ME31B, a putative RNA helicase belongs to the DEAD box family. A subclass of the DEAD box family is growing around ME31B. The members are DHH1 (S. cerevisiae), ste 13 (S. pombe), and rck/p54 (human and murine). In a conserved region of approximately 390 amino acids, these proteins all share greater than 70% identity with ME31B. The construct pOTS-Nco12-ME31B was used to produce an easily discernible quantity of insoluble ME31B. Following treatment with 8 M urea, ME31B was allowed to renature. Approximately 66% of the ME31B protein remained soluble during these manipulations. This soluble protein was purified on an HPLC hydrophobic column with a purification yield from total protein loaded of 35% soluble ME31B. With this soluble pure protein, I was able to investigate ME31B's ability to hydrolyze ATP. In an ATPase assay, ME31B hydrolyzed ATP in the presence and absence of an RNA substrate.

3$\beta$-Hydroxy-cholest-5-en-7-one (15) has been reported to lower 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in both mouse liver cells and whole animals. In experiments with rats, studies have shown that 15 is rapidly metabolized and excreted from the body. We synthesized 3$\beta$-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one (19) with the hope that blocking the sidechain metabolism at C-25 and C26 would allow sufficient quantities to accumulate in vivo and possibly lower serum cholesterol levels. 19 was synthesized via 25,26,26,26,27,27,27-heptafluorocholest-5-en-3$\beta$-ol (3) by employing the reagents chromic anhydride and 3,5-dimethylpyrazole (DMP) in methylene chloride (66% yield; $>$99% pure by NMR). A thorough characterization of 19 and its nonfluorinated counterpart 15 is presented. Several byproducts from the synthesis of 3 and 19 were discovered. Their preliminary characterizations are also reported.

I am interested in understanding a mechanism by which multicellular organisms sense number of cells of the same type. The model selected is the conditioned medium factor secreted by Dictyostelium discoideum. In submerged monolayer culture, Dictyostelium cells can differentiate into prespore and prestalk cells at high cell densities in response to cAMP but this process does not occur at low cell densities. However, cells at low densities will differentiate in medium taken from developing cells starved at a high density. The putative factor in the medium was designated CMF for Conditioned Medium Factor (Mehdy and Firtel 1985). In this report, we show that the CMF activity can be separated into high and low molecular weight fractions. The large conditioned medium factor can be purified to a single 80 kD protein with N- and O-linked glycosylation and has CMF activity at a concentration of $\sim$4 pM (0.3 ng/ml). This 80 kD CMF can undergo size reduction to a $\sim$100-fold more active set of smaller peptides with molecular weight less than 10 kD. Glycosylation is required for the activity of the low molecular weight CMF. Starvation triggers the release of CMF from a precursor pool already present in vegetative cells, and diffusion calculations indicate that the CMF level in the vicinity of a single isolate will not accumulate to the threshold concentration $\sim$0.3 ng/ml. CMF antisense transformants do not aggregate, whereas normal development is restored by the addition of purified 80 kD CMF. These results suggest that CMF is a secreted factor that functions in vivo as an indicator of cell density in starved cells. The developing cells simultaneously secrete CMF and monitor its extracellular level. When a majority of the cells in a given area have starved as indicated by the high level of CMF, aggregation is triggered to ensure the onset of development is synchronized. When present below a threshold concentration, the expression of genes required for early development is blocked or not induced. This factor plays an essential role in the regulatory pathway necessary for cells to obtain the developmental competence to induce prestalk and prespore gene expression in response to cAMP.

Much recent emphasis in vascular biology has focused on endothelial cells which form the inner lining of blood vessels. This unique arterial location exposes these cells directly to mechanical forces resulting from blood flow and the transmission of the pressure wave through a compliant vessel. In this study, the effects of the cyclical expansion and relaxation of the vessel wall on endothelial cell metabolism have been modeled using a uniaxial strain device. A normal range of physiological strains for large arteries was examined. The production rates of prostacyclin (PGI$\sb2$), endothelin, tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-type 1 (PAI-1) by endothelial cells were constant over 24 hour periods. The production of both PGI$\sb2$ and endothelin was enhanced by cells exposed to a high level of cyclical strain compared to controls, while t-PA production was unaltered. These results were true for human and bovine endothelial cells. The stimulation of endothelin production was dose dependent with the level of strain while PGI$\sb2$ stimulation required a minimal level of strain before increases over controls were observed. Human endothelial cells subjected to cyclical strain showed elevated production of PAI-1 compared to controls. The possibility that cyclical strain could be used to regulate cell function was investigated. Cyclical strain was applied in an on-off-on manner over a 36 hour period with a 12 hour division between initial application of strain and final reapplication of strain. When the strain was stopped, PGI$\sb2$ production rapidly returned to control levels while endothelin production remained elevated but at a level significantly below the initially stimulated rate. Reapplication of cyclical strain caused a return of the endothelin production rate to a level essentially the same as that during the initial stimulation period. This study initiated a quantitative investigation of cyclical strain effects on endothelial cell production of the mRNA levels of prostaglandin H (PGH) synthase gene, the enzyme of which is involved in production of PGI$\sb2$. Preliminary results using a quantitative reverse transcription-polymerase chain reaction technique suggested that mRNA levels of PGH synthase are not altered in response to cyclical strain.

Initial aspects of the molecular mechanisms of cell-substrate interactions were studied under physiologically relevant shear conditions using a parallel-plate flow chamber. The adhesion process was dissected into the initial events of arrest and stabilization using the methodology developed in this study employing video microscopy coupled to digital image processing on a SPARC 2 workstation. The cell-substrate interactions were studied using human melanoma (i.e. parental (MeWo) and two variants selected for resistance to wheat germ agglutinin cytotoxicity (3s5 and 70w)) and Chinese hamster ovary (CHO) cells expressing different levels of the fibronectin integrin, $\alpha\sb5\beta\sb1$, on their surface. These cells allowed investigation of the roles of transglutaminase (TGase) and the integrin surface receptors in the arrest and stabilization events. TGase inhibitors, monodanyslcadaverine and INO-3178, used in the studies with the melanoma cells showed inhibition of the stabilization event with no measurable effect on the arrest event when adhesion to immobilized fibronectin was investigated under flow conditions. Besides fibronectin, other immobilized proteins were evaluated using the melanoma cells such as laminin, vitronectin, type I and type IV collagen, von Willebrand factor, wheat germ agglutinin (WGA), and Peptite-2000. The results showed the arrest and stabilization events are not necessarily mediated to the same degree by the same molecular components. For example, arrest to WGA was very favorable relative to arrest to fibronectin but stabilization was very poor. Some substrates such as human laminin showed both favorable arrest and stabilization behavior relative to fibronectin. Polyclonal antibodies to the fibronectin and vitronectin integrin receptors on the melanoma cells were used to investigate the role of these integrins in the arrest and stabilization events. It appeared that both receptors are involved to some degree in the arrest and stabilization events to both substrates. The adhesion studies of the CHO cells confirmed these findings that increased fibronectin integrin enhanced arrest and stabilization to both fibronectin and vitronectin. In summary, these results suggest that the stabilization of cells to immobilized proteins is in part attributed to transglutaminase cross-linking integrin receptors on the surface of tumor cells to proteins via lysine-glutamine linkages.

CEACAM1 (CarcinoEmbryonic Antigen-related Cell Adhesion Molecule 1), is expressed at the surface of new blood vessels of tissues undergoing proliferation. In human tumors, CEACAM1 expression is associated with early stages of angiogenesis. CEACAM1 is a known pro-angiogenic factor, increasing VEGF activity in vivo; however, the role of CEACAM1 in angiogenesis warrants further investigation. Excisional wounds were used as an experimental model, as many of the processes that occur in healing wounds also take place in tumor growth - epithelial hyperproliferation, inflammation, and angiogenesis. 6-mm diameter skin wounds were inflicted on the dorsal side of Ceacam1-/- and wild-type mice. Upon histological examination, it was shown that wound healing in Ceacam1-/- mice is indeed delayed. In Ceacam1-/- wounds, re-epithelialization is decreased significantly at 3 and 7 days post-injury. Inflammation in Ceacam1-/- wounds is also altered: the infiltration of F4/80+ macrophages into the wound at 7 and 10 days post-injury is significantly decreased, as is the influx of mast cells at 7 days post-injury. Vascular density in Ceacam1-/- wounds is also significantly decreased at 7 and 10 days post-injury; however, VEGF expression in the wound is not altered. The results of this study not only confirm CEACAM1's role as an important factor in angiogenesis, but further expand its role as a mediator of epithelial growth and inflammation. / CEACAM1 (CarcinoEmbryonic Antigen-related Cell Adhesion Molecule 1) est exprimé à la surface de nouveaux vaisseaux sanguins de tissus en prolifération ou de tumeurs humaines, et est associé aux stades précoces de l'angiogenèse. De plus, CEACAM1 semble être un puissant facteur angiogénique en potentialisant l'activité du VEGF in vivo. Malgré de nombreuses observations, le rôle de CEACAM1 dans l'angiogenèse normale reste à définir. Dans ce contexte, nous avons réalisé des essais de cicatrisation cutanée dans des souris Ceacam1-/-. Des plaies de 6 mm ont été effectuées sur le dos des souris. La vitesse de cicatrisation a été suivie sur 10 jours, et les plaies ont été examinées en histologie 3, 7 et 10 jours après l'essai. D'un point de vue macroscopique, la délétion de CEACAM1 n'a pas d'effet sur la cicatrisation des plaies. En revanche, des analyses plus précises en histologie montrent que la cicatrisation des souris Ceacam1 -/- est différée. En effet, le mécanisme de re-épithélisation des plaies des souris Ceacam1 -/- est retardé aux points 3 et 7 jours après blessure. De plus, l'inflammation des plaies des souris Ceacam1 -/- est également affectée : l'infiltration des macrophages F4/80+ au sein des plaies 7 et 10 jours après blessure, ainsi que celle des mastocytes au point 7 jours après blessure sont significativement diminuées. Enfin, la densité vasculaire des plaies Ceacam1-/- est également réduite de façon significative. En revanche, l'expression de VEGF au sein des plaies ne semble pas altérée. Les résultats de cette étude confirment le rôle de CEACAM1 dans l'angiogenèse, mais présentent de façon plus importante CEACAM1 comme un facteur clé dans le mécanisme de cicatrisation des plaies cutanées.

The Met receptor tyrosine kinase was originally identified as a rearranged oncogene, Tpr-Met. Both Met and its ligand, hepatocyte growth factor (HGF) regulate epithelial cell dispersal and morphogenesis and are deregulated in several human tumors. At the onset of this thesis, little was known about the signals that regulate these events. In this thesis I have defined the involvement of the Crk adapter protein in Met-dependent anchorage independent growth, cell spreading, cell dispersal, invasion and morphogenesis. The disruption of focal adhesion signaling in Met transformed fibroblasts is associated with the loss of Crk/p130Cas and Crk/Paxillin complexes. In contrast, Crk associates with Cbl and Gab1 in Met transformed fibroblasts. A role for Crk/Gab1 coupling in anchorage independent growth is proposed based on the retention of these complexes in Met transformed fibroblasts grown in suspension and their ability to enhance JNK activity. Using MDCK epithelial colonies, I demonstrated that mutants of Crk lacking the amino terminal SH3 domain inhibit HGF-stimulated lamellipodia formation, cell spreading and breakdown of adherens junctions. Moreover, when overexpressed, wild type Crk can promote all of these events in the absence of HGF. Consistent with the elevated Rac activity observed in cells overexpressing Crk, the ability of Crk to promote these events is Rac-dependent. The overexpression of Crk results in the formation of a molecular complex containing Crk, Paxillin, GIT2 (an ARF-GAP) and beta-PIX (a Rac exchange factor) that relocalizes to focal complexes in cells at the edge of the colony. The formation of this complex is critical for the ability of Crk to mediate lamellipodia formation and cell spreading, as mutants of Paxillin that fail to associate with Crk or GIT2, or that do not target to focal adhesions, inhibit these processes. Consistent with the involvement of Crk in HGF cell dispersal, the coupling of Gab1 with Crk is requi

Apoptosis is a genetically programmed highly regulated form of cellular suicide that plays an essential role in the development and tissue homeostasis of multicellular organisms. As a variety of pathological states such as cancer, autoimmune and neurodegenerative diseases can be ascribed to the deregulation of the apoptotic program, understanding the molecular mechanisms underlying this process is a necessary first step to therapeutic intervention. The BCL-2 family of proteins is of paramount importance in the regulation of apoptosis through their control of caspases, a family of cysteine proteases responsible for cellular demolition. The p53 tumour suppressor protein is a transcription factor that eliminates potentially dangerous cells via activation of the apoptotic program through the regulation of various genes, including those of the BCL-2 family. I found that BIK, a member of the pro-apoptotic BH3-only class of BCL-2 homologues, is upregulated by p53. Unlike all other BH3-only proteins however, BIK was found to be uniquely localized to membranes of the endoplasmic reticulum. BIK was induced in response to several stress stimuli, including genotoxic stress (radiation; doxorubicin) and over-expression of E1A or p53, but not by ER stress pathways resulting from protein misfolding. Using siRNA technology, I showed that BIK plays a critical role in p53-induced cell death by acting at the ER to trigger Ca2+ release, mitochondrial fission, BAX/BAK activation, cytochrome c release, caspase activation and apoptosis. BIK also stimulated the (BCL-2 inhibited) accumulation and oligomerization of BAK at ER membranes. Cells doubly deficient of both BAX and BAK were resistant to ER Ca2+ release and apoptosis by ectopic expression of both BIK and p20BAP31, suggesting that these multidomain pro-apoptotic BCL-2 proteins may serve as a common checkpoint at the ER for varying modes of stress stimuli. Thus, p53 appears to employ BIK as part of its apopto

Arginine methylation is a post-translational modification occurring in higher eukaryotes that results in the addition of one or two methyl group on the nitrogen in the side chain of arginines. The enzymes responsible for protein arginine methylation have been classified in three groups. Type I enzymes promote the formation of both NG-monomethylated and asymmetric o-NG,NG-dimethylated arginines (aDMA). Type II enzymes catalyze the formation of monomethylated and symmetrical o-N G,N'G-dimethylated arginines (sDMA). The type III enzyme found in yeast catalyzes the monomethylation of the delta-guanidino nitrogen atom of the arginine residue. Although some abundant proteins have been described as being substrates for arginine methyltransferases for some time, there are still few known proteins to bear this modification. The primary goal of the work presented in this thesis was to identify new arginine methylated proteins and functionally characterize the roles of arginine methylation in new cellular processes. First, we generated four arginine methyl-specific antibodies: ASYM24 and ASYM25 are specific for aDMA whereas SYM10 and SYM11 recognize sDMA. Cell extracts were used to purify the protein complexes recognized by each of the four antibodies and the proteins were identified by microcapillary reverse-phase liquid chromatography coupled on line with electrospray ionization tandem mass spectrometry (LC/MS/MS). The analysis of 2 tandem mass spectra for each methyl-specific antibody resulted in the identification of 247 proteins, of which 197 are putatively arginine methylated. / The DNA repair MRE11/RAD50/NBS1 (MRN) complex was purified using one of the aDMA specific antibody. Since a role of protein arginine methylation in DNA damage checkpoint control and DNA repair had not been previously reported we chose to investigate the consequence of MRE11 methylation in DNA damage. Our results show that the MRE11 checkpoint protein is arginine methylated as determined by mass spectrometry and methylarginine-specific antibodies. The glycine-arginine rich (GAR) domain of MRE11 was specifically methylated by protein arginine methyltransferase 1 (PRMT1). Mutation of the arginines within MRE11 GAR domain severely impaired the exonuclease activity of MRE11. Cells treated with methyltransferase inhibitors displayed a DNA damage-resistant DNA synthesis phenotype and prevented the re-localization of the MRN complex to sites of DNA damage. Downregulation of PRMT1 with small interfering RNAs (siRNA) also yielded a damage-resistant DNA synthesis phenotype that was rescued with the methylated MRE11 complex. Taken together, the work presented in this thesis allowed the identification of many new potentially arginine methylated proteins and demonstrated a novel role for arginine methylation in the regulation of DNA repair enzymes and of the intra-S phase DNA damage checkpoint.

Telomeres are protective structures at the end of eukaryotic chromosomes essential for indefinite cell proliferation. Their disruption causes activation of DNA repair pathways, growth arrest and/or cell death. In normal cells telomere shortening during cell division has been proposed to act as a tumor suppressor mechanism to block the proliferation of cells at risk of undergoing malignant transformation. Overcoming this proliferative block by activating a mechanism to maintain telomeres is a necessary requirement for unlimited proliferation and tumor progression. Human cells have two mechanisms for telomere maintenance: a more common one based on telomerase and a rarer one based on recombination called ALT. / Here we report the isolation of an immortal human cell line that maintains short telomeres in the absence of biologically active telomerase and key features of active ALT. Our results suggest that the mechanisms of telomere maintenance in human cells may be more diverse than previously thought and have important implications for the development of anti-cancer strategies based on the inhibition of telomere maintenance. / Due to widespread distribution of telomerase in human tumors and its absence in most normal cells, telomerase is the main target of these anti-cancer strategies. However, targeting telomerase per se or in combination with anti-cancer drugs is not sufficient to trigger rapid cell death of tumor cells. On the other hand, disturbances in telomere capping do not require telomere shortening to induce growth arrest and may act more quickly. Our goal was to investigate the feasibility of a new approach based on the combination of telomere destabilization and chemotherapeutic drugs. Our results show that interfering with telomere maintenance enhances the susceptibility of human tumor cells to anti-cancer drugs independently of their telomere lengths and mechanisms to maintain them. / Finally, given the involvement of telomeres in maintaining genomic stability, we investigated the mechanism by which mutations in the telomerase RNA subunit contribute to autosomal dominant dyskeratosis congenita, a premature ageing disease associated with mutations in the telomerase holoenzyme. Our data strongly indicate that the clinical manifestations of this disease may be caused by telomere shortening due to haploinsufficiency of telomerase activity and provide a direct correlation between disturbances in telomere length maintenance and human disease.