La matrice extracellulaire est un réseau tridimensionnel complexe qui joue le rôle de support aux cellules ainsi que de réservoir de molécules bioactives régulant le comportement cellulaire. Elle est composée de 1027 protéines chez l’Homme (Naba et al., Matrix Biol. 2016), 274 protéines constituant le matrisome et 753 associées (facteurs de croissance et protéines régulatrices de la matrice extracellulaire) et de 6 glycosaminoglycanes dont 5 sulfatés. La matrice extracellulaire est impliquée dans de nombreuses pathologies (Bonnans et al., Nat. Rev. Mol. Cell Biol. 2014). La lysyl oxydase, responsable de la réticulation des collagènes et de l’élastine est impliquée dans de nombreux cancers. La matrice extracellulaire est un réservoir de fragments bioactifs, nommés matricryptines, qui sont libérés par protéolyse des biomolécules de la matrice et régulent de nombreux processus biologiques tels que l’angiogenèse et l’adipogenèse (Ricard-Blum et Vallet Matrix Biol. 2017). Nous avons exprimé en cellules humaines plusieurs matricryptines dont les ectodomaines des collagènes membranaires XIII, XVII, XXIII et XXV et identifié leurs partenaires extracellulaires. Nous avons caractérisé le propeptide de la lysyl oxydase par SEC-MALS, diffusion dynamique de la lumière et par SAXS et avons modélisé à partir des données de SAXS sa structure tridimensionnelle. Nous avons identifié 17 nouveaux partenaires de ce fragment et analysé le mutant Arg158Gln dépourvu d’activité biologique. Cette mutation identifiée chez l’Homme inhibe les activités anti-prolifératives du propeptide et est associée à un risque accru de cancer du sein (Min et al., Cancer Res. 2009). Nous avons exprimé la lysyl oxydase mature et modélisé sa structure tridimensionnelle en utilisant toutes les données disponibles. Les interactions identifiées au cours de ce travail ont été associées à celles obtenues par curation manuelle de la littérature pour construire la première version de l’interactome extracellulaire humain / The extracellular matrix is an intricate tridimensional network supporting cells and a bioactive molecule reservoir involved in the regulation of cell behavior. It is composed of 1027 proteins in humans (Naba et al., Matrix Biol. 2016), including 274 of the core matrisome and 753 associated proteins (growth factors and extracellular matrix regulators) and 6 glycosaminoglycans including 5 sulfated. The extracellular matrix is altered in numerous pathologies (Bonnans et al., Nat. Rev. Mol. Cell Biol. 2014). The lysyl oxidase is responsible for the cross-linking of collagens and elastin and is involved in many cancers. The extracellular matrix is a reservoir of bioactive fragments named matricryptins which are released by proteolysis of extracellular matrix proteins and regulate numerous biological processes like angiogenesis and adipogenesis (Ricard-Blum et Vallet, Matrix Biol. 2017). We have expressed under a recombinant form in human cells some matricryptins including the ectodomains of the membrane collagens XIII, XVII, XXIII and XXV and have identified their extracellular partners. We have characterized the propeptide of lysyl oxidase by SEC-MALS, dynamic light scattering, and SAXS and have built a coarse-grained 3D model by SAXS-derived constraints. We have identified 17 new partners of this fragment and analyzed the mutant Arg158Gln which has no biological activity. This mutation has been identified in humans and inhibits the propeptide anti-proliferative properties. It is associated to an increased risk of breast cancer (Min et al., Cancer Res. 2009). We have expressed the mature lysyl oxidase and modelled its tridimensional structure using available data. All the interactions identified in this study were associated to manually curated interactions described in the literature to build the first version of the human extracellular interactions network

No description available.

No description available.

Made available in DSpace on 2015-07-22T20:50:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-05-25. Added 1 bitstream(s) on 2015-08-11T03:26:12Z : No. of bitstreams: 1 Publico-12837a.pdf: 1258726 bytes, checksum: a7e0f280ec3b88f2f775c2b325704cd7 (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:12Z : No. of bitstreams: 2 Publico-12837a.pdf: 1258726 bytes, checksum: a7e0f280ec3b88f2f775c2b325704cd7 (MD5) Publico-12837b.pdf: 2043061 bytes, checksum: 4edc37b57c73112ae7353868a78233ca (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:12Z : No. of bitstreams: 3 Publico-12837a.pdf: 1258726 bytes, checksum: a7e0f280ec3b88f2f775c2b325704cd7 (MD5) Publico-12837b.pdf: 2043061 bytes, checksum: 4edc37b57c73112ae7353868a78233ca (MD5) Publico-12837c.pdf: 1773766 bytes, checksum: 56addbd5601cb4e7336afb640ed56fe8 (MD5) / Objetivo: O prolapso de órgãos pélvicos (POP) resulta da falha na sustentação do assoalho pélvico, e anormalidades do tecido conjuntivo podem estar envolvidas na etiologia e/ou na progressão da disfunção. Analisar-se-á a expressão diferencial de genes e de proteínas que participam da biossíntese do colágeno e da elastina: lisil oxidases (LOXs), fibulina-5, fibrilinas-1 e -2 e pró-colágeno C proteinase (PCP/BMP1), no tecido vaginal de mulheres sem e com POP acentuado consoante seu estado hormonal. Casuística e Métodos: Durante a histerectomia total, biópsias de parede vaginal anterior foram obtidas de mulheres caucasianas na pré-menopausa (fase proliferativa do ciclo menstrual) e na pós-menopausa com POP acentuado (POPQ estadio III e IV), e de controles assintomáticas (POPQ 0). RNAm e proteínas totais foram extraídos usando Trizol e RIPA Buffer, e os genes e proteínas de interesse quantificados por RT-PCR em tempo real e Imunobloting, respectivamente. As seguintes análises comparativas foram realizadas: (1) expressão dos genes e das proteínas da família LOX (LOX e LOXL1-4), fibulina-5 e fibrilinas- 1 e -2 em pacientes na pré-menopausa com e sem POP; (2) expressão do gene e da proteína PCP/BMP1 em pacientes na pré- e pós-menopausa com POP, e respectivos controles. Os testes de Wilcoxon signed-rank e Fisher foram usados para as análises estatísticas (p<0.05). Resultados: Obtivemos amostras de 15 pacientes e 11 controles na pré-menopausa para o estudo (1), e 39 pacientes na pré-menopausa (POP=23 e Controle=16) e 18 na pósmenopausa (POP=13 e Controle=5) para o estudo (2). A partir das análises, observamos (1) diminuição significativa na expressão dos genes LOX, LOXL1 e LOXL3, bem como nas proteínas LOX e LOXL3 no tecido vaginal de pacientes POP na pré-menopausa comparadas com mulheres assintomáticas (p<0.05); (2) hipoexpressão do gene PCP/BMP1 nos tecidos vaginais de mulheres com POP acentuado comparadas com controles, tanto na prémenopausa como na pós-menopausa (ambos p=0.01); redução significativa das isoformas 130kDa, 92,5kDa e 82,5kDa da PCP/BMP1 no tecido vaginal de pacientes na pósmenopausa (p=0.01), bem como hiperexpressão da isoforma 130kDa nas mulheres com POP acentuado na pré-menopausa (p=0.009), comparadas com as respectivas controles. Conclusão: As expressões das enzimas LOXs e pró-colágeno C proteinase estão alteradas no tecido vaginal de mulheres com POP, e são moduladas pelo estado hormonal. A alteração na regulação destas enzimas, envolvidas na biossíntese da matriz extracelular, pode contribuir para deficiente síntese do tecido conjuntivo e do suporte vaginal, e estar envolvida no desenvolvimento do POP. / Objective: Pelvic organ prolapse (POP) results from the failure of pelvic floor support, and connective tissue abnormalities may be involved in the etiology and/or progression of the dysfunction. We aimed to analyze the differential expression of genes and proteins related to the collagen and elastin biogenesis: lysyl oxidases (LOXs), fibulin-5, fibrillin -1 and -2, and procollagen C proteinase (PCP/BMP1) in vaginal tissue of women without and with advanced POP controlled by hormonal status. Materials and Methods: During total hysterectomy, anterior vaginal wall biopsies were obtained from Caucasian premenopausal women (proliferative phase of menstrual cycle) and postmenopausal women with severe POP (POPQ stage III and IV) and asymptomatic controls (POPQ 0). Total mRNA and protein were extracted using Trizol and RIPA buffer, and the genes and proteins of interest were quantified by real-time RT-PCR and Immunoblotting, respectively. The following analysis were performed: (1) expression of LOX family genes and proteins (LOX and LOXL1-4), fibulin-5, fibrillin-1 and -2 in premenopausal women with and without POP; (2) PCP/BMP1 gene and protein expression in vaginal tissue of pre- and postmenopausal POP women, and respective controls. Wilcoxon signed-rank and Fisher tests were used for statistical analysis (p<0.05). Results: Samples from 15 premenopausal patients and 11 controls were obtained for study (1); 39 premenopausal (POP=23 and Control=16) and 18 postmenopausal women samples (POP=13 and Control=5) for study (2). We observed: (1) significant decrease in expression of LOX, LOXL1 and LOXL3 genes, as well as LOX and LOXL3 proteins in vaginal tissue of premenopausal POP patients compared with asymptomatic women (p<0.05); (2) PCP/BMP1 gene downregulation in the vagina of women with severe POP compared with controls, in both premenopausal and postmenopausal phase (both p=0.01); significant reduction of 130 kDa, 92.5 kDa and 82.5 kDa PCP/BMP1 isoforms in vaginal tissue of postmenopausal patients (p=0.01), and 130 kDa isoform upregulation in premenopausal women with severe POP (p=0.009), compared with their respective controls. Conclusion: The expression of LOXs enzymes and PCP/BMP1 are altered in vaginal tissue of women with severe POP, and are modulated by hormonal status. Dysregulation of these enzymes involved in the extracellular matrix biogenesis may contribute to impaired tissue and vaginal support, and may be involved in POP development. / TEDE / BV UNIFESP: Teses e dissertações