No description available.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references (leaves 79-89).

GBF1 a émergé autant que facteur cellulaire nécessaire pour la réplication de plusieurs virus à ARN. Au cours de l’infection par le virus de l’hépatite C (VHC), GBF1 est essentiel pour les étapes précoces de la réplication, bien qu’il soit dispensable lorsque celle-ci est établie. Afin de mieux comprendre la fonction de GBF1 dans la régulation de l'infection par le VHC, nous avons tenté d’explorer les interactions entre GBF1 et les protéines du VHC. Ainsi, grâce à l’approche du double hybride en levure et par co-immunoprécipitation et par PLA (proximity ligation assay), nous avons pu montrer que NS3 interagit avec GBF1. De plus, NS3 semble interférer avec la localisation subcellulaire de GBF1 dans des cellules exprimant NS3. Cette interaction a été retrouvée entre le domaine protéase de NS3 et Sec7, le domaine catalytique de GBF1. Un crible sur des mutations altérant l’interaction GBF1-NS3, par double hybride en levure, a permis révéler un mutant NS3 (N77D de la souche Con1) qui est non-réplicatif malgré une activité protéase bien conservée. De plus, le résidu muté est exposé à la surface, ce qui suggère qu’il pourrait appartenir à la zone d’interaction de NS3 avec GBF1. La mutation correspondante dans la souche JFH1 produit le même phénotype que la souche Con1 du VHC. L’ensemble des résultats révèlent l’existence d’une interaction entre GBF1 et NS3 et suggèrent qu’une altération de cette interaction est délétère pour la réplication du VHC. / GBF1 has emerged as a host factor required for the replication of RNA viruses of different families. During the hepatitis C virus (HCV) life cycle, GBF1 performs a critical function at the onset of replication, but is dispensable when the replication is established. To better understand how GBF1 regulates HCV infection, we have looked for interactions between GBF1 and HCV proteins. NS3 was found to interact with GBF1 in yeast two-hybrid, in co-immunoprecipitation and in proximity ligation assays, and to interfere with GBF1 function and alter GBF1 intracellular localization in cells expressing NS3. The interaction was mapped to the Sec7 domain of GBF1 and the protease domain of NS3. A yeast two-hybrid screen for mutations altering NS3-GBF1 interaction yielded an NS3 mutant (N77D, Con1 strain) that is non-replicative despite conserved protease activity. The mutated residue is exposed at the surface of NS3, suggesting it could be part of the domain of NS3 that interacts with GBF1. The corresponding mutation in JFH-1 strain (S77D) produces the same phenotype. Our results provide evidence for an interaction between NS3 and GBF1 and suggest that an alteration of this interaction is detrimental to HCV replication.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available via the Internet as viewed 10 July 2008.

Thesis (Ph.D.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2009/06/09). Includes bibliographical references.

No description available.

Protein trafficking between the endoplasmic reticulum (ER) and Golgi complex is regulated by the activity of ADP-ribosylation factors (Arfs). Arf activation by guanine nucleotide exchange factors (GEFs) leads to the recruitment of the coatomer protein COPI and vesicle formation. By using fluorescently-tagged proteins in live cells, we have been able to identify novel functions for Arfs and the Arf-GEF GBF1 at the ER-Golgi intermediate compartment (ERGIC) and mitotic Golgi clusters. We first focused on Arf function at the ERGIC after observing both class I (Arf1) and class II (Arfs 4 and 5) Arfs at this structure. We discovered that class II Arfs remain bound to ERGIC membranes independently of GBF1 activity following treatment with brefeldin A (BFA). Further characterization of the class II Arfs using additional pharmacological agents such as Exo1 and inactive mutant forms of Arf4 demonstrated that the class II Arfs associate with the ERGIC membrane via receptors distinct from GBF1. Our work suggests that GBF1 accumulation on membranes in the presence of BFA is due to loss of Arfs from the membrane rather than the formation of an abortive complex with Arf and GBF1. Next, while studying GBF1 in live cells, we unexpectedly observed GBF1 localizing to large fragmented structures during mitosis. We identified these structures as mitotic Golgi fragments that are positive for GBF1 and COPI throughout mitosis. Again using live cells treated with BFA and Exo1, we demonstrated that GBF1 concentrates on these mitotic fragments suggesting that they are derived from Golgi membranes. By colocalization studies and fluorescence recovery after photobleaching, we demonstrated that these mitotic fragments maintain a cis-to-trans subcompartmental Golgi polarization and membrane dynamics of GBF1 similar to interphase cells. Interestingly, inactivation of GBF1 and loss of COPI from the membranes of the mitotic Golgi fragments did not delay progressing through mitosis. Our results from our second project indicate for the first time that the mitotic Golgi clusters are bona fide Golgi structures that exist throughout mitosis with a functional COPI machinery.

ADP Ribosylation Factor 1 (ARF1) is a universal small GTP binding protein which has an important role in vesicular trafficking between endoplasmic reticulum and Golgi. ARF1 is a basic component of Coat Protein I (COPI) vesicles which have functions in both formation of coatomer complex and recruitment of cargo proteins. In this study, the expression ARF1 was analyzed in pea (P. sativum L. cv. Araka) grown at different developmental stages. Because of the differential hormonal levels at corresponding stages, the effects of hormones on ARF1 expression were also studied. The results of present research show that ARF1 expression in embryos and 2 days grown plants after germination is lower when compared to 6 days grown plants. In order to see the hormonal effect, 3 weeks old plants were supplied with 50&micro / M of each hormone for 3 times on alternate days. Protein extraction, cell fractionation,Western blot was carried out and immunoblot analysis was conducted with AtARF1 polyclonal antibodies. It was shown that, in pea shoots, abscisic acid and gibberellin increases the inactive GDP bound ARF1 by hydrolyzing ARF-GTP through activating ARFGTPase activating protein (ARF-GAP) or partially inhibiting ARF-Guanine Nucleotide Exchange Factor (ARF-GEF). In roots, ARF-GDP (cytosolic fraction), ARF-GTP (microsomal fraction) and total amount of ARF1 (13.000 x g supernatant fraction) were down regulated by ~11, ~19 and ~11 fold respectively with the application of gibberellin / and by ~11, ~7 and ~3 fold respectively with the application of abscisic acid / when compared to control plants. These results indicate the importance of plant hormones in the regulation of ARF1 in pea.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 106-116.

La capacité des cellules à être invasives et métastasiques est une caractéristique fondamentale de la malignité tumorale. Nous avons récemment montré que le facteur d’ADP-ribosylation 1 (ARF1) est surexprimé dans les lignées cellulaires hautement invasives du cancer du sein et que la stimulation du récepteur au facteur de croissance épidermique (EGFR) peut activer cette isoforme pour contrôler la migration ainsi que la prolifération. Cependant, le rôle de cette GTPase dans la régulation du processus d’invasion cellulaire et les mécanismes moléculaires associés demeure inconnu. Nous avions comme objectifs dans cette thèse, de définir les voies de signalisation sous le contrôle d’ARF1 dans les cellules de cancer du sein et démontrer que l’expression et l’activation de cette GTPase est associée à un phénotype hautement invasif. Nos études démontrent que la modulation de l'expression et l'activité d’ARF1 affecte la capacité des cellules MDA-MB-231 (pour M. D. Anderson-metastatic breast-231), une ligne hautement invasive, à dégrader la matrice extracellulaire via l'activité de la métalloprotéinase MMP-9. ARF1 contrôle les deux principales structures impliquées dans l'invasion, en jouant sur la maturation d’invadopodes ainsi que la relâche de microvésicules membranaires. D’un point de vue mécanistique, l'axe de signalisation ARF1, RhoA-RhoC et la chaine légère de la myosine (MLC) explique ces phénomènes. De plus, nous démontrons que l'un des mécanismes par lequel ARF1 régule la migration est en contrôlant l'assemblage des points adhésions focaux et ce, dans plusieurs types de cellules cancéreuses du sein. ARF1, en étant un membre du complexe d’adhésion, réglemente le recrutement et l’activité de protéines clés à la β1-intégrine tels que la paxilline, la talin et la kinase d’adhésion focale (FAK). Pour finir, nous rapportons que ARF1 et ARF6 ont un rôle majeur dans la transition épithélio-mésenchymateuse. ARF1 est retrouvé fortement exprimé dans les tissus de sous-types les plus agressifs et les plus avancés de cancer du sein. Dans un modèle murin, la modulation à la baisse de l’expression d’ARF1 dans les cellules MDA-MB-231 corrèle avec la diminution de croissance des tumeurs primaires et l’installation des métastases pulmonaires. De plus, nous rapportons que la surexpression des ARF dans des cellules non invasives, les MCF7 (pour Michigan Cancer Foundation-7), permet la nidification de métastases. En effet, dans les MCF7, ARF1 contrôle l’adhésion intercellulaire via la β-caténine et l’E-cadhérine, promeut l’activation de l’oncogène Ras (pour Rat Sarcoma/ Rat Fibrosarcoma virus) et l’expression de plusieurs inducteurs de transition épithélio-mésenchymateuse comme snail et slug. De plus, ARF1 contrôle l’invasion, la prolifération cellulaire et même la résistance à certains agents chimio-thérapeutiques. Globalement, nos études identifient ARF1 comme un interrupteur moléculaire de la progression tumorale et suggèrent que la limitation de son expression/activité pourrait améliorer le devenir des patients atteints du cancer du sein. / Invasive and metastatic chapacities are fundamental features for tumor malignancy. We have recently shown that the ADP-ribosylation factor 1 (ARF1) is over-expressed in highly invasive breast cancer cell lines and stimulation of the epidermal growth factor receptor (EGFR) may activate this isoform to regulate migration and proliferation. However, the role of this GTPase in regulating cell invasion process and related molecular mechanisms remain unknown. In this thesis, we had as objectives, to define the signaling pathways under the control ARF1 in breast cancer cells and show that the expression and activation of the GTPase is associated with highly invasive phenotype. Our studies show that the modulation of the expression and activity of ARF1 affect the ability of MDA-MB-231 cells (M. D. Anderson-metastatic breast-231), a highly invasive line, to degrade the extracellular matrix via the activity of the metalloproteinase MMP-9. ARF1 controls the two main structures involved in the invasion, playing on invadopodia maturation and shedding of membrane microvesicles. The molecular mechanisms involve the regulation of RhoA and RhoC activity by ARF1 and the following downtream events associated with and the myosin light chain (MLC) phosphorylation. Furthermore, we demonstrate that ARF1 also regulates migration by controlling the assembly of focal adhesion complexes in many types of breast cancer cells. ARF1, also prensent in adhesion complexes, regulates the recruitment and activity of key proteins such as paxillin, talin and focal adhesion kinase (FAK) to β1 integrin. Finally, we report that ARF1 and ARF6 play a major role in the epithelial-mesenchymal transition (EMT). ARF1 is found highly expressed in tumor tissue of the most aggressive and advanced subtypes of breast cancer. Lowered expression of ARF1 in vivo in the MDA-MB-231 cells impars tumor growth in primary tumors and inhibits lung metastasis. We report that upregulation of the ARF in non-invasive cells, MCF7 (Michigan Cancer Foundation-7) induce metastasis nidification. Indeed, we show in MCF7 that ARF1 controls intercellular adhesion via the β-catenin and E-cadherin, promotes Ras (Rat Sarcoma/ Rat Fibrosarcoma virus) oncogene activation, and conrols expression of several epithelial-mesenchymal transition markers such as snail and slug. Moreover, we demonstrate that ARF1 controls invasion, proliferation and even resistance to certain chemo-therapeutic agents, in MCF7 cells. Overall, our studies identify ARF1, as a molecular switch of tumor progression and suggest that limiting its expression / activity could improve the outcome of breast cancer patients.