The RasGAP—SH3-domain Binding Protein (G3BP1) is an endoribonuclease that links Ras-signalling pathways and mRNA stability. Though implicated in many cellular roles, a well defined function for G3BP1 has yet to be determined. The aim of the following dissertation is to further implicate G3BP1 in mammalian cellular responses to environmental stress, most notably that of Ultraviolet Radiation. We observe G3BP1 transverse from the cytoplasm to the nucleus upon treatment of HeLa cells with apoptotic levels of UVC. Interestingly, forcing its nuclear accumulation in the absence of stress is sufficient to induce apoptotic characteristics. Preliminary observations suggest that plasma membrane signalling and the resultant activation of stress activated protein kinases, JNK and p38 MAPK, and CASPases might play a role in this process. Thus, we further elaborate G3BP1's role in cellular responses to stress and suggest a new function for the protein, that being the promotion of apoptosis. / "RasGAP—SH3-domain Binding Protein (G3BP)" est un endoribonucléase qui associe la voie de signalisation de Ras et la stabilité d'ARN messager. On ne connaît pas une fonction bien défini pour cette protéine, même si c'est impliquée dans plusieurs rôles cellulaire. Le but de cette dissertation est d'appuyer l'implication du G3BP1 aux réponses cellulaire mammalien au stress environmental, particulièrement à la radiation ultraviolet. On observe le G3BP1 transverse du cytoplasm au noyau au traitement des cellules HeLa avec des doses UV apoptotique. La chose fascinante est quand on introduit s'accumulation du noyau en l'absence du stress est suffisant de provoquer les charactéristiques apoptotique. Les observations au premier degré suggèrent que les signalisations d'origine de membrane plasmique et l'activation resultant du JNK, p38 MAPK, et CASPases peuvent jouer un rôle en ce processus. Ainsi, on a élaboré le rôle du G3BP1 en réponse cellulaire au stress et suggère une nouvelle fonction pour la protéine : la promotion de l'apoptose.

Mu and D108 are temperate bacteriophages which can follow two possible modes of growth after infection of Escherichia coli. The bacteriophages can follow the lytic cycle, in which the bacteriophage genome is replicated and progeny phages are produced, or the lysogenic cycle, in which the bacteriophage remains dormant in the E. coli genome. In this case, the bacteriophage's DNA is replicated along with the host chromosome. Central to the lytic/lysogenic switch, is the Ner protein. Ner is a small, basic, DNA-binding protein. To better understand the mechanism by which bacteriophage D108 regulates its lytic/lysogenic switch, a plasmid containing the D108 left end was mutagenized by hydroxylamine treatment or by transformation into a mutator strain of E. coli, in order to isolate Ner proteins which would be deficient in repressor activity. / The 3-dimensional structure of the Ner protein from bacteriophage Mu has been resolved by NMR spectroscopy. / Homologs of the Ner proteins have been identified in the Enterobacteriaceae and in humans. (Abstract shortened by UMI.)

The interferon (IFN)-inducible protein kinase PKR is thought to mediate the biological effects of IFNs through its ability to phosphorylate eukaryotic initiation factor 2 and to inhibit protein synthesis. Additional reports have implicated PKR in transcriptional regulation. In this study, a role for PKR in the regulation of signal transduction pathways that are required for IFN action was examined. It is demonstrated that PKR negatively regulates signaling pathways induced by IFNs and dsRNA. This regulatory effect is attributed to a protein-protein interaction between PKR and STAT1, the signaling component common to these pathways, that impairs STAT1 transcriptional function. The interaction between PKR and STAT1 is not a kinase-substrate relationship since PKR does not phosphorylate STAT1 and both wildtype and catalytically inactive PKR can interact with STAT1. It is also shown that PKR regulates STAT1 protein expression. The regulatory effect does not occur at the transcriptional or post-transcriptional level since STAT1 mRNA transcription and stability are unaffected by PKR. Regulation of STAT1 expression is specific for STAT1 since expression of other STATs is not affected by PKR. The ability of PKR to regulate STAT1 function and expression has important implications in the control of gene expression, viral immunity, and cell growth.

Bone homeostasis results from the delicate balance between bone formation, by osteoblasts, and bone resorption, by osteoclasts in a process termed remodeling which occurs throughout adult life. The tumor suppressor PTEN, which antagonizes the activity of PI3Kinase by dephosphorylating the D3 position of phosphatidyl inositol (3,4,5)P3, has been shown to be implicated in many of the same signaling pathways that are important in the differentiation and activation of both osteoclasts and osteoblasts. Very few studies have attempted to elucidate the role that PTEN may play in bone biology. Here we use a murine conditional knockout approach to specifically target PTEN ablation to osetoclasts, using a CD11b-Cre, and to osteoblasts, with the Col-1α(I)-Cre. Our results indicate that PTEN may be playing an essential in vivo role in cell survival of osteoclast precursors and regulating the ability of osteoblasts to support osteoclastogenesis as well as confirming its role in bone mass accumulation. / L'homéostasie osseuse est le résultat de deux processus opposés mais complémentaires qui se poursuivent au cours de la vie adulte des vertébrés, celle de la minéralisation par les ostéoblastes et celle de la résorption par les ostéoclastes. PTEN est un suppresseur de tumeur qui oppose la fonction et les effets de la PI3Kinase en agissant comme phosphatase sur la position 3 du phosphatidyl inositol (3,4,5)P3. De cette manière, il agit sur plusieurs des mêmes voies de signalisation qui ont été démontré importantes dans la différentiation et la survie des cellules osseuses. Jusqu'à présent, peu d'études ont été effectuées pour démontrer le rôle précis que joue PTEN dans ces cellules. Nous avons utilisé l'approche d'ablation conditionnelle chez la souris afin de cibler la perte d'activité de PTEN dans les ostéoclastes, avec la souris CD11b-Cre, et dans les ostéoblastes, avec la souris Col-1α(I)-Cre. Nos résultats indiquent que PTEN joue un rôle important in vivo pour la survie des précurseurs des ostéoclastes et dans la régulation de la fonction de différentiation des ostéoclastes par les ostéoblastes.

PTP-PEST is a soluble intracellular phosphatase that is ubiquitously expressed in mammalian tissues. The biological importance of this enzyme is highlighted by the fact that it is essential for murine embryogenesis and that it has been implicated in actin dynamics as well as other processes such as immunoreceptor signalling and cell death. Having five proline-rich regions in its C-terminal tail, PTP-PEST has a large potential for protein:protein interactions, several of which are known. To better understand the physiological importance of PTP-PEST, we explored the possibility that other substrates or binding partners existed. To this end, we carried out a modified yeast two-hybrid assay using full-length PTP-PEST with the D199A substrate trapping mutation as bait and screened a library of cDNA clones from 17-day-old mouse embryos. Screening identified the cytosolic adaptor protein, SKAP-HOM, as a novel substrate of PTP-PEST and GST pull-down assays confirmed this interaction in HeLa299 cells. We showed that PTP-PEST's PTP-domain and proline-1 region are essential for this interaction, as are SKAP-HOM's Y260 residue and SH3 domain. Further mutagenesis studies revealed that PTP-PEST residues S39 and S434 play a role in inhibiting and promoting the interaction with SKAP-HOM, respectively. Preliminary evidence was also obtained suggesting that p59fyn kinase plays a role in promoting the PTP-PEST D199A:SKAP-HOM interaction through a mechanism other than SKAP-HOM phosphorylation. Understanding the biological significance of this interaction could lead to novel discoveries in the fields of cell metastasis and inflammation. / PTP-PEST est une phosphatase soluble intracellulaire qui est exprimée de façon ubiquitaire dans les tissus de mammifères. L'importance biologique de cette enzyme relève du fait qu'elle est essentielle durant l'embryogénèse chez la souris, et qu'elle est de plus impliquée dans l'organisation dynamique du cytosquelette d'actine, ainsi que dans d'autres processus tels la signalisation par les immunorécepteurs et la mort cellulaire. Avec ses cinq régions riches en motifs proline dans la région C-terminale, PTP-PEST possède un large potentiel d'interactions protéine : protéine, parmi lesquelles plusieurs sont déjà identifiées. Afin de mieux comprendre l'importance physiologique de PTP-PEST, nous avons exploré la possibilité de l'existence d'autres substrats ou ligands. À cette fin, nous avons développé un test modifié de levures à deux-hybrides, utilisant une construction pleine longueur de PTP-PEST insérée d'une mutation de piégeage de ligands D199A comme appât. Suite à l'analyse d'une banque d'ADNc provenant d'embryons de souris de 17 jours, nous avons identifié une protéine adaptatrice cytosolique, SKAP-HOM, en tant que nouveau substrat de PTP-PEST. L'essai de capture de la protéine GST a de plus confirmé cette interaction dans les cellules HeLa299. Nos résultats démontrent que le domaine PTP de PTP-PEST, ainsi que la région proline-1, sont essentiels à cette interaction, tout comme le résidu Y260 et le domaine SH3 de SKAP-HOM. Une analyse de mutagénèse plus approfondie a identifié les résidus S39 et S434 de PTP-PEST comme jouant respectivement un rôle dans l'inhibition et dans la promotion de l'interaction avec SKAP-HOM. Nous avons aussi obtenus des résultats préliminaires suggérant que la protéine kinase p59fyn joue aussi un rôle dans la promotion de l'interaction PTP-PEST D199A:SKAP-HOM, par un mécanisme autre que la phosphorylation de SKAP-HOM. La compréhension de l'importance biologique de cette interaction pour

High fidelity chromosome segregation in all cells requires the formation of bi-oriented attachments between spindle microtubules (MT) and chromosomes. The kinetochore provides a bridge between the MTs and chromosomes. Ame1 is an essential but undercharacterized component of the central kinetochore COMA sub-complex (Ctf19, Okp1, Mcm21, Ame1). In order to characterize Ame1, I used two conditional alleles of the COMA, ame1-4 and okp1-5. I examined the role of Ame1 in the context of the kinetochore and in the maintenance of the spindle assembly checkpoint (SAC) and the formation and repair of kinetochore-MT attachments. I found that ame1-4 cells have a compromised COMA. In contrast, the COMA is disrupted in okp1-5 cells, but Ame1 remains localized to the kinetochore. Nonetheless, the stability of DNA binding and MT binding kinetochore complexes remains intact in ame1-4 and okp1-5 cells. I used the difference between okp1-5 and ame1-4 to further delineate the relationship between Ame1 and the COMA. ame1-4 cells exhibit defective sister chromatid attachments that are not repaired, and are unable to maintain a checkpoint arrest. We find that disruption of the COMA results in a failure to maintain the localization of Sli15 to the kinetochore. In turn, Sli15 functions in checkpoint maintenance and spindle passenger protein migration. Indicative of the loss of passenger protein migration, ame1-4 cells exhibit a cytokinesis defect. Finally, over-expression of OKP1 in ame1-4 cells restores localization of ame1-4p and re-establishes checkpoint maintenance but does not restore Sli15 kinetochore localization. A synthetic genetic screen (SGA) was carried out to identify genetic interactors of the ame1-4 allele. Thirty-three (33) genes were found to interact with ame1-4 to produce a synthetic sick/lethal phenotype. Many genes identified were common interactors with other kinetochore subunits. Comparing the genetic interaction network of the COMA genes, ame1-4 shared many interac / Dans toute cellule en division, la haute fidélité de la ségrégation des chromosomes passe par la formation d'attachements bi orientés entre les microtubules du fuseau (MT) et les chromosomes. Le kinétochore est la structure faisant le lien entre les MT et les chromosomes. Le complexe COMA (Ctf19, Okp1, Mcm21, Ame1) est un sous-complexe central du kinétochore, et Ame1 en est un composant essentiel quoique peu caractérisé. Dans le but de caractériser Ame1, j'ai utilisé deux allèles du complexe COMA: ame1-4 et okp1-5. J'ai examiné le rôle d'Ame1 dans le cadre du kinétochore et de la maintenance du point de contrôle de l'assemblage du fuseau ainsi que dans la formation et la réparation des attachements entre kinetochore et MT. J'ai constaté que, dans les cellules ame1-4, le complexe COMA est compromis. Dans les cellules okp1-5, le complexe COMA est également perturbé, alors qu'Ame1 est localisé au kinétochore. Toutefois, dans les cellules ame1-4 comme dans les cellules okp1-5, la stabilité de la liaison à l'ADN et aux MT des complexes du kinétochore demeure intacte. J'ai utilisé la différence entre les mutants okp1-5 et ame1-4 pour mieux comprendre la relation entre Ame1 et le complexe COMA. Dans les cellules ame1-4, les attachements des chromatides soeurs sont déficients. Ceux-ci ne sont pas réparés et sont incapables de maintenir l'arrêt de croissance suite à l'activation du point de contrôle. Nous avons constaté que l'inactivation du complexe COMA provient de l'incapacité à localiser Sli15 au kinétochore. Par ailleurs, Sli15 joue un rôle dans la maintenance du point de contrôle et la migration des protéines passagères du fuseau. Le fait que les cellules ame1-4 présentent une cytokinèse déficiente indique une perte de migration des protéines passagères. Enfin, la surexpression d'OKP1 dans les cellules ame1-4 rétablit la localisation de ame1-4 et la maintenance du point de contrôle mais pas la localisation de Sli15 au$

Patterns of methylation at the 5-position of cytosine are postulated to be involved in several mammalian processes such as the regulation of gene expression, genomic imprinting and X chromosome inactivation. Disruptions in methylation patterns affect embryonic development and are involved in carcinogenesis, indicating the importance of regulating these patterns. The establishment of methylation patterns is believed to be initiated during gametogenesis and continue during early embryonic development. DNA (cytosine-5)-methyltransferase (EC2.1.1.37) (DNA methyltransferase) is the only known mammalian enzyme that catalyzes the formation of 5-methyl cytosine. Here, DNA methyltransferase is proposed to play a key role in establishing and maintaining methylation patterns during male gametogenesis and is predicted to be regulated during spermatogenesis. The regulation of DNA methyltransferase mRNA during testicular development in mouse and rat was first established as preliminary evidence for the regulation of DNA methyltransferase during spermatogenesis. Highly enriched populations of spermatogenic cell types were then isolated from mouse testes and examined for DNA methyltransferase mRNA and protein expression. Expression of DNA methyltransferase protein was proportional to the levels of a 5.2 kb mRNA. Substantial levels of protein and 5.2 kb mRNA were observed in mitotic types A and B spermatogonia, preleptotene spermatocytes, meiotic leptotene/zygotene spermatocytes and postmeiotic round spermatids but barely detectable in pachytene spermatocytes in which a large decrease in 5.2 kb mRNA levels and the appearance of a 6.2 kb transcript was observed. The subcellular localization of DNA methyltransferase in germ cells was also regulated. These findings reveal DNA methyltransferase to be highly regulated during spermatogenesis and support a role for the enzyme in events associated with germ line methylation changes such as developmental gene regulation, genomic imprintin

The cap binding protein complex (elF-4F) has been shown to play an important role in the regulation of gene expression. The cloning of the genes encoding the large subunit of eIF-4F in yeast and mammals (p150 and p220 respectively) have provided the opportunity to characterize this little understood polypeptide. In this study, two functions of the yeast homolog, TIF4631 (p150), were analyzed: (1) complex formation with eIF-4E, and (2) RNA binding. Interaction with eIF-4E was found to require the presence of an internal region of p150 surrounding an evolutionarily conserved 9 amino acid motif. The RNA-binding capacity of p150 was also demonstrated by UV-induced crosslinking and by binding to a poly-uridylic acid resin. Deletion analysis identified the presence of two RNA-binding domains, one within the N-terminal portion of the protein and the other immediately C-terminal to the eIF-4E binding region. A sequence resembling the RGG-box RNA binding motif was found in the N-terminal domain, whereas the internal RNA binding domain is localized around the previously identified RS-motif-like region.

The proximal promoter of the rat carbamyl phosphate synthetase I gene contains 4 cis-acting regulatory regions, designated sites GAC, I, II, and III. Transient transfection assays revealed that the GAG element functions as the main activator element, whereas sites I and III bind repressor proteins and site II binds an anti-repressor. We propose that sites I and III repress the CPS I promoter either by quenching the sole activator(s) bound to the GAG element, or by directly repressing the basal transcription apparatus. Footprint analyses of the promoter with and without a mutation within site II suggested that the site II-anti-repressor may interfere with the site I and site III repressors by a similar quenching mechanism. Although the elements within the proximal promoter exhibit the potential for complex regulation, they are unable to confer tissue-specific promoter activity when analyzed either by in vitro transcription employing nuclear extracts from tissues that do not express the endogenous gene (i.e. brain, kidney, and spleen), or by transient transfection assays in non-CPS I-expressing cells (i.e. Rat1 and C6). However, we identified a cell-type specific enhancer that lies 10 kb upstream of the promoter. Unexpectedly, the enhancer functions as well in a gene-specific manner because enhancer-stimulated promoter activity was exclusively dependent on the GAG element within the CPS I proximal promoter. As a result, the GAG element is required to couple enhancer activity to the proximal promoter and exemplifies an efficient method whereby enhancers can be constrained to activate only their cognate promoters.

This project will attempt to characterize the OPN secreted from our human embryonic kidney cells. Since we believe that tissue-specific post-translational modifications, such as phosphorylation and glycosylation, play a crucial role in the physiological properties of OPN with regards to its function, the focus will be to obtain some insight into the post-translational modifications occurring to OPN in our renal expression system. We believe that these modifications are comparable to what is occurring to OPN in vivo. / Some of these post-translational modifications, namely phosphorylation, appear to influence the activity of OPN as an inhibitor of nucleation, growth and aggregation of crystals. Therefore in addition to characterizing OPN, an attempt will be made to look at some of the hormones which could potentially play a role in regulating the post-translational processes occurring to this protein. Kidney stone disease being a calcium dependent process, this project will focus on two hormones, PTH and 1,25(OH)2D3, because of their involvement in calcium regulation in the body.