The modulation of biosynthetic patterns of human articular chondrocytes by cytokines and growth factors was investigated in vitro by analysing the gene expression of cartilage matrix components at the mRNA level. The effects of IL-1$ beta$, TGF-$ beta$, and IGF-1, individually or in combination on expression of mRNAs for type II collagen, aggrecan, stromelysin, collagenase, and tissue inhibitor of metalloproteinases (TIMP) were quantitatively determined by competitive PCR. The results revealed that each of these mRNA species had an unique response pattern to these external agents. TGF-$ beta$ enhanced expression of mRNAs for type II collagen, aggrecan, and TIMP, while it depressed the expression of metalloproteinases. In contrast, IL-1 regulated expression of these mRNAs in an opposite fashion. The magnitude of response to IL-1 and TGF-$ beta$ varies for different target mRNAs. A prolonged incubation with TGF-$ beta$ effectively reversed the IL-1-induced depression of TIMP-1 mRNA level. IGF-1 increased the mRNA levels for aggrecan and type II collagen, and effectively reversed the inhibitory effects of IL-1 on these two genes. This study indicates how physiologically-relevant cytokines and growth factors regulate the expression of structural and degradative components by articular chondrocytes. The understanding of the factors regulating the biosynthetic behaviors of articular chondrocytes under various physiological and pathological conditions is important if effective repair of the tissue is to be achieved.

Mammalian DNA sequences (ors), enriched for origins of DNA replication, have been isolated by extruding nascent DNA from replication bubbles activated at the beginning of S phase in CV-1 monkey cells. To determine whether these sequences had origin function, their ability to enable pBR322 plasmids to replicate autonomously in mammalian cells was examined. Using the DpnI-resistance assay and density labeling with bromodeoxyuridine, plasmids containing four different ors (ors 3, 8, 9 and 12) were found to replicate after transfection in CV-1, COS-7 and HeLa cell lines. These results were verified using electron microscopy for ors 8 and ors 12 plasmids, and the position of the replication bubbles in ors 12 plasmids was mapped to the ors sequence. Plasmids containing ors 3, 8, 9 and 12 were also shown to replicate in a cell-free replication system using extracts from HeLa cells. Origin mapping experiments in the in vitro system showed that, early in the replication reaction, incorporation of nucleotides occurs preferentially at ors-containing fragments, indicating ors-specific initiation of replication. / All of the ors sequences, and all known origins of replication from prokaryotes and viruses of eukaryotes, contain inverted repeat sequences with the potential to form cruciform (stem-loop) structures. Transient cruciform formation at the origin has been postulated to be involved in the initiation of DNA replication, but the detection of such structures in vivo has been difficult. With the aim of developing a tool for the detection and functional analysis of cruciforms in vivo, two monoclonal antibodies, 2D3 and 4B4, were raised against a stable DNA cruciform structure. By gel retention assays it was shown that 2D3 and 4B4 bind not only to the cruciform that was used as immunogen, but also to another stable DNA cruciform of unrelated sequence, and to T-shaped double-stranded DNA molecules containing a single stem-loop; they do not bind to linear double-stranded DNA identical in sequence to the cruciform DNA, nor to linear single-stranded DNA, to single-stranded DNA with a stem-loop, or tRNA. Using T7 endonuclease 3, the binding site of these antibodies was mapped to the base of the cruciform stem regardless of sequence. It was concluded that 2D3 and 4B4 recognize conformational determinants specific to the elbow-like structures at the base of the stem of DNA cruciforms.

In the middle of the 1990's, Dr Jaw Ming Cherng, a previous student in the laboratory discovered the presence of an RNA molecule whose 5' end lies in the core origin of polyomavirus. This RNA molecule was transcribed in the same orientation as the late transcripts of polyomavirus and was named ori RNA. / The ori RNA was detected by run-on transcription. In this technique, the viral transcription complexes---the DNA molecule and the RNA polymerases bound to it---are isolated from nuclei of virus infected cells and the nascent transcripts are extended in vitro with radioactive nucleotides. The RNA transcripts of a desired region of polyomavirus are isolated by hybridization with a single stranded DNA and then RNAase treated and finally analyzed after migration on a polyacrylamide gel. The ori RNA was shown to be transcribed by the RNA polymerase II as its synthesis was diminished after treatment with alpha-amanitin, an inhibitor of this class of RNA polymerase. However, the hybridization and nuclease treatment induce some degradation of the RNA and may lead to an incorrect interpretation of the results. Before proceeding further in the characterization of the ori RNA, it was therefore necessary to optimize the treatment needed to isolate ori RNA. / To achieve this, we tested different conditions of nuclease treatment of the hybrids. An RNA molecule covering the origin region was transcribed in vitro and hybridized to three different single stranded DNAs of various lengths. We optimized the hybridization conditions, the treatment of hybrids with nucleases, and the conditions for elution of trimmed RNAs from the single stranded DNA.

The human beta-globin LCR is a tissue-specific enhancer composed of 4 major erythroid-specific DNaseI hypersensitive sites (HS 1 to 4). The beta-globin LCR has previously been reported to confer high-level, position independent expression onto globin transgenes. Each HS has been shown to have some enhancer activity individually; however, there is evidence that they may work by different mechanisms. In this study, we have explored this possibility by assessing what type of position effects arise in individual clones having integrated either muLCR, HS2 or HS3 linked to the human beta-globin gene at different integration sites. We used Fluorescence Activated Cell Sorting (FACS) in conjunction with immunochemistry to measure human beta-globin expression in single murine erythroid leukemia cells (MEL). MEL cells (c88 strain) were induced to produce human beta-globin by treatment using 2% DMSO or by 10 nM TSA treatment so as to gain further insight on the molecular mechanism of these enhancers. Our results suggest that (1) HS3 is generally a stronger enhancer than HS2. (2) HS2 is exclusively affected by a graded position effect whereas HS3 (and the [LCR to a lesser extent) is prone to position effect variegation (PEV). (3) PEV was alleviated in HS3 and muLCR clones when we used 10nM TSA as the induction method but did not change HS2 position effects. (4) The expression per expressing cell seemed copy number dependent for HS3 whereas it was not for HS2 This data taken together with evidence from the literature suggests that HS2 and HS3 function by different mechanisms whereby HS2 would play a role in acetylating histones/protection from DNA methylation and open chromatin whereas HS3 would increase the total amount of transcripts in each expressing cell.

We purified HIV-1 reverse transcriptase (RT) enzymes that contain E44D and V118I either alone or in a background of different combinations of AZT-resistance conferring mutations in order to study the underlying biochemical mechanisms. We found that enzymes containing E44D in a background of these latter mutations increase the efficiency of excision of 3TC-MP, while V118I-containing enzymes show reductions in rates of incorporation of AZT-MP and 3TC-MP. The V118I mutant is also associated with diminished rates of ATP-dependent primer unblocking. The additional presence of AZT-mutations can partially neutralize this deficit, which helps to explain the concurrent presence of these changes in resistant isolates. These biochemical data suggest that mutations E44D and V118I play distinct mechanistic roles in dual resistance to AZT and 3TC. Our findings are consistent with an increasing number of clinical studies suggesting that E44D- and V118I-containing clusters are sometimes associated with HIV resistance to multiple nucleotide analogue RT inhibitors.

The nuclear factor kappaB (NF-kappaB) transcription factors are key regulators of immunomodulatory genes regulation. NF-kappaB activity is regulated through the phosphorylation of inhibitory proteins (IkappaBs) by the IkappaB kinase (IKK) complex (IKK alpha/beta/gamma), leading to IkappaB degradation and NF-kappaB translocation to the nucleus where they promote transcription of immunoregulatory genes. Moreover, cRel and p65 activities are also regulated by direct phosphorylation of their transactivation domain. Recently, two IKK non-canonical homologues, IKKepsilon and TBK-1 (TANK binding kinase-1) have been identified with functions distinct from the classical IKKalpha/IKKbeta. TBK-1/IKKepsilon trigger antiviral immunity through direct phosphorylation of the IRF3/IRF7 transcription factors, which are key regulators of the interferon response. Since IKKepsilon modulates the activity of IRF3/IRF7, it is of interest to assess whether IKKepsilon/TBK-1 also regulates the transactivation activity of NF-kappaB. Our hypothesis was that IKKepsilon/TBK-1 modulates the activity of cRel by direct phosphorylation of its transactivation domain (TD). In this study, we demonstrate that IKKepsilon and TBK-1 directly phosphorylate cRel in vitro and in vivo. Two of the three consensus sequences recognized by IKKepsilon/TBK-1 in the cRel TD are directly phosphorylated by IKKepsilon. cRel was translocated to the nucleus in cells expressing wild type versus kinase dead variant. The expression of IKKepsilon increases c-Rel transactivation in reporter gene assays. Serine to alanine mutation was further used to characterize the function of this phosphorylation at the level of nuclear translocation and transactivation potential using immunofluorescence and reporter gene assay. Furthermore, co-expression studies revealed that IKKepsilon and not the kinase dead variant is responsible for cRel degradation in a dose-dependent manner and this effect is partially revert

Prolactin (PRL) is a polypeptide hormone synthesized by the anterior pituitary as well as by a number of extrapituitary tissues. The hormone is an essential regulator of important biological processes. The best characterized role for PRL is its participation in the development of the lobuloalveolar system in the mammary gland. The biological actions of PRL are mediated in the cell by the membrane-bound prolactin receptor (PRLR), which is a member of the cytokine family of receptors. Binding of PRL to the PRLR induces receptor dimerization and activation of the constitutively associated Jak2 kinase, which leads to the tyrosine phosphorylation of the kinase as well as phosphorylation of tyrosine residues on the PRLR creating recruitment sites for SH2 containing signaling molecules. Among the signaling molecules recruited to the PRLR/Jak2 complex are the signal transducers and activators of transcription 5 (Stat5), the protein tyrosine phosphatase SHP-2 and the inhibitory protein SOCS-1. Work in this thesis was performed in order to understand the molecular basis behind the biological responses to PRL trough investigating the intracellular signaling mechanisms emanating from the PRLR. / Tyrosine phosphorylation of the PRLR, particularly of the most C-terminal tyrosine, was shown to play a critical role in the induction of PRL responsive genes. The mechanism of regulation of signaling activities by the C-terminal tyrosine of the PRLR was not known. The aim of the doctoral work was to investigate the signaling mechanisms controlled by the C-terminal tyrosine of the PRLR. / The signal transducer and activator of transcription 5 (Stat5) is an important mediator of PRLR signaling leading to the activation of PRL responsive genes. Work in this thesis established that Stat5 tyrosine phosphorylation is independent of PRLR tyrosine phosphorylation, interestingly however, Stat5 nuclear translocation was directly regulated by the C-terminal tyrosine of the PRLR. Additionally, the studies demonstrated that the C-terminal tyrosine is a site for the recruitment of the protein tyrosine phosphatase SHP-2. A second site for the SHP-2 recruitment was determined to be the adaptor protein Gab2 that is tyrosine phosphorylated in response to PRL stimulation. Furthermore, the studies illustrate that the C-terminal tyrosine, through recruiting SHP-2 phosphatase, regulates the phosphorylation state of tyrosine 1007 of Jak2 kinase. Dephosphorylation of tyrosine 1007 in the activation loop of Jak2 by SHP-2 prevents the association between Jak2 and the inhibitory protein SOCS-1 leading to the maintenance of Jak2 kinase activity and the prevention of the ubiquitination and proteasomal degradation of Jak2 as well as the PRLR. / Collectively, work in this thesis resolves some of the molecular mechanisms implicated in PRLR signaling, specifically through the most C-terminal tyrosine of the PRLR. The findings are not only important for realizing the molecular basis of PRL physiological actions, such as mammary gland development, but also for understanding the signaling schemes utilized by members of the cytokine receptor superfamily.

Granulins are small cysteine-rich peptide growth factors that, in mammals, are derived from a common glycoprotein precursor (progranulin) containing one half and seven non-identical tandemly repeated granulin domains. While there is evidence for only one progranulin gene in mammalian genomes, work presented in this thesis demonstrates that granulins form an extended gene family in teleost fish. Two zebrafish genes that constitute likely co-orthologues to mammalian progranulin, progranulin-a and progranulin-b, encode precursors that encode 10 and 9 copies of the granulin motif, respectively. Two additional genes in zebrafish, designated progranulin-1 and progranulin-2, each give rise to precursors consisting of one and one-half granulin-like repeats only. The progranulin-1 and progranulin-2 genes are organized in tandem, and possess exonic complementarity to a single non-coding RNA gene transcribed in the antisense orientation from the complementary DNA strand. A cDNA encoding a chimeric precursor consisting of the amino-terminal progranulin-1 followed by the carboxyl-terminal region of progranulin-2 was characterized and is likely generated through the mechanism of splicing in trans between the two primary transcripts for progranulin-1 and progranulin-2. Chromosomal assignment of the zebrafish progranulin genes indicates that progranulin-a, but not progranulin-b, is located on a chromosome that displays syntenic correspondence to mammalian progranulin. Cumulatively, the data suggest that an ancestral progranulin gene was duplicated at the base of the vertebrate radiation to generate precursors with distinct architectures and that one was lost in the lineage leading to tetrapods after the split between sarcopterygians and actinopterygians. Like their mammalian counterpart, the expression of the zebrafish progranulins is widespread in adult tissues. During development, the maternal expression of progranulin-a and progranulin-b parallels that

The hepatocyte growth factor (HGF)/Met receptor tyrosine kinase (RTK), regulates a wide variety of biological responses including cell migration, proliferation and invasion and deregulation of these processes can lead to cancer development. At the start of this PhD thesis, the concept that deregulation of RTKs can occur through loss of negative regulatory mechanisms, had emerged. RTKs are down-regulated through the endocytic pathway, which involves receptor ubiquitination. Work from this thesis demonstrates that uncoupling the Met RTK from Cbl-mediated ubiquitination (Met Y1003F) results in oncogenic activation of Met both in vitro and in vivo. Met ubiquitination is not required for internalization but is essential for lysosomal degradation. Loss of Met ubiquitination abrogates association with endocytic sorting machinery for degradation and leads to enhanced stability and retention of Met Y1003F within an intracellular compartment. Here, MetY1003F induces sustained activation of the Ras-MAPK pathway, required for cell transformation. Fusion of a mono-ubiquitin moiety to the C-tail of MetY1003F, is sufficient to restore engagement with endocytic machinery and down-regulation of Met and consequently inhibit sustained activation of MAPK as well as cell transformation. / Numerous modes of RTK internalisation have been identified, including clathrin mediated endocytosis, yet little is known about the signals determining which route is taken and the contribution of each to RTK signalling and stability. Work from this thesis demonstrates that Met induces and is localised to a form of membrane protrusion, called dorsal ruffles. Internalisation of Met from these protrusions delivers Met to the endocytic pathway in manner which promotes efficient degradation. I have established a requirement for the Gab1 scaffold protein for dorsal ruffle induction. Moreover, a novel Gab1-Nck complex is a common and essential requirement for dorsal ruffle formation downstream from the Met, EGF and PDGF RTKs. My data supports a role for dorsal ruffles in cell migration and epithelial morphogenesis. Met is active within dorsal ruffles and we propose that these membrane structures function as signalling microdomains in a manner similar to endosomes. Importantly, disruption of dorsal ruffles alters Met RTK signalling and biological response. These studies illustrate the critical relationship between RTK localisation and signalling and underscore the importance of understanding how receptors traffic. / Le récepteur à activité tyrosine kinase (RTK) Met (ou récepteur au HGF), régule une large variété de réponses biologiques, dont la migration, la prolifération et l'invasion cellulaires; la dérégulation de ces processus pouvant conduire au développement de cancers. Lorsque j'ai débuté cette thèse, une notion venait d'émerger selon laquelle la suractivation des RTK dans les cellules cancéreuses pouvait être la conséquence d'une perte de régulation négative. Les RTK sont négativement régulés par endocytose, un processus impliquant l'ubiquitination du récepteur. Le travail réalisé dans cette thèse démontre que le découplage entre le récepteur Met et son ubiquitination par la protéine Cbl (Met Y1003F) conduit à l'activation oncogénique de Met à la fois in vivo et in vitro. L'ubiquitination de Met n'est pas nécessaire à son internalisation, mais est essentielle à sa dégradation par le lysosome. La perte de l'ubiquitination de Met empêche son association avec la machinerie de l'endosome de triage, conduisant à une plus grande stabilité et au maintien de Met 1003F dans un compartiment intracellulaire. Met 1003F provoque alors une activation prolongée de la voie de signalisation Ras-MAPK, impliquée dans la transformation cellulaire. La fusion d'une molécule de mono-ubiquitine à l'extrémité C-terminale de Met 1003F est suffisante pour restaurer l'engagement vers la voie endocytique et la régulation négative de Met, et par conséquent inhibe l'activation prolongée de MAPK ainsi que la transformation cellulaire. fr / Plusieurs modes d'internalisation des RTKs ont été identifiés, dont l'endocytose rapide par l'entremise des clathrines. Par contre, les signaux déterminant leur choix et la contribution de chacun dans la signalisation et la stabilité des RTKs restent peu connus. J'ai identifié que Met stimule la formation de protrusions membranaires appelées "protrusions dorsales" et s'y localise. L'internalisation de Met à partir de ces protrusions permet de l'intégrer à la voie d'endocytose et de promouvoir sa dégradation de manière efficace. J'ai pu établir que la protéine d'échaffaudage Gab1 était nécessaire à l'induction de ces protrusions dorsales. De plus, j'ai identifié que la formation d'un nouveau complexe entre les protéines Gab1 et Nck est un pré-requis essentiel pour la formation de ces protrusions membranaires en aval des récepteurs Met, EGFR et PDGFR. Mes résultats montrent aussi que ces protrusions dorsales jouent un rôle dans la migration cellulaire et la morphogénèse épithéliale. Met étant actif dans ces protrusions dorsales, nous proposons ici l'idée selon laquelle ces structures membranaires fonctionnent comme des microdomaines de signalisation d'une manière similaire aux endosomes. D'ailleurs, la signalisation et la réponse biologique du récepteur Met sont altérées sans ces protrusions dorsales. Cette étude illustre le lien étroit entre la localisation des RTK et leur signalisation, et souligne l'importance d'une meilleure compréhension de la circulation intracellulaire des récepteurs. fr

Protein tyrosine phosphatase 1B (PTP1B) has been studied extensively as a modulator of metabolic signaling. In addition to the insulin receptor, which is a key physiological target of PTP1B, a number of other receptor and receptor-associated tyrosine kinases have also been reported as PTP1B substrates. Although hyperactivation of several of these kinases is associated with cancer, mice lacking PTP1B are not prone to tumorigenesis. In fact, PTP1B deficiency confers resistance to particular types of cancer; however, the mechanisms responsible for this and other cellular effects of PTP1B remain poorly characterized. / With the aim of better understanding its functions at a molecular level, two phosphoproteins, cortactin and STAM2, were identified as substrates of PTP1B. Cortactin regulates actin cytoskeletal dynamics in various contexts while STAM2 is involved in sorting ubiquitinated receptor tyrosine kinases (RTKs) to the lysosomal degradative pathway following their activation. In both cases, the phosphorylation of specific tyrosine residues was regulated by PTP1B, and mutation of these sites affected the cellular response to different stimuli. These novel targets could each contribute to the known effects of PTP1B deficiency on processes including RTK signaling, cell migration, and tumorigenesis. / PTP1B and other members of the PTP enzyme family are considered promising drug targets. However, to date, the most effective in vitro PTP inhibitors have tended to be highly charged, limiting cellular permeability. As an alternative approach that could aid our functional studies, a novel, uncharged, competitive inhibitor of PTP1B was characterized. This compound effectively inhibited PTP1B in cell-based assays as well as a subset of other PTPs in vitro. A series of derivatives were tested and molecular docking studies were performed in order to understand its important functional groups and identify its likely site of action. / Through the identification of cortactin and STAM2 as PTP1B targets, this thesis research has contributed significantly to our understanding of the molecular events regulated by PTP1B. Furthermore, the characterization of a small-molecule inhibitor provides a biochemical tool for probing PTP function, which could also be used as a scaffold for future drug development. / La protéine tyrosine phosphatase 1B (PTP1B) a été longtemps étudiée en tant que modulateur de signalisation métabolique. En plus du récepteur de l'insuline, qui est une cible physiologique clée de PTP1B, un grand nombre de recepteurs et de tyrosine-kinases associées aux recepteurs ont été rapportés comme substrats de PTP1B. Bien que l'hyperactivation de plusieurs de ces kinases est associée au cancer, les souris déficientes en PTP1B ne sont pas prédisposées au développement de tumeurs. En fait, la déficience en PTP1B confère une résistance à certains types de cancers, mais les mécanismes responsables de ceci ainsi que d'autres effets cellulaires de PTP1B ne sont pas bien caractérisés. / En étudiant les fonctions de PTP1B au niveau moléculaire, deux phosphoprotéines ont été identifiées comme substrats : la cortactine et STAM2. La cortactine régule la dynamique du cytosquelette d'actine alors que STAM2 est impliqué dans le tri des récepteurs tyrosine kinases (RTKs) ubiquitinés vers la voie de dégradation lysosomale. Pour les deux substrats, la phosphorylation de certains résidus tyrosines était régulée par PTP1B et la mutation de ces sites affectait la réponse cellulaire à différents stimuli. Ces nouveaux substrats de PTP1B peuvent contribuer aux effets connus de déficience en PTP1B notamment la signalisation des RTKs, la migration cellulaire et le développement de tumeurs. / PTP1B ainsi que les autres membres de la famille d'enzymes PTP sont considérés comme des cibles prometteuses pour le développement de médicaments. Par contre, les inhibiteurs de PTP les plus efficaces à ce jour sont fortement chargés ce qui limite la perméabilité cellulaire. Comme approche alternative qui pourrait aider nos études de fonction, un nouvel inhibiteur compétitif et non-chargé de PTP1B a été identifié. Ce composé inhibe efficacement PTP1B dans des essais cellulaires ainsi qu'un sous-ensemble de PTPs in vitro. Une série de dérivés ont été testés et des études de docking moléculaire ont été faites afin de mieux comprendre les groupes fonctionnels importants de cet inhibiteur et d'identifier son site d'action probable. / En identifiant la cortactine et STAM2 comme substrats de PTP1B, cette thèse de recherche a contribuée significativement à notre compréhension d'événements moléculaires régulés par PTP1B. En outre, la caractérisation d'un inhibiteur fourni un outil biochimique pour étudier la fonction des PTP et peut servir d'échafaudage pour le développement de médicaments.