Micro satellite instability (MSI) is a form of genomic instability that has recently been implicated in the pathogenesis of thyroid cancer. The purpose of this study is to further define the distribution of microsatellite instability in both normal and neoplastic thyroid follicular epithelium. / Using laser capture microdissection, cells from both normal and tumor tissue were individually collected. PCR amplification of the DNA was then performed using six dinucleotide and two mononucleotide microsatellite markers. / Forty benign and malignant thyroid tumors were compared with their adjacent normal thyroid follicular tissue and were analyzed for MSI. 9/14 papillary thyroid carcinomas and 10/16 of follicular thyroid carcinomas demonstrated MSI at >30--40% of loci tested. For benign follicular adenomas, 9/10 demonstrated microsatellite stability or low-frequency MSI. / Microsatellite instability appears to play a role in thyroid pathogenesis as evidenced by the high frequency of MSI in malignant thyroid neoplasms. In addition our study showed a significant difference in MSI frequency between follicular adenomas and follicular carcinomas. More importantly, the technique of laser capture microdissection allows for more accurate selection of benign, malignant and normal DNA.

The Rho GTPases are members of the Ras superfamily of small monomeric GTP binding proteins that regulate multiple cellular processes affecting both cell proliferation and cytoskeletal dynamics. They are positively and negatively regulated by the guanine nucleotide exchange factors (GEFs) and the GTPase-activating proteins (GAPS), respectively. In principle, extracellular signals such as growth factors and other agents bind to plasma membrane receptors and modulate Rho GTPase function through intracellular mechanisms that affect GEF or GAP activities. There is now compelling evidence that both receptors and cytoplasmic proteins implicated in Rho GTPase signaling are modulated by post-translational modifications such as phosphorylation. / CdGAP (Cdc42 GTPase- activating protein) is a negative regulator of Rac1 and Cdc42 and the netrin-1 receptor DCC (deleted in colorectal cancer) is an upstream activator of these GTPases during axon guidance. To date, very little is known about the biochemical regulation of these two proteins. Given that CdGAP protein migrates higher than its expected molecular weight and contains a number of consensus phosphorylation sites for different kinases, and that the DCC ortholog in C. elegans UNC-40 is tyrosine phosphorylated, it is likely that both proteins are regulated through mechanisms involving phosphorylation. / In the first part of this thesis, we have demonstrated that CdGAP phosphorylation in vivo is stimulated by external cues that activate the Ras-MEK-ERK pathway. We also found that CdGAP is phosphorylated in vivo on serine, threonine but not tyrosine residues. Phosphorylation of Thr776 by ERK and that this phosphorylation is important in regulating CdGAP's activity towards Rac1. / In the second part, we found that the human ortholog of CdGAP is also active both in vitro and in vivo on Cdc42 and Rac1 but not RhoA. Furthermore, we found that the human protein is also highly phosphorylated in vivo on serine and threonine residues but not tyrosine. / In the last part, we have combined classical biochemical approaches and the dissection of rat primary commissural neurons and found that DCC is phosphorylated in vivo upon netrin-1 stimulation on serine, threonine and tyrosine residues. We have also found that the phosphorylation and function of DCC in axon outgrowth and guidance is mainly regulated by the Src family tyrosine kinases.

Abstract 4E-T, a novel binding partner of eIF4E protein, transports 4E from the cytoplasm to the nucleus. This protein is also involved in mRNA degradation. 4E-T binds to eIF4E and carries eIF4E-protein along with the mRNA to P-bodies, which is the site of mRNA degradation. In this study, we show that 4E-T is a phospho-protein and is phosphorylated on multiple sites. We tried to identify some of the phosphorylation sites on this protein. By using overlapping PCR, a triple phospho mutant of this protein was made. We also investigated if 4E-T is phosphorylated by the S6 kinase (S6K). Our results indicate that even though 4E-T contains the phosphorylation motif of S6 kinase, S6K is not a kinase of this protein. Moreover, we examined if 4E-T phosphorylation on Ser213, 353 resides changes the interaction between this protein and 4E-HP. We could not find any evidence showing that the binding of these proteins together is dependent of phosphorylation of 4E-T on Ser213, 353 amino acids. Finally, we show that the interaction between 4E-T and 4E proteins is not regulated by phosphorylation. The Phospho-triple mutant of 4E-T protein can be used a strong tool to elucidate the function of this protein and how the interaction between this protein and its partners is regulated. / Résumé 4E-T, un nouveau partenaire de liaison à la protéine 4E, transporte 4E du cytoplasme au noyau. Il a aussi été démontré que cette protéine est impliquée dans la dégradation de l'ARNm. 4E-T lie 4E et la transporte, ainsi que l'ARNm, au P-bodies, site de la dégradation des ARNm. Ici, nous démontrons que 4E-T est une phosphoprotéine et qu'elle est phosphorylée en plusieurs endroits. Nous avons tenté d'identifier certains des sites de phosphorylation de cette protéine. En utilisant l'overlapping PCR, un phospho-mutant triple de cette protéine a été créé. Nous avons aussi vérifié si 4E-T est phosphorylée par la kinase S6 (S6K). Nos résultats indiquent que, même si 4E-T contient le motif de phosphorylation de la kinase S6, S6K n'est pas une kinase de cette protéine. De plus, nous avons examiné si la phosphorylation de 4E-T change l'interaction entre cette protéine et 4E-HP. Nous n'avons trouvé aucune preuve que la liaison de ces protéines entre elles serait phospho-dépendante. Finalement, nous démontrons que l'interaction entre 4E-T et 4E n'est pas régulée par la phosphorylation. Le phospho-mutant triple de 4E-T peut être utilisé comme un puissant outil pour élucider la fonction de cette protéine et comment l'interaction entre cette protéine et ses partenaires est régulée.

Rheumatoid arthritis (RA) is a chronic, autoimmune inflammatory disorder that exhibits both articular and systemic manifestations. Prominent among molecular mediators of RA is prostaglandin E2 (PGE2) and the inducible rate-limiting enzyme involved in its biosynthesis, cyclooxygenase-2 (COX-2). Long regarded as inflammogenic, the so-called "COX-2/PGE2 axis" is now likened to a homeostatic inflammomodulatory axis poised to regulate the initiation, magnitude, duration and resolution of the inflammatory response. The COX-2/PGE2 pathway's regulatory role in maintaining normal inflammatory homeostasis (i.e., "inflammostasis") is dependent on its tight temporal and inflammation phase-specific regulation; yet, the mechanisms governing the timing and dynamics of this pathway's activity during inflammatory episodes are ill-defined. The data presented in this thesis demonstrate that the COX-2/PGE2 axis is subject to a series of regulatory "control points" implicating various effector mechanisms and positioned at different levels along the axis to ensure tight temporal regulation of its activity during an inflammatory response. Our data expose a novel control point involving proteolytic cleavage-induced potentiation of human synovial fibroblast (HSF) COX-2 catalytic activity following proinflammatory stimulation. Cleavage is limited, site-specific and quantitatively tightly correlated to PGE2 production (R2 = 0.941). Pharmacological COX-2 inhibitors impede enzyme cleavage, while inhibition of calpain and cathepsin proteases blocks PGE2 biosynthesis in HSFs; in both cases, PGE2 production and COX-2 cleavage remain tightly correlated. Subsequent to this first control point, the COX-2/PGE2 axis falls subject to feedback regulation by PGE2 at the level of COX-2 gene expression. We show that PGE2 induces the phosphorylation of tristetraprolin (TTP), a well-studied mRNA destabilizing protein. Phosphorylation (likely p38 MAPK-mediated) promotes TTP's nuclear export/cytoplasmic sequestration and concomitantly inhibits its mRNA destabilizing capacity, culminating in coupled and coordinated COX-2 promoter transactivation and mRNA stabilization. The consequent increase in COX-2 mRNA/protein levels establishes a self-sustaining cycle of PGE2 production that ensures the expression of downstream PGE2 target genes implicated in terminating inflammation. Among such genes is the p38 MAPK phosphatase DUSP-1, whose expression in HSFs is controlled transcriptionally (partly via the p38 MAPK pathway) and post-transcriptionally (by TTP). Like COX-2, coupling of DUSP-1 gene transcription and mRNA stabilization ensures sustained DUSP-1 levels required for the shutdown of p38 MAPK activity and subsequent inflammatory resolution. / La polyarthrite rhumatoïde (PR) est une maladie auto-immune, inflammatoire chronique des articulations avec des manifestations systémiques. Parmi les médiateurs moléculaires de cette maladie se retrouve la prostaglandine E2 (PGE2) et l'enzyme inductible responsable de sa synthèse durant l'inflammation, la cyclooxygénase-2 (COX-2). Longtemps considéré comme inflammogénique, l'axe COX-2/PGE2 est maintenant reconnu pour ses activités homéostatiques et inflammomodulatrices et son rôle régulateur de l'initiation, l'ampleur, la durée et la résolution de la réponse inflammatoire. La conservation de l'homéostasie inflammatoire (i.e., "inflammostasie") par l'axe COX-2/PGE2 nécessite que son activité soit temporellement réguleé par des mécanismes qui sont spécifiquement adaptés à chaque phase de la réponse inflammatoire; cependant, les modes régulateurs de cet axe durant des épisodes inflammatoires restent mal définis. Mes données de thèse démontrent que l'axe COX-2/PGE2 est régulé à plusieurs niveaux (i.e., "points de contrôle") par des mécanismes effecteurs différents et conçus pour assurer une régulation temporelle de l'axe durant une réponse inflammatoire. Nos données révèlent un point de contrôle antérieurement inconnu impliquant la potentialisation de l'activité enzymatique de la COX-2 par son clivage protéolytique dans les fibroblastes synoviaux humains (HSFs) stimulés avec des agents proinflammatoires. Ce clivage est limité, site spécifique et quantitativement corrélé avec la synthèse de PGE2 (R2 = 0.941). Les inhibiteurs pharmacologiques de la COX-2 bloquent son clivage, alors que l'inhibition des protéases calpaïnes et cathepsines empêche la synthèse de PGE2 dans les HSFs; dans chacun des cas, la production de PGE2 et le clivage de la COX-2 restent fortement corrélés. Suite à ce premier point de contrôle, l'axe COX-2/PGE2 est assujetti au rétrocontrôle par la PGE2 au niveau de l'expression de la COX-2. Nous illustrons que la PGE2 induit la phosphorylation de tristetraprolin (TTP), une protéine déstabilisatrice de l'ARN. Cette phosphorylation (probablement mediée par la voie MAPK p38) cause l'export nucléaire/séquestration cytoplasmique de TTP, conduisant à l'inhibition de ses capacités déstabilisatrices et, ultimement, à l'activation transcriptionnelle et stabilisation de l'ARN couplée et coordonné de la COX-2. Les niveaux élevés conséquents de l'ARN/protéine COX-2 établissent un cycle autoreproducteur pour la synthèse de PGE2 qui assure l'expression des gènes cibles de la PGE2 impliquées dans la résolution de l'inflammation. Parmi ces gènes se retrouve la phosphatase DUSP-1 ("dual-specificity phosphatase"), donc l'expression dans les HSFs est contrôlée au niveau transcriptionnel, en partie par la voie MAPK p38, et post-transcriptionnel par la TTP. Comme pour la COX-2, le couplage de ces deux mécanismes régulateurs assure l'expression soutenue de la DUSP-1 requise pour l'inactivation de la voie p38 et la résolution éventuelle de l'inflammation.

PTEN, the most frequently mutated gene in human prostate cancer, is atumour suppressor that inhibits angiogenesis by regulating the expression ofseveral pro- and anti-angiogenic factors. The goal of this project was to identifynovel angiogenic factors and other secreted proteins that are regulated by PTEN.Proteomics techniques were employed to identify proteins that were differentiallyexpressed in the conditioned media of PTEN-null and PTEN-expressing humanprostate cancer cells. 16 proteins were identified, 4 of which were up-regulatedby PTEN and 12 of which were down-regulated by PTEN. Gene expressionanalysis and polysome profiling revealed that 2 proteins were transcriptionallyregulated by PTEN and 5 proteins were regulated at the level of translation.Three of the PTEN-regulated factors identified in this study, spondin 2 (SPO2),Zn-alpha-2-glycoprotein (ZAG), and cystatin C (CST3) are up-regulated invarious cancers and could be useful serum biomarkers for the diagnosis ofprostate cancer. / PTEN est un gène supresseur de tumeur. Il est fréquemment muté chez leshumains ayant un cancer de la prostate. Un de ses rôles est d'inhiberl'angiogénèse en régulant l'expression de plusieurs facteurs pro- et antiangiogéniques.L'objectif de cette étude était d'identifier de nouveaux facteursangiogéniques et de nouvelles protéines secretées qui sont régulés par cetteprotéine. L'utilisation de différentes techniques protéomiques, sur le milieu deculture de cellules prostatiques cancéreuses, nous a permis d'identifier 16protéines ayant des niveaux d'expression différents selon que la protéine al'étude, PTEN, était ou non exprimée. Parmi elles, 4 ont un niveau d'expressionmoindre en présence de la protéine PTEN alors que les 12 autres présentent unniveau d'expression plus élevé. L'analyse du profil de polysomes et del'expression génique pour 7 d'entre elles montrent que la protéine PTEN enrégule deux au niveau transcriptionnel et cinq au niveau traductionnel. La forteexpression de trois protéines identifiées dans cette étude, spondin 2 (SPO2), Znalpha-2-glycoprotein (ZAG), et cystatin C (CST3), dans plusieurs cancers pourraitles qualifier comme nouveaux biomarqueurs dans le diagnostique du cancer de laprostate.

Estrogens are steroid hormones responsible for the proper function of a variety of mammalian physiological processes. In addition to their central role in reproduction, estrogens also affect the cardiovascular, skeletal, immune and nervous systems and play a role in the initiation and progression of breast cancer. The biological actions of estrogens are mediated by the products of two genes within the nuclear receptor superfamily (NRs), estrogen receptor alpha (ER(alpha) and estrogen receptor beta (ERbeta). Upon interaction with estrogens, ERs recognize specific DNA sequences and modulate the transcription of neighboring target genes via recruitment of coactivators. A diverse group of proteins has emerged as potential coactivators for ERs. Among the best characterized ER coactivators, the p160 family of coactivators consists of three members, SRC1/NCOA1, TIF2/GRIP1 and SRC3/AIB1/RAC3, which associate with p300 and CBP, coactivators with HAT activity. Recruitment of HAT activity at the promoter regions of the target genes results in histone modification and chromatin remodeling to facilitate transcription initiation. / Most coactivator proteins contain one or more copies of a helical LXXLL motif, called NR-box, in which L denotes leucine and X denotes any amino acids. Distinct LXXLL motifs display varying degrees of selectivity for nuclear receptors, and artificial LXXLL peptides interacting ERbeta but not ERalpha were identified. Here we have investigated using the Bioluminescence Resonance Energy Transfer and GST pull down assays the role of flanking charged amino acids in LXXLL motifs in interaction with ERalpha and ERbeta. The BRET assay was more sensitive than the GST pull down assay for detection of the interaction between estrogen receptors and LXXLL motif derivatives, which was promoted by 17beta-estradiol (E2) in an H12-dependent manner and at physiological concentration of ligands, but prevented by ER antagonists 4-hydroxy-tamoxifen (OHT) and ICI182, 780. Two Lys residues in the first motif of AIB1 play a crucial role, while Lys at position -2 in the second TIF2 motif only modulates the interaction with ERalpha. The differential effect of mutations at position -2 on interaction with ERalpha and ERbeta suggests that the flanking sequences of LXXLL motifs may modulate selectively binding to the two ERs.

The endoplasmic reticulum (ER) plays a vital role in synthesis, folding and sorting of newly synthesized secretory cargo proteins. Quality control mechanism in the ER is a key regulator of this process, which separates correctly folded proteins from immature or misfolded secretory proteins, and ultimately retains and disposes the latter before they can exit the ER. Numerous diseases have been associated with defects in the regulation of the egress of the nascent membrane proteins out of ER to the cell surface. BAP31, a polytopic integral membrane protein at the ER, has been implicated as a putative quality control factor and/or cargo protein in regulating the export of specific sets of nascent membrane proteins. Here, I demonstrate BAP31 to be an important player in regulating and maintaining functional integrity of the cell surface. Both truncated (p20BAP31) and deletion (BAP31-null) mutants of BAP31 significantly abrogated expression of tetraspanins at the cell surface, and thus integrin-mediated cell adhesion and survival due to BAP31 effects on tetraspanin transport from the ER. Consistent with its role as a chaperone or cargo receptor, BAP31 was found to interact with Sec61beta and TRAM, components of the nascent protein translocation machinery and to form an important part of the ribosome-translocon complexes at the ER. In addition to its predicted role in quality control mechanism in the ER, BAP31, a BCL-2 associated protein, is recognized as an important regulator of apoptosis, an essential physiological mechanism of cell suicide in development and homeostasis in all multicellular organisms. Here, I characterized SPIKE, a new proposed pro-apoptotic BCL-2 protein and interacting partner of BAP31. SPIKE is a novel and untypical pro-apoptotic BH3 only protein capable of inducing apoptosis without binding to anti-apoptotic BCL-2 partners and endogenous BAP31, whose role in specific apoptotic pathway remains to be elucidated. Thus, BAP31 plays important, but distinct roles in both normal cell physiology and apoptosis.

Translational control is crucial to balancing the cell's protein output and genetic expression. The substrate of the translational machinery---messenger RNA (mRNA)---is itself subject to regulation. The lifetime of an mRNA is limited and therefore mRNA decay is a critical step in the regulation of gene expression. Translation and mRNA decay are intimately related processes as they both handle the cell economy. mRNAs are generally in a balancing act between the translational and the repression/decay machinery, which ultimately decides the fate of an mRNA and its protein expression rate. In fact, translation affects the rate of mRNA decay. For instance, aberrant messages which contain a premature-termination codon (PTC) require ribosome scanning in order to read the message, discover the mistake, and essentially prompt its destruction. Here, a relationship between the nuclear translation-like factor---eIF4AIII, the nuclear import factor of cIF4E---4E-T, and mRNA decay was discovered. eIF4AIII is a nuclear protein that interacts physically or functionally with translation initiation factors eIF4G and eIF4B, respectively, and shares strikingly high identity with the initiation factors eIF4AI/II. This work demonstrates that eIF4AIII but not eIF4AI is required for nonsense-mediated decay (NMD). NMD is a surveillance mechanism in eukaryotes which degrades the mRNA when a PTC is present. NMD is a splicing and translation-dependent event in mammalians. We show eIF4AIII is deposited at the exon-exon junction during splicing, is a shuttling protein, and is necessary for NMD. At steady state, 4E-T is predominantly cytoplasmic and is concentrated in bodies that conspicuously resemble the recently described Processing bodies (P-bodies), which are sites of mRNA decay. We demonstrate that 4E-T colocalizes with mRNA decapping factors in bona fide P-bodies and that its binding partner, eIF4E, is tethered to P-bodies in a 4E-T dependent manner. Moreover, 4E-T controls mRNA half life. We demonstrate that 4E-T interaction with eIF4E represses translation, which is thought to be a prerequisite for targeting of mRNAs to P-bodies. Hence, analysis of prospective translation factors has led to elucidation of mRNA decay pathways.

Two important steps of translation initiation include the recognition of the mRNA cap structure by eIF4E and the recycling of erF2. Each step is thought to be regulated independently through the interaction of eIF4E with 4E-BPs and the phosphorylation of the a subunit of eIF2 at serine 51. Phosphorylation of eIF2alpha by dsRNA-dependent protein kinase PKR inhibits protein synthesis in cells subjected to virus infection; therefore, most viruses have evolved mechanism to overcome the deleterious effects of PKR. The human papillomavirus (HPV) E6 oncoprotein contributes to virus-induced pathogenicity through multiple mechanisms including the inhibition of apoptosis and the blockade of interferon action. This study demonstrates a novel function of PKR providing a link between the two mechanisms of regulation of translation initiation. Activation of PKR induces the PI3K-PKB/Akt and FRAP/mTOR pathways leading to S6 and 4E-BP1 phosphorylation upon stress conditions and in response to growth stimuli. Induction of the PI3K pathway antagonizes the apoptotic effects of PKR activation, but does not intervene with its translational inhibitory activity. Investigating functional interaction of HPV E6 and PKR, we determined that BPV-18 E6 protein synthesis is regulated by eIF2alpha phosphorylation. On the other hand, E6 oncoprotein is able to rescue cells from PKR-mediated inhibition of protein synthesis and induction of apoptosis by promoting eIF2alpha dephosphorylation through physical association with GADD34/PP1 holophosphatase complex. These findings demonstrate, for the first time, the ability of PKR to activate a growth-stimulatory pathway; PI3K. Furthermore, it demonstrates role of oncogenic E6 in antagonizing signaling pathways induced by PKR including eIF2alpha phosphorylation and PI3K pathway.

Angiostatin is one of the most widely studied inhibitors of angiogenesis and encompasses the first 3 or 4 kringle domains of human plasminogen (Plg). Of particular interest, is the fifth kringle of human Plg (KS), which displays higher anti-angiogenic potency than angiostatin. In fact, kringles 1 to 5 exhibit greater inhibitory activity against endothelial cells than angiostatin, and this finding suggests that K5 domain acts synergistically to enhance the anti-angiogenic effect of angiostatin. Therefore, we proposed that the K5 domain---on its own---could act as a potent anti-angiogenic and anti-tumor protein within the context of a cancer gene therapy strategy. / In our first study, we assessed the angiostatic properties of the K5 peptide domain in an orthotopic brain cancer model. We demonstrated that the disulfide bridging conformation of K5, necessary to maintain its functionality, is conserved upon secretion by gene-modified mammalian cells. Kringle 5 retrovirally gene-engineered human U87 glioma cells produced functional soluble K5 protein capable of suppressing growth factor-induced endothelial cell migration in vitro and inhibiting glioma-induced angiogenesis in vivo. Interestingly, secreted K5 protein blocked the recruitment of tumor-associated CD45+Mac3 +Grl- macrophages in vivo and inhibited the migration of CD206+ human monocyte-derived macrophages in vitro. Moreover, in a clinically relevant orthotopic glioma model, soluble K5 induced long-term survival in a majority of test animals. Thus, these findings validate the use of a gene therapy approach to deliver Plg K5 protein and suggest that K5 acts as a novel 2-pronged anti-tumor agent, mediating its inhibitory effect via its action on host-derived endothelial cells and tumor-associated macrophages. / To determine if K5 mediated its anti-tumor effect by modulating other immune effector cells, we tested the use of soluble K5 in a murine DA/3 mammary adenocarcinoma model. Soluble K5 produced by retrovirally gene-modified DA/3 cells led to long-term survival (over 1 year) of immunocompetent BALB/c mice however, we failed to observe tumor rejection in immunodeficient NOD-SCID and BALB/c nude mice. Further analysis revealed that K5 enhances the recruitment of tumor-infiltrating CD3+ lymphoid cells, in particular the natural killer T (NKT)-lymphocyte phenotype. Consistent with our previous findings, we demonstrated that K5 led to a significant decrease in tumor-associated microvessel length and density. Interestingly, K5 tumors were characterized by a robust neutrophilic infiltrate. This may be explained by the ability of K5 to act as a strong chemotactic agent for human neutrophils in vitro as well as its ability to promote CD64+ activation within the CD11b+ neutrophil phenotype. These findings confirm that K5 acts as a potent angiostatic agent and possesses a novel pro-inflammatory role via its ability to recruit tumor-associated neutrophils and NKT-lymphocytes, leading to a strong anti-tumor response. / Tumor-associated macrophages (TAM) are key immune effector cells implicated in promoting tumor progression and metastasis. It would thus be desirable to explore strategies to reduce TAM infiltration within the tumor microenvironment. In our first study, we demonstrated that soluble K5 protein blocks macrophage recruitment. In addition, the recent observation that angiostatin reduces macrophage infiltration in an atherosclerosis model prompted our laboratory to further explore the use of angiostatin as an anti-macrophage agent. We demonstrated that angiostatin suppresses the in vitro migration of both murine peritoneal macrophages and human monocyte-derived CD206 + macrophages. Furthermore, we showed that angiostatin led to a decrease in the gelatinolytic activity of macrophage-produced matrix metalloproteinase-9, which may explain, in part, the observed angiostatin-mediated inhibition of migration. Additionally, we detected the presence of the beta-subunit of ATP synthase on the cell-surface of macrophages. ATP synthase was previously found to be a receptor for angiostatin on the cell-surface endothelial cells. We propose that the presence of ATP synthase on the surface of macrophages may promote interaction with angiostatin and prevent migration, similar to what has been reported with endothelial cells. Our findings suggest that angiostatin holds promise as an inhibitory agent against macrophages.