Made available in DSpace on 2018-08-01T23:25:58Z (GMT). No. of bitstreams: 1 tese_10204_Artigo Mestradro Nathália - Defesa 01.08 (1) (1).pdf: 7254929 bytes, checksum: 36eb94c32d1fc4c52933a3189e3caba0 (MD5) Previous issue date: 2016-08-19 / À medida que o processo de odontogênese avança, dando início à deposição de tecidos mineralizados da coroa, ocorre de forma paralela o processo de erupção dentária, que permite ao dente atravessar as barreiras teciduais que o circundam até que ele possa emergir na cavidade oral. Esse processo envolve a degradação da lâmina própria e a reabsorção da parte superior da cripta óssea que envolve o dente por ação de metaloproteinases (MMPs).O objetivo desta pesquisa foi identificar a expressão de MMP-2 em germes dentários de molares de ratos e nos tecidos circunjacentes ao longo do processo eruptivo. A detecção de MMP-2 foi realizada por imuno-histoquímica em 24 amostras de animais com idades entre 04 a 16 dias. A expressão de MMP-2 foi bservada no tecido ósseo basal e apical e em regiões do germe dentário: papila dentária, ameloblastos, odontoblastos, retículo estrelado efolículo dentário. Os esultados desta pesquisa indicam que a forte expressão de MMP-2 em ameloblastos e odontoblastos pode indicar um papel dessa enzima nos processos de síntese, mineralização e maturação dos tecidos mineralizados do dente. Por outro lado, a expressão de MMP-2 no folículo dentário e osso ao redor do germe podem indicar a participação de MMP-2 nos processos de remodelação óssea necessários para o prosseguimento do processo de erupção dentária.

Damage to cardiac contractile proteins such as myosin light chain 1 (MLC1), during oxidative stress is mediated by reactive oxygen species such as peroxynitrite (ONOO-), resulting in impairment of cardiac systolic function. The purpose of this study is to investigate the effects of the increased level of ONOO- on MLC1 degradation by the proteolytic enzyme matrix metalloproteinase-2 (MMP-2) during oxidative stress which ultimately decreases cardiac function.<p> In the present study two distinct models were utilized to demonstrate the mechanism by which MLC1 is modified by ONOO- and how these post-translational modifications lead to its increased degradation by MMP-2. In a model of newborn hypoxia-reoxygenation in piglets we demonstrated that ONOO--induced nitration and nitrosylation of tyrosine and cysteine residues of MLC1 increase its degradation by MMP-2. Furthermore, we found nitration of a tyrosine residue located adjacent to the cleavage site for MMP-2. We verified these results by using a model of isolated rat heart myocytes to determine that the same mechanism responsible for cardiac dysfunction in newborn piglets occurs in isolated myocytes and that the MMP-2 involved in degradation of MLC1 is located within the myocytes. Moreover, we were able to determine that this mechanism occurs during ischemia itself before the onset of reperfusion. Furthermore, we have found that pharmacological intervention aimed at inhibition of MLC1 nitration/nitrosylation during ischemia by the ONOO- scavenger FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), or inhition of MMP-2 activity with phenanthroline, provides an effective protection of cardiomyocyte contractility. The work presented here provides new evidence on the mechanisms of regulation of contractile proteins during the development of contractile dysfunction.

Damage to cardiac contractile proteins such as myosin light chain 1 (MLC1), during oxidative stress is mediated by reactive oxygen species such as peroxynitrite (ONOO-), resulting in impairment of cardiac systolic function. The purpose of this study is to investigate the effects of the increased level of ONOO- on MLC1 degradation by the proteolytic enzyme matrix metalloproteinase-2 (MMP-2) during oxidative stress which ultimately decreases cardiac function.<p> In the present study two distinct models were utilized to demonstrate the mechanism by which MLC1 is modified by ONOO- and how these post-translational modifications lead to its increased degradation by MMP-2. In a model of newborn hypoxia-reoxygenation in piglets we demonstrated that ONOO--induced nitration and nitrosylation of tyrosine and cysteine residues of MLC1 increase its degradation by MMP-2. Furthermore, we found nitration of a tyrosine residue located adjacent to the cleavage site for MMP-2. We verified these results by using a model of isolated rat heart myocytes to determine that the same mechanism responsible for cardiac dysfunction in newborn piglets occurs in isolated myocytes and that the MMP-2 involved in degradation of MLC1 is located within the myocytes. Moreover, we were able to determine that this mechanism occurs during ischemia itself before the onset of reperfusion. Furthermore, we have found that pharmacological intervention aimed at inhibition of MLC1 nitration/nitrosylation during ischemia by the ONOO- scavenger FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), or inhition of MMP-2 activity with phenanthroline, provides an effective protection of cardiomyocyte contractility. The work presented here provides new evidence on the mechanisms of regulation of contractile proteins during the development of contractile dysfunction.

As metaloproteinases da matriz (MMPs) são enzimas proteolíticas dependentes de zinco que degradam componentes da matriz extracelular, facilitando a remodelação tecidual e a migração celular. MMPs secretadas por osteoclastos exercem papel central na absorção óssea fisiológica e estão também associadas a processos de degradação patológica do osso. No entanto, o papel essencialmente degradador de osso das MMPs, particularmente da MMP-2, vem sendo questionado em anos recentes por estudos que evidenciam sua importância na diferenciação de células da linhagem osteoblástica e na formação de tecido ósseo em cultura. Neste sentido, é possível que a MMP-2 exerça um papel importante na formação de tecido ósseo em processo de reparação. O objetivo do presente trabalho foi investigar a pretensa ação osteo-estimulatória da rhMMP-2 ligada à monoleína (usada como carreador) implantada em defeito confeccionado na calvária de ratos. Foram confeccionados defeitos ósseos unilaterais de 4 mm de diâmetro na calvária de ratos adultos; nos animais controles o defeito ósseo foi mantido com o preenchimento natural de coágulo sanguíneo e nos animais implantados o defeito foi preenchido com monoleína ou com rhMMP-2 ligada à monoleína. Os animais foram eutanasiados após 2 e 4 semanas e a taxa de neoformação óssea foi estimada em cortes histológicos por um método histométrico de contagem diferencial de pontos. A taxa de neoformação óssea foi semelhante nos animais dos grupos controle e monoleína e significativamente maior nos animais do grupo MMP-2, em ambos os períodos analisados. Os resultados permitem concluir que a monoleína não interferiu com o processo reparacional e pareceu eficaz como carreador da rhMMP-2, e adicionam evidências á hipótese da importância da atividade da MMP-2 para a formação óssea, em um modelo experimental in vivo de reparo ósseo. / Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes that degrade extracellular matrix components, facilitating cell migration and tissue remodeling. MMPs secreted by osteclasts are important in the physiological bone resorption as in pathological bone degradation. However, the essentially bone absorbing hole of MMPs, particularly of the MMP-2, has been questioned in recent years by studies that show its importance in osteoblastic cells differentiation and in vitro bone formation. Therefore, the MMP-2 may have also an important hole in reparational bone formation. The purpose of the present study was to investigate the pretense osteostimulatory effect of the rhMMP-2 linked to monoolein (used as a carrier) implanted into rat calvarial defects. Bone defects of 4mm in diameter were created unilaterally in rats calvaria and filled with natural blood clot (control), monoolein or rhMMP-2 linked to monoolein. The animals were killed 2 and 4 weeks postoperatively and the rate of new bone formation was estimated in histological sections by a histometric differential point-counting method. The rate of reparational bone formation was similar in the animals from control and monoolein groups and was significantly greater in the MMP-2 group, in both periods. From the results it may be concluded that monoolein did not interfere with the reparacional process and seemed effective as a rhMMP-2 carrier. In addition, the results add evidence to the importance of MMP-2 activity for bone formation, in an in vivo bone healing experimental model.

As metaloproteinases da matriz (MMPs) são enzimas proteolíticas dependentes de zinco que degradam componentes da matriz extracelular, facilitando a remodelação tecidual e a migração celular. MMPs secretadas por osteoclastos exercem papel central na absorção óssea fisiológica e estão também associadas a processos de degradação patológica do osso. No entanto, o papel essencialmente degradador de osso das MMPs, particularmente da MMP-2, vem sendo questionado em anos recentes por estudos que evidenciam sua importância na diferenciação de células da linhagem osteoblástica e na formação de tecido ósseo em cultura. Neste sentido, é possível que a MMP-2 exerça um papel importante na formação de tecido ósseo em processo de reparação. O objetivo do presente trabalho foi investigar a pretensa ação osteo-estimulatória da rhMMP-2 ligada à monoleína (usada como carreador) implantada em defeito confeccionado na calvária de ratos. Foram confeccionados defeitos ósseos unilaterais de 4 mm de diâmetro na calvária de ratos adultos; nos animais controles o defeito ósseo foi mantido com o preenchimento natural de coágulo sanguíneo e nos animais implantados o defeito foi preenchido com monoleína ou com rhMMP-2 ligada à monoleína. Os animais foram eutanasiados após 2 e 4 semanas e a taxa de neoformação óssea foi estimada em cortes histológicos por um método histométrico de contagem diferencial de pontos. A taxa de neoformação óssea foi semelhante nos animais dos grupos controle e monoleína e significativamente maior nos animais do grupo MMP-2, em ambos os períodos analisados. Os resultados permitem concluir que a monoleína não interferiu com o processo reparacional e pareceu eficaz como carreador da rhMMP-2, e adicionam evidências á hipótese da importância da atividade da MMP-2 para a formação óssea, em um modelo experimental in vivo de reparo ósseo. / Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes that degrade extracellular matrix components, facilitating cell migration and tissue remodeling. MMPs secreted by osteclasts are important in the physiological bone resorption as in pathological bone degradation. However, the essentially bone absorbing hole of MMPs, particularly of the MMP-2, has been questioned in recent years by studies that show its importance in osteoblastic cells differentiation and in vitro bone formation. Therefore, the MMP-2 may have also an important hole in reparational bone formation. The purpose of the present study was to investigate the pretense osteostimulatory effect of the rhMMP-2 linked to monoolein (used as a carrier) implanted into rat calvarial defects. Bone defects of 4mm in diameter were created unilaterally in rats calvaria and filled with natural blood clot (control), monoolein or rhMMP-2 linked to monoolein. The animals were killed 2 and 4 weeks postoperatively and the rate of new bone formation was estimated in histological sections by a histometric differential point-counting method. The rate of reparational bone formation was similar in the animals from control and monoolein groups and was significantly greater in the MMP-2 group, in both periods. From the results it may be concluded that monoolein did not interfere with the reparacional process and seemed effective as a rhMMP-2 carrier. In addition, the results add evidence to the importance of MMP-2 activity for bone formation, in an in vivo bone healing experimental model.

Made available in DSpace on 2014-08-20T14:30:17Z (GMT). No. of bitstreams: 1 Dissertacao_Eliana_do_Nascimento_Torre.pdf: 1908566 bytes, checksum: 7f4e5292cab07c5cd5840870b92b0df1 (MD5) Previous issue date: 2011-07-19 / Adhesive fillings are satisfactory on short-term evaluation. However, when the evaluation period of longevity of these fillings is longer, problems with the stability of the polymer formed by the adhesive system and the degradation of collagen forming the hybrid layer provoke a large decrease in the durability of this type of filling. The extracellular matrix metalloproteinases (MMPs) are enzymes, which have been associated with the degradation of collagen present at the hybrid layer. Therefore, the possibility of inhibiting the activity of these enzymes has been considered an important strategy to maintain and increase the longevity of adhesive fillings. Hence, the aim of this work is to evaluate the inhibitory potential of MMPs through the addiction of the monomers with promising characteristics reported in previous studies. These monomers have molecules known as ―kidnappers‖ of bivalent cations. This characteristic is important because the catalytic place of MMPs has zinc and calcium in its constitution. Therefore, the coordination of molecules with the catalytic place of the enzyme would be able to inhibit the activity of the MMPs. Dentine from teeth recently pulled will be used to obtain and purify the MMPs of dentine.. The analysis of MMPs inhibition will be carried out through a zymography. In case of positive results for the inhibition of MMPs, cytotoxicity and genotoxicity tests will be carried out in cellular lineages of human pulp fibroblasts apart from an immortalized lineage of fibroblasts of 3T3/NIH mice. The MTT (bromide 3-(4.5-dimethylthiazol-2-ilo)-2.5-diphenyltetrazolium) colorimetric test will be used to measure the cytotoxicity of the products tested and the test of micronucleus will be used to measure genotoxicity. In all groups the 10mM concentration induced 100% cell death. There was no difference in sensitivity in both strains. Statistically, in relation to the control group/untreated, the monomers were more cytotoxic in HPFs of groups 4 and 5. Similar results were found for the 3T3 lineage, except for group 2, which also showed statistical significance for all concentrations. There was a higher number of micronucleated cells in the groups where the two monomers of intermediate chains longer (PEG200 and PEG400 DMA) were used when compared to control. The results suggest that these two monomers are more cytotoxic and genotoxic. Most of the methacrylate monomers showed considerable but not total inhibition of MMP-2 by zymography. The exception was the PEG200 DMA, which only the concentration of 5 mM inhibited the activity of this gelatinolitic MMP / Restaurações adesivas apresentam desempenho satisfatório em avaliações de curto prazo, porém quando o período de avaliação da longevidade dessas restaurações é maior, problemas com a estabilidade do polímero formado pelo sistema adesivo e com a degradação do colágeno formador da camada híbrida, provocam uma queda expressiva na durabilidade desse tipo de restauração. As metaloproteinases da matriz extracelular (MMPs) são enzimas que têm sido associadas com a degradação do colágeno formador da camada híbrida e, por isso, a possibilidade de inibir a atividade dessas enzimas tem sido considerada uma estratégia importante para a manutenção e aumento da longevidade das restaurações adesivas. O objetivo do presente estudo foi avaliar o potencial inibitório da MMP-2 por monômeros com características promissoras relatadas na literatura relacionadas ao seu potencial inibidor de MMPs, sendo estes, EGDMA (1), TEGDMA (2), T4GDMA (3), PEG200 DMA (4) e PEG400 DMA (5). Dentina humana derivada de dentes recentemente extraídos foi utilizada para a obtenção e purificação das MMPs. O ensaio de inibição da MMP-2 foi realizado por zimografia. Testes de citotoxicidade e genotoxicidade foram realizados com uma linhagem primária de fibroblastos pulpares humanos (FPH), além de uma linhagem imortalizada de fibroblastos de camundongos 3T3/NIH. O teste colorimétrico MTT (brometo de 3-(4,5-dimetiltiazol-2-ilo)-2,5-difeniltetrazólio) foi usado para medir a citotoxicidade dos produtos testados, enquanto que o teste de formação de micronúcleos foi usado para avaliar a genotoxicidade. Em todos os grupos a concentração de 10mM induziu a 100% de morte celular. Não houve diferença de sensibilidade nas duas linhagens estudadas. Estatisticamente, em relação ao grupo controle/não tratado, os monômeros foram mais citotóxicos nos FPHs nos grupos 4 e 5. Resultados semelhantes foram encontrados na linhagem 3T3, à exceção do grupo 2, que apresentou diferença estatística em todas as suas concentrações, nesta célula. Houve maior número de células micronucleadas nos grupos onde foram utilizados os dois monômeros de cadeias intermediárias mais longas (PEG200 DMA e PEG400 DMA) quando comparados ao controle, sugerindo os resultados, que estes monômeros são mais citotóxicos e genotóxicos. A maioria dos monômeros metacrilatos apresentou considerável, mas não total inibição da MMP-2 em todas as concentrações através dos ensaios de zimografia. A exceção foi o PEG200DMA, o qual somente a concentração de 5mM inibiu a atividade gelatinolítica da referida MMP

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Leukemia is a hematological neoplasm with abnormal genetic mutation or chromosomal translocation in the myeloblast or lymphoblast, and characterized by accumulation of immature cells and malfunction of lymphocytes and myeloid-derived cells. The prognosis of treatment depends on genetic mutation, chromosomal aberration, disease progression and age of patients. Currently, bone marrow transplantation is a useful therapeutic strategy, but the success in therapy is limited by the bone marrow of donors and life-threatening events such as immune repulsion. Although chemotherapy improves leukemia treatment, long-term chemotherapy usually leads to the production of drug-resistant cancer cells. Thus, the development of new modality in overcoming drug-resistant should be beneficial for in leukemia therapy. In this thesis, Naja nigricollis toxin £^, piceatannol, caffeine, and Bungarus multicinctus protease inhibitor-like protein 1 (PILP-1) are employed to investigate the molecular mechanisms in regulating apoptosis and invasion of leukemic cell lines K562 and U937. Hopefully, the signaling pathways elicited by these treatments may be aid in identifying new targets in treating leukemia. Toxin £^ inducing cell death is found to evoke p38 MAPK-mediated Bcl-2 down-regulation, which facilitates mitochondria dysfunction, ROS generation and cytiochrome c release. Finally, activation of caspases leads to apoptotic death of toxin £^-treated cells. Piceatannol elicits Ca2+/p38£\ MAPK- mediated c-Jun and ATF-2 phosphorylation, leading to up-regulation of Fas/FasL protein expression and autocrine Fas-mediated death pathway activation. Caffeine treatment down-regulates MMP-2/-9 down-regulation via Ca2+/ROS-mediated inactivation of ERK/c-Fos and activation of p38 MAPK/c-Jun pathway. Consequently, caffeine treatment suppresses invasion of leukemia cells. PILP-1-induced ADAM17 down-regulation suppresses Lyn-mediated Akt phosphorylation, resulting in death of PILP-1-treated leukemia cells. Taken together, the results of the present study elucidate the signaling pathways responsible for apoptosis and invasion of leukemia cells. Moreover, these findings might suggest new targets in developing therapeutic strategy in treating leukemia.

Cardiovascular disease is one of the main causes of mortality and one of the significant burdens to society. Major cardiovascular diseases such as acute myocardial infarction (heart attack), heart failure and cardiac arrhythmia often result in the development of ischemia/reperfusion (I/R) injury. Untreated I/R injury is known to cause cardiac contractile dysfunction. It is established that matrix metalloproteinase-2 (MMP-2) is activated and degrades contractile proteins during I/R, and many other factors including metabolic enzymes, kinases and structural proteins are affected by I/R. However, the molecular mechanisms responsible for these changes are unclear. Since MMP-2 is known to its broad spectrum of action, I hypothesize that, in addition to contractile proteins, proteins related to regulation of energy metabolism are MMP-2 targets during I/R, and protein kinase such as myosin light chain kinase (MLCK) is also involved in this process. The use of proteomics in studying heart injury triggered by I/R will reveal new potential targets for pharmacological protection of heart from I/R induced contractile dysfunction. In addition, selective inhibition of MMP-2 using MMP-2 siRNA protects the heart from I/R injury. In this study, we investigated the protein modulation during I/R using proteomic approach. In order to study the effect of protein kinases (MLCK) and MMP-2, their selective inhibitors were used to inhibit those factors and evaluate the changes in energy metabolic proteins during I/R. Proteomic analysis revealed that six proteins are involved in energy metabolism: ATP synthase β subunit, cytochrome b-c1 complex subunit 1, 24-kDa mitochondrial NADH dehydrogenase, NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, cytochrome c oxidase subunit, and succinyl-CoA ligase subunit, resulting in decreased levels in I/R hearts. The data suggests that energy metabolic proteins, especially the metabolic enzymes involved in the electron transport chain in the mitochondria may contribute to I/R injury. In addition, our data provides evidence that the right and left ventricles of the heart respond differently to I/R injury, in terms of the regulation of contractile proteins and energy metabolic enzymes. Studies using MLCK inhibitor, ML-7, and MMP-2 inhibitor, MMP-2 siRNA to investigate the effect of myosin light chain kinase (MLCK) and MMP-2 in energy metabolic proteins have shown that succinyl-CoA ligase and ATP synthase are affected by MLCK and MMP-2 respectively. These results demonstrate that the effect of inhibition of the MLCK and MMP-2 involves optimization of energy metabolism in I/R injury, likely resulting in increased energy production. Hence, the observed proteins increase in cardiac recovery after I/R. Also, inhibition of MLCK and MMP-2 by ML-7 and MMP-2 respectively shows cardio protective effect during I/R. In summary, this study provides a novel pathogenesis in the development of I/R-induced cardiac contractile dysfunction. Moreover, we suggest a new therapeutic approach whereby using MMP-2 siRNA can be a promising gene therapy in the development of new preventive or treatment strategies against I/R injury.

Abstract Bladder cancer when superficial has a good prognosis but it has a high recurrence risk and about 10–15% of the superficial carcinomas will progress into muscle invasive or metastatic type. The most powerful factor for predicting the behavior of bladder carcinoma is the stage of the tumor. Invasion to the lamina propria increases the risk of recurrence and progress to muscle-invasive tumor. Also grade of the tumor and tumor multiplicity associates with high risk for recurrence. New markers are still needed to find those patients who need more and better treatments to avoid the recurrence and progress. The need for new non-invasive markers to diminish the need for frequent cystoscopy in follow-up is also obvious. Gelatinases MMP-2 and MMP-9 are known to associate to tumor invasion and progression. Also their tissue inhibitors TIMP-1 and TIMP-2 take part in these diversified processes and metastasis formation. In the present work the expression and clinical value of gelatinases MMP-2 and MMP-9 and their tissue inhibitors TIMP-1 and TIMP-2 were evaluated in bladder carcinoma. Primary tissue samples of 121 patients were analyzed for expression of MMP-2 and/or MMP-9 using immunohistochemistry. The serum samples of 87 patients who were treated in the Oncology Department of Oulu University Hospital were collected and studied with ELISA. The control group consisted of 44 healthy volunteers. Overexperssion of MMP-2 protein correlated significantly to disease-specific survival and showed an independent prognostic value as a biomarker. High MMP-9 expression instead correlated to favorable overall survival of bladder cancer patients. Circulating proMMP-2, TIMP-2 and MMP-2:TIMP-2 complex levels were lower in cancer patients than in healthy volunteers in control group. High levels of all these three markers correlated with better prognosis in bladder cancer patients.