Les peptides et les protéines jouent un rôle central dans les processus biologiques. Les méthodes de production de ces molécules se sont fortement développées dans le but de déterminer leur structure, comprendre leurs fonctions et développer de nouvelles thérapies. En particulier, la synthèse chimique des protéines s’est fortement développée dans les années 1960 avec l’introduction de la chimie peptidique en phase solide (SPPS) par B. Merrifield. Depuis les années 1990, la combinaison de méthodes de ligation chimique natives et de stratégies d’assemblage séquentielles et convergentes ont permis la synthèse de nombreuses protéines. Cependant, la synthèse de protéines de haut poids moléculaires reste un défi synthétique. Il est donc important de développer de nouveaux systèmes de ligation chimique et des stratégies d’assemblage plus performantes. Une nouvelle méthode de ligation native, la ligation SEA, repose sur la capacité des segments bis(2-sulfanylethyl)amido (SEA) à réagir en milieu aqueux avec des cystéinyl peptides. Différents outils chimiques basés sur l’utilisation du groupement SEA ont été développés dans cette thèse. La première partie de ce manuscrit présente une méthode d’assemblage séquentiel de segments peptidiques en solution s’effectuant du N-terminal vers le C-terminal. Cette méthode a permis la synthèse du domaine N-terminal du facteur de croissance des hépatocytes (HGF). Afin de surmonter les limitations de l’assemblage en solution, la seconde partie de cette thèse porte sur le développement d’un procédé de synthèse de protéines par ligation native séquentielle en phase solide du N-terminal vers le C-terminal. Enfin, une dernière partie exploite la réactivité des segments bis(2-selanylethyl)amido (SeEA) et leur potentiel pour la synthèse de nouveaux échafaudages peptidiques. / Peptides and proteins play a crucial role in all fundamental biological processes. Chemical methods have been developed for the production of peptide and proteins which allows understanding their structures, functions and the development of novel therapies. In particular, the introduction of the Solid Phase Peptide Synthesis (SPPS) by Merrifield in the 60s, followed by the emergence of peptide ligation methods in the 90s have opened the way to the preparation of synthetic proteins. Recently, the developments of sequential and convergent assembly methods give access to large synthetic proteins. However, the synthesis of high molecular weight proteins remains a challenging task. Therefore, it is necessary to develop novel peptide ligation methods and assembly scheme strategies. Central to this PhD work is the recently developed bis(2-sulfanylethyl)amido (SEA) native peptide ligation method. The first part of this manuscript describes an efficient sequential assembly method in the N-to-C direction for protein synthesis in solution which was used for producing a functional N domain of Hepatocyte Growth Factor‎ (HGF). We next examined also a solid phase method for the sequential native ligation of unprotected peptide segments in the N-to-C direction to overcome the limitations in solution. The last part of the manuscript reports the chemicals properties of bis(2-selanylethyl)amido (SeEA) peptide segments and their usefulness for building novel peptide scaffolds.

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.

Hepatocyte growth factor (HGF) signaling drives epithelial cells to scatter by breaking cell-cell adhesions and migrating as solitary cells, a process that parallels epithelial-mesenchymal transition. HGF binds and activates the c-Met receptor tyrosine kinase, but downstream signaling required for scattering remains poorly defined. This study addresses this shortcoming in a number of ways.A high-throughput in vitro drug screen was employed to identify proteins necessary in this HGF-induced signaling. Cells were tested for reactivity to HGF stimulation in a Boyden chamber assay. This tactic yielded several small molecules that block HGF-induced scattering, including a calcium channel blocker. Patch clamping was used to determine the precise effect of HGF stimulation on Ca2+ signaling in MDCK II cells. Cell-attached patch clamping was employed to detect Ca2+ signaling patterns, and channel blockers were used in various combinations to deduce the identity of Ca2+ channels involved in EMT. The results of these experiments show that HGF stimulation results in sudden and transient increases in calcium channel influxes. These increases occur at predictable intervals and rely on proper tubulin polymerization to appear, as determined through the use of a tubulin polymerization inhibitor. Though multiple channels occur in the membranes of MDCK II cells, noticeably TRPV4 and TrpC6, it is TrpC6 that is specifically required for HGF-induced scattering. These HGF-induced calcium influxes through TrpC6 channels drive a transient increase in NFAT-dependent gene transcription which is required for HGF-induced EMT. This was determined through the use of luciferase-based NFAT reporter assays and confirmed through confocal immunofluorescence. Using a small-molecule inhibitor of WNK kinase, it was determined that loss of WNK kinase function is sufficient to prevent HGF-induced EMT. Furthermore, patch-clamp analysis demonstrated that WNK kinase significantly increases channel opening at the surface of MDCK cells, indicating a possible mechanism of action for c-Met inhibition, but leaving doubt as to whether WNK kinase is in fact normally involved in c-Met signaling, or whether it is simply permissive.

Androgens are the main regulators of human hair growth stimulating larger, terminal hair development e.g. beard and causing scalp balding, androgenetic alopecia. Hair disorders cause psychological distress but are poorly controlled. Androgens probably act by altering regulatory paracrine factors produced by the mesenchyme-derived dermal papilla. This study aimed to investigate paracrine factors involved in androgen-regulated alopecia, particularly hepatocyte growth factor (HGF) family members, by investigating their in vivo status. Balding and non-balding scalp hair follicles and their component tissues were isolated and analysed by molecular biological methods (reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative PCR and DNA microarray analysis), cell culture and immunohistochemistry. Scalp follicles expressed a range of paracrine messenger genes. The dermal papilla, cultured dermal papilla cells and dermal sheath expressed several HGF family genes, while matrix cells only produced the receptor RON suggesting autocrine roles for HGF and MSP, but a paracrine route only for MSP. Comparing balding and non-balding follicles from the same individuals revealed the expected reduction in several keratin and keratin-related protein genes supporting this approach's validity. There were also significant differences in paracrine factors previously implicated in androgen action by in vitro studies. Several factors believed to increase during androgen stimulation of larger, darker follicles, e.g. IGF-I and SCF, were lowered in balding follicles, while putative inhibitory factors, e.g. TGFß-1, were increased. HGF and MSP and their receptors, c-Met and RON, were significantly reduced. These results increase our understanding of androgen action in human hair follicles; this could lead to better treatments for hair disorders. / Saudi government

Os insulinomas são os tumores neuroendócrinos pancreáticos funcionantes mais frequentes, entretanto, os aspectos moleculares envolvidos em sua tumorigênese precisam ser melhor esclarecidos. As características morfológicas e histoquímicas dos insulinomas não conseguem predizer completamente seu comportamento biológico, apenas o fenótipo invasivo local e a presença de metástase são as formas confiáveis do diagnóstico maligno. A presente investigação teve por objetivos analisar a expressão gênica por reação em cadeia da polimerase (PCR) em tempo real e a expressão protéica por imuno-histoquímica dos componentes da via de sinalização do fator de crescimento hepatocítico (HGF) e seu receptor (c-MET) em 27 amostras de insulinomas, sendo: 16 tumores benignos grau 1 (G1), seis tumores benignos grau 2 (G2), dois insulinomas malignos grau 3 (G3) e três metástases hepáticas. Além disso, realizou-se a pesquisa de mutações somáticas no gene MET. Observou-se (1) o aumento da expressão dos genes HGF, MET e ST14 (codificante para a matriptase) e a baixa expressão do gene HAI-1 (codificante para a protease inibidora tipo-kunitz do tipo 1) nos insulinomas malignos e metástases quando comparados aos insulinomas benignos G1; (2) uma correlação positiva entre a expressão do mRNA do gene MET e o gene ST14 e índice proliferativo Ki-67, bem como uma correlação inversa entre a expressão do mRNA do gene HAI-1 e os genes MET, HGF, ST14 e o índice mitótico; (3) uma correlação positiva entre a expressão do gene ST14 e a expressão do mRNA do gene HGF; (4) maior expressão protéica de c-MET nos insulinomas malignos G3 em relação aos insulinomas G1/G2 e (5) ausência de mutações nos éxons 2, 10, 14, 16, 17 e 19 do gene MET. Concluiu-se que os genes HGF, MET, ST14 e HAI-1 estão diferencialmente expressos entre insulinomas malignos e benignos, o que pode ter implicações diagnósticas e terapêuticas / In an attempt to better understand the molecular processes involved in the tumourigenesis of islet beta-cells, the present study evaluated the expression of genes belonging to the hepatocyte growth factor and its receptor (HGF/MET) system, namely, MET, HGF; HGFAC and ST14 (encode HGF activator and matriptase, respectively, two serine proteases that catalyze conversion of pro-HGF to active HGF); and SPINT1 and SPINT2 (encode serine peptidase inhibitors Kunitz type 1 and type 2, respectively, two potent inhibitors of HGF activator and of matriptase) in 27 sporadic insulinomas; 16 grade 1 (G1), six grade 2 (G2), two grade 3 (G3) and three hepatic metastases. Quantitative reverse-transcriptase polymerase chain reaction was employed to assess RNA expression of the target genes and immunohistochemical analysis was used to evaluate the expression of MET and SPINT1. Somatic mutations of MET gene were searched by direct sequencing of exons 2, 10, 14, 16, 17 and 19. Overexpression of MET was observed in grouped G3 insulinomas and metastases concomitantly with upregulation of the genes encoding HGF and matriptase and downregulation of SPINT1. Positive correlations were observed between MET RNA expression and Ki-67 proliferation index while a negative correlation was detected between SPINT1 expression and the mitotic index. No somatic mutations were found in MET gene. The final effect of the increased expression of HGF, its activator (matriptase) and its specific receptor (MET) together with a decreased expression of one potent inhibitor of matriptase (SPINT1) is probably a contribution to tumoural progression and malignancy in insulinomas

Os insulinomas são os tumores neuroendócrinos pancreáticos funcionantes mais frequentes, entretanto, os aspectos moleculares envolvidos em sua tumorigênese precisam ser melhor esclarecidos. As características morfológicas e histoquímicas dos insulinomas não conseguem predizer completamente seu comportamento biológico, apenas o fenótipo invasivo local e a presença de metástase são as formas confiáveis do diagnóstico maligno. A presente investigação teve por objetivos analisar a expressão gênica por reação em cadeia da polimerase (PCR) em tempo real e a expressão protéica por imuno-histoquímica dos componentes da via de sinalização do fator de crescimento hepatocítico (HGF) e seu receptor (c-MET) em 27 amostras de insulinomas, sendo: 16 tumores benignos grau 1 (G1), seis tumores benignos grau 2 (G2), dois insulinomas malignos grau 3 (G3) e três metástases hepáticas. Além disso, realizou-se a pesquisa de mutações somáticas no gene MET. Observou-se (1) o aumento da expressão dos genes HGF, MET e ST14 (codificante para a matriptase) e a baixa expressão do gene HAI-1 (codificante para a protease inibidora tipo-kunitz do tipo 1) nos insulinomas malignos e metástases quando comparados aos insulinomas benignos G1; (2) uma correlação positiva entre a expressão do mRNA do gene MET e o gene ST14 e índice proliferativo Ki-67, bem como uma correlação inversa entre a expressão do mRNA do gene HAI-1 e os genes MET, HGF, ST14 e o índice mitótico; (3) uma correlação positiva entre a expressão do gene ST14 e a expressão do mRNA do gene HGF; (4) maior expressão protéica de c-MET nos insulinomas malignos G3 em relação aos insulinomas G1/G2 e (5) ausência de mutações nos éxons 2, 10, 14, 16, 17 e 19 do gene MET. Concluiu-se que os genes HGF, MET, ST14 e HAI-1 estão diferencialmente expressos entre insulinomas malignos e benignos, o que pode ter implicações diagnósticas e terapêuticas / In an attempt to better understand the molecular processes involved in the tumourigenesis of islet beta-cells, the present study evaluated the expression of genes belonging to the hepatocyte growth factor and its receptor (HGF/MET) system, namely, MET, HGF; HGFAC and ST14 (encode HGF activator and matriptase, respectively, two serine proteases that catalyze conversion of pro-HGF to active HGF); and SPINT1 and SPINT2 (encode serine peptidase inhibitors Kunitz type 1 and type 2, respectively, two potent inhibitors of HGF activator and of matriptase) in 27 sporadic insulinomas; 16 grade 1 (G1), six grade 2 (G2), two grade 3 (G3) and three hepatic metastases. Quantitative reverse-transcriptase polymerase chain reaction was employed to assess RNA expression of the target genes and immunohistochemical analysis was used to evaluate the expression of MET and SPINT1. Somatic mutations of MET gene were searched by direct sequencing of exons 2, 10, 14, 16, 17 and 19. Overexpression of MET was observed in grouped G3 insulinomas and metastases concomitantly with upregulation of the genes encoding HGF and matriptase and downregulation of SPINT1. Positive correlations were observed between MET RNA expression and Ki-67 proliferation index while a negative correlation was detected between SPINT1 expression and the mitotic index. No somatic mutations were found in MET gene. The final effect of the increased expression of HGF, its activator (matriptase) and its specific receptor (MET) together with a decreased expression of one potent inhibitor of matriptase (SPINT1) is probably a contribution to tumoural progression and malignancy in insulinomas

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.

1 Abstract Liver fibrosis is a condition described by extensive accumulation of scar tissue in the liver. With further progression, it leads to cirrhosis or even to hepatocellular carcinoma. Liver fibrosis accompanies every chronic liver disease and its prevalence in adult European population is estimated to be around 4%. During my dissertation work, I studied the function of three members of Metzincin family of metalloproteinases - ADAM17, ADAM10 and MMP-19, in liver fibrosis and liver regeneration using mouse genetic models. ADAM17 and ADAM10 are important regulators of signalling pathways which are involved in immune response as well as differentiation. Both proteases are able to cleave ectodomains of their substrates from cell membrane, affecting bioavailability of ligands and functionality of receptors. Several of their substrates are involved in liver pathologies. MMP-19 on the other hand, is a metalloprotease mainly involved in extracellular matrix cleavage, important process in fibrosis development, as well as resolution of fibrosis. Our results demonstrate that ablation of ADAM10 results in increased susceptibility to liver fibrosis in mice, both spontaneous and toxin induced. ADAM10 deficiency affected biliary epithelium, as we detected higher markers of biliary damage in serum of ADAM10 deficient...

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