Inflammatory and neuropathic pains arise in part from sensitization at nociceptive synapses in the spinal cord. Activity-dependent signaling cascades converge onto the tyrosine kinase Src, which participates in augmenting the function of N-methyl-D-aspartate receptors (NMDARs) and thus potentiates the nociceptive system. Src is capable of these effects because it is anchored to the NMDAR complex via an adaptor protein called NADH dehydrogenase subunit 2 (ND2). There is evidence that this interaction occurs between amino acids 40-49 of Src and amino acids 310-321 of ND2. I have determined that a peptide consisting of amino acids 310-321 of ND2, and affixed to the HIV Tat domain for cell permeability, is capable of alleviating tactile allodynia induced by Complete Freund's Adjuvant (CFA) in rats. Src40-49Tat was not effective in two models of inflammatory pain. This work further implicates the Src-ND2 interaction in pain hypersensitivity and suggests that Tat ND2 310-321 may alleviate it.

Inflammatory and neuropathic pains arise in part from sensitization at nociceptive synapses in the spinal cord. Activity-dependent signaling cascades converge onto the tyrosine kinase Src, which participates in augmenting the function of N-methyl-D-aspartate receptors (NMDARs) and thus potentiates the nociceptive system. Src is capable of these effects because it is anchored to the NMDAR complex via an adaptor protein called NADH dehydrogenase subunit 2 (ND2). There is evidence that this interaction occurs between amino acids 40-49 of Src and amino acids 310-321 of ND2. I have determined that a peptide consisting of amino acids 310-321 of ND2, and affixed to the HIV Tat domain for cell permeability, is capable of alleviating tactile allodynia induced by Complete Freund's Adjuvant (CFA) in rats. Src40-49Tat was not effective in two models of inflammatory pain. This work further implicates the Src-ND2 interaction in pain hypersensitivity and suggests that Tat ND2 310-321 may alleviate it.

Histone deacetylase inhibitors (HDIs) are a group of chemo-preventive and chemo-therapeutic agents that have generated significant attention in clinical trials, given their ability to selectively induce cell cycle arrest, differentiation and/or apoptosis of tumor cells. Presently, these agents are proposed to function by altering gene expression levels, primarily by promoting histone hyperacetylation and gene transcription. However, in this thesis, HDIs are reported to control the expression of genes from the c-Src kinase family and p21WAF1 by means other than transcriptional activation. <p>Overexpression and activation of c-Src, a 60kDa non-receptor tyrosine kinase, has been implicated in the development, growth, progression, and metastasis of several human cancers, especially those of the colon. Butyrate and the more specific histone deacetylase inhibitor trichostatin A (TSA) were both found to effectively inhibit the expression of c-Src mRNA and protein in a number of tumor cell lines, including those of the colon, liver and breast. Expression of the SRC oncogene is alternatively regulated by the SRC1A and SRC1 promoters. HDIs were shown to repress c-Src expression by inhibiting transcription of both of these promoters, independent of any new protein synthesis. Furthermore, butyrate and TSA similarly regulated the expression of the c-Src family kinase (SFK) members Yes, Fyn, Lyn and Lck in human colon cancer cell lines. In addition, TATA binding protein (TBP) associated factor 1 (TAF1) was shown to be necessary for basal transcription of the SRC1A, YES and LYN promoters, but was not required for HDI mediated repression. <p>Induction of the potent cyclin dependent kinase inhibitor p21WAF1 has been identified to be a key feature of HDI mediated cell cycle arrest. The level of p21WAF1 expression has been extensively reported to be directly upregulated by HDIs in a p53 independent manner that requires Sp family binding sites in the p21WAF1 proximal promoter to induce transcription. However, HDIs were shown to be capable of inducing p21WAF1 gene expression, dependent on new protein synthesis, by increasing mRNA stability. To date, p21WAF1 mRNA stability has been extensively studied and a number of cis-acting elements in the 3 untranslated region (UTR) of the p21WAF1 mRNA have been implicated in the regulation of mRNA stability, such as AU rich elements (AREs) and a 42 nucleotide HuD/Elav binding element. Similarly, in this work, two novel cis-acting elements were identified in the 3 UTR of p21WAF1 and were shown to facilitate basal and HDI induced post-transcriptional regulation of p21WAF1 mRNA stability in HepG2 cells. Collectively, these studies highlight the intricacy of HDI mediated effects and challenge the preconceptions regarding the molecular mechanism of these anti-tumor agents.

Τα αρνητικά και θετικά κατά Gram βακτήρια E. coli και S. αureus αντίστοιχα αναγνωρίζονται και δεσμεύονται στην επιφάνεια των αιμοκυττάρων της C. capitata. Η πρόσδεση των βακτηρίων ενεργοποιεί τόσο τις β1 ιντεγκρίνες, όσο και σηματοδοτικά μονοπάτια που περιλαμβάνουν τα μόρια μεταγωγής σήματος Ras, FAK, Src και MAP κινάσες. Οι παραπάνω ενεργοποιήσεις, σε συνδυασμό με τη συμμετοχή του κυτταροσκελετού της ακτίνης και της τουμπουλίνης, καταλήγουν στην επαγωγή της έκκρισης μορίων απαραίτητων για την κυτταροφαγία των βακτηρίων που είναι και το τελικό αποτέλεσμα των παραπάνω διαδικασιών. Τα σφαιρίδια λάτεξ -αλλά και πιθανόν και άλλοι αβιοτικοί παράγοντες- παρότι δεν έχουν καμία προηγούμενη εξελικτική σχέση με τα αιμοκύτταρα, ως σύγχρονο προϊόν της ανθρώπινης γνώσης, αναγνωρίζονται και δεσμεύονται στην επιφάνεια των αιμοκυττάρων από άγνωστους μέχρις στιγμής υποδοχείς. Η κυτταροφαγία τους προωθείται μέσω ενεργοποίησης σηματοδοτικών μονοπατιών που περιλαμβάνουν την ενεργοποίηση των μορίων FAK, Src και MAP κινασών καθώς και με τη συμμετοχή του κυτταροσκελετού της ακτίνης και της τουμπουλίνης. Ο LPS αναγνωρίζεται και δεσμεύεται στην επιφάνεια των αιμοκυττάρων, ενεργοποιεί άγνωστους μέχρις στιγμής υποδοχείς και διαμέσου σηματοδοτικών μονοπατιών που περιλαμβάνουν τις Ras, ενεργοποιεί τις MAP κινάσες και το σύστημα της έκκρισης. Αν και ενεργοποιεί και τις τρεις MAP κινάσες, μόνο η ERK και η p38 απαιτούνται τόσο στη διαδικασία της έκκρισης, όσο και στη διαδικασία της ενδοκυττάρωσής του. Η FAK, αν και ενεργοποιείται από τον LPS, δεν εμπλέκεται στην διαδικασία της ενδοκυττάρωσής του. Τα παραπάνω δείχνουν ότι τα αιμοκύτταρα έχουν αναπτύξει διακριτούς μηχανισμούς για την κυτταροφαγία των παθογόνων, των μικρομορίων και των αβιοτικών παραγόντων, γεγονός που δείχνει την ικανότητα εξέλιξης των εντόμων έτσι ώστε να καλύπτουν τις ανάγκες της επιβίωσή τους. / Gram negative and positive bacteria E. coli and S. αureus respectively, are recognized and binded on C. capitata hemocyte surface. After binding, they activate β1 integrins and intracellular signalling pathways involving the kinases Ras, FAK, Src and MAP. This signal transduction, with the participation of the cytoskeleton of actin and tuboulin, leads to a regulated secretion that is a prerecuisite for phagocytosis. Latex beads and probably other abiotic factors, despite having no previous evolutionary relation to the hemocytes being a new product of human knowledge, are recognized and binded on hemocyte surface by so far unknown receptors. They activate intracellular signalling pathways that involves FAK, Src and MAP kinases and promote -with the participation of actin and tuboulin cytoskeleton- their phagocytosis. LPS is recognized and binded on hemocyte surface and activates so far unknown receptors and through unknown intracellular signalling pathways involving Ras, activates the MAP kinases and the regulated secretion. Although it activates all three MAPKs, only the ΕΡΚ and p38 are required not only for the secretion, but also for its internalization. Although FAK is activated by LPS, does not get involved in the process of its internalization. All the above mentioned results indicate that the hemocytes have developed distinct mechanisms for phagocytosis of pathogens, micromolecules and abiotic factors, a fact that underlines insects evolutionary adaptations, so that they can survive.

Over-expression of both hepatocyte growth factor (HGF) and its receptor Met frequently occurs in invasive human breast cancer, suggesting that the establishment of an HGF “autocrine loop” may be linked to breast tumour progression. We have recently shown a novel activating function of two signaling molecules, Src tyrosine kinase and the signal transducer and activator of transcription-3 factor (Stat3), on HGF expression in breast epithelial cells. Interestingly, Stat3 is also important in normal breast development,but this function does not require Src. In addition, β1-integrin adhesion occurs minimally in differentiated breast epithelium, but is upregulated during oncogenic progression and is required for transformation by Src. We therefore hypothesize that β1-integrin engagement is necessary for Src/Stat3-dependent activation of HGF transcription and breast tumourigensis. Using specific inhibitors of Src (Dasatinib) and Stat3 (CPA7) we demonstrated that autocrine HGF expression is linked to activation of Src/Stat3 in a malignant breast cell line. Phenotypic reversion (e.g., cell rounding and loss of filopodial extensions) and inhibition of pY705Stat3, HGF and pYMet expression as determined by immunofluorescence was achieved with both inhibitor treatments separately, and a synergistic effect was observed with combined treatment. Furthermore, β-catenin localization was nuclear in malignant cells, but shifted to cortical cytoplasmic following inhibitor treatment, similar to non-malignant mouse breast epithelial cells (EPH4). We are currently extrapolating these findings to a 3D Matrigel culture model in which EPH4 cells form acini-like spheroids with hollow lumen surrounded by a well-polarized outer layer of cells. Under these conditions, Stat3 levels are decreased followed by a reduction in cyclin D1 expression, while Src activation remains at a low baseline level. Interestingly,expression of Stat5, which has a reciprocal relationship with Stat3 in breast development and involution, is increased concomitant with elevated β-casein expression. Moreover, Fibronectin and HGF in combination stimulate tubular outgrowths with lumen filling. These findings suggest that aberrant changes in extracellular matrix milieu may stimulate integrin cross talk resulting in a switch of HGF/Met signaling to a transformation phenotype. Information from this study may lead to novel cancer therapies through targeting the HGF/Met and integrin signaling cascades. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-09-19 18:19:22.744

Cell migration and invasion are essential physiological processes required for the growth and development of all multicellular organisms. However, they have also been implicated in the pathogenesis of certain vascular system diseases and invasive cancers. In this study, we investigate two proteins involved in cell proliferation and survival signaling, p53 and Stat3, which have been found misregulated in atherosclerosis and cancer, to establish what effect they have on the development of Src-induced invasive phenotypes in aortic vascular smooth muscle cells (VSMC) and NIH 3T3 fibroblasts. In the first stage of this experiment, we investigated the tumor suppressor p53. Once believed to act primarily as a regulator of the cell cycle, DNA repair, senescence and apoptosis, current evidence suggests that p53 can also regulate cell migration and invasion. For our study, we stably transduced VSMC and NIH 3T3 fibroblasts with constitutively active Src (SrcY527F) to generate invasive cell lines with pronounced podosome and rosette formation. We established for the first time that p53 suppresses Src-induced podosome and rosette formation, extracellular matrix degradation, cell migration and invasion in these cells. We also present novel data showing that p53 suppresses these invasive phenotypes, at least in part, by up-regulating the expression of caldesmon, an actin binding protein which stabilizes stress fibers and inhibits podosome and rosette formation. In the second part of this study, we show that Stat3, a pro-survival and pro-metastatic transcription factor, is required downstream of Src for the promotion of invasive phenotypes in VSMC and NIH 3T3 fibroblasts. Interestingly we have also shown for the first time that Stat3 can localize to podosomes and rosettes in these cells. The exact physiological reasoning for this localization, however, remains to be determined. This study provides strong evidence suggesting that mutual antagonism between the anti-invasive ‘p53-caldesmon’ and pro-invasive ‘Src-Stat3’ pathways dictates the outcome of Src-induced invasive phenotypes in VSMC and NIH 3T3 fibroblasts. / Thesis (Master, Biochemistry) -- Queen's University, 2010-01-09 21:57:30.056

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

La phosphatidylinositol-3 kinase (PI3-K) est un complexe comportant une sous-unité catalytique (C) et régulatrice (R). Trois isoformes R (p85?, p85? et p55?) et quatre isoformes C (p110?, p110?, p110? et p110?) sont connues. Nous avons démontré préalablement chez les entérocytes humains que la PI3-K performe des rôles différenciation-spécifiques dans la suppression d'anolkose médiée par la signalisation intégrines ? 1 /Fak/Src. Cependant, comme dans la plupart des études concernant la PI3-K, les considérations pour les distinctions entre les isoformes sont négligées. L'hypothèse de la présente étude est donc que les isoformes PI3-K performent des rôles distincts au niveau de la régulation de la survie et de la suppression d'anoïkose médiées par la signalisation intégrines ?1/Fak/Src et ce, selon l'état de différenciation entérocytaire. Les objectifs de l'étude étaient les suivants : 1) Confirmer, valider et compléter la détermination des profils d'expression des isoformes et de complexes isoformes PI3-K chez les entérocytes indifférenciés et différenciés; 2) Analyser l'engagement de chacun des complexes isoformes PI3-K par la signalisation intégrines ? 1 /Fak/Src selon l'état de différenciation entérocytaire; 3) Analyser les contributions de chacune des isoformes au niveau de l'activation d' Akt-1 et de la promotion de la survie selon l'état de différenciation entérocytaire; et 4) Analyser l'impact d'une surexpression des isoformes R chez les entérocytes indifférenciés au niveau de l'activation d'Akt-1 et de la résistance à l'anoikose. Nos résultats indiquent que: 1) les profils d'expression d'isoformes régulatrices et catalytiques PI3-K sont distincts selon l'état de différenciation; 2) des profils distincts de complexes isoformes PI3-K prédominants sont également retrouvés selon l'état de différenciation; 3) des complexes isoformes PI3-K également distincts sont recrutés/engagés par la signalisation Fak/Src; .4) l'inhibition spécifique (pharmacologique ou via siARN) de complexes isoformes PI3-K influence distinctement sur l'activation d'Akt-1, l'effecteur principal de la PI3-K et ce, selon l'état de différenciation; 5) l'inhibition spécifique (pharmacologique ou via siARN) de complexes isoformes PI3-K induit 1'apoptose/anoikose de manière isoforme-distincte et différenciation-spécifique; et 6) la surexpression de certaines isoformes régulatrices confère une certaine mesure de résistance à l'apoptose, et peut même, dans certains cas conférer une mesure additionnelle de résistance à l'anoikose. Mises ensembles, les données de cette étude supportent fortement le principe d'une participation de la PI3-K qui varie selon l' isoforme et en fonction de l'état de différenciation dans la signalisation intégrines ? 1 /Fak/Src de promotion de survie et de suppression d' andikose, chez les entérocytes humains.

Thesis (Ph. D.)--Cornell University, May, 1998. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [78]-93).