional assessment of entirely in-vitro generated CD8 T cells derived from bone marrow hematopoietic stem cells (BM-HSCs) has not been established and remains controversial. Here we demonstrate that a phenotypic, molecular, and functional signature of in vitro-derived CD8 T cells is akin to that of ex vivo CD8 T cells. Transfer of in vitro-derived CD8 T cells into syngeneic and immunodeficient host mice showed no graft-versus-host response, while a robust homeostatic proliferation was observed, respectively. These findings, along with a diverse and broad TCR repertoire expressed by the in vitro-derived CD8 T cells, allowed for the successful generation of antigen (Ag)-specific T cells to be obtained from an entirely in vitro-generated CD8 T cell pool, which calls for further tailoring of their use against viral infections or malignancies. Furthermore, I demonstrate that Notch signaling regulates the expression of the cytolytic molecule Granzyme A in CD8+ T cells. This is supported by the inability of Notch-deprived TCR-signaled CD8 T cells to express Granzyme A, while CD8 T cells that received Notch signals readily expressed Granzyme A, suggesting that Notch signaling is a prerequisite for induction of this cytolytic molecule. We further demonstrate that Notch signaling by OP9 cells allows for efficient differentiation of conventional effector CD8 T cells from SAP-/- BM-derived HSCs and restricts differentiation of innate CD8 T cells while allowing for differentiation of IL17-producing CD8 T cells from BM-HSCs isolated from Itk-/-Rlk-/- (DKO) mice. Moreover, we reveal that the process of positive and negative selection in vitro is constrained by peptide-MHC (pMHC) class I expressed by the OP9 cells and disclose that the commitment of DP precursors to the CD8 T cell lineage is facilitated by Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout intrathymic T cell differentiation.

The generation of the cytotoxic CD8 T cell response is dependent on the functional outcomes imposed by the intrathymic constraints of differentiation and self-tolerance. Although thymic function can be partly replicated in vitro using OP9-DL1 cell cultures to yield CD8 αβ T cell receptor (TCR)-bearing cells from hematopoietic progenitor cells, a comprehensive and functional assessment of entirely in-vitro generated CD8 T cells derived from bone marrow hematopoietic stem cells (BM-HSCs) has not been established and remains controversial. Here we demonstrate that a phenotypic, molecular, and functional signature of in vitro-derived CD8 T cells is akin to that of ex vivo CD8 T cells. Transfer of in vitro-derived CD8 T cells into syngeneic and immunodeficient host mice showed no graft-versus-host response, while a robust homeostatic proliferation was observed, respectively. These findings, along with a diverse and broad TCR repertoire expressed by the in vitro-derived CD8 T cells, allowed for the successful generation of antigen (Ag)-specific T cells to be obtained from an entirely in vitro-generated CD8 T cell pool, which calls for further tailoring of their use against viral infections or malignancies. Furthermore, I demonstrate that Notch signaling regulates the expression of the cytolytic molecule Granzyme A in CD8+ T cells. This is supported by the inability of Notch-deprived TCR-signaled CD8 T cells to express Granzyme A, while CD8 T cells that received Notch signals readily expressed Granzyme A, suggesting that Notch signaling is a prerequisite for induction of this cytolytic molecule. We further demonstrate that Notch signaling by OP9 cells allows for efficient differentiation of conventional effector CD8 T cells from SAP-/- BM-derived HSCs and restricts differentiation of innate CD8 T cells while allowing for differentiation of IL17-producing CD8 T cells from BM-HSCs isolated from Itk-/-Rlk-/- (DKO) mice. Moreover, we reveal that the process of positive and negative selection in vitro is constrained by peptide-MHC (pMHC) class I expressed by the OP9 cells and disclose that the commitment of DP precursors to the CD8 T cell lineage is facilitated by Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout intrathymic T cell differentiation.

No description available.

Résumé: La réactivation de la voie Notch est une des caractéristiques les plus redondantes observées au cours de la carcinogenèse pancréatique. L’expression aberrante de la protéine HES1, cible de la signalisation Notch, est observée dès les premières phases de la carcinogenèse (Miyamoto et al., 2003). L’utilisation d’inhibiteurs de gamma-sécrétase semble être une stratégie thérapeutique prometteuse soutenue par de nombreuses études in vitro et in vivo (Plentz et al. 2009). Cette étude vise à démontrer que l’inhibition de la voie Notch et particulièrement de sa protéine cible, HES1, permet de réduire les propriétés tumorigéniques des cellules cancéreuses pancréatiques humaines. Pour cela, nous avons utilisé deux lignées cellulaires issues d’adénocarcinomes pancréatiques humains : MIAPaCa-2 et BxPC-3 ; celles-ci ont été traitées avec des inhibiteurs de gamma-sécrétase et infectées par des lentivirus codant pour des shARNs ciblant de manière spécifique les ARNm de Notch1 ou Hes1. Nos RÉSULTATS démontrent que les cellules BxPC-3 présentent un plus fort niveau d'activation de NOTCH1 qui corrèle avec un plus fort niveau d'expression d’HES1 comparativement aux cellules MIAPaCa-2. L’inhibition de la voie Notch réduit la prolifération des cellules BxPC-3 sans affecter celle des cellules MIAPaCa-2 ni même la survie cellulaire des deux lignées. De plus, on observe une diminution de la migration des cellules BxPC-3 suite à l’inhibition de la voie Notch. Notre étude suggère, d'une part, que l’efficacité des inhibiteurs de gamma-secrétase ne dépend pas seulement de l’expression des récepteurs NOTCHs mais principalement de leur niveau d'activation. Et, d’autre part, une implication directe et majeure de la protéine cible HES1 dans la médiation des effets de la voie Notch.||Abstract:

Notch signalling plays a key role in physiological development and tumourigenesis. The recent discovery and characterisation of Notch ligand Delta-like 4 (D114), which is predominantly expressed in endothelial cells, have underscored the role of Notch signalling in angiogenesis. This thesis investigates the regulation and function of D114-Notch signalling in angiogenesis and tumourigenesis. First, the D114-Notch pathway interacted with the cellular hypoxia-sensing pathway. In human umbilical vein endothelial cells (HUVECs), D114 overexpression repressed hypoxic induction, and the repression was mediated by Notch target gene Hey2. In the breast cancer cell line MCF7, hypoxia induced Notch target gene Hey1 and ligand Jagged2 via hypoxia inducible factor 1. The hypoxic induction of Hey1 was also dependent on Notch signalling. Second, D114 expression in HUVECs was up-regulated by several pathways. Notch signalling, activated by receptor overexpression, ligand stimulation or cell-cell contact, induced D114 expression. Treatment with vascular endothelial growth factor (VEGF) or hypoxia also induced D114 expression via Notch signalling. In addition, VEGF promoted Notch signalling and D114 expression in tumours. Third, D114 expression in HUVECs was up-regulated by co-culturing with cancer cells. B16 mouse melanoma cells and human breast cancer cell lines induced D114 expression in HUVECs via Notch signalling. B16 cells also induced D114 expression via soluble factors independent of Notch signalling. Finally, D114-Notch signalling regulated tumour growth in vivo. D114 overexpression in cancer cell lines activated Notch signalling in the stroma of xenograft tumours, and promoted the growth of human U87 (glioblastoma) and PC3 (prostate cancer) xenografts. In addition, D114-overexpressing U87 tumours were resistant to anti- VEGF treatment during later stages; however, they did respond to anti-Notch treatment. Altogether, the D114-Notch pathway is tightly regulated and plays an important role in physiological and tumour angiogenesis. Inhibiting this pathway may be a viable therapy for cancers resistant to VEGF inhibition.

No description available.

No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains ix, 128 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 118-127).