Thesis (Ph.D. in Cell Biology, Stem Cells, and Development) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 102-114). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Les peptides constituent une famille de biomolécules dont l’utilisation dans différents domaines thérapeutiques (cancer, diabète, sida) s’est fortement développée ces dernières années. Le défi pour les chimistes consiste à y accéder grâce à de nouvelles méthodes fiables et efficaces. La première partie de notre travail a d’abord été orientée vers le développement deux méthodes de ligations non natives efficaces et complémentaires de celles existant. La première méthode, appelée ligation thiocarbamate, permet d’obtenir des peptides alkylthiocarbamate avec de très bons rendements, alors que la seconde, appelée ligation azaGly, aboutit à la formation d’un azaGlypeptide. La seconde partie de cette thèse traite de la conception et synthèse de nouveaux peptides susceptibles d’inhiber la signalisation HGF/SF-MET. Le récepteur à activité tyrosine kinase MET et son ligand, l’HGF/SF (Hepatocyte Growth Factor/Scattor Factor), sont des cibles de choix pour une thérapie anti-cancéreuse. La ligation thiocarbamate, précédemment décrite, et la ligation thioéther plus classique ont été utilisées pour préparer une chimiothèque de peptides sulfonatés d’inhiber cette signalisation de façon extracellulaire. La capacité de liaison des composés de la chimiothèque avec le domaine extracellulaire de MET a été évaluée grâce à la technologie biopuces. L’activité biologique (tests MTT, d’activité kinase) des meilleurs produits a été ensuite évaluée. / Use of peptides as biomolecules have been extensively applied to various therapeutic fields (cancer, diabetes, AIDS). The challenge for chemists consists in development of new reliable and efficient strategies. Our work especially focused on the conception of two innovative non native chemical ligations bringing an additionnal asset to the existing state of the art.The first ligation, i.e. thiocarbamate ligation, affords alkylthiocarbamate peptides with remarkable yields. Regarding the second one, the azaGly ligation allows the straightforward synthesis of azaGlypeptides. On the other hand, this thesis deals with the design of new peptides suitable to inhibit the signaling pathway of the tyrosine–kinase MET receptor and its ligand HGF/SF (Hepatocyte Growth factor/Scattor factor). Indeed interfering with MET signaling appears to be a promising therapeutic approach.The thiocarbamate ligation disclosed previously along with a classical thioether ligation have been employed for the chemical library design of sulfonated peptids in order to inhibit extracellular interactions. Binding activities assessment of the chemical libray toward the MET extracellular domain has been achieved using a microarray technology. Biological activities (MTT tests and kinase activity) have also been investigated.

In response to extra-cellular cues, cells activate signal transduction pathways to elicit a biological response. Cell surface growth factor receptors such as the Met receptor tyrosine kinase (RTK) activate signals that result in cellular proliferation, survival, migration, as well as epithelial morphogenesis. In order for signal transduction to occur, docking proteins are recruited to the activated RTK, become phosphorylated on tyrosine residues, which then serve as docking sites for the recruitment of other signaling proteins. Docking proteins function to diversify the signal by assembling multi-protein complexes. The Gab1 docking protein is the most tyrosine phosphorylated protein upon Met receptor activation and is required for Met mediated signaling and biology. / Gab1 belongs to a family of docking proteins including the highly related Gab2 protein. Gab1 promotes signals for epithelial morphogenesis downstream of the Met receptor, however Gab2 is unable to do so. Insertion of the Gab1 Met binding Motif (MBM) which confers direct binding to the Met receptor, as well as membrane targeting of Gab2 is sufficient to switch the capacity of Gab2 to activate the morphogenic program, cell scatter and lamellipodia formation. This is achieved via activation of sustained signaling pathways, and redistribution of the Gab protein, and associated molecules to sites of lamellipodia formation at the peripheral edge of the cell. / Activation of the Met RTK, promotes the formation of dorsal ruffles on the apical surface of epithelial cells. The Met receptor, Gab1 and Gab1 associated molecules Shp2, Crk, and p8S subunit of PI3K, are localized to these structures, however only the Gab1erk complex is required to drive dorsal ruffle formation. Gab1 is required for Met induced dorsal ruffles as well as downstream the PDGF and EGF RTKs. These are a signaling micro-environment which results in enhanced receptor degradation. Inhibition or enhancement of Met mediated dorsal ruffle formation correlates with receptor stability. / Dorsal ruffle formation downstream of Met requires the enzymatic activity of PI3K and PLCgamma, both enzymes that metabolize PIP2, and form complexes with Gab1 downstream of Met. PLCgamma and the PIP3 lipid product of PI3K are co-localized with Gab1 in dorsal ruffles. Gab1 engages with elements of the cytoskeleton, actin and cortactin, providing a link between growth factor signaling and remodeling of the actin cytoskeleton. Gab1 is localized to membrane protrusions of the basal surface in organoid cultures and is required for actin protrusions of the basal surface of breast cancer cells.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

No description available.

Breast cancer is a heterogeneous disease comprised of distinct biological entities that correlate with diverse clinical outcomes. Gene expression profiling has divided this heterogeneity into luminal, ERBB2+ and basal molecular subtypes. Basal breast cancers are difficult to treat as they lack expression of candidates suitable for targeted therapies and are associated with poor outcome. / Elevated protein level of the hepatocyte growth factor receptor, MET, is observed in 20% of human breast cancers and correlates with poor prognosis. However, the role of MET in mammary tumorigenesis is poorly understood. To address this, we generated a murine model that expresses weakly oncogenic mutants of Met (Metmt) in the mammary epithelium under the transcriptional control of the mouse mammary tumor virus promoter. We demonstrate that Metmt induces mammary carcinomas with diverse phenotypes and used gene expression microarrays to elucidate gene expression changes induced by Met. Since mammary tumors contained variable contents of epithelium and stroma, we used laser capture microdissection to procure epithelial cells for microarray analysis. Based on immunohistochemistry and expression profiling, we show that Metmt produces tumors with luminal or basal characteristics. From hierarchical clustering, Metmt-induced basal tumors clustered with murine models that share features of epithelial to mesenchymal transition and human basal breast cancers. Moreover, Metmt basal tumors clustered with human basal breast cancer. The status of MET among the human breast cancer subtypes has not previously been addressed. We demonstrate that MET levels are variable across molecular subtypes but show elevation in the basal subtype and correlates with poor outcome. We used a candidate gene approach derived from microarray data to gain an understanding of signals required for Met-dependent tumorigenesis. We investigated Nck adaptor proteins and demonstrate a role for Nck in cell motility and actin dynamics of Met-dependent breast carcinoma cells and show elevated expression in human basal breast cancers. By generating a unique mouse model in which Met is expressed in mammary epithelia, with the examination of MET levels in human breast cancer, we have established a novel link between MET and basal breast cancer. This work identifies poor outcome basal breast cancers that may benefit from anti-MET therapies.

Orientadores: Laura Sterian Ward, Antônio Hugo José Fróes Marques Campos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T01:02:11Z (GMT). No. of bitstreams: 1 Rocha_AngelicaGomes_M.pdf: 1644333 bytes, checksum: e3503fb6e2c8209f7a6696df111d6a78 (MD5) Previous issue date: 2014 / Resumo: Marcadores de malignidade, especialmente capazes de distinguir lesões de padrão folicular, que sejam de fácil implantação na rotina do diagnóstico de nódulos tireoidianos, continuam sendo extremamente necessários, dado o crescente aumento de nódulos tireoidianos diagnosticados nos últimos anos. A via HGF/c-Met/STAT3 está relacionada com desenvolvimento e progressão tumoral, sendo que a expressão de c-Met, HGF e de STAT3 foram descritas em grande parte dos carcinomas papilíferos de tireoide (CPT), mas não em tecido tireoidiano normal, sugerindo sua relação com o desenvolvimento e progressão do CPT. Para avaliar a utilidade da expressão proteica de c-Met, HGF, STAT3, e de sua proteína fosforilada (pSTAT3) no diagnóstico e no prognóstico de pacientes com nódulos tireoidianos, analisamos 356 tecidos tireoidianos, sendo 153 carcinomas papilíferos (CPT), dos quais 95 eram clássicos (CPC), 47 carcinomas papilíferos variante folicular (CPVF), e 11 carcinomas papilíferos de células altas (CPCA); 34 carcinomas foliculares (CFT), 34 adenomas foliculares (AF), 124 bócios e 11 tecidos normais. Todos os pacientes foram tratados e acompanhados de acordo com um mesmo protocolo padrão por 1-10 anos (Mo=5 anos). Áreas representativas do tecido foram selecionadas para a construção de uma lâmina de tissue microarray (TMA) que foi submetida à técnica de imunoistoquímica e analisada pelo score de Allred. A expressão citoplasmática de c-Met foi capaz de diferenciar nódulos malignos de benignos (p<0,0001, sensibilidade 86%, especificidade 76%, VPP 77%, VPN 86%); CPT de CF (p=0,0003, sensibilidade 96%, especificidade 31%, VPP 87%, VPN 63%); variante folicular de CPT de CF (p=0,0232 sensibilidade 93%, especificidade 31%, VPP 66%, VPN 77%); assim como variante folicular de CPT de AF (p=0,0003, sensibilidade 93%, especificidade 50%, VPP 68%, VPN 87%). Além disso, a expressão de c-Met se correlacionou com tireoidite (p<0,0001) e multifocalidade (p=0,0028), mas não com presença de cápsula, invasão, tamanho do tumor, estadiamento TNM, e presença de metástase no diagnóstico e na evolução. A expressão nuclear de STAT3 diferenciou os nódulos benignos dos malignos (p<0,0001, sensibilidade 83%, especificidade 74%, VPP 75%, VPN 83%); CF de AF (p=0,0457, sensibilidade 80%, especificidade 52%, VPP 65%, VPN 71%); bócios de CPT variante folicular (p<0,001, sensibilidade 89%, especificidade 65%, VPP 91%, VPN 60%); bócio de CF (p<0,0001, sensibilidade 89%, especificidade 80%, VPP 95%, VPN 60%); bócio de AF (p=0,0005, sensibilidade 89%, especificidade 80%, VPP 95%, VPN 60%). Além disso, a expressão de STAT3 se correlacionou com tireoidite (p=0,0095) e multifocalidade (p<0,0001), mas não com presença de cápsula, invasão, tamanho do tumor, estadiamento TNM, e presença de metástase no diagnóstico e na evolução. A expressão de pSTAT3 e HGF não auxiliou no diagnóstico dos nódulos, e tampouco se correlacionou com características de agressividade dos tumores. Conclui-se que as proteínas c-Met e STAT3 podem ser consideradas marcadores clínicos úteis na rotina de laboratórios, uma vez que foram capazes de diferenciar os nódulos malignos dos benignos, alguns tipos histológicos dos nódulos, além de se correlacionarem com fatores de agressividade dos tumores / Abstract: Malignancy markers, especially the ones that are capable of distinguishing follicular lesions and with easy deployment in the routine diagnosis of thyroid nodules are much needed, given the increasing number of thyroid nodules in recent years. The HGF/c-Met/STAT3 pathway is related to the development and progression of many types of cancers, and c-Met, HGF and STAT3 expression were described in most papillary thyroid carcinomas (PTC), but not in normal thyroid tissue, suggesting it is related with the development and progression of PTC. To evaluate the usefulness of c-Met, HGF, STAT3, and its phosphorylated form (pSTAT3) protein expression in the diagnosis and prognosis of patients with thyroid nodules, we analyzed 356 thyroid tissues, including 153 papillary carcinomas (PTC), 95 classical type, 47 follicular variants of papillary carcinoma, and 11 tall cells carcinomas; 34 follicular carcinomas (FC), 34 follicular adenomas (FA), 124 goiters and 11 normal tissues. All patients were treated and monitored according to the same standard protocol for 1-10 years (Mo = 5 years). Representative tissue areas were selected for the construction of a tissue microarray (TMA) which was subjected to immunohistochemistry and analyzed by the Allred score. The cytoplasmic expression of c-Met was able to differentiate malignant from benign nodules (p <0.0001, sensitivity 86%, specificity 76%, PPV 77%, 86% NPV); PTC from FCT (p = 0.0003, sensitivity 96%, specificity 31%, PPV 87%, 63% NPV); follicular variant of PTC from FCT (p = 0.0232 sensitivity 93%, specificity 31%, PPV 66%, NPV 77%); as well as follicular variant of CPT from FA (p = 0.0003, sensitivity 93%, specificity 50%, PPV 68%, 87% NPV). Furthermore, c-Met expression was correlated to the presence of thyroiditis (p<0.0001) and multifocality (p=0.0028), but not with the presence of capsule, invasion, tumour size, TNM staging, and metastasis at diagnosis or evolution of the disease. The nuclear expression of STAT3 differentiated benign from malignant nodules (p <0.0001, sensitivity 83%, specificity 74%, PPV 75%, NPV 83%); FCT from FA (p = 0.0457, sensitivity 80%, specificity 52%, PPV 65%, NPV 71%); goiter follicular variant of PTC (p <0.001, sensitivity 89%, specificity 65%, PPV 91%, 60% NPV); goiter from FCT (p <0.0001, sensitivity 89%, specificity 80%, PPV 95%, NPV 60%); goiter from FA (p = 0.0005, sensitivity 89%, specificity 80%, PPV 95%, NPV 60%). In addition, STAT3 expression was associated with thyroiditis (p=0.0095) and multifocality (p<0.0001), but not with the presence of capsule, invasion, tumour size, TNM staging, and metastasis at diagnosis or evolution of the disease. The expression of pSTAT3 and HGF did not help the diagnostic of nodules and was not correlated with any tumour characteristic of aggressiveness. We concluded that c-Met and STAT3 could be useful as molecular markers in laboratory routine, helping to differentiate malignant from benign nodules, and some histological types of nodules, and was also associated with some tumour characteristics of aggressiveness / Mestrado / Ensino em Saúde / Mestra em Clínica Médica

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