The extracellular matrix (ECM) offers a protective shelter for cells and provides signaling paths important for cell to cell communication. ECM consists of basement membranes (BM) and interstitial matrix. BMs provide mechanical support for parenchymal cells, influence cell proliferation, survival, migration and differentiation. They are also important for tissue integrity. Laminins (LM) are the major non-collagenous component of BMs. Cell-ECM interactions, mediated by receptors, are indispensable during embryonic development, wound healing, remodeling and homeostasis of tissues. The integrins are the major cell-adhesion receptors. The expression of alpha11 integrin chain in the cornea is of great interest, as it is part of the alpha11beta1 integrin receptor for collagen type I, the predominant component of the corneal stroma. The aims were to thoroughly characterize the ECM in the developing and adult human eye, with particular focus on the cornea, LM and alpha11 integrin chains, and to examine alpha11 integrin chain in an animal model of corneal wound healing and remodeling. Human fetal eyes, 9-20 weeks of gestation (wg), and adult human corneas with different diagnosis were treated for immunohistochemistry with specific antibodies against LM and alpha11 integrin chains. Normal and knockout (ko) mice were treated with laser surgery to create a deep wound in the corneal stroma. The wound healing process was followed at different time points. The cellular source of alpha11 integrin chain was studied in cell cultures. In the fetal eyes, the BM of the corneal epithelium, the Descemet’s membrane (DM) and the Bruch’s membrane each had their specific combinations of LM chains and time line of development, whereas the lens capsule and the internal limiting membrane showed constant LM chain patterns. The epithelial BMs of normal and diseased adult corneas contained similar LM chains. The normal morphology of the epithelial BM was altered in the different diseases, particularly when scarring was present. In the scarred keratoconus corneas there were excessive LM chains. The majority of keratoconus corneas also expressed extra LM chains in the DM. At 10-17 wg alpha11 integrin chain was present in the human corneal stroma, especially in the anterior portion, but it was scarce at 20 wg, in normal adult corneas and in Fuchs’ endothelial dystrophy. In contrast, it was increased in the anterior portion of the stroma in keratoconus corneas with scarring. Alpha11 integrin ko mice had a defective healing with subsequent thinner corneas. Alpha11 integrin expression correlated to the presence of alpha-smooth muscle actin in vivo as well as in vitro. The distinct spatial and temporal patterns of distribution for alpha11 integrin and each of the LM chains suggest that they play an important role in human ocular differentiation. The selectively affected LM composition and the novel expression of alpha11 integrin chain in scarred keratoconus corneas as well as the pathologic healing in ko mice, indicate that alpha11 integrin and LM chains also play an important role in the process of corneal healing, remodeling and scarring and might participate in the pathogenesis of corneal disease. This knowledge is of practical importance for future topical therapeutic agents capable of modulating the corneal wound healing processes.

Les patients atteints de MICI (maladie de Crohn et recto-colite hémorragique) présentent souvent des lésions des cellules de l'épithélium intestinal. La rémission de ces maladies nécessite à la fois un arrêt de l'inflammation et une migration des entérocytes pour réparer les dommages épithéliaux. Cette migration cellulaire appelée restitution intestinale requiert des adhérences cellule-MEC et cellule-cellule réalisées par les complexes protéiques associés aux intégrines et cadhérines. Le but de cette thèse a été d'étudier l'impact deSaccharomyces boulardii (Sb) sur la réparation de l'épithélium intestinal lésé. Nous avons montré que le surnageant de Sb contenait des facteurs modulant la restitution de l'épithélium intestinal in vivo et in vitro sans affecter la prolifération des cellules épithéliales. Ces effets motogéniques du surnageant de Sb s'exercent via la modulation des molécules d'adhérence. En effet, le surnageant de Sb augmente l'affinité de l'intégrine α2β1 pour son ligand le collagène de type I mais entre en compétition avec les intégrine αvβ5, pour inhiber l'adhérence des entérocytes sur la vitronectine. Ces modifications de l'adhérence avec la matrice extracellulaire entraînent une régulation des voies de signalisation émanant des intégrines et une réorganisation des plaquesd'adhérence. Ces évènements vont accroître la migration des entérocytes. De plus, nos résultats préliminaires portant sur Sb et l'adhérence cellule-cellule durant la restitution intestinale ont montré une implication de la E-cadhérine dans la migration induite par Sb. / Intestinal epithelial cell damage is frequently seen in IBD patients with ulcerative colitis or Crohn's disease. The remission of these diseases requires both the cessation of inflammation and the migration of enterocytes torepair the damaged epithelium. Adhesions with the ECM and the adjacent cells using complex of proteins associated with integrins and cadherins are necessary for this cell migration called intestinal restitution. Theaim of this thesis was to study the effect of S.boulardii on the resealing of a wound in intestinal epithelia. First of all, we demonstrated that the supernatant of S.boulardii contains factors that modulate intestinal epithelial cell restitution both in vitro and in vivo without affecting cell proliferation. We showed that the motogenic factors of S.boulardii act by modulating adhesion molecules. Indeed, the supernatant of S.boulardii increase the the affinity between 21 and it ligand the collagen type I, but also compete with integrin v5 to block theadhesion of enterocytes on vitronectin. These modifications of adhesion on extracellular matrix lead to aregulation of signaling pathway mediated by integrins, and a reorganization of focal adhesions. These eventscontribute to an increase of the migration of enterocytes. Add to this, our preliminaries results on S.boulardiiand cell-cell adhesion during intestinal restitution show an involvement of E-cadherin in the migrationS.boulardii-induced. With this work, we have demonstrated that heat-sensitive motogenic factors secreted by S.boulardii can enhance intestinal restitution with a dynamic regulation of adhesion between integrin and the ECM.

As integrinas constituem uma família de receptores de membrana que tem como função primária a adesão de células a proteínas da matriz extracelular e alguns de seus membros estão envolvidos nos processos de diferenciação osteoblástica e formação óssea, eventos diretamente relacionados à osseointegração de implantes de titânio (Ti). Sabe-se que superfícies de Ti com nano e microtopografia podem favorecer a diferenciação osteoblástica e a mineralização da matriz extracelular. No entanto, os mecanismos celulares envolvidos nesses processos não são completamente entendidos. Neste contexto, os objetivos deste estudo foram: (1) caracterizar as superfícies de Ti com nano (Ti-Nano) e microtopografia (Ti-Micro), (2) investigar a participação da integrina V na diferenciação osteoblástica induzida pelo Ti-Nano e (3) investigar a participação da integrina β3 na diferenciação osteoblástica induzida por Ti-Nano e Ti-Micro. Para isso, discos de Ti-Nano e Ti-Micro foram preparados por ataque ácido com H2SO4/H2O2 ou com HNO3/H2SO4 / HCl, respectivamente, e caracterizados quanto à topografia, rugosidade e composição química de superfície. Discos de Ti usinados foram usados com controle (Ti-Controle) em alguns experimentos. Células-tronco mesenquimais derivadas de medula óssea de ratos foram cultivadas sobre as três superfícies de Ti e foi avaliada a expressão gênica de componentes envolvidos na via de sinalização das integrinas por PCR array. Com base nos resultados do PCR array, as integrinas αV e β3 foram selecionadas e silenciadas por RNA de interferência (shRNA) ou CRISPR/Cas9, respectivamente, em células pré-osteoblásticas da linhagem MC3T3-E1 para investigarmos a participação dessas integrinas na diferenciação osteoblástica induzida por superfícies de Ti com diferentes topografias. Os resultados deste estudo mostraram que os tratamentos empregados foram eficientes para a produção de superfícies de Ti com topografias nas escalas nano e micrométrica. Além disso, foi demonstrado que o maior potencial osteogênico do Ti-Nano se deve, ao menos em parte, à integrina αV, uma vez que seu silenciamento reduziu a diferenciação osteoblástica induzida pela nanotopografia. Por fim, também demonstramos que a via de sinalização ativada pela integrina β3 exerce um papel fundamental no potencial osteogênico do Ti-Nano, mas não do Ti-Micro. O silenciamento da integrina β3 reduziu a diferenciação osteoblástica, concomitantemente com a regulação negativa da expressão de vários componentes das vias de sinalização de Wnt e de BMP, apenas nas células crescidas sobre a nanotopografia. Em conjunto, nossos resultados revelam um novo mecanismo para explicar a maior diferenciação osteoblástica induzida pelo Ti-Nano, que envolve uma complexa rede regulatória ativada pela maior expressão das integrinas αV e β3, esta última gerando ativação da transdução de sinal das vias de Wnt e de BMP / Integrins are a family of membrane receptors that primarily mediate cell adhesion to extracellular matrix proteins and some members are involved in the process of osteoblast differentiation and bone formation, key events of titanium (Ti) implant osseointegration. It is well known that Ti surfaces with nano and microtopography may favor osteoblast differentiation and matrix mineralization. However, the cellular mechanisms involved in this process are not entirely understood. In this context, the aims of this study were: (1) to characterize the Ti surfaces with nano (Ti-Nano) and microtopography (Ti-Micro), (2) to investigate the participation of integrin V on osteoblast differentiation induced by Ti-Nano and (3) to investigate the participation of integrin β3 on osteoblast differentiation induced by Ti-Nano and Ti-Micro. Discs of Ti-Nano and Ti-Micro were prepared with acid etching with H2SO4/H2O2 or with HNO3/H2SO4 / HCl, respectively, and characterized in terms of surface topography, roughness and chemical composition. Machined Ti discs (untreated) were used as control (Ti-Control) in some experiments. Mesenchymal stem cells from rat bone marrow were cultured on Ti discs with the three different surfaces and the gene expression of members of the integrin signaling pathway was evaluated by PCR array. Based on PCR array results, the integrins αV and β3 were selected and silenced using RNA interference (shRNA) or CRISPR-Cas9, respectively, in pre-osteoblastic cell line MC3T3-E1 to investigate the participation of these integrins in osteoblast differentiation induced by Ti with different surface topographies. The results showed that the treatments used were efficient to generate Ti surfaces with topographies at the nano and micrometric scales. We showed that the higher osteogenic potential of Ti-Nano may be, at least in part, due to the integrin &alphaV, since its silencing reduced the osteoblast differentiation induced by nanotopography. We also demonstrated that the signaling pathway triggered by integrin β3 plays a key role in the osteogenic potential of Ti-Nano, but not of Ti-Micro. The silencing of integrin β3 reduced the osteoblast differentiation concomitantly with the negative regulation of the gene expression of several Wnt and BMP signaling components only in cells grown on Ti-Nano. Taken together, our results uncover a novel mechanism to explain the higher osteoblast differentiation induced by Ti-Nano that involves a complex regulatory network triggered by integrins αV and β3 upregulation, with the integrin β3 activating the Wnt and BMP signal transductions

O objetivo desse estudo foi investigar a participação de integrina &alpha;1&beta;1 e microRNAs (miRs) no potencial osteogênico de superfícies de titânio (Ti) com nanotopografia. Discos de Ti previamente polidos foram tratados quimicamente com H2SO4/H2O2 para obtenção de nanotopografia, que foi observada por microscopia eletrônica de varredura. Para o estudo da participação da integrina &alpha;1&beta;1, células-tronco mesenquimais (CTMs) de ratos foram cultivadas em condições osteogênicas e não osteogênicas sobre superfícies de Ti com nanotopografia e sem tratamento químico (controle). O resultados mostraram que a nanotopografia de Ti aumentou a proliferação celular, a atividade de fosfatase alcalina (Alp) e regulou positivamente a expressão gênica de marcadores da diferenciação osteoblástica em CTMs cultivadas tanto em condições osteogênicas quanto em condições não osteogênicas. Além disso, uma maior expressão gênica para as integrinas &alpha;1 e &beta;1 foi observada em culturas crescidas sobre nanotopografia em condições não osteogênicas em relação ao Ti controle. O uso de obtustatina, um inibidor de integrina &alpha;1&beta;1, reduziu os efeitos da nanotopografia sobre os marcadores osteoblásticos, indicando a participação da via de sinalização dessa integrina nos efeitos da nanotopografia sobre CTMs. Para investigar a participação de miRs no efeito osseoindutor da nanotopografia de Ti, foram utilizadas CTMs humanas e células préosteoblásticas de camundongos da linhagem MC3T3-E1. A análise em larga escala da expressão de miRs revelou que 60 miRs foram regulados positivamente (no mínimo, 2x maior), enquanto 58 miRs foram regulados negativamente (no mínimo, 2x menor) em CTMs crescidas sobre a nanotopografia. Três desses miRs, miR-4448, -4708 e -4773, cuja expressão foi significativamente reduzida pela nanotopografia de Ti (no mínimo, 5x menor), afetaram a diferenciação osteoblástica de CTMs. Esses miRs atuam diretamente sobre SMAD1 e SMAD4, proteínas transdutoras da sinalização da proteína óssea morfogenética 2 (Bmp-2), conhecida por sua capacidade osseoindutora. Além disso, verificou-se que a sobreexpressão de miR-4448, -4708 e -4773 em células pré-osteoblásticas MC3T3-E1 inibiu a expressão gênica e proteica de SMAD1 e SMAD4 e, consequentemente, a expressão gênica de marcadores ósseos. Esses dados sugerem a influência do circuito miR-SMAD-Bmp-2 sobre o efeito osseoindutor da nanotopografia. Conjuntamente, os achados do presente estudo mostraram que o efeito da nanotopografia de Ti sobre a diferenciação osteoblástica resulta de um mecanismo regulatório complexo, do qual fazem parte as vias de sinalização da integrina &alpha;1&beta;1 e da Bmp-2, com a participação de miRs. Esses resultados podem representar um avanço para o desenvolvimento de novas modificações de superfície, com o objetivo de acelerar e/ou melhorar o processo de osseointegração. / The aim of this study was to investigate the role of the &alpha;1&beta;1 integrin and microRNAs (miRs) on the osteogenic potential of titanium (Ti) with nanotopography. Polished Ti discs were chemically treated with H2SO4/H2O2 to generate nanotopography, which was observed under scanning electron microscopy. For the study related to the &alpha;1&beta;1 integrin, rat mesenchymal stem cells (MSCs) were cultured under osteogenic and non-osteogenic conditions on Ti with nanotopography and non-treated Ti discs (control). Nanotopography increased cell proliferation and alkaline phosphatase (Alp) activity and upregulated the gene expression of bone markers in cells cultured under osteogenic and non-osteogenic conditions. Furthermore, the gene expression of &alpha;1 and &beta;1 integrins was higher in cells cultured on nanotopography under non-osteogenic conditions compared with control. Obtustatin, an inhibitor of &alpha;1&beta;1 integrin, reduced the higher gene expression of the bone markers induced by nanotopography. These results indicate that &alpha;1&beta;1 integrin signaling pathway determines the osteoinductive effect of nanotopography on MSCs. The role of miRs in the osteogenic potential of Ti with nanotopography was evaluated using human MSCs and MC3T3-E1 mouse pre-osteoblastic cells. The miR sequencing analysis revealed that 60 miRs were upregulated (> 2 fold), while 58 miRs were downregulated (< 2 fold) in MSCs grown on nanotopography. Three miRs, miR-4448, -4708 and -4773, which were significantly downregulated (< 5 fold) by nanotopography, affected the osteoblast differentiation of MSCs. These miRs directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (Bmp-2) osteogenic signal, which were upregulated by nanotopography. Overexpression of miR-4448 - 4708 and 4773 in MC3T3-E1 cells noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and by targeting them, these miRs repressed gene expression of key bone markers. These results suggest that a miR-SMAD-Bmp-2 circuit acts in the Ti nanotopography-mediated osteoblast differentiation. Taken together, our data showed that the osteoblast differentiation induced by Ti with nanotopography is governed by a complex regulatory network involving a crosstalk between &alpha;1&beta;1 integrin and Bmp-2 signaling pathways with participation of miRs.

Made available in DSpace on 2015-03-04T18:50:49Z (GMT). No. of bitstreams: 1 dissertacao_aline.pdf: 3429789 bytes, checksum: 82cf452f7244c55bd99e241aadcec89f (MD5) Previous issue date: 2007-05-07 / Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior / The extracelullar matrix (ECM) is formed by an assembly of proteins and glycoproteins which surrounds the cells, in various tissues. The laminin is a glycoprotein localized in the ECM that consists of three polypeptidic chains cross- shaped. It functions by anchoring epithelial cells to basal lamin, through associations with integrins, collagen, elastin and fibronectin. Integrins are adhesion receptors localized on cellular surface, that mediate interactions between cells and ECM. The interactions between proteins of ECM and cellular proteins are crucial for many normal biological processes such as cell differentiation, signal transduction, immune responses, wound healing and metastasis formation. The nature of interactions between laminin and integrin has not been fully identified yet. The present work aims to analyze, in an molecular scale, the interaction between human α6β1 integrin and laminin 111. In this work we conducted many studies, including: i) structural and sequential alignments of β-propeller and βA regions in lacking I-domain integrins; ii) model building of β-propeller e βA regions of α6β1 integrin complexed with small ECD or RGD peptide antagonists; iii) sequential alignment of various laminin LG domains; iv) model building of laminin 111 LG1 domain; and v) model building of the first described complex between the N-terminal portion of α6β1 integrin and laminin 111 LG1 domain. In order to do this, homology modeling and molecular dynamics techniques were applied, together with alignments between integrins α and β chains, and laminin LG domains. Our initial results show that the loop between blades 3 and 4 of α6 integrin subunit discriminates ligands by electrostatic interactions. Therefore we assumed that α6β1 integrin preferentially interacts with ECDF based peptides. It was demonstrated that the laminin 111 LG1 domain interacts with α6β1 integrin by the contact of this H β strand and α6 β-propeller. Further, by its electrostatic function and proximity to H β strand, LG1 residue Asp82 adheres to Mg+2 containing MIDAS in α6β1 integrin. This interaction appears to be indispensable for α6β1 and laminin 111 binding. / A matriz extracelular (ECM) é definida como um complexo de proteínas e glicoproteínas que envolve as células nos mais diversos tecidos. A laminina é uma glicoproteína que é formada por três cadeias polipeptídicas dispostas em cruz. Sua função é a de ancorar as células epiteliais à ECM, da qual faz parte, através de associações a outras proteínas como as integrinas, o colágeno, a elastina e a fibronectina. As integrinas são receptores de adesão localizadas na superfície celular, que medeiam a interação célula-ECM. Estas interações são cruciais para diversos processos biológicos, tais como a diferenciação celular, a transdução de sinais, a resposta imunológica, a cicatrização de ferimentos e a formação de metástases. Porém a forma estrutural com que a interação entre a integrina e a laminina ocorre ainda não foi esclarecida. Neste contexto este trabalho visa analisar, em escala molecular, a forma com que a integrina α6β1 e a laminina 111 humanas interagem. Assim, foram conduzidos vários estudos, entre eles: i) alinhamentos estruturais e seqüenciais das regiões β-propeller e βA de integrinas não-possuidoras de domínio I; ii) construção do modelo das regiões β-propeller e βA da integrina α6β1 em complexo com pequenos inibidores peptídicos do tipo ECD ou RGD; iii) alinhamento entre os domínios LG de lamininas; iv) construção do modelo do domínio LG1 da laminina 111; e v) construção do primeiro modelo descrito do complexo formado pela porção N- terminal da integrina α6β1 e o domínio LG1 da laminina 111. Para tanto, foram aplicadas as técnicas de modelagem por homologia e dinâmica molecular, além de alinhamentos entre as cadeias α e β de integrinas, e dos domínios LG de lamininas. Inicialmente os resultados mostraram que o loop que corresponde à região entre os subdomínios D2 e D3 da cadeia α6 discrimina ligantes por interações eletrostáticas, e a partir disso, que a integrina α6β1 possui interação preferencialmente com peptídeos do tipo ECDF. Foi mostrado que o domínio LG1 de laminina 111 interage com a integrina α6β1 pelo contato da fita β H com o β-propeller de α6. Além disso, por seu caráter eletrostático e proximidade à fita β H , o resíduo Asp82 de LG1 se adere ao íon Mg+2 do MIDAS da integrina α6β1, e que esta interação é indispensável à ligação entre as duas proteínas.

Repair of the adult mammalian spinal cord is prohibited by several extrinsic and intrinsic factors. As the CNS matures, growth-promoting proteins such as integrins are developmentally downregulated resulting in a reduced capacity for axonal outgrowth. Integrins are heterodimeric receptors involved in cell-cell and cell-matrix interactions. Specifically, within mature corticospinal tract (CST) axons, integrins are not transported into the axonal compartment. One integrin heterodimer, α9β1, is of particular interest for its ability to promote neurite outgrowth when bound to a component of the injury-induced milieu, tenascin-C. This project aimed to increase integrin expression within the CNS using induced pluripotent stem cell-derived human neural progenitor cells (iPSC-hNPCs). Using immunocytochemistry and western blotting, endogenous integrin expression within iPSC-hNPCs was determined. In addition, overexpression of α9 integrin was achieved using transfection and lentiviral transduction. The capacity of wild type (WT) and α9-hNPCs to extend neurites on tenascin-C was assessed using neurite outgrowth assays. Results revealed increasing α9 integrin expression in hNPCs significantly promoted neurite outgrowth when cultured on tenascin-C. Interestingly, increasing the concentration of human tenascin-C, resulted in increasingly longer neurites from WT hNPCs suggesting hNPCs could actively upregulate integrin expression. Subsequently, WT and α9-hNPCs were transplanted into layer V of the neonatal rat sensorimotor cortex, which projects to the CST. WT and α9-hNPCs survived up to 8 weeks post-transplantation and produced projections along white matter tracts, including areas of the CST. Additionally, hNPCs retained α9-eYFP protein expression in vivo over time and was localised within axonal projections. These results highlight the capabilities of iPSC-hNPCs to promote integrin expression within the rodent CNS presenting one potential avenue to target neuronal replacement following spinal injury. Future research should focus on assessing the regenerative capacity of WT and α9-hNPCs within an injury model concentrating on the ability of these cells to adapt within an injured environment.

Alterations to the tumour microenvironment is a common feature of many cancers, including breast cancer, and there is increasing evidence that alterations to the microenvironment, including; increased integrin expression, ECM deposition and protease activity, promote cancer progression. Most invasive breast cancers arise from a preinvasive stage, ductal carcinoma in situ (DCIS). Previous work in our laboratory has shown the microenvironment of DCIS is altered, such that myoepithelial cells (MECs) switch to a tumour-promoting phenotype, associated with upregulation of integrin αvβ6 and fibronectin (FN) expression. Mechanisms by which integrin αvβ6 and FN expression are regulated is unclear. We show DCIS progression into invasion is accompanied by an increase in MEC expression of integrin αvβ6 and periductal FN deposition, and their expression were associated in DCIS. These findings were modelled in isolated primary DCIS-MECs, primary normal MECs and MEC lines, with and without integrin αvβ6 expression, where integrin αvβ6-positive MECs upregulating FN expression. We identified integrin αvβ6-positive DCIS ducts were larger than integrin αvβ6-negative DCIS ducts, and mechanical stretching of primary normal MECs and a normal MEC line led to upregulation of integrin αvβ6 expression and FN deposition in a TGFβ-dependent manner. We further show upregulation of integrin αvβ6 and FN by MECs mediate TGFβ-dependent upregulation of MMP13 which promotes breast cancer cell invasion in vitro. These data show altered tissue mechanics in DCIS and MEC expression of integrin αvβ6 and FN deposition are linked, and implicate TGFβ in their activation. These findings suggest integrin αvβ6 and FN may be used as markers to stratify DCIS patients.

Orientador: Miriane de Oliveira / Resumo: Introdução: O hormônio triiodotironina (T3) influencia o metabolismo e desenvolvimento do tecido adiposo (TA), modulando a proliferação e diferenciação de adipócitos, podendo agir sobre os reguladores do processo de adipogênese, como o receptor ativado por proliferador de peroxissomo (PPARy). O TA está envolvido na regulação da energia corporal, sintetizando e secretando substâncias denominadas adipocinas, dentre elas a adiponectina e leptina. A adiponectina está relacionada ao aumento da sensibilidade à insulina, enquanto a leptina está envolvida com o gasto energético. O T3 pode desencadear ações por ativação de vias extranucleares, dentre elas a via MAPK/ERK e integrina αVβ3. Objetivo: Verificar a ação do T3, com participação das vias extranucleares MAPK/ERK e integrina αVβ3, na modulação de adiponectina e leptina, além de avaliar os parâmetros relacionados ao perfil adipogênico e dano de DNA. Métodos: Adipócitos, 3T3-L1, foram tratados com T3 (10nM) por uma hora, na ausência ou presença dos inibidores de MAPK/ERK – PD98059 (PD, 50uM) e da integrina αvβ3 – ácido tetraiodotiroácetico (Tetrac, 10-4M). A ausência de qualquer tratamento foi considerada grupo controle (C). Após o período de tratamento foi realizado PCRq-RT para analisar a expressão de mRNA de adiponectina e leptina, e Western Blot para expressão proteica de adiponectina, leptina, PPARy, pAKT e pERK; a viabilidade celular foi realizada pelo ensaio de MTT; a quantificação do acúmulo lipídico pelos ens... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: The hormone triiodothyronine (T3) influences the metabolism and development of adipose tissue (TA), modulating the proliferation and differentiation of adipocytes, and can act on regulators of the adipogenic differentiation process, such as the peroxisome proliferator activated receptor). TA is involved in the regulation of body energy, synthesizing and secreting substances called adipokines, among them adiponectin and leptin. Adiponectin is related to increased insulin synaptic, since leptin is involved in energy expenditure. T3 can trigger actions by activation of extranuclear pathways, including MAPK / ERK and integrin α Vβ3. Objective: Given the role of T3 in TA and the importance of adipokines, the objective of this study is to verify the action of T3 with the participation of extranuclear pathways in the modulation of adiponectin and leptin and the parameters related to the adipogenic profile. Methods: Adipocytes, 3T3-L1, were treated with a physiological dose of T3 (10nM) for one hour, in the absence or presence of MAPK / ERK-PD98059 (PD) and integrin αvβ3 - tetraiodothyrocetic (Tetrac) integrin inhibitors. The absence of any treatment was considered as a control group (C). After the treatment period PCRqRT was performed to analyze the expression of leptin and adiponectin mRNA, and Western Blot for protein expression of adiponectin, leptin, PPARγ, pAKT and pERK; cell viability was performed by the MTT assay; the quantification of lipid accumulation by the... (Complete abstract click electronic access below) / Mestre

Cyclobutanes represent an underutilised structural feature in medicinal chemistry, partially due to difficulties in forming them in an easy and controlled manner. Herein is described their application to a drug discovery project and development of the enamine [2+2] cyclisation; a straightforward synthesis of functionalised cyclobutanes. A library of 30 cyclobutane based integrin antagonists have been designed and synthesised to explore the SAR around the hit dual β3 integrin antagonist ICT9055. Several of which were shown to be highly potent antagonists inhibiting cancer cell adhesion, migration and invasion while remaining non-toxic. ICT9072 had comparable β3 activity to hit compound ICT9055 but also had activity against αvβ5 and therefore showed greater inhibition of migration of DLD-1 cells. This showed the ability to modify this scaffold for multi integrin antagonism and potential benefit of this. Synthetic studies towards the marine natural product providencin has led to the development of a previously unknown intramolecular enamine [2+2] cyclisation which has been shown to proceed in a diastereoselective manner. This reaction has been applied to the synthesis of a highly functionalised enatiopure cyclobutene suitable for inclusion into the total synthesis. A model furyl cyclobutane has also been synthesised to exemplify the route from the enantiopure cyclobutene through to the furyl cyclobutane fragment of providencin.

Förster Resonance Energy Transfer (FRET)-based techniques are gaining an increasing importance in cell biology and cell-matrix adhesion studies because they allow both the detection of conformational changes of target proteins and their localisation in cells. Frequency Domain-Fluorescence Lifetime Microscopy (FD-FLIM) is currently considered one of the most reliable methods to measure FRET in live cells. However, due to its dependence on many technical prerequisites, its use is not yet widespread. The purpose of this work was to first establish FD-FLIM measurements of FRET on a new FD-FLIM microscope module. Then we aimed to apply FD-FLIM-FRET measurements to the study of conformational changes of the cell matrix-adhesion proteins vinculin and integrin and of the growth factor receptor Tie-2. In the first part of the work, published FRET probes including distance-sensors and two sets of vinculin-based probes were extensively tested with FD-FLIM, sensitised emission and ratiometric FRET. FD-FLIM was shown to be the most accurate method in approximating molecular distances between fluorophores. Moreover this study unveiled specific caveats associated with both existing vinculin FRET probes. FD-FLIM was then used to study conformational changes of the extracellular matrix receptor alphavβ3 integrin and of the angiopoietin receptor Tie-2 using specific FRET probes designed by us. While data showed that the alphav-integrin-FRET probe localised to adhesion sites, more experiments will be required to evaluate its full functionality. The Tie-2-FRET probe was fully functional and, upon ligand binding, allowed the detection of a bending movement of the extracellular domain towards the cell membrane. Finally, a combination of FRET, immunofluorescence and tension release experiments were used to show that intracellular tension is not required to maintain integrins in their activated conformation. However, intracellular tension is required to recruit other key proteins such as vinculin, talin and tensin to adhesions sites. Overall this work demonstrates the importance of FD-FLIM-FRET as a tool to investigate conformational changes of adhesion proteins and transmembrane receptors within the cell environment.