The ability to sense and respond to fluctuating environmental conditions is a conserved and essential feature of all living organisms. Cellular stress responses sense and transmit the demands of specific stressors to appropriate homeostatic effectors, which can then implement appropriate cellular adaptation mechanisms. This study examines two distinct and highly conserved eukaryotic signaling responses that are implemented in response to unfolded protein in the endoplasmic reticulum, a situation known as ER stress. We find that Schizosaccharomyces pombe calnexin (Cnx1p) is regulated by phosphorylation at a conserved serine residue in its cytoplasmic tail. Our analysis of Cnx1p-S553 phosphomutants suggests that this post-translational modification regulates the association of calnexin with ER membrane-bound ribosomes. We also find that this regulation of Cnx1p is important for cell size control and ER stress tolerance. Next, we identify and characterize a number of novel small-molecule inhibitors of the Ire1-dependent ER homeostatic mechanisms in Saccharomyces cerevisiae. We extend these findings beyond this yeast model by showing that one of these small-molecules also inhibits Ire1 signaling in mammalian cells. This compound, designated UPRM8, was used as a chemical probe to study the importance of Ire1-dependent ER homeostatic responses in constitutively ER-stressed multiple myeloma cells. UPRM8 elicited an apoptotic cell death in RPMI-8226 multiple myeloma cells due to its impairment of cytoprotective, Ire1-dependent ER homeostatic mechanisms. Lastly, this study also examines the roles of a cell polarity scaffold protein, called Bem1p, in the responses to pheromone and cell wall stressors in Saccharomyces cerevisiae. A novel separation of function mutant of Bem1, called Bem1-s3, was identified and it revealed that the tandem SH3 domains of Bem1p are required for efficient pheromone signaling. Furthermore, we used mass spectrometry to identify novel Bem1p-interacting proteins and post-translational modifications. These novel physical interactions, as well as the phenotypes of Bem1 null and phosphomutants, suggested a novel role for Bem1p in the adaptive response to cell wall stressors. In summary, this thesis describes stress signaling mechanisms and some methods of manipulating them in order to exert control over cell fate. / La capacité à percevoir et à répondre aux fluctuations des conditions de l'environnement est une caractéristique essentielle et conservée chez tous les organismes vivants. Les réponses aux stress cellulaires sont sensibles aux exigences de facteurs de stress spécifiques qu'elles transmettent à des effecteurs homéostatiques appropriés, et qui à leur tour induisent des mécanismes cellulaires d'adaptation. Cette étude examine deux réponses distinctes et hautement conservées qui sont mises en oeuvre en réaction à la présence de protéines incorrectement repliées dans le Réticulum Endoplasmique (RE), une situation appelée "stress réticulo-endoplasmique". Nous avons démontré que la calnexin (Cnx1p) de Schizosaccharomyces pombe est régulée par la phosphorylation d'un résidu sérine conservé et localisé à l'extrémité cytoplasmique de la protéine. Notre analyse du phospho-mutant Cnx1p-S553 suggère que cette modification post-traductionnelle régule l'association de la calnexine aux ribosomes liés à la membrane du RE. Nous avons également constaté que la régulation de la phosphorylation de la calnexine est importante pour le contrôle de la dimension cellulaire et la tolérance au stress réticulo-endoplasmique. Ensuite, nous avons identifié et caractérisé un certains nombre de nouvelles petites molécules qui sont des inhibiteurs des mécanismes d'homéostasie du RE dépendant d'Ire-1 chez Saccharomyces cerevisiae. Ces résultats ont été étendu au-delà du modèle de la levure, en montrant que l'une de ces petites molécules inhibe également la voie de signalisation d'Ire-1 chez des cellules de mammifères. Ce composé, désigné par UPRM8, a été utilisé comme test chimique pour étudier l'importance de la réponse homéostatique au stress réticulo-endoplasmique constitutivement présent chez les cellules de myélomes multiples. UPRM8 induit une apoptose chez les cellules de myélomes multiples RPMI-8226 en empêchant les mécanismes cyto-protectifs dépendants d'Ire-1. Finalement, cette étude a également examiné les rôles d'une protéine impliquée dans la polarité cellulaire, appelée Bem1p, dans la réponse à une phéromone et à un facteur de stress de la paroi cellulaire chez Saccharomyces cerevisiae. Un nouveau mutant de Bem1, nommé Bem1-s3, a été identifié et a révélé que les domaines SH3 en tandem de Bem1p sont requis pour une signalisation efficace des phéromones. De plus, nous avons utilisé la spectrométrie de masse pour identifier de nouvelles protéines qui interagissent avec Bem1p ainsi que des modifications post-traductionnelles. Ces nouvelles interactions ainsi que les phénotypes des mutants Bem1 et Bem1p ont suggéré un nouveau rôle pour Bem1p dans la réponse adaptative à des facteurs de stress de la paroi cellulaire. En résumé, cette thèse décrit des mécanismes de signalisation du stress et des moyens mises en œuvre par la cellule afin de composer avec les facteurs de stress et d'exercer un contrôle sur son devenir.

A 186-bp fragment of ors8, a mammalian DNA replication origin, was previously identified as the minimal sequence required for origin function in vivo and in vitro. The 186-bp origin contains, among other features, an octamer motif, serving as the binding site for the transcription factor Oct-1, and an A3/4-homologous region, serving as the binding site for the origin binding protein, OBA. The research objective of this thesis is to investigate the role of Oct-1 and OBA in mammalian in vitro DNA replication. / Depletion of the Oct-1 protein inhibited in vitro DNA replication to basal levels. The inhibition was partially reversed upon the exogenous addition of Oct-1 POU protein. Site-directed mutagenesis of the octamer motif in the origin resulted in a mutant clone that lacked Oct-1 binding but replicated efficiently. The results suggest that Oct-1 is an enhancing component in DNA replication but its effect is not caused through direct DNA binding, but rather through protein-protein interactions. / OBA was purified from HeLa cells through its ability to interact with the 186-bp origin. OBA binds to A3/4, a 36-bp mammalian origin sequence. The DNA binding subunit of OBA is identical to the 86 kDa subunit of Ku antigen. Depletion of OBA/Ku, by inclusion of anti-Ku antibodies to the in vitro replication reaction of p186, inhibited DNA replication. Furthermore, addition of the A3/4 oligonucleotide to the replication reaction of mammalian origin-containing plasmids (p186, pors12, and pX24) inhibited replication. This inhibition was reversed upon addition of OBA. In contrast, SV40 in vitro replication remained unaffected. OBA also possessed DNA helicase activity. By co-immunoprecipitation analyses, OBA was found to associate with DNA polymerases delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, and Oct-1. / The replication activity of xrs-5, a derivative of Chinese hamster ovary (CHO) K1 cell line defective in Ku86, was also examined. Total and cytoplasmic extracts from xrs-5 cells replicated p186 DNA as efficiently as CHO K1 extracts. In contrast, xrs-5 nuclear cell extracts lacked DNA replication activity, which was restored upon addition of OBA. / The data implicate OBA/Ku in a direct role in the initiation of mammalian DNA replication as an origin-specific binding protein with helicase activity. The physical association of OBA/Ku with Oct-1 and the replication machinery reveals a possible mechanism by which these proteins are recruited to DNA replication origins. A cytoplasmic Ku-like origin-specific binding protein likely compensates for the absence of the endogenous OBA/Ku in xrs -5 cells. / These studies provide a better understanding of the proteins that bind to mammalian origins, thereby aiding in understanding the mechanisms that regulate the initiation of DNA replication.

The follicle stimulating hormone receptor (FSHR) is an essential G-protein-coupled receptor expressed in granulosa cells of the ovary and Sertoli cells of the testis. Upon hormone binding, the FSHR activates G-proteins, initiates gonadotropin signals and triggers granulosa cells and oocyte or Sertoli cells and germ cells communication that is required for gonadal development and maturation. This hormone signaling also participates in the biosynthesis of progesterone and estradiol in females and testosterone in males. The expression of FSHR reaches higher levels in mature follicles and in stages XIII-I of the seminiferous epithelial cycle. Little is known on the mechanisms by which cell- and stage-specific expression of FSHR is regulated. In the present studies, we have characterized four cis-elements in an ovine FSHR (oFSHR) promoter, and identified the corresponding DNA binding proteins. The first cis-element at +32 to +54 of the proximal oFSHR promoter was characterized as an E-box overlapping an orphan receptor response element (ORRE) to which upstream stimulatory factors (USFs) and chicken ovalbumin upstream promoter transcription factors (COUP-TFs) can compete for their binding. The second element was mapped at -46 to -67 as a CACC box that Krupple-like transcription factors recognize. The third cis-element at -117 to -197 was found to be a composite activator protein-1 (AP-1) binding site embedding an E-box and an ORRE that a group of proteins including AP-1, steroidogenic factor-1 (SF-1) and COUP-TFs can competitively occupy depending on their availability and activity. The fourth element from -284 to -303 was identified as a composite retinoic acid response element (RARE) that is recognized by retinoic acid receptors (RARs) and SF-1. Functional studies revealed that USFs, AP-1 and SF-1 were activators whereas COUP-TFs and RARs were repressors in the presence of retinoic acid (RA). Our data suggest a mechanism by which activators and repressors compete for

While replication origins, cis-acting sequences directing the initiation of DNA synthesis, have been well-characterized in many model organisms, the multiple sequence and protein components present at the chromosomal origins of higher eukaryotic organisms have not yet been fully defined. Genetic assays that identify origin function in cloned DNA fragments would provide a useful approach for the isolation and analysis of mammalian DNA replication origins. / In this thesis, (1) cloned fragments from a known mammalian origin, the ori$ beta$ of the hamster 3$ sp prime$ DHFR region, are demonstrated to replicate autonomously, both following transfection into human cells, and when used as templates in an in vitro replication system based on human cell extracts; (2) larger scale versions of these two assay methodologies are used to isolate over 40 novel putative origins of DNA replication from anticruciform purified human genomic DNA libraries; (3) transfection and in vitro autonomous replication assays are applied to demonstrate the potential origin function of a mitochondrial DNA sequence implicated in the insertional mutagenesis of a human genomic locus; (4) an origin mapping strategy based on the in vitro assay is used to provide evidence for the existence of a replication origin in a cloned and sequenced portion of the human 15q11q13 chromosomal subdomain, a region associated with allele-specific replication timing, genomic imprinting, and genetic disease; and (5) some of these autonomously replicating origins are cloned into a selectable YAC vector and are shown to permit the long term episomal maintenance, in human cells, of the transfected plasmid constructs. / These results consistently demonstrate that short mammalian genomic DNA fragments can replicate autonomously, supporting the applicability of the replicon model in humans, and could be extended to the search for an origin core consensus element, to the investigation of higher order organization and temporal control of human DNA replication origins, and to the construction of a complete human artificial chromosome.

The response of target cells to the polypeptide hormone, prolactin, is initiated by the inaction of the hormone with a cell surface receptor that belongs to the class of receptors with a single membrane-spanning domain, designated as the cytokine/GH/PRL receptor family. The mechanism of signal transduction for this family of receptors is not yet determined. Cloning and functional analysis of different forms of the prolactin receptor (PRL-R) were carried out to elucidate the mechanism of signal transduction. The protein sequence deduced from cDNAs isolated from a rat ovarian cDNA library revealed the existence of a long form of the PRL-R in the rat. The mature form has a predicted size of 591 amino acids and shows strong overall sequence similarity with previously cloned long forms of the PRL-R from other species. Another form of the PRL-R was characterized in Nb$ sb2$ cells, a rat lymphoma cell line, dependent on PRL for mitogenesis. This receptor is a deleted form of the long form of the PRL-R missing 198 amino acids in the cytoplasmic domain. The ability of the different forms of the PRL-R to transmit a lactogenic signal was tested in functional systems. The systems consisted of the transient co-transfection of chinese hamster ovary (CHO) cells with a fusion gene containing a milk protein gene promoter linked to the chloramphenicol acetyl transferase (CAT) gene coding region and an expression vector containing various forms of PRL-R cDNAs under the control of the SV-40 early promoter. CAT activity was measured three days after transfection. Results indicate that the long form of the PRL-R is capable of transmitting PRL's signal and activating the milk protein gene promoter. However, when the short form of the receptor was transfected, there was no induction of CAT activity. Interestingly, the Nb$ sb2$ form of the PRL-R was fully capable of transmitting PRL's signal, when examined in the above functional system. This implies that the 198 amino acids segment of t

lck is a member of the src family of tyrosine protein kinase genes expressed predominantly in T-lymphocytes. Its product, p56$ sp{ rm lck}$ is physically and functionally associated with the CD4 and CD8 T-cell surface antigens, suggesting a possible role in CD4/CD8-mediated tyrosine phosphorylation signals during the process of antigen stimulation. Activation of p56$ sp{ rm lck}$ by mutation of tyrosine 505 was prevented by further mutation of either the site of autophosphorylation, tyrosine 394 to phenylalanine, or the site of myristylation, glycine 2 to alanine, as determined by the effects of overexpression of these p56$ sp{ rm lck}$ mutants in rodent fibroblasts. The role of activation of p56$ sp{ rm lck}$ in T-cells was examined by overexpression of the tyrosine 505-to-phenylalanine mutant in a CD4-negative, MHC-class II restricted T-cell hybridoma. The enhancement of T-cell responsiveness observed with expression of activated Lck was qualitatively similar to the effect of expression of CD4 in these cells, providing evidence that p56$ sp{ rm lck}$ positively regulates T-cell functions and that it mediates some of the functions of CD4 and CD8 during antigen stimulation.

<p> <i>myo</i>-Inositoloxygenase (MIOX) catalyzes the first step in <i>myo</i>-inositol catabolism. MIOX has not been annotated in <i> Drosophila melanogaster</i>, but the protein encoded by the <i> CG6910</i> gene is similar to the mouse MIOX protein. <i>CG6910 </i> "knocked-down" expression was explored using RNAi. "Knock-down" flies did not survive on inositol defined media, indicating that <i> CG6910</i> encodes MIOX. Survival of these flies on sucrose defined media suggest that MIOX is not essential for development. Biochemical assays demonstrated that <i>D. melanogaster</i> has MIOX activity. Computational analyses revealed potential miRNA sites, and that a number of essential components are conserved. <i>MIOX</i> genes found in other drosopholids are highly similar to <i>D. melanogaster MIOX</i>, and analyses of the syntenic regions concur with established evolution. Western blot analyses showed differential expression amongst <i>D. melanogaster</i> from different geographic locations and between species. These studies may contribute to understanding the role of inositol catabolism in fruit fly development and diabetes.</p>

There is increasing evidence that CUX1 can act as an oncogene in various cell types. First of all, it was found to be overexpressed in different human tumors, such as in uterine leiomyomas, breast tumors and pancreatic tumors. Both p75 and p110 were also found to be overexpressed in tumor cell lines of different origins. My research project has focused on characterizing the oncogenic potential of short CUX1 isoforms through the study of transgenic mice. The first model we used was to overexpress p75 CUX1 under the control of the cytomegalovirus enhancer-chicken β-actin promoter. Interestingly, these mice developed polycystic kidneys at variable penetrance and severity, correlating with transgene expression levels. Cystic kidneys were found to display enhanced proliferation and also showed an upregulation of c-myc and downregulation of p27KIP1. The second model we used was to overexpress p75 or p110 CUX1 under the control of the mouse mammary tumor virus-long terminal repeat in the hypoxanthine phosphorybosyltransferase locus. The first phenotype we observed was that p75 CUX1 transgenic virgin female mice of the first backcross generations developed a myeloproliferative-disease-like myeloid leukemia. These results indicate that overexpression of p75 CUX1 can alter the proliferation and differentiation of some hematopoietic cells. The second phenotype we observed in these mice was that both p75 CUX1 and p110 CUX1 mice of pure FVB background developed mammary tumors of various histopathologies. Metastasis to the lung was observed in the p75 CUX1 transgenic mice only. Comparisons between tumors and adjacent normal mammary glands revealed that transgenes were overexpressed in most but not all tumors, yet in all cases tested CUX1 DNA binding was increased. Interestingly, higher expression of erbB2 mRNA was seen in most solid carcinomas, while adenosquamous carcinomas displayed higher expression of various Wnt / De plus en plus d'études suggèrent que le facteur de transcription CUX1 soit un oncogène. Premièrement, CUX1 est surexprimé dans certaines tumeurs humaines, dont les léiomyomes utérins, les tumeurs du sein et les tumeurs du pancréas. Les isoformes courtes CUX1 p75 et p110 sont de plus surexprimées dans plusieurs lignées cellulaires provenant de différents types de tumeurs humaines. L'objectif de mon projet de recherche était de vérifier le potentiel oncogénique des isoformes courtes de CUX1 dans des souris transgéniques. Dans le premier modèle de souris, CUX1 p75 était exprimé sous le contrôle du promoteur du Cytomégalovirus. Ces souris ont développé des reins cystiques avec un degré de pénétrance et de sévérité directement proportionnel aux niveaux d'expression du transgène. Les cellules épithéliales des reins cystiques proliféraient davantage que les cellules normales et montraient une expression élevée de c-myc mais réduite de p27KIP1. Dans le second modèle de souris, CUX1 p75 ou p110 était exprimé sous le contrôle du promoteur du virus MMTV (Mouse Mammary Tumor Virus), spécifiquement intégré dans le locus hprt (hypoxanthine phosphoribosyltransferase). Des rétrocroisements successifs dans la souche FVB ont permis d'uniformiser le bagage génétique des souris transgéniques. Les souris CUX1 p75 des premières générations, donc de bagage génétique mixte, ont développé des leucémies caractérisées par une hyperprolifération de cellules myéloïdes neutrophiles. Ces résultats suggèrent que CUX1 p75 peut affecter la prolifération et la différenciation de certaines cellules hématopoïétiques. Dans la souche FVB pure, les souris CUX1 p75 et p110 ont développé des tumeurs mammaires de diverses histopathologies. Des métastases aux poumons ont été observées dans trois cas de souris CUX1 p75 avec des carcinomes solides mammaires. Les transgènes é

Mouse biliary glycoproteins (Bgp) are members of the carcinoembryonic antigen (CEA) family, a subfamily of the immunoglobulin superfamily. Nine similar but distinct mouse Bgp cDNA clones have been isolated and characterized. These Bgp isoforms encode polypeptides that contain multiple Ig domains, a single transmembrane domain and a short or long intracytoplasmic domain. Sequence analyses have demonstrated that Bgp isoforms are generated through complex alternative splicing of a single gene and allelic variation. Mouse Bgp proteins are highly expressed in the epithelial cells of the liver and the intestine and are implicated in diverse cellular functions including cell adhesion and bile acid transport, and are postulated to be involved in signal transduction. Investigation into the expression of mouse Bgp isoforms in normal and malignant tissues revealed that Bgp expression is downregulated in tumors. Furthermore, Bgp expression is influenced by transcriptional control mechanisms involving DNA methylation of the Bgp gene upstream regulatory regions.

Bacteriophage D108 is a temperate, transposable, mutator phage similar to phage Mu. These heteroimmune phages have 37 kb linear, double-stranded DNA genomes which are over 90% homologous. / We have isolated four independent insertions of an entire thermoinducible D108 c ts10 phage genome in the low-copy plasmid pSC101. We also characterized a previously isolated Mu c ts62 insertion in the same plasmid replicon. Fine-structure analysis of these insertions showed that lytically transposing Mu and D108 genomes caused 5 bp duplications of the target site. In addition, we cloned and sequenced the terminal 800 bp of the right end of D108, a region important for the transposition of the phage genome. This analysis has delineated the critical DNA sequences required for D108 transposition, and has also indicated that these sequences are not entirely homologous to those of phage Mu, despite the fact that the phages can transpose each other's DNA. Moreover, a 520 bp insertion of DNA found in the right end of D108, but not Mu, has also been characterized, and has begins 72 bp from the right end of D108, immediately adjacent to the cis -acting sequences required for D108 transposition. This insertion does not appear to be an insertion element, but rather an extra gene in the right end of D108 whose function is apparently non-essential for D108 growth. The sequence differences and rearrangements between Mu and D108 in this region indicate a complex evolutionary relationship between these phages at their right extremities. / In addition to characterizing the DNA differences between Mu and D108, we have also examined a difference occurring at the protein level. The L gene products of Mu and D108 have different molecular weights, but are encoded from a region of homology between the two phages. We cloned a region of D108 DNA that was able to rescue amber mutations in the L gene of Mu by homologous recombination, but not complement Mu L amber phages in vivo. This region produced a gene product which was truncated at the carboxy terminus, but was able to cross-react with anti-Mu phage serum. The production of this truncated protein was lethal to growing Escherichia coli cells, apparently interfering with cell wall biosynthesis.