Μελετήθηκε η επίδραση ουραιμικού ορού στο σύστημα των μεταλλοπρωτεϊνασών MMP-2 και MMP-9 και των αναστολέων τους TIMP-1 και TIMP-2 καθώς και σε βασικές βιολογικές δράσεις των ενδοθηλιακών κυττάρων σε καλλιέργειες HUVEC. Ο ορός συλλέχθηκε από ασθενείς που υποβάλλονταν σε συνεδρίες αιμοκάθαρσης (πριν και μετά τις συνεδρίες). Διαπιστώθηκε πως ο ουραιμικός ορός μειώνει την ικανότητα πολλαπλασιασμού, μετανάστευσης και επούλωσης τρύματος, ενώ επάγει την απόπτωση. Επιπλέον, ο ουραιμικός ορός επάγει τις μεταλλοπρωτεϊνάσες MMP-2 και MMP-9, καταστέλλει τους αναστολείς τους TIMP-1 και TIMP-2 και μειώνει την παραγωγή κολλαγόνου τύπου IV και ελαστίνης. / -

Made available in DSpace on 2014-06-11T19:27:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-12-17Bitstream added on 2014-06-13T19:15:14Z : No. of bitstreams: 1 tiano_gc_me_araca.pdf: 753020 bytes, checksum: 112d8897b7c84b5e590dd8fe0fffdb91 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O declínio mundial da cárie dentária é atribuído ao uso abrangente do flúor. Embora esse elemento seja capaz de proteger os dentes, seu uso excessivo pode levar a uma ação citotóxica causando a inibição do crescimento celular, da síntese de proteínas e até mesmo a morte celular. O primeiro objetivo deste estudo foi investigar a concentração ideal do NaF (NaF) capaz de ativar os fibroblastos gengivais de camundongos sem induzir morte celular. Observou-se que, nesses fibroblastos, a concentração de 40 μg F/mL induziu morte celular de 62,6 %. Na concentração de 20 μg F/mL a morte celular foi de apenas 22,1%. Com base nesses resultados, optou-se por utilizar a concentração de 20 μg F/mL como dose máxima para investigar os mecanismos envolvidos na ativação dos fibroblastos gengivais. Dessa forma, avaliou-se a capacidade do NaF induzir a expressão de MMP-2 e MMP-9 pelos fibroblastos gengivais de camundongos na presença ou ausência de LPS, assim como a produção da quimiocina CCL-3/MIP-1α e óxido nítrico. Avaliou-se também a participação das vias de sinalização intracelular p44/42, p38, PI3K e NF-кB envolvidas durante essa ativação, por meio da utilização dos respectivos inibidores PD98059 (50 μM), SB202190 (10 μM), LY294002 (30 μM) e dexametasona (10 μM). Observou-se que o NaF foi capaz de estimular os fibroblastos gengivais a expressarem MMP-9, mas não MMP-2, na concentração de 20 μg F/mL com pico máximo 6 horas após, retornando aos níveis normais 24 horas após. A produção da quimiocina CCL3/MIP-1α pelos fibroblastos estimulados pelo NaF também foi observada com a concentração de 20 μg F/mL com pico máximo 6 horas após estímulo. Na presença de LPS, observou-se uma potenciação da expressão de MMP-9 e produção de CCL3/MIP-1α na concentração de 20 μg F/mL, 6 horas após. / The worldwide decline of the dental caries is attributed to the widespread use of fluoride. Although this element is capable of protecting the teeth, its excessive use, can lead to a cytotoxic action, causing an inhibition of the cell growth, of the protein synthesis and even the cellular death. Based on these results, we have chosen to use a concentration of 20 μ g F/mL as maximum concentration to investigate the mechanisms involved in the activation of the gingival fibroblasts. It was observed that, on those fibroblasts, the concentration of 40 μg F/mL has resulted in a death cellular index of 62.6%. In the concentration of 20 μg F/mL the cellular death was of 22.1% only. Based on these results, the concentration of 20 μg F/mL has been chosen as maximum concentration to investigate the mechanisms involved in the activation of the gingival fibroblasts. Later, the ability of NaF to induce the expression of MMP-2 and MMP-9 in gingival fibroblasts of mice in the presence or absence of LPS has been assessed, as well as the production of chemokine CCL-3/MIP-1a and nitric oxide. It also evaluated the participation of intracellular signaling pathways p44/42, p38, PI3K e NF-kB involved in this activation, through inhibitors PD98059 (50 μM), SB202190 (10 μM), LY294002 (30 μM) e dexamethasone (10 μM). It was observed that the NaF was capable to stimulate the gingival fibroblasts to express MMP-9, at the concentration of 20 μgF/mL with maximum peak 6 hours after, returning to normal levels 24 hours after. The expression of MMP-2 was not observed. The production of chemokine CCL3/MIP-1α was also observed with the concentration of 20 μgF/mL with maximum peak 6 hours after the stimulation. In the presence of LPS, it was observed an intensification in the expression of MMP-9 and also in the production of CCL3/MIP-1α at the concentration of 20 μgF/mL, 6 hours later.

Objetivo: analisar a influÃncia do uso de inibidores de metaloproteinases da matriz [epigalocatequina-3-galato (EGCG) e digluconato de clorexidina (CHX)] utilizados como prÃ-tratamento dentinÃrio sobre a resistÃncia de uniÃo de um sistema adesivo autocondicionante em dentina hÃgida (H) e erosivamente desmineralizada (ED). Materiais e MÃtodos: terceiros molares humanos hÃgidos extraÃdos tiveram o terÃo oclusal seccionado, expondo a superfÃcie dentinÃria subjacente ao esmalte. A microdureza de superfÃcie (Knoop) da dentina exposta foi usada como parÃmetro para a seleÃÃo de 36 dentes, que foram aleatoriamente distribuÃdos em 6 grupos experimentais, de acordo com o tipo de dentina e com o prÃ-tratamento empregado (n=6): HAD (HÃgida + Ãgua destilada); HCHX (HÃgida + CHX a 2%); HEGCG (HÃgida + EGCG a 0,1%); EDAD (Erosivamente desmineralizada + Ãgua destilada); EDCHX (Erosivamente desmineralizada + CHX a 2%); EDEGCG (Erosivamente desmineralizada + EGCG a 0,1%). Todos os grupos foram submetidos à formaÃÃo de uma pelÃcula adquirida com saliva humana e, em seguida, os dentes dos grupos EDAD, EDCHX e EDEGCG foram submetidos a um ciclo erosivo (Ãcido cÃtrico 1%, pH 3,75; 3x/dia por 5 dias), enquanto que nos grupos HAD, HCHX e HEGCG, a soluÃÃo Ãcida foi substituÃda por Ãgua destilada. No perÃodo entre os ciclos, os espÃcimes foram armazenados em saliva artificial durante 2 h. ApÃs os ciclos, as superfÃcies dentinÃrias (hÃgidas e erosivamente desmineralizadas) foram prÃ-tratadas com 15 &#956;L da soluÃÃo, ativamente, por 60 s e, posteriormente, o sistema adesivo Clearfil SE Bond (CSEB) foi aplicado e um platà de 4 mm de resina composta Filtek Z250 foi construÃdo. ApÃs estocagem em Ãgua destilada a 37 ÂC por 24 h, os dentes foram cortados em forma de palitos, os quais foram submetidos a um ensaio mecÃnico de microtraÃÃo à velocidade de 0,5 mm/min. Resultados: os valores de resistÃncia de uniÃo (MPa) foram estatisticamente avaliados por ANOVA a dois critÃrios (condiÃÃo da dentina e prÃ-tratamento) e teste de Bonferroni (entre grupos), com nÃvel de significÃncia de 5%. Os grupos tratados com CHX apresentaram valores de resistÃncia de uniÃo estatisticamente inferiores (p<0,05) em relaÃÃo aos demais grupos, independentemente da condiÃÃo de dentina. Os grupos com prÃ-tratamento com Ãgua destilada e EGCG nÃo apresentaram diferenÃas significantes entre si (p>0,05). ConclusÃo: o uso de CHX influenciou negativamente, enquanto o uso de EGCG nÃo influenciou a resistÃncia de uniÃo do sistema adesivo CSEB à dentina hÃgida (H) e erosivamente desmineralizada (ED). / Objective: to analyze the influence of the use of two matrix metalloproteinases inhibitors [epigallocatechin-3-gallate (EGCG) and chlorhexidine digluconate (CHX)] as pretreatment solutions on dentin bond strength of a self-etch adhesive system on sound (H) and erosively demineralized dentin (ED). Materials and Methods: intact human third molars were sectioned on the occlusal third, exposing the underlying dentin surface. The surface hardness (Knoop) from exposed dentin was used as a parameter for the selection of 36 teeth that were randomly divided into 6 experimental groups according to the type and dentin pretreatment employed (n=6): HAD (sound + distilled water); HCHX (sound + 2% CHX); HEGCG (sound + 0,1% EGCG); EDAD (erosively demineralized + distilled water); EDCHX (erosively demineralized + 2% CHX); EDEGCG (erosively demineralized + 0,1% EGCG). All groups were subjected to an acquired pellicle formation with human saliva and then, teeth from EDDW, EDCHX and EDEGCG groups were subjected to an erosion cycle (1% citric acid, pH 3.75, 3 3x/day, during 5 days), whereas in the groups HAD, HCHX and HEGCG the acid solution was replaced by distilled water. Between the cycles, specimens were stored in artificial saliva for 2 h. After the cycles, dentin surfaces (sound and erosively demineralized) were pretreated with 15 &#956;L of the solution, actively, for 60 s and than Clearfil SE Bond (CSEB) was applied and a 4mm resin build up was made with composite resin Filtek Z250Â. After storage in distilled water at 37 ÂC for 24 h, specimens were sectioned in sticks, which were subjected to a which were subjected to a microtensile mechanical test with speed of 0.5 mm/min. Results: bond strength values (MPa) were statistically evaluated by two-way ANOVA (dentin condition and pretreatment) and Bonferroni test (between groups), with a significance level of 5%. The groups treated with CHX showed bond strength values statistically lower (p< 0.05) compared to the other groups, regardless of the condition of dentin. Groups with pretreatment with distilled water and EGCG showed no significant differences between them (p>0.05). Conclusion: the use of CHX influenced negatively, while the use of EGCG did not affect the bond strength of CSEB to sound and erosively demineralized dentin.

O processo de reparo do tecido ósseo tem sido alvo de estudos com o intuito de promover uma melhora na qualidade e tempo de reparo. Um elemento que tem sido estudado por alguns grupos é o fluoreto, pois tem sido proposto como terapêutico para osteoporose (Europa). Deste modo, o objetivo deste trabalho foi avaliar comparativamente o efeito do fluoreto administrado na água de beber sobre o reparo ósseo alveolar de ratos. Neste estudo foram utilizados 4 grupos com ratos Wistar de 80 dias de vida (n=200), os quais receberam água de beber contendo 5, 15 e 50 ppm de fluoreto e água deionizada (controle) durante todo experimento. Esses animais tiveram o incisivo superior direito extraído. Os animais foram eutanasiados 0 hora, 7, 14, 21 e 30 e 60 dias após a extração. Amostras de plasma sanguíneo foram obtidas para análise de concentração de flúor, e a região do alvéolo dental foi coletada para as análises de microscopia (Hematoxilina Eosina e imunoistoquímica) e zimografia (metaloproteinases de matriz 2 e 9 MMP-2 e 9). A análise de concentração de flúor no plasma sangüíneo mostrou maior presença desse elemento no grupo tratado com 50 ppm de F nos períodos de 21 e 30 dias. Na análise histológica foi detectado osso neoformado em todos os animais até 60 dias, porém o grupo de 50 ppm de F apresentou menor formação óssea nos períodos de 14, 21 e 30dias. A análise morfométrica confirmou um aumento na densidade de volume de osso neoformado, entre 7 e 60 dias, nos grupos controle, 5 ppm, 15 ppm e 50 ppm de F, com concomitante diminuição na densidade de volume de coágulo sangüíneo. A expressão de RANK-L e OPG não foi alterada pela exposição crônica ao fluoreto na água de beber durante os períodos estudados (p<0,05). A atividade da MMP-2 foi mais acentuada no período inicial, enquanto a MMP-9 teve maior atividade no período final. Concluiu-se que o F, em altas concentrações pode alterar a dinâmica do reparo ósseo alveolar diminuindo a formação de novo tecido ósseo, associado a um atraso na remissão do coágulo sangüíneo. Proteínas como RANK-L, OPG, MMP-2 e MMP-9 também podem ter sua expressão alterada no tecido ósseo em reparo. / The repair of bone has been investigated with the aim of promoting an improvement in the quality and time of the repair process. One element that has been studied by some groups is fluoride, it has been proposed as a treatment for osteoporosis (Europe). Thus, the objective was to comparatively evaluate the effect of fluoride administered in drinking water on alveolar bone repair in rats. This study used 4 groups of rats of 80 days (n=200), which received drinking water containing 5, 15, and 50 ppm of fluoride, and deionized water (control) throughout the experiment. These animals had their upper right incisor extracted. The animals were euthanized 0 hours, 7, 14, 21 and 30 and 60 days after extraction. Samples of blood plasma were obtained for analysis of fluoride concentration, and the region of the alveolus was collected for microscopic analysis (Hematoxylin eosin and Immunohistochemistry) and zymography (metalloproteinases 2 and 9 MMP-2 and 9). Analysis of fluoride concentration in blood plasma showed a greater presence of this element in the group treated with 50 ppm of F in periods of 21 and 30 days. Histological analysis detected a new bone formed in all animals up to 60 days, but the group of 50 ppm F showed lower bone formation at 14, 21 and 30 days. Morphometric analysis confirmed an increase in the volume density of newly formed bone, between 7 and 60 days in control groups, 5 ppm, 15 ppm and 50 ppm F, with a concomitant decrease in the volume density of blood clot. The expression of RANK-L and OPG was not altered by chronic exposure to fluoride in drinking water during the study period (p<0.05). The activity of MMP-2 was more pronounced in the early period, while MMP-9 had higher activity in the final period. It was conclude that the F, in high concentrations change the dynamics of the alveolar bone repair by decreasing the formation of new bone, associated with a delay in treating the blood clot. Proteins as RNK-L, OPG, MMP-2 and MMP-9 may also have changed in their expression in bone tissue repair.

Orientador: Marcela Rocha de Oliveira Carrilho / Tese (doutorado) - Univerisdade Estadual de Campoinas, Faculdade de Odontologia / Made available in DSpace on 2018-08-21T15:34:54Z (GMT). No. of bitstreams: 1 Vidal_CristinadeMattosPimenta_D.pdf: 9151819 bytes, checksum: a4149cbb1649c1a20ea45484421c229c (MD5) Previous issue date: 2012 / Resumo: Há pouco mais de 15 anos, as metaloproteinases da matriz (MMPs) foram consideradas enzimas responsáveis pela degradação da matriz orgânica dentinária na progressão da cárie. Neste meio tempo, porém, poucos estudos foram publicados tendo como base esta premissa. Recentemente, foi indicado que outras proteases como as cisteíno-catepsinas (CTs) poderiam atuar nesta degradação. Os objetivos deste estudo foram de avaliar e comparar a abundância e atividade proteolítica de diferentes MMPs (MMP-2, -8 e -9) e CTs (B, K e L) na matriz orgânica dentinária sadia e cariada. A abundância e distribuição destas enzimas in situ, isto é, na dentina sadia e cariada, foi realizada por imuno-histoquímica convencional. A abundância de MMP-2, -9 e CTs B e K, assim como a avaliação da estrutura do colágeno nos tecidos sadio e cariado, também foram realizadas por imunofluorescência. Também foi realizada a extração dessas enzimas da dentina por diferenes métodos: 1) extração com hidrocloreto de guanidina associado ao EDTA (G-EDTA); 2) ácido fosfórico (AF); 3) ácido acético (AC) e 4) hidrocloreto de guanidina associado ao ácido acético (G-AC). A avaliação da abundância de MMP-2, -8 e -9 e das CTs B e K foi realizada por western blot. A atividade enzimática nos extratos obtidos pelos protocolos de extração foi analisada por zimografia e por espectrofluorimetria. Além disso, o grau de solubilização da matriz orgânica dentinária em tecido sadio e cariado foi avaliado pela quantificação de hidroxiprolina (HYP). As imagens de imuno-histoquímica convencional mostraram abundância de MMP-2, -8 e -9 assim como das CTs B, K e L especialmente em dentina profunda e na região da pré-dentina, com maior abundância no tecido cariado. Os mesmos achados foram observados para a imunofluorescência, sendo que no tecido cariado, além da maior abundância das proteases, a estrutura do colágeno mostrou-se alterada. A partir da extração de proteases da dentina sadia e cariada, o ensaio de western blot revelou a presença de MMP-2, -8 e -9 e CTs B e K nos diferentes protocolos, com maior abundância no tecido cariado. A zimografia mostrou atividade gelatinolítica correspondente a MMP-2 para todos os protocolos de extração, com variação em função do extrato avaliado. A espectrofluorimetria mostrou variação na atividade proteolítica de MMPs e CTs em dentina sadia conforme o extrato e protocolo de extração avaliados. O grau de solubilização da matriz orgânica dentinária foi maior no tecido cariado. A liberação de HYP também variou em função dos métodos de extração testados, com maior liberação observada para o protocolo de extração AC. Pode-se concluir que, além das MMP-2, -8 e -9, as CTs B, K e L estão presentes na dentina humana sadia e cariada, com maior abundância no tecido cariado, sendo que a presença e atividade dessas enzimas pode variar em função do método de extração de proteínas utilizado. Os resultados desse estudo sugerem que MMPs e CTs podem atuar em conjunto na degradação do matriz orgânica dentinária no processo de cárie e ainda reforçam a teoria para a fisiopatologia da cárie, em que um mecanismo proteolítico endógeno seria o responsável pela destruição da estrutura dental / Abstract: There are just over 15 years since the matrix metaloproteases (MMPs) were considered responsible for the degradation of dentin organic matrix in caries progression. From then on, only few studies were published based on this premise. More recently, it was showed that other proteases, like the cysteine-cathepsins (CTs), could also participate of such degradation process. The objective of this study was to evaluate and compare the abundance and proteolytic activity of different MMPs (MMP-2, -8 and -9) and CTs (B, K e L) in sound and carious dentin. Firstly, the abundance and localization of enzymes in sound and carious dentin was performed by conventional immunohistochemistry. In addition, MMP-2, -9 and CTs B and K abundance associated with evaluation of collagen structure in sound and carious dentin was done by immunofluorescence. Different dentin enzyme-extraction methods were also tested: 1) using guanidine-hydrochloride associated with EDTA (G-EDTA); 2) phosphoric acid (AF); 3) acetic acid and 4) guanidine-hydrochloride associated with acetic acid (G-AC). The presence of MMP-2, -8 e -9 and CTs B and K was evaluated by western blot. Proteolytic activity in dentin extracts was analyzed by zymography and spectrofluorometrically. The organic matrix solubilization in sound and carious dentin was evaluated by hydroxyproline assay (HYP). Immunohistochemistry showed the presence of MMP-2, -8 and -9 and CTs B, K and L, especially in deep dentin and predentin, showing more abundance in caries. The same results were observed in immunofluorescence, which also showed that the collagen structure was modified in carious dentin. The presence of MMP-2, -8 and -9 and CTs B and K was showed in western blot in all extraction protocols evaluated, with more abundance in carious when compared to sound dentin. Gelatinolytic activity corresponding to MMP-2 was showed in zymography, but it was different according to the extraction protocol. Proteolytic activity for MMPs and CTs was observed spectrofluorometrically in sound dentin and some variation was observed according to the extraction protocol. Dentin organic matrix solubilization was confirmed by HYP, with higher HYP released in AC extraction protocol. When comparing sound and carious dentin, organic matrix degradation was higher in caries. It can be concluded that, besides MMP-2, -8 and -9, the CTs B, K and L are also present in sound and carious dentin, with higher abundance in caries. However, the presence and activity may vary according to the extraction method used. The results indicate that both MMPs and CTs may act in concert in dentin organic matrix degradation in caries progression and also support the physiopathology theory for caries in which a host-derived proteolytic mechanism would be responsible for dentin degradation / Doutorado / Materiais Dentarios / Doutora em Materiais Dentários

Abstract Numerous adverse effects and health problems are associated with smoking, but the mechanisms of the adverse effects of smoking on skin are not well documented. The aim of the present study was to elucidate the effects of smoking on the structure, metabolism and appearance of skin. The study population consisted of 98 Finnish males, of whom 47 were current smokers and 51 non-smokers. The main parameters under evaluation were the appearance and physical qualities of skin, including skin wrinkling, thickness and elasticity. Biochemical analyses were performed to assess the rate of type I and III collagen biosynthesis as well as the degradation of the extracellular matrix (ECM) of skin in terms of matrix metalloproteinase levels (MMPs). To compare the morphology of skin between the groups, histological and immunohistological studies were performed, including assessments of the proportional area and width of dermal elastic fibres. The results revealed decreased synthesis of type I and III collagens in smokers as well as changes in the regulatory mechanisms which control the turnover of these and other extracellular matrix proteins. The level of matrix metalloproteinase -8 (collagenase-2), a protease degrading both type I and type III collagen, in suction blister fluid was significantly higher in smokers, indicating enhanced degradation of these collagens. In skin tissue samples, the levels of the active forms of MMP-8 and MMP-9 were significantly lower in smokers compared to non-smokers. Serum levels of MMP-8 were slightly but not significantly higher in smokers, whereas the levels of the matrix metalloproteinases MMP-2 and MMP-9 (72-kDa and 92-kDa gelatinase, respectively) were significantly higher in smokers compared to non-smokers. Salivary MMP-8 and MMP-9 were lower in smokers compared to non-smokers, but only the latter showed a statistically significant difference. The levels of the tissue inhibitor of matrix metalloproteinases (TIMP-1) were significantly lower in the suction blister fluid of smokers compared to non-smokers. In general, there were no significant differences in skin thickness and elasticity or regeneration of barrier function, nor in the amount or width of elastic fibres between the groups. We did not observe significant differences in skin wrinkling between smokers and non-smokers, but smokers looked older than their age compared to non-smokers. It can be concluded that the rate of type I and III collagen synthesis in skin is decreased and the regulation of ECM turnover is altered in smokers, which may lead to deterioration of the tensile strength and resiliency of skin in the long term, even though no morphological changes were detected in the present study.

Abstract Degradation of extracellular matrix (ECM) and basement membrane (BM) are required both in normal physiological conditions such as wound healing and in pathological tissue remodelling such as chronic ulcers and cancers. Matrix metalloproteinases (MMPs) are an enzyme family, which can cleave most ECM and BM components. They are associated with physiological and pathological processes but their exact roles are still largely unknown. The expression of MMP-8 and MMP-26 in acute and chronic human cutaneous wounds using histological and cell culture methods were investigated. MMP-8 was expressed in epithelial cells, neutrophils, and other inflammatory cells especially in chronic ulcers while in acute wounds MMP-8 expression was weak or absent. MMP-26 was temporarily present in acute wounds while it was strongly expressed in close vicinity to the BM in multiple cell types of most chronic ulcers. In vitro keratinocyte wound assay showed that MMP-8 and -26 were expressed in migrating cells. Bone formation, collagen metabolism, and inflammation in MMP8-/- mice tooth extraction wounds and also periapical lesion formation were analysed. No differences between wild type or MMP-8-deficient mice in the new bone area or periapical lesion size were found. However, type III procollagen production was increased and inflammatory cell influx was decreased in MMP8-/- mice. In addition, Fas ligand (FasL) production was increased in mandibular alveolar mucosa but decreased in alveolar bone of MMP-8 deficient mice. MMP-8 was also found to cleave FasL in vitro. A total of 90 human mobile tongue squamous cell carcinoma (SCC) samples were collected. Bryne’s malignancy scores, thickness of the SCCs, expression of microvessel density (CD31 and factor VIII), cyclooxygenase-2 (COX-2), the laminin-5 (currently termed laminin-332) γ2-chain, integrin αvβ6, estrogen receptor-α (ER-α), estrogen receptor-β (ER-β), and MMPs (-2, -7, -8, -9, -20, and -28) were analysed. The high expression of MMP-8 was associated with a better prognosis for the patients, particularly in females. In addition, tongue carcinoma formation in MMP8-/- mice was investigated. Tongue SCC developed more often in MMP8-/- female mice than wild type littermates. In addition, MMP-8 can cleave ER- α and -β and estrogen can induce MMP-8 production in vitro. A total of 22 biopsies, 10 resection sections, and three lung metastases of 25 osteosarcoma patients samples were stained with MMP-2, -8, -13, -26, and tissue inhibitor of metalloproteinase-1 (TIMP-1) using immunohistological methods. Expression of these markers was mostly present in sarcoma cells but MMP-8 was not present in lung metastases. In resection sections, chemotherapy altered MMP-2, -8, and -13 expressions compared to biopsies. However, an association between the expression and prognosis of osteosarcoma patients could not been found. In conclusion, MMP-8 seems to be an estrogen-related protective factor in tongue SCC and can regulate ECM and BM components and inflammation during wound healing. Further studies are needed to evaluate the exact function especially of MMP-8 in human osteosarcoma.

Orientadores: Sérgio Luis Felisbino, Francisco Eduardo Martinez / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T11:21:35Z (GMT). No. of bitstreams: 1 Fontanelli_BeatrizAparecidaFioruci_D.pdf: 2932311 bytes, checksum: fc7d79d2b10d42488acf176230c43c39 (MD5) Previous issue date: 2014 / Resumo: Pesquisadores têm mostrado que o consumo crônico de etanol altera a concentração do ácido retinóico, metabólito ativo da vitamina A, em muitos órgãos, incluindo a próstata. O ácido retinóico é essencial para o desenvolvimento normal da próstata e para a manutenção de sua homeostase. Alterações na concentração e no metabolismo do ácido retinóico estão relacionadas com o desenvolvimento de lesão na próstata. Adicionalmente, a atividade de metaloproteinases da matriz extracelular (MMPs), também está relacionada com o desenvolvimento de alterações na próstata. Assim, o presente trabalho teve por objetivo descrever os efeitos dos consumos, baixo e alto, de etanol sobre as proteínas envolvidas na síntese e no catabolismo do ácido retinóico (artigo I), bem como, sobre a atividade enzimática das MMPs (artigo II) nos lobos dorsais e laterais da próstata.Vinte ratos adultos (~ 90 dias de idade) de cada variedade, UChA e UChB, foram divididos nos grupos (n=10/grupo): UChA (consumo baixo de etanol, 0,2-2 g/kg/dia), UChAC (ratos que não consumiram etanol); UChB (consumo alto de etanol, > 2g/kg/dia), UChBC (ratos que não consumiram etanol).Após o período experimental (~ 150 dias de idade), os ratos foram eutanasiados por decapitação e os lobos dorsais e laterais das próstatas foram coletados e dissecados: (1) para avaliar os níveis e a localização das proteínas ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1, CYP2E1, através de western blot e imuno-histoquímica, bem como, a atividade catabólica das CYP26A1, CYP26B1, CYP2E1 por ensaio bioquímico e quantificação por HPLC-MS/MS; (2) e para avaliar a atividade da MMP-2 e da MMP-9, e os níveis dos inibidores teciduais de metaloproteinases (TIMP-1/ TIMP-2), através de zimografia e Elisa, respectivamente. No grupo UChA, a ALDH1A3 aumentou na próstata dorsal, enquanto as proteínas ALDH1A1 e ALDH1A2 diminuíram na próstata lateral. No grupo UChB, as proteínas ALDH1A1, ALDH1A2, e ALDH1A3 aumentaram na próstata dorsal, enquanto a ALDH1A3 diminuiu no lobo lateral. A concentração do ácido retinóico aumentou, indicando diminuição da atividade da CYP2E1, e diminuiu quando se avaliou a CYP26, indicando aumento de sua atividade na próstata dorsal do UChB. Além disso, o ácido retinóico diminuiu quando se avaliou a atividade de CYP total nos grupos experimentais, sendo somente aumentado na próstata lateral do UChA. O consumo baixo de etanol (grupo UChA) diminuiu a atividade das MMP-2 e MMP-9 e o nível das TIMP-2 e TIMP-1 na próstata lateral, enquanto que na próstata dorsal o etanol diminuiu a atividade de MMP-2 e o nível de TIMP-1. Por outro lado, no grupo UChB, o etanol diminuiu somente a atividade da MMP-9 na próstata lateral e não alterou os níveis de TIMP-1 e TIMP-2.Nossos resultados indicam que o etanol modula a síntese e o catabolismo do ácido retinóico na próstata do rato de modo dependente de sua concentração. Além disso, o consumo crônico e baixo de etanol diminui a atividade das metaloproteinases -2 e -9, sendo a próstata lateral o lobo afetado e, portanto, mais susceptível a estas alterações, do que o lobo prostático dorsal. / Abstract: Researchers have shown that chronic ethanol consumption alters the retinoic acid concentration, an active metabolite of vitamin A, in many organs including the prostate. The retinoic acid is essential for the normal development of prostate and for maintaining its glandular homeostasis. Changes in concentration and metabolism of retinoic acid are related to lesion development in the prostate. Additionally, the activity of matrix metalloproteinases (MMPs), also relates to development of alterations in prostate. Thus, this study aimed to describe the effects of low and high doses of ethanol consumption, on the proteins involved in the synthesis and catabolism of retinoic acid (Article I), as well as on the enzymatic activity of MMPs (Article II) the dorsal and lateral lobes of the prostate. Twenty adult rats (~ 90 days old) of each variety, UChA and UChB, were divided into groups (n = 10 / group): UChA (low ethanol consumption, 0.2-2 g /kg / day), UChAC (rats not consumed ethanol); UChB (high ethanol consumption, > 2 g/ kg/ day), UChBC (rats not consumed ethanol). After the experimental period (~ 150 days old), the rats were euthanized by decapitation and dorsal and lateral lobes of the prostates were collected and dissected: (1) for evaluate the levels and location of the proteins ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1, CYP2E1, by western blot and immunohistochemistry, as well as, catabolic activity of CYP26A1, CYP26B1, CYP2E1 by biochemical assay and quantification by HPLC¿MS/MS; (2) and to evaluate the activity of MMP-2 and MMP-9, and the levels of tissue inhibitors of metalloproteinases (TIMP-1 / TIMP-2) using zymography and ELISA, respectively. In the UChA group, ALDH1A3 increased in dorsal prostate, while the proteins ALDH1A2 and ALDH1A1 decreased in the lateral prostate. In the UChB group, the proteins ALDH1A1, ALDH1A2 and ALDH1A3 increased in the dorsal prostate, while ALDH1A3 decreased in the lateral lobe. The concentration of retinoic acid increased, indicating a decrease in the CYP2E1 activity, and decreased when evaluated CYP26, indicating increased of CYP26 activity in the UChB dorsal prostate. Furthermore, the retinoic acid decreased when assessing the CYP total activity in the experimental groups, but only increased in the lateral prostate of UChA. The low ethanol consumption (UChA group) reduced the activities of MMP-2 and MMP-9 and the levels of TIMP-2 and TIMP-1 in the lateral prostate, while dorsal prostate the ethanol decreased the MMP-2 activity and the level of TIMP-1. On the other hand, in the UChB group, ethanol only decreased the activity of MMP-9 in the lateral prostate and did not alter the levels of TIMP-1 and TIMP-2. Our results indicate that ethanol modulates the synthesis and catabolism of retinoic acid in the rat prostate in a concentration-dependent manner. In addition, the chronic and low consumption of ethanol decreases the activity of metalloproteinases -2 and -9 in the lateral lobe prostate, showing that this organ is more susceptible to these changes than dorsal lobe prostate / Doutorado / Anatomia / Doutora em Biologia Celular e Estrutural

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontics (RE) is a treatment alternative for the infected immature tooth to establish an environment in the canal that enables continued root development and the growth of pulp or pulp-like tissue within the canal. A scaffold created in the canal encourages the formation of vital tissue. The porcine sub-intestinal-submucosa (SIS) membrane, Dynamatrix®, has the potential to serve as an endodontic scaffold. Research at Indiana University School of Dentistry (IUSD) has shown that Dynamatrix® can support the growth of human dental pulp stem cells (HDPSC) and human pulp fibroblasts (HPF). Positive angiogenic cytokine profiles were seen after these cells were seeded on Dynamatrix®. Endothelial cells play an important role in the formation of blood vessels and are a source of angiogenic cytokines. Exposure of these cells to DynaMatrix® may result in a positive angiogenic profile for both cytokines and matrix metalloproteinases (MMPs). Objective: The aim of this in-vitro study was to investigate if the exposure of human endothelial cells to the DynaMatrix® membrane would result in differences in the expression of cytokines and MMPs that play roles in angiogenesis. Materials and Methods: Human endothelial cells (HUVECs) were obtained from American Type Culture Collection (ATTC, Manassas, VA) and used in this study. Groups were established as follows: (a) Group 1: HUVECs seeded in culture media only, (b) Group 2: DynaMatrix® membrane incubated alone in the serum-media without any cells, and (c) Group 3: HUVECs seeded on DynaMatrix® membranes. After 72 hours of incubation, the conditioned media were collected and analyzed for the expression of 20 angiogenic cytokines and MMPs utilizing cytokine and MMP protein arrays. The density of each cytokine and MMP expressed was measured, averaged, and statistically analyzed by ANOVA. Results: Exposure of human umbilical vein endothelial cells (HUVECs) to the DynaMatrix® membrane resulted in a positive angiogenic profile for both cytokines and MMPs. Conclusion: This work furthers the evidence for the potential of DynaMatrix® to serve as a more predictable scaffold in RE.

Matrix metalloproteinases (MMPs) are known to be the mediators of extracellular matrix remodeling. Increased levels of matrix metalloproteinases, particularly MMP-9, have been found in the aqueous humor of patients with glaucoma. However the exact role of MMP-9 in glaucomatous changes is not understood. Previous results from the West-Mays’ lab indicated that MMP-9 deficient (knockout - KO) mice exhibit elevated IOP, in the absence of distinct morphological changes in the anterior chamber. In the current thesis, I investigated whether the elevated IOP in MMP-9KO mice leads to RGC death. Wild type and KO littermates at different age groups: 2-3 months, 3-4 months, 6-8 and 9-12 months were studied. IOP was measured using TonoLab rebound tonometer. My results demonstrated that IOP was significantly increased in MMP-9KO mice compared to control littermates at all ages examined. To investigate if the elevated IOP was due to a difference in central corneal thickness (CCT), CCT measurements were made between WT and KO mice using ultrasound pachymeter. There was no difference in CCT demonstrating that the elevated IOP observed in MMP-9KO mice was not related to changes in corneal thickness. To determine whether the elevated IOP led to RGC death, the animals were sacrificed, eyes were enucleated and retinas (n=4) from both WT and KO animals were dissected and stained with Brn-3a antibody. Additional eyes were harvested from both WT and KO mice for histological and immunofluorescence studies. I found no observable difference in Brn3a+ RGC count between MMP9-WT and KO mice. Furthermore, no difference in retinal morphology, glial reactivity and laminin expression between WT and KO mice was observed. In the future it will be important to investigate whether elevated IOP in the MMP-9KO mice leads to optic nerve axonal loss and further investigate the possibility that the MMP-9KO retina is neuroprotected. / Thesis / Master of Science (MSc)