O câncer de mama é no Brasil o segundo maior causador de morte entre mulheres. Os hormônios sexuais estão dentre os vários fatores indutores ou promotores da carcinogênese. ADAMTS (uma desintegrina e metaloproteinase com domínios trombospondina) é uma enzima Zn2+/Ca2+dependente. Avaliamos a influência dos hormônios na expressão e localização da ADAMTS-1 em 3 linhagens de mama. qPCR demonstrou que a progesterona estimulou o aumento de mRNA de ADAMTS-1 na MCF-10A e na MDA-MB-231, o tratamento com estrógeno e progesterona estimulou a diminuição. No Western blot observamos nas linhagens MCF-10A e MCF-7 que os hormônios tendem a aumentar os níveis de ADAMTS-1, e o estrógeno estimulou uma acentuada secreção em MCF-7. Na linhagem MDA-MB-231, os tratamentos demonstraram uma tendência a diminuir os níveis de ADAMTS-1 intracelular. Na imunofluorescência e Western blot observamos a predominância de ADAMTS-1 nuclear. Os resultados sugerem que os hormônios regulam a expressão de ADAMTS-1 nas células normais e tumorais, e que está predominantemente presente no núcleo celular. / Breast cancer is the second largest cause of death among women in Brazil. Sex hormones are one of the several inducers factors or cancer promoters. ADAMTS (a disintegrin and metaloproteinase with thrombospondin motifs) are dependent on Zn2+/Ca2+ enzymes. We evaluated influence of hormones on ADAMTS-1 levels and localization in three different breast cell lines. qPCR showed that progesterone stimulated an increase of ADAMTS-1 mRNA in MCF-10A, however, in MDA-MB-231, the treatment by estrogen and progesterone decreased levels. Western blot analysis showed a tendency to increased ADAMTS-1 levels in MCF-10A and MCF-7 due to the hormone treatment. Treatment by estrogen led to a marked secretion in MCF-7. In MDA-MB-231 the treatment showed a tendency to decreased intracellular ADAMTS-1 protein levels. Immunofluorescence and Western Blot we observed ADAMTS-1 predominant in the nucleus. Results suggest that sex hormones regulate the ADAMTS-1 expression in normal and tumoral breast cells, ADAMTS-1 is predominant in the cellular nucleus.

Tumor necrosis factor (TNF) is upregulated in a number of cardiomyopathies. This thesis investigates TNF in triggering the expression and activation of matrix metalloproteinases (MMPs) in pressure overload cardiac disease, and explores the role of superoxide. Cardiac pressure overload was generated in adult wild-type and TNF-/- mice by transverse aortic constriction. Isolated cardiomyocytes and cardiofibroblasts from neonatal mice ventricles were treated with recombinant TNF (rTNF), and MMP induction and activation were assessed, with and without apocynin (a NADPH-oxidase inhibitor). TNF-/- mice showed less superoxide production and MMP activation, compared to wild-type mice, following pressure overload. rTNF upregulated the production of NADPH-dependent superoxide in cardiomyocytes as early as 1 hour (24 hours in cardiofibroblasts). rTNF also increased the expression of MMP-9 and MMP-12 in cardiomyocytes more than in cardiofibroblasts, and MMP-8 and MMP-13 more in cardiofibroblasts. This induction in both cardiac cell types was concomitant with superoxide production.

Sex steroids are inevitable in women. However, long-term exposure to sex steroids increases the risk of breast cancer. A complete understanding of sex steroid control of the breast and how it relates to breast cancer risk is still lacking. Angiogenesis and proteolytic enzyme activity are crucial for the process by which tumors evolve into a vascularized, invasive phenotype. Matrix metalloproteinases are potent matrixdegrading enzymes that affect several steps in tumor progression including angiogenesis. In the female reproductive organs, sex steroids regulate angiogenesis and MMP activity, yet little is known how sex steroids affect these crucial events in normal and malignant breast tissue. This thesis elucidates a link between sex steroids, MMP activity, and angiogenesis. It is shown that estradiol down-regulates while tamoxifen up-regulates the protein expression and activity of MMP-2 and MMP-9 in human breast cancer cells in vitro and in human breast cancer xenografts in vivo. The results further suggest that a biological consequence of this regulation may be modulation of tumor angiogenesis. The net effect of adding tamoxifen to estradiol treatment was an increase in extracellular levels of the endogenous angiogenesis inhibitor endostatin and decreased levels of the tumor promoter TGF-β1 compared to estradiol treatment only. This was accompanied by reduced vasculature and decreased tumor growth. Similarly, a regulatory effect of estradiol and tamoxifen on endostatin generation was observed in normal human breast tissue by whole-tissue culture and microdialysis in human breast tissue in situ. In conclusion, the results presented in this thesis suggest previously unknown mechanisms of action of estradiol and tamoxifen in breast cancer and in normal human breast tissue, and novel means by which estradiol may tip the scale to favor angiogenesis. This knowledge may be important for the understanding of sex steroid dependent breast carcinogenesis and in the future development of tissue-specific preventive as well as therapeutic strategies against breast cancer.

Matrix metalloproteinases (MMPs) network with other biological molecules to maintain the extracellular matrix (ECM) in normal physiology and perform different roles. Understanding and assigning specific role to each of 24 members of these endoproteinases is impeded because of lack of specific and efficient detection methods in biological samples. Moreover, MMP-based anti-cancer drug development has also been challenged because, currently, there is no robust methodology to distinguish the inactive pro-enzymes, active enzymes or those complexed with endogenous inhibitors in biological specimens. The objective of this project is to develop a chemical proteomics strategy based on Matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) to help identify and discriminate the various MMP forms. Firstly, a triazine dye-based ligand immobilized on chromatography beads was utilized to assess whether it binds to recombinant human MMPs (rhMMPs). The results highlighted that the ligand interacts with latent forms of MMPs in agreement with the literature. Secondly, the potential of the ligand was assessed using MALDI-MS/MS based methodology in in vitro cancer models. Cell line culture supernatants were used in amounts to emulate the availability of tumour biopsies in clinical settings. The MS/MS spectral peaks specific to MMPs (MMP-2 and MMP- 14), and two endogenous inhibitors TIMP-1 and TIMP-2 were found in affinity chromatography eluates of cell culture supernatants with higher Mascot scores for the latter. While western blot detected MMP-2 in cell extracts, MALDI-MS/MS did not detect MMPs because of amounts below the limit of detection (LOD) of the instrument. Although the ligand was found to be interacting with MMPs and detergent-free salt elution buffers improved MALDI analysis, recovery of MMPs from biological samples was sub-optimal. The dye ligand was observed to bind other enzymes and despite various strategies to reduce non-specific binding of proteins or enable selective elution did not improve MMP enrichment. Further work using methodology described in this study is required after scaling up the MMP amounts in biological specimen and to resolve the issue of non-specific binding of proteins to the ligand by understanding its structure.

In this work, a new polyurethane (PU) chain extender was developed to incorporate a Glycine-Leucine (Gly-Leu) dipeptide, the cleavage site of several matrix metalloproteinases. PUs were synthesized with either the Gly-Leu-based chain extender (Gly-Leu PU) or a phenylalanine-based chain extender (Phe PU). Both PUs had high molecular weight averages (Mw > 125,000 g/mol) and were phase segregated, semi-crystalline polymers (Tm ~ 42°C) with a low soft segment glass transition temperature (Tg < -50°C). Uniaxial tensile testing of PU films revealed that the polymers could withstand high ultimate tensile strengths (~ 8-13 MPa) and were flexible with breaking strains of ~ 870-910% but the two PUs exhibited a significant difference in mechanical properties. The Phe and Gly-Leu PUs were electrospun into porous scaffolds for degradation and cell-based studies. Fibrous Phe and Gly-Leu PU scaffolds were formed with randomly organized fibers and an average fiber diameter of approximately 3.6 µm. In addition, the Phe PU was electrospun into scaffolds of varying architecture to investigate how fiber alignment affects the orientation response of cardiac cells. To achieve this, the Phe PU was electrospun into aligned and unaligned scaffolds and the physical, thermal, and mechanical properties of the scaffolds were investigated. The degradation of the Phe and Gly-Leu PU scaffolds was investigated in the presence of active MMP-1, active MMP-9, and a buffer solution over 28 days to test MMP-mediated and passive hydrolysis of the PUs. Mass loss and structural assessment suggested that neither PU experienced significant hydrolysis to observe degradation over the course of the experiment. In cell-based studies, Phe and Gly-Leu PU scaffolds successfully supported a high density of viable and adherent mouse embryonic fibroblasts (MEFs) out to at least 28 days. Culturing murine embryonic stem cell-derived cardiomyocytes (mESCDCs) alone and with MEFs on aligned and unaligned Phe PU scaffolds revealed both architectures supported adherent and functionally contractile cells. Importantly, fiber alignment and coculture with MEFs improved the organization and differentiation of mESCDCs suggesting these two parameters are important for developing engineered myocardial constructs using mESCDCs and PU scaffolds.

No description available.

In this work, a new polyurethane (PU) chain extender was developed to incorporate a Glycine-Leucine (Gly-Leu) dipeptide, the cleavage site of several matrix metalloproteinases. PUs were synthesized with either the Gly-Leu-based chain extender (Gly-Leu PU) or a phenylalanine-based chain extender (Phe PU). Both PUs had high molecular weight averages (Mw > 125,000 g/mol) and were phase segregated, semi-crystalline polymers (Tm ~ 42°C) with a low soft segment glass transition temperature (Tg < -50°C). Uniaxial tensile testing of PU films revealed that the polymers could withstand high ultimate tensile strengths (~ 8-13 MPa) and were flexible with breaking strains of ~ 870-910% but the two PUs exhibited a significant difference in mechanical properties. The Phe and Gly-Leu PUs were electrospun into porous scaffolds for degradation and cell-based studies. Fibrous Phe and Gly-Leu PU scaffolds were formed with randomly organized fibers and an average fiber diameter of approximately 3.6 µm. In addition, the Phe PU was electrospun into scaffolds of varying architecture to investigate how fiber alignment affects the orientation response of cardiac cells. To achieve this, the Phe PU was electrospun into aligned and unaligned scaffolds and the physical, thermal, and mechanical properties of the scaffolds were investigated. The degradation of the Phe and Gly-Leu PU scaffolds was investigated in the presence of active MMP-1, active MMP-9, and a buffer solution over 28 days to test MMP-mediated and passive hydrolysis of the PUs. Mass loss and structural assessment suggested that neither PU experienced significant hydrolysis to observe degradation over the course of the experiment. In cell-based studies, Phe and Gly-Leu PU scaffolds successfully supported a high density of viable and adherent mouse embryonic fibroblasts (MEFs) out to at least 28 days. Culturing murine embryonic stem cell-derived cardiomyocytes (mESCDCs) alone and with MEFs on aligned and unaligned Phe PU scaffolds revealed both architectures supported adherent and functionally contractile cells. Importantly, fiber alignment and coculture with MEFs improved the organization and differentiation of mESCDCs suggesting these two parameters are important for developing engineered myocardial constructs using mESCDCs and PU scaffolds.

Darbo uždaviniai: 1. Nustatyti pradinės AGDD paplitimą vidutinio amžiaus (40 – 64 metų) pacientų, sergančių IŠL grupėje bei atsitiktinėje to paties amžiaus Kauno miesto gyventojų imtyje. 2. Palyginti pacientų, sergančių tik IŠL ir IŠL bei pradinės AGDD klinikinius duomenis. 3. Nustatyti MMP-2 (-735 C/T), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T) genotipų dažnį bei genotipų derinių įtaką AGDD susiformavimui. 4. Nustatyti MMP-2 (-735 C/T), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T) genotipų dažnį esant minkštoms ir kietoms drūzoms, sergant AGDD. 5. Nustatyti MMP-2 (-735 C/T), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T) genotipų dažnį bei genotipų derinių įtaką AGDD ir IŠL drauge bei tik IŠL pasireiškimui. 6. Nustatyti funkcinio kontrastinio jautrumo tyrimo rodmenis pacientams, sergantiems pradine lengva ir pradine vidutine AGDD bei spalvų juslės pokyčius sergantiems pradine AGDD, ir oftalmologiškai sveikiems pacientams. / The goals were as follows: 1. To determine the prevalence of AMD in patients with IHD and compare with the prevalence in a random sample of Kaunas population (at 40-64 yrs old). 2. To compare the main clinical characteristics of the patients exhibiting early AMD and IHD together with the patients with IHD alone. 3. To determine the frequency of the genotypes of the matrix metalloproteinases (MMP-2 (-735 C/T), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T)), and genotype combinations that have an influence on the development of early AMD. 4. To determine the frequency of the genotypes of the matrix metallo¬proteinases (MMP-2 (-735 C/T), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T)), in early AMD patients with soft or hard drusen. 5. To determine the frequency of the genotypes of the matrix metallo¬proteinases (MMP-2 (-735 C/T), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T)), and the influence of genotype combi¬na¬tions on the development of AMD and IHD together, and only on IHD development. 6. To determine the results of functional acuity contrast sensitivity test in patients with early mild and early intermediate AMD, and color contrast sensitivity in patients with AMD, and in ophthalmologically healthy patients.

Submitted by THALITA LEONE ALVES ROCHA null (thalarocha@hotmail.com) on 2016-04-18T15:51:39Z No. of bitstreams: 1 dissertação thalita final.pdf: 794073 bytes, checksum: 8f42b335b8638b50194b64ede638c446 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-04-19T16:16:26Z (GMT) No. of bitstreams: 1 rocha_tla_me_bot.pdf: 794073 bytes, checksum: 8f42b335b8638b50194b64ede638c446 (MD5) / Made available in DSpace on 2016-04-19T16:16:26Z (GMT). No. of bitstreams: 1 rocha_tla_me_bot.pdf: 794073 bytes, checksum: 8f42b335b8638b50194b64ede638c446 (MD5) Previous issue date: 2016-02-29 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As metaloproteinases de matriz extracelular (MMPs) podem limitar a vasodilatação pulmonar e os efeitos inotrópicos positivos promovidos pela adrenomedulina durante a hipertensão pulmonar. O presente estudo teve por objetivo avaliar os efeitos da administração combinada da doxiciclina (inibidor não seletivo das MMPs) e da adrenomedulina sobre as alterações hemodinâmicas observadas durante a embolia pulmonar aguda em ovinos. Alterações hemodinâmicas e respiratórias foram mensuradas em ovinos anestesiados, pré-tratados com doxiciclina (10 mg/kg por via intravenosa), submetidos à EPA induzida pela injeção intravenosa (IV) de microesferas de silicone (500 mg) e posteriormente tratados com solução salina (grupo Dox+PE) ou adrenomedulina (50 ng/kg/min) (grupo Dox+PE+Adm). Os resultados deste estudo foram comparados com grupos históricos recentemente publicados por nosso grupo de pesquisa, realizados sob as mesmas condições experimentais, onde foram utilizados ovinos anestesiados não submetidos a qualquer intervenção (grupo Sham) ou submetidos à EPA e tratados com solução salina (grupo PE) ou com adrenomedulina (50 ng/kg/min) (grupo PE+Adm). Doxiciclina não produziu efeitos adicionais sobre as diminuições significativas no índice de resistência vascular pulmonar e aumento no índice cardíaco (ambos em 25%) observadas com o uso da adrenomedulina (grupo PE+Adm). A administração da adrenomedulina (grupo PE+Adm e Dox+PE+Adm) diminuiu significativamente a pressão arterial média e o índice de resistência vascular sistêmica, levando a uma hipotensão sistêmica moderada. Reduções significativas na pressão parcial de oxigênio arterial foram observadas após a doxiciclina e a EPA, que não foram afetadas pela administração da adrenomedulina. Estes resultados demonstram que a administração combinada da doxiciclina e adrenomedulina não traz benefícios hemodinâmicos adicionais quando comparada ao uso isolado da adrenomedulina, sugerindo que está combinação não se apresenta vantajosa durante a EPA induzida por microesferas. / Matrix metalloproteinases (MMPs) may limit severely the pulmonary vasodilatory and inotropic effects of adrenomedullin during pulmonary hypertension. Hemodynamic and respiratory changes were measured in anesthetized bovine pre-treated with doxycycline (10 mg/kg intravenously), subjected to APE induced by intravenous injection of silicone microspheres (500 mg) and subsequently treated with physiological saline (Dox+PE group) or adrenomedullin (50 ng / kg / min) (Dox+PE+ Adm group). The results were compared with historical group recently published by our research group, carried out under the same experimental conditions, where anesthetized sheep were used not subjected to any intervention (Sham group) or subjected to APE, and treated with physiological saline (PE group) or with adrenomedullin (50 ng / kg / min) (PE+Adm Group). Doxycycline produced no effect on significant temporal decreases in pulmonary vascular resistance index and increases in cardiac index (both by 25%) observed with adrenomedullin. The administration of adrenomedullin significantly decreased mean arterial pressure and systemic vascular resistance index, leading to a moderate systemic hypotension. Significant decreases in arterial oxygen partial pressure were observed after doxycycline or APE, but these changes were not affected by adrenomedullin. These results demonstrate that the combined administration of doxycycline and adrenomedullin does not provide additional hemodynamic benefits when compared to isolated use of adrenomedullin, suggesting that this combination does not appear advantageous for the APE-induced microspheres. / FAPESP: 2012/12.291-7

Orientador: Josimeri Hebling / Resumo: Objetivo: Avaliar o desempenho clínico de restaurações de resina composta em lesões cervicais não cariosas (LCNC) realizadas com ou sem a aplicação de um agente de ligação cruzada (carbodiimida, EDC) durante o período de 12 meses. Métodos: 21 pacientes com no mínimo duas LCNC participaram deste estudo. Um total de 142 LCNC foram distribuídas randomicamente em dois grupos (n=71 cada): controle e experimental. Todas as LCNC foram restauradas pelo mesmo operador e com os mesmos materiais (sistema adesivo Adper Single Bond 2 e resina composta Z350), sendo que, no grupo experimental a dentina recebeu a aplicação de EDC 0,5 mol/L por 60 segundos após o condicionamento ácido e no grupo controle apenas PBS. As restaurações foram avaliadas imediatamente após sua confecção (baseline) e após uma semana, 6 e 12 meses pelo critério USPHS/Ryge por apenas um avaliador calibrado. Os escores obtidos a partir da avaliação clínica, considerando-se os fatores de confundimento, foram analisados pelos testes de Woolf e Cochran-Mantel-Haenzel (p<0,05). Resultados: As taxas de falhas acumuladas na retenção das restaurações nos períodos 7 dias, 6 e 12 meses, foram de 0%; 3,0% e 4,5%, respectivamente, para o grupo controle e de 0%; 3,0% e 7,4% para o grupo experimental (EDC). Não houve diferença estatisticamente significante entre os dois grupos para todos os critérios e períodos avaliados. A taxa de retorno dos pacientes nos períodos de 7 dias, 6 e 12 meses foi de 100%, 90,5% e 85,7%, respectivamente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: o evaluate the clinical performance of composite restorations placed in noncarious cervical lesions (NCCL) performed with or without the application of a cross-linking agent, over a period of 12 months. Methods: 21 patients with at least two NCCL, participated in this study. A total of 142 NCCL were randomly allocated into two groups (n=71): control and experimental. All NCCL were restored by the same operator and with the same materials (Single Bond 2 and Z350). In the experimental group the etched dentin was exposed to 0.5 mol/L EDC for 60 seconds while in the control group the etched dentin was exposed to Phosphate buffer solution (PBS). The restorations were evaluated at baseline, 7 days, 6 and 12 months using the USPHS/Ryge criteria, by one blinded calibrated examiner. The scores from the clinical evaluation were submitted to Woolf and CochranMantel-Haenzel tests, considering the confounding factors (p<0.05). Results: The rates of accumulated failures in the retention of restorations in the periods 7 days, 6 and 12 months were 0%; 3,0% and 4.5%, respectively, for the control group, and 0%; 3,0% and 7.4% for the experimental group. There was no statistically significant difference between the two groups for all criteria and periods. The return rate of patients in the 7-day, 6- and 12-month periods was 100%, 90.5% and 85.7%, respectively. Conclusion: The treatment of the etched dentin with with EDC prior to the application of the dentin bonding agent did not affect the performance of composite resin restorations in NCCL...(Complete abstract electronic access below) / Doutor