Orientadores: Mário Fernando de Góes, Alan Roger dos Santos Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-25T14:50:37Z (GMT). No. of bitstreams: 1 Silva_WagnerGomesda_M.pdf: 3135648 bytes, checksum: 61c724ad2dea362d9e0c9ac978529067 (MD5) Previous issue date: 2014 / Resumo: A cárie relacionada à radioterapia (CRR), também conhecida como "cárie de radiação", é uma grave toxicidade bucal que acomete pacientes oncológicos submetidos à radioterapia (RDT) na região de cabeça e pescoço. Apesar de ser atribuída principalmente aos efeitos indiretos da RDT, como a hipossalivação, a CRR também é associada a efeitos diretos da radiação sobre o esmalte, a dentina e a polpa dental. Desta forma, o objetivo deste estudo foi testar a hipótese nula de que a RDT não é capaz de gerar danos diretos aos componentes micromorfológicos da matriz inorgânica e orgânica dos dentes de pacientes com câncer de cabeça e pescoço (CCP). Um total de 39 dentes, incluindo 22 que receberam RDT in vivo e 17 dentes não irradiados provenientes de pacientes com CCP, foram agrupados em dois eixos experimentais que avaliaram cortes histológicos preparados por desgaste (grupo experimental I) e por desmineralização (grupo experimental II). No grupo I, investigou-se a preservação da micromorfologia dos tecidos dentários; bem como a atividade gelatinolítica por meio da zimografia in situ. No grupo II, investigou-se a preservação da micromorfologia dos tecidos dentários; bem como a expressão imunoistoquímica de MMP-2, MMP-9 e MMP-20. Foi constatada preservação estrutural de todos os componentes micromorfológicos analisados, alta atividade gelatinolítica em dentina desmineralizada e intensa expressão de MMP-2, MMP-9 e MMP-20 em diferentes áreas de todos os espécimes avaliados. Contudo, não foi possível identificar diferenças significativas entre os espécimes irradiados e não irradiados em nenhuma das análises estatísticas. A hipótese nula teste não foi rejeitada e os resultados deste estudo sugerem que a ação direta da RDT não é capaz de gerar destruição radiogênica da matriz inorgânica ou orgânica dos dentes de pacientes com CCP e que, possivelmente, os efeitos diretos da RDT sobre a estrutura dentária não são determinantes para o início ou progressão da CRR / Abstract: Radiation-Related Caries (RRC), also known as "radiation caries" is a severe oral toxicity that affects cancer patients who have undergone radiotherapy (RDT) in the head and neck area. Although mainly attributed to the indirect effects of RDT, such as hyposalivation, RRC is also associated with direct effects of radiation on the enamel, dentin and dental pulp. Therefore, the aim of this study was to test the null hypothesis that RDT is not able to generate direct damage to the micromorphological components of inorganic and organic matrix of teeth from patients with head and neck cancer (HNC). A total of 39 samples, including 22 teeth that received in vivo RDT and 17 teeth from non-irradiated patients with HNC were grouped into two experimental groups that evaluated ground sections (experimental group I) and histological sections prepared by demineralization (experimental group II). In group I, the preservation of the micromorphological components of dental tissues as well as the gelatinolytic activity by in situ zymography were investigated. In group II, the preservation of micromorphological components of dental tissues and the immunohistochemical expression of MMP-2, MMP-9 and MMP-20 were investigated. Structural preservation of all micromorphological components analyzed, a high gelatinolitic activity in demineralized dentin and intense expression of MMP-2, MMP-9 and MMP-20 were found in different areas of all specimens evaluated. However, it was not possible to identify significant differences between irradiated and non-irradiated specimens in any of the statistical analyses. The null hypothesis was not rejected and the results of this study suggest that direct effects of RDT are not able to generate morphological radiogenic destruction of inorganic or organic teeth matrix of HNC patients, and, possibly, the direct effects of RDT on the dental tissues are not decisive to the onset and progression of RRC / Mestrado / Patologia / Mestre em Estomatopatologia

Orientador: Oslei Paes de Almeida / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-07-25T08:18:42Z (GMT). No. of bitstreams: 1 DellaColetta_Ricardo_D.pdf: 6321143 bytes, checksum: fee6575189b6054e21beeaa54cc06f5a (MD5) Previous issue date: 1999 / Resumo: Fibromatose gengival hereditária (FGH) é uma condição rara caracterizada por um aumento gengival generalizado com crescimento lento e progressivo. Para elucidar algumas das características regulatórias que resultam nesta condição, quatro linhagens celulares de fibroblastos provenientes de indivíduos de uma mesma família com FGH foram isoladas e caracterizadas em relação ao comportamento prol proliferativo, produção, e expressão de colágeno e suas proteínas chaperones, expressão de MMPs e TIMPs e produção de citocinas. Fibroblastos de gengiva normal (GN) e FGH em condições de subconfluência celular apresentaram típicas características morfológicas, mas em condições de saturação da densidade, os fibroblastos de FGH apresentaram dimensões menores que as células controle. Cinco diferentes ensaios de proliferação celular mostraram que a relação de proliferação foi significantemente maior em fibroblastos de FGH. A produção e expressão de Hsp47 é também aumentada em fibroblastos de FGH em paralelo com o aumento na produção de colágeno. Em adição, a expressão de Hsp47 é regulada por estresse celular e pró-peptídeos correspondendo as regiões da porção N-terminal das cadeias 'alfa'1 (I). A produção de citocinas foi detectada por ELlSA utilizando anticorpos específicos para TGF-' beta '31, IL-6, IL-1 ' beta ' e TNF- 'alfa '. Em fibroblastos de FGH a produção de TGF-' beta ' 1 e IL-6 foi estatisticamente maior que em fibroblastos normais (p<0,013 e p< 0,0035 respectivamente), enquanto que IL-' beta ' 1 e TNF- 'alfa ' não foram detectadas em todos as linhagens celulares. A expressão e produção de MMP-1 e MMP-2, como revelada por RT -PCR e zimografia, foram significantemente menores em fibroblastos de FGH comparando com células de GN. Por outro lado, a expressão de TIMP-1 e TIMP-2 foi ligeiramente maior em fibroblastos de GN. A neutralização de TGF- ' beta ' 1 com anticorpos específicos elevou os níveis de expressão e produção de MMP-1 e dramaticamente reduziu os níveis de MMP-2, enquanto que os de níveis de TIMPs não foram alterados. Estes resultados sugerem que a pato gênese do aumento gengival de indivíduos com FGH é possivelmente resultado de uma associação de fatores incluindo alterado comportamento proliferativo, exacerbada síntese de colágeno e Hsp47, desregulação no balanço proteolítico e elevada produção de citocinas / Abstract: Characterization of cellular proliferation and expression of collagen and its molecular chaperones, cytokines and matrix metalloproteinases and its tissue inhibitors in gingival fibroblasts from pacients with hereditary gingival fibromatosis Hereditary gingival fibromatosis (HGF) is a rare oral condition characterized by a slow and progressive enlargement of the gingiva, involving both the maxillaand mandible. To further elucidate some of the regulatory features resulting in this condition, the culture characteristics of the four cell lines of gingival fibroblasts derived from patients of the some family with HGF were isolated and characterized on proliferation index, collagen and its molecular chaperones production and expression, MMPs and TIMPs expression, and cytokines production. HGF and normal gingiva (NG) fibroblasts in subcofluent culture densities showed typical morphologic characteristics, but in saturation density, HGF fibroblasts were shorter than NG cells. Five different cell proliferation assays showed that the cell proliferation rate was significantly higher in HGF fibroblasts. The production and expression of Hsp47 were significantly higher in HGF fibroblasts than in NG fibroblasts, along with increased producto?n of collagen. In addition, Hsp47 is coordinately regulated by stress and by feedback mechanism mediated by N-terminal procollagen propeptides corresponding to residues of a1 (I)-chains. The cytokines production was detected by ELlSA using specific antibodies for TGF-' beta ' 1 , IL-6, IL-1 ' beta ' and TNF- 'alfa '. In HGF fibroblasts, the production of TGF-' beta ' 1 and IL-6 was higher than fibroblasts from normal tissues (p<0.013 and p<0.0035 respectively), whereas IL-1' beta ' and TNF-'alfa ' were not detected in ali cell lines. RT-PCR and enzymographic analysis clearly demonstrated that the expression and production of MMP-1 and MMP-2 were significantly lower in fibroblasts from HGF than from NG. Interestingly, TIMP-1 and TIMP-2 expression from NG cells was shown to be slightly higher than those from HGF. The use of neutralizing antibodies to TGF-' beta ' 1 caused a slight increase in MMP-1 and a decrease in MMP-2 expression, whereas that no change TIMPs levels. These results suggest that the pathogenesis of gingival outgrowth in HGF patients is possibly resulted of a association of factors such as increased proliferative potential, altered synthesis of collagen and Hsp47, dysregulated proteolytic balance and increased cytokines production / Doutorado / Biologia e Patologia Buco-Dental / Doutor em Odontologia

Made available in DSpace on 2014-08-20T14:30:18Z (GMT). No. of bitstreams: 1 Dissertacao_ Fabricio_Aulo_Ogliari.pdf: 587095 bytes, checksum: 8bb56a09354ea01200df793cf0e60ef5 (MD5) Previous issue date: 2009-07-10 / The aim of this study was to synthesize and to investigate the influence of the 2,3-epithiopropyl methacrylate (ETMA) in a dental adhesive. Synthesis of the ETMA was performed using glycidyl methacrylate as starting reagent, catalyzed by cerium ammonium nitrate in presence of NH4SCN. After purification, ETMA was characterized by 1H NMR and FTIR spectroscopy. ETMA monomer was used as an additive agent in a commercial available dental adhesive (Scotchbond Multipurpose, 3M ESPE) at three different concentrations (0.1, 1 and 10 wt %). Adhesive without ETMA was used as control group. Degree of conversion of the polymerized adhesives was accomplished by FTIR analysis. Blend copolymerization was investigated by 1H NMR spectroscopy analysis. To investigate immediate and long-term properties of the adhesives, microtensile bond strength to human dentin and SEM fractographic analysis were performed. ETMA was synthesized through the conversion of GMA with NH4SCN and high yield was obtained (90%). One-step s synthetic route and a simple manner of purification were described. ETMA presence didn t affect the degree of conversion of the adhesive resins. The monomer that composed the adhesive resins presented copolymerization among them, not presenting ETMA release in 0.1 and 1% groups. Experimental adhesives had statistically higher microtensile bond strength values than the no ETMA adhesive (p<0.05), independently of the water storage. All groups were stable over time / O objetivo deste estudo foi sintetizar e avaliar a influência do 2,3-epitiopropil metacrilato (ETMA) utilizado como monômero funcionalizado sobre a resistência de união imediata e longitudinal de um adesivo comercial. O ETMA foi sintetizado através da transformação do glicidil metacrilato na presença de tiocianato de amônio catalisado por nitrato de cérico de amônio. O produto obtido, após destilação, foi confirmado e caracterizado por ressonância magnética nuclear de prótons e por espectroscopia no infravermelho. ETMA foi utilizado como aditivo na quantidade de 0,1, 1 e 10% em massa a um adesivo comercial (Scotchbond Multiuso - SBMU, 3MESPE). Como controle SBMU sem ETMA foi testado. Foi avaliada a capacidade de copolimerização do ETMA com os outros monômeros do adesivo comercial assim como a sua influência no grau de conversão e na resistência de união à microtração em dentina. A resistência de união a microtração em dentina foi avaliada após 24h, 6 meses e 12 meses de armazenagem em água destilada. As amostras fraturadas do ensaio de microtração foram avaliadas por microscopia eletrônica de varredura e microscopia ótica para a análise do modo de fratura. Foi possível a síntese do ETMA através da conversão de GMA utilizando NH4SCN como reagente. Um rendimento global de 90% foi atingido utilizando-se um método simples de purificação. A adição de ETMA não influenciou os valores de grau de conversão e estes sistemas monoméricos demonstraram capacidade de copolimerização ente si, não apresentando traços de liberação de ETMA nos grupos de 0,1 e 1%. Os adesivos contendo ETMA apresentaram valores de resistência de união significativamente maiores que o grupo controle sem ETMA (p<0.05). Os valores de resistência de união do grupo controle e dos grupos experimentais permaneceram estáveis mesmo após 12 meses de armazenagem, sendo que os grupos experimentais apresentaram valores mais elevados de resistência de união em comparação ao controle independentemente do período de avaliação

Abstract Malignant tissue growth induces marked biochemical and structural changes in the extracellular matrix of the tumour and its surrounding tissues. In the present study, we evaluated the prognostic value of the serum concentration of the markers of synthesis of type I collagen (PICP, PINP) and type III collagen (PIIINP) as well as the marker of type I collagen degradation (ICTP) and compared them with the conventional indicators of prognosis (clinical stage, grade of differentiation, histological subtype, residual tumour load and the age of the patient). The prognostic value of peritoneal cytological findings at operation was an additional object in our studies. High preoperative serum ICTP (>5.6μg/L) and PIIINP (>3.2μg/L) concentrations and a low PICP:PINP ratio (>2) correlated with poor prognosis in ovarian carcinoma in univariate analysis and in multivariate analysis when each variable was analyzed separately with the conventional factors. However, ICTP concentration was the only prognostic variable in multivariate analysis including PIIINP, PINP, ICTP and CA125. When analyzed with conventional prognostic factors (clinical stage, grade, residual tumour, presence of ascites, histology), clinical stage and ICTP were independent indicators of prognosis. In addition, malignant cells in the peritoneal fluid aspirate at primary operation, grade and the age of the patient predicted poor prognosis in multivariate analysis. Postoperative serum ICTP concentration 9-months after the operation was the strongest prognostic factor as compared to the preoperative ICTP and CA125 values and clinical variables. These results indicate that serum collagen metabolites, especially ICTP, are indicators of prognosis in epithelial ovarian cancer. The present ICTP-test does not detect the degradation products of immature type I collagen, the dominating form in ovarian cancer tissue. Therefore, the excess ICTP in invasive ovarian cancer might originate through the degradation of trivalently matured collagens in non-malignant tissues surrounding the malignancy. ICTP may thus be an indicator of invasive properties of the tumor and its determination opens up new perspective to predict the clinical outcome of ovarian cancer.

Les tiques du genre Ixodes englobent un grand nombre d’espèces dont Ixodes ricinus, objet de cette étude. Ixodes ricinus peuple les sous-bois de nos régions et la plupart des grandes forêts de l’Europe centrale et de l’est. Au cours de leur cycle parasitaire, les tiques réalisent un repas sanguin au détriment de petits mammifères (mulots, lièvres,…), mais aussi de grands mammifères tels que le cerf, la biche ou le sanglier. Elles deviennent particulièrement voraces au début du printemps et en automne, c’est pourquoi à ces époques de l’année, les promeneurs et les animaux domestiques (principalement, le chien) peuvent en être infestés. Pour mener à bien leur repas sanguin, les tiques mettent en place une série de mécanismes les rendant non seulement indécelables par l’animal parasité mais aussi leur donnant la capacité de contrecarrer les défenses de l’hôte. En effet, lors de sa fixation et durant tout le repas sanguin, la tique injecte un cocktail de facteurs bioactifs qui inhibent la sensation de douleur au moment de l’ancrage dans le derme, mais également les défenses immunitaires de l’hôte parasité. De plus, certains de ces facteurs possèdent des propriétés anti-hémostatiques garantissant la fluidité du sang pendant tout le repas.<p><p>Le Service de Biologie Moléculaire des Ectoparasites (anciennement Service de Génétique Appliquée) a identifié un grand nombre de séquences d’ARNm induites spécifiquement dans les glandes salivaires de la tique au cours de son repas sanguin. Deux d’entres-elles ont servi de point de départ à ce travail :il s’agit de seq16 (renommée Metis1) et de seq7 (renommée Ir-CPI).<p><p>Le travail sur la séquence seq16 a conduit à l’identification d’une nouvelle famille de 5 métalloprotéases (nommée Metis pour Metalloprotease from Ixodes ricinus). L’analyse des séquences a permis de les associer à certaines métalloprotéases hémorragiques de venin de serpent. Bien que les 5 métalloprotéases décrites possèdent toutes les mêmes caractéristiques au niveau de leur séquence et de leur profil d’expression dans les glandes salivaires, les résultats d’analyse phylogénétique, d’étude de variation antigénique, de leur mode d’activation et de leur spécificité d’action permettent de les diviser en 3 sous-familles. L’utilisation de la technique d’ARN interférence en in vivo et l’analyse vaccinale soulignent le rôle essentiel de la famille Metis dans les premières heures du repas de la tique et de manière générale dans la réussite du repas sanguin. Enfin, des études d’activité ont montré que certains membres de la famille ont une activité fibrino(gèno)lytique ;plus particulièrement, la protéine Metis4 possède une activité protéolytique envers la gélatine, la caséine, la fibronectine et le fibrinogène. Ces études ont montré également un mode d’activation original ;plutôt que d’être activée par une pro-séquence en amino-terminal, Metis4 s’active spontanément en perdant un peptide à son extrémité carboxy-terminale. <p><p>Le travail sur la séquence seq7 a conduit à la caractérisation d’un nouvel inhibiteur de sérines protéases. Cette protéine, nommée Ir-CPI pour « Ixodes ricinus - Contact Phase Inhibitor », possède un motif kunitz et est capable d’inhiber de manière importante la voie intrinsèque de la coagulation et dans une moindre mesure la fibrinolyse. Grâce à la technique d’ARN interférence, l’activité de la protéine recombinante a été corrélée à l’activité de la protéine native exprimée dans les glandes salivaires. Ir-CPI inhibe dans un plasma humain l’activation réciproque du facteur XII, de la prékallikréine et du facteur XI, et se fixe spécifiquement à la forme activée de ces trois facteurs. De plus, Ir-CPI est capable de bloquer la fibrinolyse en inhibant spécifiquement la plasmine. Enfin, les résultats obtenus sur deux modèles animaux indépendants établissent l’effet d’Ir-CPI comme agent anti-thrombotique en empêchant la formation de caillot et de manière remarquable sans altérer l'équilibre hémostatique.<p><p>En conclusion, ce travail a identifié des protéines anti-hémostatiques qui agissent soit comme inhibiteur de protéases empêchant la coagulation soit comme protéases facilitant la lyse du caillot. Cette redondance de protéines anti-hémostatiques illustre remarquablement la capacité de la tique à agir en synergie sur des facteurs essentiels aux mécanismes de défense de l’hôte.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Abstract The dentin-pulp complex comprises mineralized dentin and the vital soft tissues encased inside dentin, i.e. odontoblasts and pulp tissue. During caries progression, the dentinal minerals are dissolved and eventually the collagenous organic matrix is degraded. However, the exact mechanisms and enzymes responsible for the organic matrix breakdown remain unknown. Matrix metalloproteinases (MMPs), a family of endopeptidases capable of degrading in concert virtually all extracellular matrix components, are expressed during normal dentin-pulp complex formation and maintenance. MMP activity has also been suggested to contribute to the organic matrix degradation during dentin caries progression and to the repair and defense reactions elicited by caries in the dentin-pulp complex cells. The aim of the study was to further elucidate the role of host MMPs in dentin caries progression and the origin of MMPs in carious dentin as well as the possible changes in MMP expression in the cells of the dentin-pulp complex in response to caries. MMP inhibitors decreased the area of dentin caries lesions in vivo, suggesting the involvement of host MMPs in dentin caries pathogenesis. When the overall MMP gene expression was examined by cDNA microarray, pooled pulp samples demonstrated a high level of MMP-13 expression, but failed to show any unequivocal changes in MMP expression due to caries. MMP-13 expression is rare among normal human adult tissues. Real-time quantitative PCR of individual pulp and odontoblast samples demonstrated a rather large variation in relative MMP-13 mRNA expression between samples, especially pulp samples. Protein expression of MMP-13 was detected in pulp and odontoblasts without any major differences between the tissues of sound and carious teeth. This was also the case with the MMP-20 (enamelysin) protein, which was demonstrated in odontoblasts and the pulp tissue of fully developed human teeth. MMP-20, MMP-8, and gelatinases (especially MMP-2) were demonstrated in human dentin, and dentinal MMPs exhibited activity against native and denatured type I collagen when released. The study demonstrates the presence of MMPs in the soft and hard tissue compartments of the dentin-pulp complex. These enzymes may also contribute to dentin caries progression and response reactions to caries.

Abstract Sepsis is a major challenge for healing responses maintaining homeostasis. Coagulation and inflammation are activated at the whole-body level, even in undamaged tissues. Despite constantly growing knowledge and advances in care, high mortality in severe sepsis remains. It was hypothesised that tissue regeneration processes may also be altered in severe sepsis. The study population consisted of 44 patients with severe sepsis and 15 healthy controls. Serum samples were obtained during ten days of severe sepsis and twice again, three months and six months later. Experimental suction blisters were performed twice during severe sepsis and at 3 and 6 months. Serum samples were obtained and suction blisters were induced once in controls. Biochemical analyses were performed to assess the level of procollagen I and III aminoterminal propeptides (PINP, PIIINP), reflecting the synthesis of corresponding collagens; in serum and suction blister fluid. In addition collagen I degradationproduct in serum was measured. Physiological measurements of transepidermal water loss and blood flow were done in order to evaluate the re-epithelisation rate and blood flow in an experimental wound. Levels of matrix metalloproteinases (MMPs) 2, 8 and 9 were measured from serum and suction blister fluid. Decrease in water evaporation from an experimental blister wound was slower in sepsis than in controls. On the fourth day the sepsis patients had higher blood flow in the blister wound than the controls (both in the healing wound and in the newly induced wound). The procollagen III aminoterminal propeptide (PIIINP) levels were increased in serum in severe sepsis, whereas procollagen I aminoterminal propeptide (PINP) levels were not, making up a pronounced PIIINP/PINP ratio. PIIINP and PINP levels were associated with disease severity and outcome. In addition, collagen I degradation measured with ICTP assay was increased in severe sepsis and PINP/ICTP ratio was lower. Furthermore, the overall protein concentration and PINP and PIIINP levels were low in suction blister fluid, which implies that the balance of the extracellular matrix consistence is disturbed in uninjured skin in severe sepsis. Then again in survivors the levels of PINP and PIIINP were up-regulated at three months but returned to normal by six months. MMP-9 levels in serum and skin blister fluid were lower in severe sepsis than in controls during the ten days studied. The MMP-2 levels were found to be increased both in serum and in skin blister fluid in severe sepsis in comparison to the controls and MMP-2 was associated with disease severity and outcome. MMP-8 was increased in serum and in skin blister fluid. In conclusion, the balance of collagen turnover is altered in severe sepsis in serum and skin and epidermal re-epithelisation is delayed. The levels of MMP-2 and MMP-8 are increased whereas levels of MMP-9 are depressed.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-04-15T13:55:03Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Lianne versão final.pdf: 1813010 bytes, checksum: 7347cf54c764e95be94ff262dc30ccb5 (MD5) / Made available in DSpace on 2016-04-15T13:55:03Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Lianne versão final.pdf: 1813010 bytes, checksum: 7347cf54c764e95be94ff262dc30ccb5 (MD5) Previous issue date: 2015-08-11 / CAPEs / O câncer do colo uterino é o quarto tipo de câncer mais comum entre as mulheres mundialmente, e o sétimo no geral. No Brasil, esta neoplasia é a terceira mais frequente no gênero feminino, com mais de 15 mil casos novos a cada ano. A infecção persistente pelo papilomavírus humano dos tipos de alto risco oncogênico está associada ao câncer cervical, porém uma pequena percentagem de mulheres infectadas por estes vírus desenvolve lesões pré-malignas que podem evoluir à invasão. Portanto, outros cofatores são necessários para a persistência viral que, após 2 anos sem intervenção, leva à expressão desregulada e concomitante das oncoproteínas transformantes E6 e E7. As enzimas degradadoras de matriz extracelular conhecidas como metaloproteinases da matriz tem sido extensamente estudadas devido ao seu papel na invasão, metástase, angiogênese e recidiva tumoral. Nas lesões cervicais, um aumento na expressão das metaloproteinases foi identificado, porém, seu papel na progressão das lesões pré-invasivas e sua interação com a infecção pelo papilomavírus ainda não está bem esclarecida. Neste intuito, o presente estudo visou avaliar, através de técnica imuno-histoquímica, a associação dos níveis de expressão das metaloproteinases da matriz 2 e 9 com os graus de lesões cervicais. Para isso, foram utilizadas amostras de biopsias parafinadas, diagnosticadas e arquivadas entre 2011 e 2015 no Laboratório de Anatomia Patológica do Hospital das Clínicas de Recife, Pernambuco, Brasil, incluindo 115 biopsias de lesões cervicais, obtidas por conização, bem como 14 casos de tecidos cervicais sem lesão. Os resultados obtidos neste trabalho demonstraram um aumento da expressão das metaloproteinases 2 e 9 nos casos de invasão em relação a condições pré-malignas, tanto no citoplasma quanto no núcleo de células epiteliais escamosas, sendo a intensidade da expressão nuclear das metaloproteinases estudadas mais significativa nas células cancerosas. Também foi observada diminuição da expressão das metaloproteinases 2 e 9 no citoplasma das células glandulares adjacentes na presença do carcinoma escamoso invasor, quando comparadas ao controle. Mais estudos precisam ser realizados para avaliar o valor prognóstico das metaloproteinases 2 e 9 nas condições pré-malignas e malignas do colo uterino. / The cervical cancer is the fourth most common cancer among women worldwide, and seventh overall. In Brazil, this cancer is the third most common in women, with more than 15,000 new cases each year. Persistent infection with human papillomavirus types of high oncogenic risk is associated with cervical cancer, but a small percentage of women infected by these viruses develop precancerous lesions that may progress to invasion. Therefore, other cofactors are required for viral persistence which, after 2 years without intervention, leads to unregulated and concomitant expression of transformants E6 and E7 oncoproteins. The extracellular matrix degrading enzymes known as matrix metalloproteinases has been extensively studied because of its role in invasion, metastasis, angiogenesis and tumor recurrence. In cervical lesions, an increase in the expression of metalloproteinases was identified, however, its role in the progression of pre-invasive lesions and their interaction with the papillomavirus infection is not well understood. To this end, the present study evaluated, through immunohistochemical technique, the association of the expression levels of matrix metalloproteinases 2 and 9 with grades of cervical lesions. For this, we used paraffined biopsy samples, diagnosed and filed between 2011 and 2015 at the Pathology Laboratory of the Hospital das Clínicas of Recife, Pernambuco, Brazil, including 115 biopsies of cervical lesions, obtained by conization, and 14 cases of tissues without cervical injury. The results of this study demonstrated an increased expression of metalloproteinases 2 and 9 in invasion compared to pre-malignant conditions, in both cytoplasm and nucleus of squamous epithelial cells, and a more significant intensity of the nuclear expression of metalloproteinases studied in cancer cells. It was also observed decreased expression of metalloproteinases 2 and 9 in the cytoplasm of adjacent glandular cells in the presence of invasive squamous cell carcinoma, when compared to control. More studies are needed to assess the prognostic value of metalloproteinases 2 and 9 in premalignant and malignant conditions of the cervix.

Orientador: Pedro Duarte Novaes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-27T00:17:50Z (GMT). No. of bitstreams: 1 Peroni_LeonardoVieira_M.pdf: 2591831 bytes, checksum: cb9ff6e79f251a6f1318c8e8daa209f7 (MD5) Previous issue date: 2015 / Resumo: A radioterapia causa efeitos adversos em grande parte dos tecidos nos quais é utilizada. Na cavidade oral, tem sido evidenciado que o periodonto é sensível à radiação em altas doses. Além de causar mudanças histofisiológicas, a radiação ionizante pode propiciar o desenvolvimento de alterações estruturais nos tecidos de suporte, incluindo osso e ligamento periodontal. As metaloproteinases de matriz (MMPs) estão presentes no ligamento periodontal e são enzimas que atuam na remodelação da matriz extracelular, degradando alguns de seus componentes. Acredita-se que sua expressão possa alterar a estrutura do ligamento periodontal. Sendo assim, neste trabalho teve-se como objetivo avaliar o efeito da radiação ionizante no ligamento periodontal do dente incisivo de rato, por meio da microscopia de polarização, do teste mecânico e da zimografia, estabelecendo possíveis correlações entre os testes. A amostra constituiu-se de 63 ratos machos da linhagem Wistar (Rattus norvegicus albinus) divididos em 3 grupos: o grupo controle, sendo o único grupo não irradiado (n=21), o grupo de animais mortos 14 dias após a irradiação (n=21) e grupo de animais mortos 28 dias após a irradiação (n=21). Os grupos irradiados foram submetido à sessão única de radioterapia com dose de 15. A análise dos resultados mostrou que o teste de força e a microscopia de polarização apresentaram diferença estatisticamente significante entre os grupos (p<0,001), o que não ocorreu para os resultados da zimografia. Os coeficientes de correlação de Pearson (R) detectaram forte correlação diretamente proporcional entre os testes de força e polarização (R=0,808), e moderada correlação inversamente proporcional entre a polarização e a zimografia de MMP-2 na forma ativa (R=-0,446). Portanto, concluiu-se que a radioterapia pode levar à diminuição da resistência à força de intrusão e provocar a desorganização do colágeno no ligamento periodontal; além disso, a expressão das metaloproteinases 2 e 9 não foi influenciada pela radiação ionizante e apenas a organização do colágeno apresentou correlação com a resistência das fibras do ligamento periodontal e com a expressão da metaloproteinase de matriz 2 na sua forma ativa / Abstract: Radiotherapy causes adverse effects in most tissues in which it is used. In the oral cavity, it has been shown that the periodontium is sensitive to high doses of radiation. Besides causing histophysiological changes, ionizing radiation can promote the development of structural changes in the supporting tissues, including bone and periodontal ligament. The matrix metalloproteinases (MMPs) are in the periodontal ligament and are enzymes that remodel extracellular matrix by degrading some of its components. Structure of periodontal ligament may be changed by its expression. Thus, this study aimed to evaluate the effect of ionizing radiation on the periodontal ligament of the rat incisor, by polarizing microscopy, strength and zymography test, establishing correlations among these tests. The sample consisted of 63 male Wistar rats (Rattus norvegicus albinus) divided into 3 groups: control group, the only group that wasn¿t irradiated (n=21); a group of animals killed 14 days after irradiation (n=21) and a group of animals killed 28 days after irradiation (n=21). Irradiated groups were submitted to a single session of radiotherapy with 15 Gy dose. The results showed that only the strength test and the polarization microscopy had a statistically significant difference between groups (p<0.001), differently from zimography results. The Pearson correlation coefficients (R) detected strong direct correlation between the strength tests and polarization (R= 0.808), and moderate inverse correlation between polarization and MMP-2 zymography in the active form (R= -0.446). Thereby, the conclusion was that radiotherapy can lead to a diminished resistance to the intrusion strength and to a disorganization of the periodontal¿s ligament collagen. The expression of metalloproteinases 2 and 9 was not affected by radiotherapy and only collagen organization had correlation with periodontal ligament fibers strength and expression of matrix metalloproteinase 2 in its active form / Mestrado / Radiologia Odontologica / Mestre em Radiologia Odontológica

O objetivo deste trabalho foi analisar a relação entre estrutura e função dos domínios não catalíticos do HF3, uma metaloproteínase da classe P-III do veneno da Bothrops jararaca com atividades hemorrágica e pró-inflamatória. Mostramos que proteínas recombinantes contendo o domínio rico em cisteínas (domínios tipo-disintegrina/rico em cisteínas, DC, e domínio rico em cisteínas, C) são capazes de aumentar o rolamento de leucócitos na microcirculação e de inibir a agregação plaquetária induzida pelo colágeno. Por outro lado, a proteína D e a proteína DC contendo a mutação Asp/Ala no motif Glu-Cys-Asp não apresentaram estas atividades. Peptídeos derivados da região hiper variável (HVR) do domínio rico em cisteínas também promoveram o rolamento de leucócitos, sendo esta atividade foi inibida por anticorpos anti-aMb2, e ainda inibiram a agregação plaquetária. Em conjunto, estes resultados sugerem que o domínio rico em cisteínas do HF3 e sua HVR desempenham um papel em sua atividade pró-inflamatória mediada pela integrina aMb2, e na inibição da agregação plaquetária. / This aim of this study was analyze the relationship between structure and function of the non-catalytic domains of HF3, a hemorrhagic and pro-inflammatory metalloproteinase of the P-III class, from Bothrops jararaca venom. Here we show that recombinant proteins of HF3 containing the cysteine-rich domain (disintegrin-like/cysteine rich and cysteine-rich proteins) but not the disintegrin-like protein and a disintegrin-like/cysteine rich protein carrying the mutation Asp/Ala in the Glu-Cys-Asp motif were able to significantly increase leukocyte rolling in the microcirculation and to inhibit collagen-induced platelet aggregation. Peptides from the hyper variable region (HVR) of the cysteine-rich domain also promoted leukocyte rolling and this activity was inhibited by anti-aMb2 antibodies. HVR peptides also inhibited platelet-aggregation. Taken together, these results suggest that the cysteine- rich domain of HF3 and its HVR play a role in triggering pro-inflammatory effects mediated by integrin aM/b2 and in the inhibition of platelet-aggregation.