This master thesis deals with the analysis of the ECM market solutions and trends in this area. The theoretical part of the thesis focuses on defining theoretical assumptions of studied subject. This part is concluded by the assessment of trends in the ECM field. The practical part of the thesis focuses not only on the analysis of the ECM market but also on detailed analysis of selected suppliers and their ECM solutions, which are analyzed according to selected criteria. Last part of the thesis provides the summary of the performed analysis and evaluation of selected providers.

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Work in our lab has resulted in the development of a novel approach to creating a more developmentally plastic human dermal fibroblast (hDF) phenotype that allows for the study of molecular mechanisms involved in cell-fate conversion. Culturing hDF under defined culture conditions (5% O2 and supplementation with fibroblast growth factor FGF2) induces induced the regeneration competent (iRC) phenotype that is characterized by stem cell gene expression, and increased life-span in vitro. The work presented in this thesis further characterizes the system, and describes an overall shift in extracellular matrix and adhesion molecules in human dermal fibroblasts (hDF) undergoing the transition to a more developmentally plastic phenotype (iRC). This work suggests that we create the initiation phase of Mesenchymal-to-Epithelial Transition (MET) during conversion to the iRC phenotype. This transition is marked by loss of integrin alpha-11 (α11) and its binding partner Collagen-I (COL-I). Moreover, we describe the mechanism for the down-regulation of α11 that is mediated by FGF2 activation of ERK1/2 through systematic investigation of several potential molecular mechanisms. The body of work presented here shows that the ERK 1/2 mediated down-regulation of α11 is independent of activation of TGF-β1-mediated regulation of α11. In addition to down-regulation of α11, an overall shift in the transcript levels of other adhesion molecules is observed, which demonstrates that iRC are most likely transitioning their attachment to a laminin and fibronectin-based matrix. These results suggest that iRC may be producing a more “pro-regenerative matrixâ€�. We hypothesize that the changes in integrin expression profile and interaction with ECM serve as a feedback loop during the iRC phenotype shift. Our findings suggest that this “pro-regenerativeâ€� shift in attachment of iRC as well as the ERK 1/2 mediated down-regulation of α11 could be exploited in wound healing biology and fibrosis research. Manipulation of the dynamic relationship between TGF-β1 and FGF2 has the potential to reduce scar deposition. Further identification of molecular mechanisms controlling this phenotype conversion will allow development of strategies for in situ manipulation of wound healing outcomes.

Aging is characterized by a progressive impairment of cardiac structure and function. The cardiac remodeling involves loss of cardiac myocytes, reactive hypertrophy of the remaining cells, and increased extracellular matrix (ECM) and fibrosis in the aging heart. In contrast, exercise training not only improves cardiac function, but also reduces the risk of heart disease. However, the ability of exercise training to modulate ECM and remodeling in the aging heart remains unknown. Therefore, the purpose of this study was to determine the effects of exercise training on ECM remodeling in the aging heart. We hypothesized that (1) exercise training would attenuate age-related changes in left ventricle morphology including extramyocyte space and collagen contents, and (2) exercise training would ameliorate age-induced changes in ECM-related factors including MMPs, TIMPs, TNF-α, TGF-β1, and α-SMA in the heart. Three and 31 month old Fischer 344 × Brown Norway F1 hybrid rats were assigned to four groups: young sedentary (YS), young exercise-trained (YE), old sedentary (OS), and old exercise-trained (OE). Exercise training groups walked briskly on a treadmill for 45 min/day (12° incline) at 20m/min (young) or 10 m/min (old), 5 d/wk for 12 wk. We found that endurance exercise training might ameliorate the ageinduced increase in extramyocyte space and collagen contents of the left ventricle. Exercise training might protect against age-induced fibrosis by increasing MMP-2, MMP-14 in the soluble fraction and MMP-1, MMP-3, MMP-14 in the insoluble fraction of old rat hearts. Conversely, exercise training might reduce the fibrosis by decreasing TIMP-1 in the soluble fraction of old rat hearts. Further, exercise training reduced potential upstream pro-fibrotic mediators including TNF-α and TGF-β1 in the aging rat hearts. These results are the first to demonstrate that exercise training has a protective effect against age-induced extracellular collagen matrix remodeling in the aging heart, associated with increased MMP-1, -2, -3, -14 and decreased TIMP-1, TNF-α, and TGF- β1.

Glomerular filtration is a vital physiological process removing waste products from the circulation and this process occurs across the glomerular filtration barrier (GFB). The cells and extracellular matrix (ECM), which form this barrier, are exposed to forces during ultrafiltration and special adaptation is required to withstand these forces. Dysfunction in cellular adhesion machinery or ECM assembly within the GFB causes loss of selective glomerular filtration, however, the mechanisms governing these processes are poorly understood. To this end we sought to characterise the glomerular ECM and adhesion machinery using high throughput mass spectrometry (MS)-based proteomics. MS of human glomerular ECM identified a highly complex extracellular niche, revealing the potential involvement of novel ECM proteins in glomerular development and disease processes. Furthermore we identified that glomerular cells in culture had distinct ECM proteomes and interestingly, coculture experiments demonstrated that the ECM proteome was influenced by cellular crosstalk and had a closer resemblance to glomerular ECM in vivo. Protein network analyses of in vivo and in vitro ECM datasets revealed a common core of highly connected structural ECM proteins that may be important for glomerular ECM assembly. To understand how this ECM proteome altered in disease, we studied mice with mild glomerular dysfunction. Here, transcriptomic and proteomic analyses identified alterations in ECM composition and 3D electron microscopy revealed striking ultrastructural changes in glomerular ECM. MS-based proteomics was next applied to the analysis of glomerular podocyte adhesion complexes, leading to the discovery that the actin cytoskeletal regulators and trafficking machinery are recruited to adhesions sites in an ECM-ligand dependent manner. Furthermore, these differences functionally altered cell shape and adhesion strength. These same analyses were applied to podocyte cell-cell junctions, revealing an unexpected overlap of cell-ECM and cell-cell adhesion machinery. Overall, these findings demonstrate for the first time the complexity of the glomerular ECM and adhesion signalling complexes and reinforce the benefits of global, unbiased experimental approaches. In addition the results suggest that glomerular ECM composition, organisation and adhesion signalling are context dependent, and therefore, represent potential therapeutic targets.

Integrinas são glicoproteínas transmembrânicas envolvidas na adesão célula-célula e célula-proteina da matriz extracellular (ECM) que participam da migração e fusão de células trofoblásticas gigantes (TGC) com células do epitélio materno no placentoma bovino e interagem com inibidores teciduais de metalloproteinases (TIMPs), considerados importantes reguladores das metaloproteinases de matriz (MMPs), que são reconhecidas como passo limitande para ação de enzimas que atuam no remodelamento da ECM na implantação e na gestação. Uma vez que as integrinas são consideradas responsáveis pela manutenção da arquitetura tecidual e que as MMPs podem regular a degradação e a reconstituição da ECM necessária para a invasão do trofoblasto, temos como objetivo verificar uma possível relação das integrinas com alterações placentárias comumente observadas em gestações de animais clonados (SCNT). Para avaliar a funcionalidade placentária, a expressão do lactogênio placentário (PL) e do antígeno Ki-67 foram também verificadas. Placentomas bovinos foram coletados e divididos em 3 grupos: 1) início (n = 6) e 2) final (n = 3) da gestação de animais derivados de monta natural (n=9) e final da gestação de animais clonados (n=8), clones machos (n=4) e clones fêmeas (n=4). As proteínas foram localizadas por imuno-histoquímica indireta, exceto as subunidades de integrina &alpha;6, &beta;1, &alpha;V e &beta;3 que foram localizadas por imunofluorescência. A especificidade dos anticorpos e expressão protéica foi confirmada por Western blot e a expressão do RNAm foi avaliada por meio de RT-PCR e PCR em tempo real quantitativo. As células positivas para a laminina, TIMP-2, Ki-67 e lactogênio placentário (PL) foram quantificadas e para comparação entre os grupos. A proteína das subunidades &alpha;6 do receptor de integrina e &beta;1 foi observada nos animais clonados e não clonados nos estromas materno e fetal e porção basal dos epitélios materno e fetal e foi co-localizada com a laminina. A proteína das subunidades &alpha;V e &beta;3 do receptor de integrina foi observado nos estromas materno e fetal e foi co-localizado com a fibronectina. O TIMP-2 foi exclusivamente e especificamente localizado nas TGCs no início e final da gestação de animais clonados e não clonados, mas não houve diferença significativa em sua expressão. O PL apresentou a mesma localização do TIMP-2 em todos os grupos analisados. Houve diferença significativa (p<0,05) entre 270d e clones ao se comprar a expressão relativa do RNAm da subunidade de integrina &beta;1 e do lactogênio placentário e ao se comparar a expressão protéica por western blot da laminina e do TIMP-2. Foi possível observar diferença significativa (p<0,05) entre o grupo controle e o grupo de animais clonados ao se quantificar o número de TGCs positivas para a laminina e para o lactogênio placentário. Apesar de algumas diferenças terem sido observadas na expressão de integrinas e de proteínas da ECM, sugere-se que a interação das integrinas com seus ligantes da ECM a termo não é um determinante da sobrevivência neonatal de bovinos clonados. Contudo, outros estudos são necessários para esclarecer se estas proteínas representam elementos chave na placentação de bovinos clonados em início de gestação. As diferenças observadas na expressão de integrinas e principalmente do PL entre clones fêmeas e machos sugerem possível influência de genes associados ao cromossomo sexual ou imprinting. / Integrins are transmembrane glycoproteins involved in cell-cell and cell-extracellular matrix (ECM) adhesion and signal transduction. They participate in the migration and fusion of trophoblast giant cells (TGC) with uterine epithelial cells in bovine placentomes, and interact with tissue inhibitors of metalloproteinases (TIMPs), considered important regulators of matrix metalloproteinases (MMPs). MMPs are rate-limiting enzymes degrading ECM proteins during tissue remodeling around embryo implantation, pregnancy and parturition. Since integrins are considered to be responsible for the maintenance of the architecture within tissues and MMPs may also regulate degradation and reconstitution of ECM required for trophoblast invasion, we aimed to verify a potential relation of integrins with common placental alterations observed in cloned animals (SCNT). To verify the placental functionality, expression of placental lactogen (PL) and Ki-67 antigen was also assessed. Bovine placentomes were collected and divided into 3 groups: 1) early and 2) late gestation derived from natural mating (n=9) and 3) late gestational bovine clones (n=8), also divided into male clones (n=4) and female clones (n=4). All proteins were localized by indirect immunohistochemistry except for integrin subunits &alpha;6, &beta;1, &alpha;V and &beta;3 that were shown by immunofluorescence. The antibody specificity and protein expression were confirmed by Western blot. Expression of mRNA was evaluated using RT-PCR and quantitative Real Time PCR. Positive cells for laminin, TIMP-2, Ki-67 and placental lactogen were quantified for comparisons among groups. The protein of integrin receptor subunits &alpha;6 and &beta;1 was observed in both cloned and non-cloned animals in the fetal and maternal stroma and at the basal membrane of maternal and fetal epithelial cells, co-localized with laminin and with collagen IV. The integrin receptor subunits &alpha;V and &beta;3 were observed in the fetal and maternal stroma, co-localized with fibronectin. TIMP-2 was specifically and exclusively localized in TGC in the early and term gestation in both cloned and non-cloned animals. PL has shown the same localization of TIMP-2 in all analyzed samples. Statistically significant differences (p<0,05) between term gestation of non-cloned and cloned animals were observed comparing the mRNA expression of integrin &beta;1 subunit and placental lactogen and the protein expression of laminin and TIMP-2. There were also statistically significant differences (p<0,05) between non-cloned and cloned animals in the number of laminin and placental lactogen positive cells. Despite some differences in integrin and ECM proteins expression, our results suggest that the interaction between integrins and their ECM ligands in term gestation is not a determinant for the survival rate of newborn cloned calves. However further studies are necessary to elucidate if integrins represent key elements in the early placentation of SCNT bovines. The differences found in the integrin and principally in the placental lactogen expression between female and male cloned calves suggests the influence of sex-chromosome associated genes or imprinting.

Integrinas são glicoproteínas transmembrânicas envolvidas na adesão célula-célula e célula-proteina da matriz extracellular (ECM) que participam da migração e fusão de células trofoblásticas gigantes (TGC) com células do epitélio materno no placentoma bovino e interagem com inibidores teciduais de metalloproteinases (TIMPs), considerados importantes reguladores das metaloproteinases de matriz (MMPs), que são reconhecidas como passo limitande para ação de enzimas que atuam no remodelamento da ECM na implantação e na gestação. Uma vez que as integrinas são consideradas responsáveis pela manutenção da arquitetura tecidual e que as MMPs podem regular a degradação e a reconstituição da ECM necessária para a invasão do trofoblasto, temos como objetivo verificar uma possível relação das integrinas com alterações placentárias comumente observadas em gestações de animais clonados (SCNT). Para avaliar a funcionalidade placentária, a expressão do lactogênio placentário (PL) e do antígeno Ki-67 foram também verificadas. Placentomas bovinos foram coletados e divididos em 3 grupos: 1) início (n = 6) e 2) final (n = 3) da gestação de animais derivados de monta natural (n=9) e final da gestação de animais clonados (n=8), clones machos (n=4) e clones fêmeas (n=4). As proteínas foram localizadas por imuno-histoquímica indireta, exceto as subunidades de integrina &alpha;6, &beta;1, &alpha;V e &beta;3 que foram localizadas por imunofluorescência. A especificidade dos anticorpos e expressão protéica foi confirmada por Western blot e a expressão do RNAm foi avaliada por meio de RT-PCR e PCR em tempo real quantitativo. As células positivas para a laminina, TIMP-2, Ki-67 e lactogênio placentário (PL) foram quantificadas e para comparação entre os grupos. A proteína das subunidades &alpha;6 do receptor de integrina e &beta;1 foi observada nos animais clonados e não clonados nos estromas materno e fetal e porção basal dos epitélios materno e fetal e foi co-localizada com a laminina. A proteína das subunidades &alpha;V e &beta;3 do receptor de integrina foi observado nos estromas materno e fetal e foi co-localizado com a fibronectina. O TIMP-2 foi exclusivamente e especificamente localizado nas TGCs no início e final da gestação de animais clonados e não clonados, mas não houve diferença significativa em sua expressão. O PL apresentou a mesma localização do TIMP-2 em todos os grupos analisados. Houve diferença significativa (p<0,05) entre 270d e clones ao se comprar a expressão relativa do RNAm da subunidade de integrina &beta;1 e do lactogênio placentário e ao se comparar a expressão protéica por western blot da laminina e do TIMP-2. Foi possível observar diferença significativa (p<0,05) entre o grupo controle e o grupo de animais clonados ao se quantificar o número de TGCs positivas para a laminina e para o lactogênio placentário. Apesar de algumas diferenças terem sido observadas na expressão de integrinas e de proteínas da ECM, sugere-se que a interação das integrinas com seus ligantes da ECM a termo não é um determinante da sobrevivência neonatal de bovinos clonados. Contudo, outros estudos são necessários para esclarecer se estas proteínas representam elementos chave na placentação de bovinos clonados em início de gestação. As diferenças observadas na expressão de integrinas e principalmente do PL entre clones fêmeas e machos sugerem possível influência de genes associados ao cromossomo sexual ou imprinting. / Integrins are transmembrane glycoproteins involved in cell-cell and cell-extracellular matrix (ECM) adhesion and signal transduction. They participate in the migration and fusion of trophoblast giant cells (TGC) with uterine epithelial cells in bovine placentomes, and interact with tissue inhibitors of metalloproteinases (TIMPs), considered important regulators of matrix metalloproteinases (MMPs). MMPs are rate-limiting enzymes degrading ECM proteins during tissue remodeling around embryo implantation, pregnancy and parturition. Since integrins are considered to be responsible for the maintenance of the architecture within tissues and MMPs may also regulate degradation and reconstitution of ECM required for trophoblast invasion, we aimed to verify a potential relation of integrins with common placental alterations observed in cloned animals (SCNT). To verify the placental functionality, expression of placental lactogen (PL) and Ki-67 antigen was also assessed. Bovine placentomes were collected and divided into 3 groups: 1) early and 2) late gestation derived from natural mating (n=9) and 3) late gestational bovine clones (n=8), also divided into male clones (n=4) and female clones (n=4). All proteins were localized by indirect immunohistochemistry except for integrin subunits &alpha;6, &beta;1, &alpha;V and &beta;3 that were shown by immunofluorescence. The antibody specificity and protein expression were confirmed by Western blot. Expression of mRNA was evaluated using RT-PCR and quantitative Real Time PCR. Positive cells for laminin, TIMP-2, Ki-67 and placental lactogen were quantified for comparisons among groups. The protein of integrin receptor subunits &alpha;6 and &beta;1 was observed in both cloned and non-cloned animals in the fetal and maternal stroma and at the basal membrane of maternal and fetal epithelial cells, co-localized with laminin and with collagen IV. The integrin receptor subunits &alpha;V and &beta;3 were observed in the fetal and maternal stroma, co-localized with fibronectin. TIMP-2 was specifically and exclusively localized in TGC in the early and term gestation in both cloned and non-cloned animals. PL has shown the same localization of TIMP-2 in all analyzed samples. Statistically significant differences (p<0,05) between term gestation of non-cloned and cloned animals were observed comparing the mRNA expression of integrin &beta;1 subunit and placental lactogen and the protein expression of laminin and TIMP-2. There were also statistically significant differences (p<0,05) between non-cloned and cloned animals in the number of laminin and placental lactogen positive cells. Despite some differences in integrin and ECM proteins expression, our results suggest that the interaction between integrins and their ECM ligands in term gestation is not a determinant for the survival rate of newborn cloned calves. However further studies are necessary to elucidate if integrins represent key elements in the early placentation of SCNT bovines. The differences found in the integrin and principally in the placental lactogen expression between female and male cloned calves suggests the influence of sex-chromosome associated genes or imprinting.

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