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Orientador: José Eduardo Tanus dos Santos / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-11-07T16:45:40Z (GMT). No. of bitstreams: 1 Gomes_ValeriaAguiar_D.pdf: 9967733 bytes, checksum: 0f5a9488cc56b0e1272b80bd9cb6ea0b (MD5) Previous issue date: 2012 / Resumo: A síndrome do ovário policístico (SOP) é a endocrinopatia mais comum em mulheres na idade reprodutiva e está frequentemente associada a alguns fatores de risco cardiovascular. A grande maioria das doenças cardiovasculares (DCV) ocorre inicialmente com o remodelamento vascular, em que as metaloproteinases de matriz (MMPs) são os principais mediadores. Sendo assim, o objetivo do presente estudo foi comparar os níveis plasmáticos da MMP-2 e da MMP-9 e dos inibidores teciduais de MMPs (TIMPs) das pacientes com SOP com as controles saudáveis e examinar se os níveis desses biomarcadores estão associados com às características clínicas e bioquímicas da SOP. Além disso, avaliar o efeito do anticoncepcional oral sobre os níveis plasmáticos de MMPs e respectivos inibidores endógenos nas mulheres com SOP. Para isso, na primeira parte do estudo, avaliamos 65 controles ovulatórias e 80 pacientes com SOP. As concentrações plasmáticas de MMP-8, MMP-9, TIMP-1, TIMP-2 foram medidas por Elisa e, as de MMP-2, por zimografia. Os níveis de MMP-2, MMP-8, MMP-9 e TIMP-1 não foram significativamente diferentes entre os grupos (p? 0,05). Pacientes com SOP apresentaram menores níveis plasmáticos de TIMP-2 do que as controles saudáveis (182,30 ± 5,60 vs. 204,20 ± 7,28 ng/ml; p ?0,05). Além disso, a testosterona foi preditor independente dos níveis de TIMP-2 (estimativa = -0,35, p = 0,04) e da razão MMP-9/TIMP-1 (estimativa = 0,01, p = 0,04). Para avaliar se a redução do hiperandrogenismo iria promover alguma alteração no perfil das MMPs, foram analisadas 20 mulheres com SOP que queriam contracepção hormonal (grupo SOP- ACO), 20 mulheres ovulatórias que desejavam contracepção hormonal (grupo controle- ACO) e 15 mulheres ovulatórias que desejavam contracepção não-hormonal (grupo controle). O tratamento com ACO contendo 30 mcg de etinilestradiol/2mg de acetato de clormadinona durante 6 meses reduziu significativamente as concentrações plasmáticas de MMP-2 no grupo controle ( de 1,44 ± 0,11 unidades arbitrárias no tempo basal para 1,22 ± 0,07 unidades arbitrárias após 6 meses; p = 0,01), e no grupo SOP ( de 1,43 ± 0,08 unidades arbitrárias no tempo basal para 1,25 ± 0,09 unidades arbitrárias após 6 meses; p = 0,007). O ACO reduziu as concentrações de TIMP-2 e TIMP-1 no grupo controle (todos p ?0,05), mas não teve efeitos na MMP-9 plasmática e nas razões MMP-2/TIMP-2 e MMP-9/TIMP-1 (todos p? 0,05) nos grupos avaliados. Os achados do presente estudo indicam que as mulheres com SOP possuem um desequilíbrio nas razões MMP-2/TIMP-2 e MMP-9/TIMP-1, bem como níveis reduzidos de TIMP-2. Parte desses achados estão relacionados ao hiperandrogenismo presente nessas mulheres. Na segunda parte do estudo, observamos que a redução do hiperandrogenismo, promovido pelo tratamento em longo prazo com o ACO, reduziu as concentrações plasmáticas de MMP-2. Considerando o desequilíbrio no perfil das MMPs apresentado pelas mulheres com SOP e, as possíveis consequências decorrentes desse cenário, o tratamento com ACO se mostra benéfico nessas pacientes, podendo reduzir os riscos de futuras complicações cardiovasculares / Abstract: The polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age and it is often associated with some cardiovascular risk factors. The majority of cardiovascular disease (CVD) occurs initially with vascular remodeling in which matrix metalloproteinases (MMPs) are key mediators. Therefore, the aim of this study was to compare plasma levels of MMP-2 and MMP-9 and tissue inhibitors of MMPs (TIMPs) of PCOS patients with healthy controls and to examine whether the levels of these biomarkers are associated with clinical and biochemical characteristics of PCOS. In addition to it, our goal was to evaluate the effect of oral contraceptives on plasma levels of MMPs and their endogenous inhibitors in women with PCOS. In order to prove it, in the first part of the study we evaluated 65 controls and 80 patients with ovulatory PCOS. The plasma concentration of MMP-8, MMP-9, TIMP-1 and TIMP-2 were measured by Elisa, and MMP-2 by zymography. The levels of MMP-2, MMP-8, MMP-9 and TIMP-1 were not significantly different between groups (p? 0.05). PCOS patients had lower their plasma levels of TIMP-2 than healthy controls ones (182,30 ± 5,60 vs. 204,20 ± 7,28 ng/ml; p = 0,02). Furthermore, testosterone was an independent predictor of the levels of TIMP-2 (estimate = -0.35, p = 0.04) and the MMP-9/TIMP- 1 ratio (estimate = 0.01, p = 0.04). To assess whether the reduction of hyperandrogenism would promote a change in the profile of MMPs, we analyzed 20 women with PCOS who wanted to hormonal contraception (OC-PCOS group), 20 ovulatory women who required hormonal contraception (OC-control group) and 15 ovulatory women who wanted non-hormonal contraception wanted a nonhormonal contraception (non-OC control group). Treatment with OC containing 2 mg chlormadinone acetate/30 ?g ethinylestradiol for 6 months significantly reduced plasma MMP-2 concentrations in the OC-control (from 1.44 ± 0.11 arbitrary units at baseline to 1.22 ± 0.07 arbitrary units after 6 months; p = 0.01) and the PCOS groups (from 1.43 ± 0.08 arbitrary units at baseline to 1.25 ± 0.09 arbitrary units after 6 months; p = 0.007) and TIMP-2 and TIMP-1 levels (448.0 ± 66.3 ng/mL versus 349.0 ± 40.9 ng/mL; p = 0.009) in the OC-control group (all p ?0.05) but had no effects on MMP-9 concentrations or on MMP-2/TIMP-2 and MMP-9/TIMP- 1 ratios (all p? 0.05) in any group. The results of this study indicate that women with PCOS have an imbalance in the MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios and reduced levels of TIMP-2. Parts of these findings are also related to hyperandrogenism presence in these women. In the second part of the study, we observed that the reduction of hyperandrogenism promoted by long-term treatment with the OC reduced plasma concentrations of MMP-2. Given the imbalance in the profile of MMPs presented by women with PCOS and the possible consequences of this scenario, treatment with OC shows beneficial in these patients may reduce the risk of future cardiovascular complications / Doutorado / Farmacologia / Doutora em Farmacologia

INTRODUÇÃO: O aneurisma da aorta abdominal (AAA) tem grande importância clínica em função de sua incidência e das complicações que pode acarretar, entretanto sua etiopatogenia não está completamente esclarecida. A associação entre tabagismo e desenvolvimento de AAA tem sido repetidamente confirmada. Apesar de o AAA ter sido inicialmente atribuído à aterosclerose, observou-se associação negativa entre diabetes (um dos principais fatores de risco para aterosclerose) e doença vascular aneurismática. O estudo biomecânico e histológico da parede aórtica pode contribuir para a elucidação da etiopatogenia dos aneurismas. OBJETIVOS: Avaliar as propriedades biomecânicas e histológicas da aorta abdominal de ratos em três situações: exposição à fumaça do cigarro, induzidos ao desenvolvimento do diabetes mellitus e com a associação desses dois fatores. MÉTODOS: Setenta e cinco ratos Wistar foram distribuídos em quatro grupos: controle (GC), tabagista (GT), diabético (GD), diabético e tabagista (GDT). Os ratos dos GT e GDT foram expostos à fumaça de cigarro por 30 minutos ao dia, 5 dias por semana. O diabetes foi induzido por injeção endovenosa de estreptozotocina. Após 16 semanas, os animais foram sacrificados para a coleta da aorta abdominal. Testes de tração uniaxiais destrutivos foram realizados para a obtenção das seguintes propriedades biomecânicas: força, tensão, estresse, deformação e energia de deformação. A análise histológica desses fragmentos consistiu na avaliação das fibras colágenas e elásticas e verificação da deposição de elementos da matriz extracelular na túnica média e avaliação da sua composição. Através da zimografia foi quantificada a atividade da metaloproteinase-2 nos espécimes aórticos obtidos. RESULTADOS: Foram analisados os testes biomecânicos válidos de 52 espécimes, sendo que 11 pertenciam ao GC, 10 ao GD, 16 ao GT e 15 ao GTD. A análise biomecânica dos fragmentos não revelou diferença entre os grupos controle, GD, GT e GDT. A deposição de colágeno também não apresentou diferença estatística significativa entre os grupos estudados. A contagem total de lâminas elásticas foi maior nos ratos diabéticos (GD e GDT) quando comparados aos do GT. Foi observada resposta inflamatória mais intensa, com significância estatística, em todos os grupos estudados quando comparados ao GC. A atividade da MMP-2 apresentou diminuição no GD em relação ao GDT, com significância estatística. CONCLUSÕES: As propriedades biomecânicas da parede da aorta de ratos relacionadas à resistência e elasticidade não apresenta diferença entre o GC e os GD, GT e GDT. As alterações histológicas relacionadas à contagem total e fragmentação das lâminas elásticas, deposição de matriz pericelular e perda/substituição celular na túnica média são significativas na parede da aorta do GD, GT e GDT em relação ao GC. A atividade da MMP-2 na aorta do GD é menor que na aorta do GDT / INTRODUCTION: Abdominal aortic aneurysm (AAA) is of great clinical importance due to its incidence and complications, but its etiopathogenesis is not fully understood. The association between smoking and AAA development has been repeatedly confirmed. Although AAA was initially attributed to atherosclerosis, there was a negative association between diabetes (a major risk factor for atherosclerosis) and aneurysmal vascular disease. The biomechanical and histological study of the aortic wall may contribute to the elucidation of the etiopathogeny of the aneurysms. OBJECTIVES: To evaluate the biomechanical and histological properties of the abdominal aorta of rats in three situations: exposed to cigarette smoke, induced to the development of diabetes mellitus, and the association of these two factors. METHODS: Seventy-Five Wistar rats were divided into four groups: control (CG), smoker (GT), diabetic (GD), diabetic and smoker (GDT. The GT and GDT rats were exposed to cigarette smoke for 30 minutes a day, 5 days a week. Diabetes was induced by intravenous injection of streptozotocin. After sixteen weeks, the animals were sacrificed for collection of the abdominal aorta. Uniaxial destructive tensile tests were performed to obtain the following biomechanical properties: maximal force, failure stress, failure tension, failure strain and failure strain energy. The histological analysis of these fragments consisted in the evaluation of the collagen and elastin and verification of the deposition of elements of the extracellular matrix in the tunica media and evaluation of its composition. The activity of metalloproteinase-2 in the aortic specimens obtained was quantified by zymography. RESULTS: A total of 52 strips were studied (11 from GC, 10 from GD, 16 from GT and 15 from GDT. The biomechanical analysis of the fragments was not different between the control group and the GD, GT and GDT groups. Collagen deposition also did not present a statistically significant difference between the studied groups. The total of elastic fibers was higher in diabetic rats (GD and GDT) when compared to GT. A higher inflammatory response was observed, with statistical significance, in all groups studied when compared to CG. The activity of MMP-2 showed a decrease in GD in relation to GDT, with statistical significance. CONCLUSIONS: The biomechanical properties of the aortic wall of rats related to resistance and elasticity do not present a difference between GC and GD, GT and GDT. Histological changes related to total count and fragmentation of the elastic lamina, pericellular matrix deposition, and cell loss / substitution in the tunica media are significant in the aorta wall of GD, GT and GDT in relation to GC. The activity of MMP-2 in the GD aorta is smaller than in the GDT aorta

A diminuição da biodisponibilidade do óxido nítrico (NO) e o aumento na atividade das metaloproteinases da matriz extracelular (MMPs) são alguns dos principais mecanismos fisiopatogênicos envolvidos nas doenças cardiovasculares (DCV). Foi demonstrado que o NO pode reduzir a expressão e atividade de MMPs em células musculares lisas vasculares, células mesangiais, entre outras. Em outro estudo, foi mostrado que drogas inibidoras da NO sintase (NOS) podem aumentar a expressão de MMPs. Apesar de o NO apresentar-se diminuído e as MMPs aumentadas durante as DCV, não há evidência clara de que os níveis de NO possam modular diretamente a atividade de MMPs no aparelho cardiovascular. Também não se sabe se este possível efeito seria mediado pelo NFB, nem se este possível efeito é dependente da ativação da guanilato ciclase [que promove a formação de GMP cíclico (GMPc)]. Desta maneira, este estudo teve como objetivos: A) investigar os efeitos de drogas ativadoras da via NO-GMP sobre os aumentos da atividade e expressão de MMP-9 em células endoteliais que acontecem sob efeito de phorbol 12-miristato 13-acetato (PMA, droga indutora da expressão de MMP-9); e B) determinar se a inibição de NOS em células endoteliais é acompanhada por aumento da atividade e expressão de MMPs, e C) determinar se estes efeitos são dependentes da ativação de NFB ou da formação de GMPc. Células endoteliais de veia umbilical humana (HUVECs) foram cultivadas em DMEM e tratadas por 24 horas com 10 nmol/L de PMA ou diferentes concentrações de drogas ativadoras da via NO-GMP ou de inibidor da NOS. Meio de cultura condicionado ou lisado celular foram coletados e submentidos aos ensaios de zimografia, ELISA, immunoblotting ou análise da concentração de nitrito. Os tratamentos com detanonoato, SNAP, atorvastatina e nitrito de sódio diminuíram os aumentos da atividade gelatinolítica e expressão de MMP-9 estimulados por PMA sem afetar as concentrações do inibidor tecidual da metaloproteinase da matriz-1 (TIMP-1). Esses efeitos não foram modificados pelos tratamentos com ODQ (inibidor da guanilato ciclase solúvel) ou 8- bromo-cGMP (um análogo de GMPc) ou hemoglobina (um sequestrador de NO). Enquanto o PMA aumentou a concentração de fosfo-NFB p65, os tratamentos com SNAP, atorvastatina ou nitrito não apresentaram influência sobre esse efeito. O tratamento com L-NAME, um inibidor da NOS, não apresentou efeito sobre a atividade gelatinolítica de MMP-9. Em conclusão, foram demonstrados que os efeitos inibitórios de drogas ativadoras da via NO-GMPc sobre a produção estimulada de MMP-9 em células endoteliais são independentes de mecanismos mediados por GMPc e NFB, e a inibição da NOS não altera a atividade de MMP-9. / Impaired nitric oxide (NO) bioavailability and imbalanced matrix metalloproteinases (MMPs) activity have important roles in the pathophysiological mechanisms involved in cardiovascular disease (CVD). It was shown that NO can reduce MMPs expression and activity in vascular smooth muscle cells, mesangial cells, and others. In another study, a NO synthase (NOS) inhibitor has increased MMPs expression. Although NO was decreased and MMPs was increased during CVD, there is clear evidence that NO levels can directly modulate MMPs activity in the cardiovascular system. Also, it is not known whether this effect would be mediated by NFB, nor whether this effect is dependent on guanylate cyclase activity (which promotes the formation of cyclic GMP). Thus, this project aims to study whether A) the effect of NO donors might decrease MMP-9 activity and expression in endothelial cells stimulated by phorbol 12-myristate 13-acetate (PMA) (a well-known inducer of MMP-9), and B) the effect of NOS inhibitors might increase MMPs activity and expression in endothelial cells, and C) to determine whether those effects are mediated by the NFB activation or cGMP levels. Endothelial cells from human umbilical vein (HUVECs) were grown in modified DMEM and were treated for 24 hours with 10 nmol/L PMA or different concentrations of NO-GMPc pathway stimulating drugs or NOS inhibitor. Conditioned medium or cell lysate were collected after treatments and analyzed by zymography, ELISA, immunoblotting or to determine nitrite concentration. Detanonoate, SNAP, atorvastatin or sodium nitrite treatments attenuated PMA-induced increases in MMP- 9 gelatinolytic activity and expression, but they had no effect on tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) concentrations. These effects were not modified by ODQ (a soluble guanylate cyclase inhibitor), or 8-bromo-cGMP (cGMP analogue), or hemoglobin (a NO scavenger). While PMA increased phospho-NFB p65 concentration, SNAP, atorvastatin or nitrite had no influence on this effect. The treatment with L-Name, a NOS inhibitor, had no effect on MMP-9 activity. In conclusion, this study shows that the inhibitory effects of NO-GMPc pathway stimulating drugs on MMP-9 production by endothelial cells are independent of cGMP- and NFB-mediated mechanisms, and NOS inhibitor had no effect on MMP-9 levels

Rupture of atherosclerotic plaque is one of the primary causes of death due to cardiovascular disease. The factors directing plaque progression to instability are poorly understood. It is well-known that arteries respond to changes in mechanical stress by remodeling, and that remodeling is mediated by the inflammatory response. Studies have shown that both mechanical stress and markers of inflammation are increased in the fibrous cap and shoulder regions of plaque, where rupture most often occurs. In this study we hypothesized that there are spatial relationships between the local mechanical environment and expression of markers of inflammation in atherosclerosis, and that these relationships are plaque-progression dependent. To test these hypotheses, we analyzed cross-sections at intervals along the length of human coronary atherosclerotic arteries. For each cross-section, a heterogeneous finite element model was developed to determine the spatial distribution of stress. In addition, novel techniques for quantifying inflammatory markers at high spatial resolution were used to determine the distributions of inflammatory markers. The distributions of stress and five markers of inflammation activated NF-kB, macrophages, MMP-1, nitrotyrosine, and microvessels - were then compared to determine whether spatial relationships exists. We demonstrated that the probability of activated NF-kB expression increases monotonically with increasing stress in all stages of plaque progression. This indicates that the relationship between mechanical stress and NF-kB activation is a player throughout the disease process. We found that the relationship between mechanical stress and macrophages is highly dependent on the state of plaque progression. In intermediate stages of progression macrophages increase with moderate stress but drop off again at very high stresses, while in the advanced stage macrophages continue to increase monotonically with stress. We found that MMP1 increases with stress in stages of progression where active remodeling is occurring, but decreases with stress in mature stable plaque. We found no relationship between mechanical stress and nitrotyrosine expression or microvessels. Taken together, these results support the role of mechanical stress in instigating and maintaining the inflammatory response, and help explain how mechanical input is able to direct the complex biological changes involved in remodeling.

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Height is a complex physical trait that displays strong heritability. Adult height is related to length of the long bones, which is determined by growth at the epiphyseal growth plate. Longitudinal bone growth occurs via the process of endochondral ossification, where bone forms over the differentiating cartilage template at the growth plate. Estrogen plays a major role in regulating longitudinal bone growth and is responsible for inducing the pubertal growth spurt and fusion of the epiphyseal growth plate. However, the mechanism by which estrogen promotes epiphyseal fusion is poorly understood. It has been hypothesised that estrogen functions to regulate growth plate fusion by stimulating chondrocyte apoptosis, angiogenesis and bone cell invasion in the growth plate. Another theory has suggested that estrogen exposure exhausts the proliferative capacity of growth plate chondrocytes, which accelerates the process of chondrocyte senescence, leading to growth plate fusion. The overall objective of this study was to gain a greater understanding of the molecular mechanisms behind estrogen-mediated growth and height attainment by examining gene regulation in chondrocytes and the role of some of these genes in normal height inheritance. With the heritability of height so well established, the initial hypothesis was that genetic variation in candidate genes associated with longitudinal bone growth would be involved in normal adult height variation. The height-related genes FGFR3, CBFA1, ER and CBFA1 were screened for novel polymorphisms using denaturing HPLC and RFLP analysis. In total, 24 polymorphisms were identified. Two SNPs in ER (rs3757323 C>T and rs1801132 G>C) were strongly associated with adult male height and displayed an 8 cm and 9 cm height difference between homozygous genotypes, respectively. The TC haplotype of these SNPs was associated with a 6 cm decrease in height and remarkably, no homozygous carriers of the TC haplotype were identified in tall subjects. No significant associations with height were found for polymorphisms in the FGFR3, CBFA1 or VDR genes. In the epiphyseal growth plate, chondrocyte proliferation, matrix synthesis and chondrocyte hypertrophy are all major contributors to long bone growth. As estrogen plays such a significant role in both growth and final height attainment, another hypothesis of this study was that estrogen exerted its effects in the growth plate by influencing chondrocyte proliferation and mediating the expression of chondrocyte marker genes. The examination of genes regulated by estrogen in chondrocyte-like cells aimed to identify potential regulators of growth plate fusion, which may further elucidate mechanisms involved in the cessation of linear growth. While estrogen did not dramatically alter the proliferation of the SW1353 cell line, gene expression experiments identified several estrogen regulated genes. Sixteen chondrocyte marker genes were examined in response to estrogen concentrations ranging from 10-12 M to 10-8 M over varying time points. Of the genes analysed, IHH, FGFR3, collagen II and collagen X were not readily detectable and PTHrP, GHR, ER, BMP6, SOX9 and TGF1 mRNAs showed no significant response to estrogen treatments. However, the expression of MMP13, CBFA1, BCL-2 and BAX genes were significantly decreased. Interestingly, the majority of estrogen regulated genes in SW1353 cells are expressed in the hypertrophic zone of the growth plate. Estrogen is also known to regulate systemic GH secretion and local GH action. At the molecular level, estrogen functions to inhibit GH action by negatively regulating GH signalling. GH treated SW1353 cells displayed increases in MMP9 mRNA expression (4.4-fold) and MMP13 mRNA expression (64-fold) in SW1353 cells. Increases were also detected in their respective proteins. Treatment with AG490, an established JAK2 inhibitor, blocked the GH mediated stimulation of both MMP9 and MMP13 mRNA expression. The application of estrogen and GH to SW1353 cells attenuated GH-stimulated MMP13 levels, but did not affect MMP9 levels. Investigation of GH signalling revealed that SW1353 cells have high levels of activated JAK2 and exposure to GH, estrogen, AG490 and other signalling inhibitors did not affect JAK2 phosphorylation. Interestingly, AG490 treatment dramatically decreased ERK2 signalling, although GH did stimulate ERK2 phosphorylation above control levels. AG490 also decreased CBFA1 expression, a transcription factor known to activate MMP9 and MMP13. Finally, GH and estrogen treatment increased expression of SOCS3 mRNA, suggesting that SOCS3 may regulate JAK/STAT signalling in SW1353 cells. The modulation of GH-mediated MMP expression by estrogen in SW1353 cells represents a potentially novel mechanism by which estrogen may regulate longitudinal bone growth. However, further investigation is required in order to elucidate the precise mechanisms behind estrogen and GH regulation of MMP13 expression in SW1353 cells. This study has provided additional evidence that estrogen and the ER gene are major factors in the regulation of growth and the determination of adult height. Newly identified polymorphisms in the ER gene not only contribute to our understanding of the genetic basis of human height, but may also be useful in association studies examining other complex traits. This study also identified several estrogen regulated genes and indicated that estrogen modifies the expression of genes which are primarily expressed in the hypertrophic region of the epiphyseal growth plate. Furthermore, synergistic studies incorporating GH and estrogen have revealed the ability of estrogen to attenuate the effects of GH on MMP13 expression, revealing potential pathways by which estrogen may modulate growth plate fusion, longitudinal bone growth and even arthritis.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb 17, 2009). Includes bibliographical references.

O tratamento radioterápico para pacientes com neoplasias de cabeça e pescoço pode trazer consequências secundárias graves como alterações da estrutura dental, com conseguinte prejuízo da função oral, a qual influencia negativamente a qualidade de vida. Recentemente trabalhos de pesquisa tem demonstrado que a radiação induz a expressão e ativação das metaloproteinases da matriz (MMPs), consideradas as principais enzimas responsáveis pela remodelação da matriz orgânica, incluindo os componentes e estruturas da junção amelodentinária (JAD). Questiona-se então se as alterações dentais observadas em pacientes pós-radioterapia poderiam ser causadas também pela ativação das MMPs que se encontram na JAD. O presente estudo apresentou três avaliações: a ativação e expressão das MMPs, a implementação de inibidores de proteases como método de inibição das MMPs e a ativação das MMPs devido a um desafio ácido. Para as medições foram utilizados 178 fragmentos dentais de molares, divididos aleatoriamente em 2 grupos (decíduos e permanentes) / 4 subgrupos experimentais (irradiados e não-irradiados). Os fragmentos foram expostos à radiacao com Co-60, com fracao de dose de 2 Gy, 5 dias consecutivos, ate atingirem a dose total de 60 Gy, com um total de 30 ciclos, durante 6 semanas. Com o objetivo de determinar a expressão e atividade das MMPs, foram realizados os ensaios de imunofluorescência e zimografia in situ, nos fragmentos dentais de 0,6mm, analisando os tecidos duros do esmalte, dentina e JAD. Para avaliar se produtos odontológicos inativam as MMPs, os dentes foram imersos em 0,5ml de digluconato de clorexidina a 0,12%, fluoreto de sódio a 0,05%, polifenol epigalocatequina 3-galato 400μM e água destilada (grupo controle), por 1 hora. Assim também com objetivo de avaliar se em um ambiente ácido, as MMPs apresentariam maior atividade, os dentes foram colocados em contato com 20μl de solucao desmineralizadora com pH de 4,8, por um minuto, e posteriormente lavados com 20μl de água deionizada, por um minuto. De maneira geral pudemos observar que a irradiação ativa as MMPs na JAD e estes efeitos foram mais evidentes nos dentes permanentes que nos decíduos. Com relação à expressão das diferentes MMPs, foi observada uma maior expressão das MMPs-9 e -20 para dentes decíduos, e para dentes permanentes as MMPs-2, -9 e -20 apresentaram expressão semelhante. Tendo em vista que a irradiação foi capaz de ativar as MMPs expressas na JAD de dentes permanentes, e em busca de soluções capazes de inibi-las, observamos que o Digluconato de Clorexidina, o Fluoreto de Sódio e o Polifenol Epigalocatequina 3-galato inibiram a atividade das MMPs na JAD em dentes permanentes. Por último ao investigar o efeito de um desafio ácido, na atividade das MMPs, observamos que a desmineralização não aumentou a atividade das MMPs em dentes não irradiados, porém aumentou a atividade das MMPs em dentes irradiados. Comparando dentes irradiados submetidos ou não à desmineralização, observou-se que a desmineralização incrementou a atividade das MMPs, já induzida pela irradiação. / Radiotherapy for patients with head and neck cancer can have serious secondary consequences such as changes in tooth structure, with consequent loss of oral function which negatively influences an individual\'s quality of life. Recently research work has shown that radiation induces the expression and activation of matrix metalloproteinases (MMPs) which are considered the major enzymes responsible for the remodeling of the organic matrix, including the components and structures of the dentinoenamel junction (DEJ). It is questionable if the dental changes observed in post-radiotherapy patients could also be caused by the activation of MMPs that are in the DEJ. The present study has three assessments: the activation and expression of MMPs, the implementation of protease inhibitors such as method of inactivating MMPs and the activation of MMPs due to an acid challenge. The measurements that were used were 178 molar dental fragments randomly divided into 2 groups (deciduous and permanent) / 4 experimental subgroups (irradiated and non-irradiated). The samples were exposed to radiation using Co-60 at a cumulative dose of 2 Gy fraction, 5 consecutive days, until they reached a total dose of 60 Gy, with a total of 30 cycles for 6 weeks. In order to determine the expression and activity of MMPs immunofluorescence assays were performed and in situ zymography, the dental fragments of 0.6mm, analyzing the DEJ in three areas of the tooth (cervical, cuspal and groove of pit). To assess whether MMPs inactivate dental products, the teeth were immersed in 0.5 ml of chlorhexidine digluconate at 0.12%, sodium fluoride 0.05%, polyphenol epigallocatechin-3 gallate 400μM and distilled water (control group) for 1 hour. To evaluate effects in an acidic environment, MMPs have higher activity, the teeth were put in contact with 20μl of demineralizing solution with pH 4.8, for a minute, and then washed with 20μl of deionized water, one minute. In general we observed that the radiation active MMPs in DEJ and these effects were more evident in the permanent teeth than in the primary teeth. Regarding the expression of different MMPs, showed the greatest expression of MMP-9 and -20 for deciduous teeth, and permanent teeth MMPs-2, -9 and -20 showed similar expression. Given that the irradiation was able to activate MMPs expressed in the DEJ permanent teeth, and looking for solutions that inactive them, we observed that the digluconate Chlorhexidine, the Sodium Fluoride and Polyphenol Epigallocatechin-3-gallate inhibited activity of MMPs in the DEJ in permanent teeth. Finally, to investigate the effect of an acid challenge, in the activity of MMPs, we observed that the demineralization did not increase the activity of MMPs in non-irradiated teeth but increased the activity of MMPs in irradiated teeth. Comparing irradiated whether subjected to demineralization teeth or not, it was found that demineralization increased activity of MMPs, induced by the radiation.

O conceito de campo de cancerização, em dermatologia, sugere que a pele fotodanificada tem maior potencial para o desenvolvimento de neoplasias cutâneas. A terapia fotodinâmica (TFD) é um método não invasivo para o tratamento de queratoses actínicas (QA) múltiplas, possibilitando a abordagem de todo o campo. Vinte e seis pacientes com múltiplas QAs na face foram submetidos a três sessões de TFD com metilaminolevulinato 16% (MAL) e luz vermelha, com intervalo de um mês. Biópsias foram realizadas antes e após três meses da última sessão e o material corado para hematoxilina-eosina e Weigert. O estudo imunohistoquímico foi feito para os marcadores: TP-53, pró-colageno I, Metaloproteinase-1 e Tenascina-C. A avaliação do fotoenvelhecimento global melhorou consideravelmente (p < 0,001) e a cura clínica das QAs foi de 89,5% ao final do estudo. Duas sessões mostraram ser equivalentes a três sessões de TFD. Diminuição significante do grau e extensão da atipia celular (p < 0,001), aumento das fibras colágenas (p = 0,001) e melhora do grau de elastose (p = 0,002) foram observadas. O estudo imunohistoquímico mostrou diminuição da expressão da TP-53 (p = 0,580), aumento de pró-colágeno I (p = 0,477) e de MMP-1 (p = 0,08), embora não houvesse diferença estatisticamente significante. Aumento significativo foi observado para Tenascina-C (p = 0,024). Múltiplas sessões de TFD com MAL induziram melhora clínica e histológica do campo de cancerização. A diminuição da severidade e extensão da atipia celular associada à menor expressão de TP-53 sugerem redução do potencial carcinogênico do campo / The field cancerization concept suggests that photodamaged skin has an increased risk for the development of malignant lesions. Topical photodynamic therapy (PDT) is a non-invasive therapeutic method for multiple actinic keratosis (AK), allowing the possibility of treating the entire surface. Twenty-six patients with photodamaged skin and multiple AKs on the face were submitted to three consecutive sessions of PDT with mehtylaminolevulinate 16% (MAL) and red light, one month apart. Biopsies were performed before and three months after the last treatment session, and stained for hematoxilin-eosin and Weigert. Immunohistochemestry study was performed for TP-53, pro-collagen I, Metalloproteinase-1 and Tenascin-C. The global score for photodamage improved considerably in all patients (p < 0.001). The AK clearance rate was 89.5% at the end of the study. Two treatments were similar to three MAL-PDT sessions. A significant decrease in keratinocytes atypia grade and amount was observed (p < 0.001). A significant increase in collagen deposition (p = 0.001) and improvement of solar elastosis (p = 0.002) were noticed. Immunohistochemical study showed decreased TP-53 expression although not statistically significant (p = 0.580), increased pro-collagen I and MMP-1 expressions (p = 0.477 and p = 0.08) again not statistically significant and a increased expression of Tenascin-C (p = 0.024), which was statistically significant. In conclusion, multiple sessions of MAL-PDT induced clinical and histological improvement of field cancerization. The decrease in severity and extension of keratinocytes atypia associated with a decreased expression of TP-53 suggest a reduced carcinogenic potential of the altered field