A hipóxia é capaz de alterar muitos mecanismos bioquímicos nas células endoteliais. Dentre eles, a indução da expressão endotelial de moléculas de adesão, como o fator de von Willebrand (FVW) que, em resposta ao estímulo, é secretado em sua forma mais ativa na interação com as plaquetas, o que pode resultar em trombose. Nas condições fisiológicas, o padrão multimérico do FVW no plasma é essencialmente determinado pela ADAMTS-13 (uma desintegrina e metaloproteinase com domínios trombospondina). Este estudo teve como objetivo verificar se a atividade da enzima ADAMTS-13, assim como as características do FVW relacionáveis a ela, poderiam estar alteradas na presença de hipoxemia comparativamente à condição de oxigenação normal. Este estudo longitudinal envolveu 56 pacientes portadores de cardiopatias congênitas cianogênicas, em idades entre um e sete anos, candidatos ao tratamento cirúrgico. Os pacientes foram avaliados no pré-cirúrgico (basal), no pós-operatório imediato (pós 48 horas) e após 30 dias de cirurgia, e foram divididos em dois grupos (A e B) baseado na saturação periférica de oxigênio (SpO2) no momento pós 30 dias. Foram determinados o antígeno do FVW e a análise das suas subunidades, a atividade da ADAMTS-13 e a presença de inibidores da ADAMTS-13. Os pacientes de ambos os grupos apresentaram aumento significante da SpO2, da concentração antigênica do FVW e da atividade da ADAMTS-13 nos momentos pós 48 horas e pós 30 dias em comparação com o momento pré (basal). As densidades normalizadas da subunidade principal do FVW (225 kDa) e do fragmento de 176 kDa apresentaram tendência ao aumento nos momentos pós 48 horas e pós 30 dias nos dois grupos. A razão entre a atividade da ADAMTS-13 e o FVW estava menor do que 1 no momento pós 48 horas, indicando consumo da enzima; entretanto, no momento pós 30 dias a razão fica 1:1, e o FVW se aproxima dos valores de referência. Verificamos ainda que 29% destes pacientes apresentaram inibidores contra a ADAMTS-13 no momento pré-operatório. Ainda explorando as variáveis SpO2, FVW:Ag, atividade da ADAMTS-13 e a composição das subunidades do FVW, foi feito um estudo de correlação linear entre estas variáveis. Observamos uma baixa correlação entre a enzima ADAMTS-13 e o FVW:Ag, e da enzima com os fragmentos do FVW de 176 e 140 kDa, principalmente no grupo B. No grupo A, esta correlação no momento pós 48 horas mostrou tendência a ser negativa. A maioria dos pacientes apresentou melhoras na saturação periférica de oxigênio. O aumento das variáveis estudadas no pós-operatório imediato pode ter ocorrido em função da cirurgia, que provavelmente ocasionou um quadro de lesão endotelial com inflamação, indicando que pode existir um equilíbrio entre o FVW e a ADAMTS-13 em níveis fisiológicos. Entretanto, este equilíbrio pode ser quebrado quando ocorre aumento do FVW, provavelmente por consumo da enzima. Parece-nos, portanto, que a ADAMTS-13 pode funcionar como um mecanismo de proteção a estes pacientes com tendência à trombose / Hypoxia has been shown to alter several biochemical mechanisms in endothelial cells. In addition, hypoxia induces the endothelial expression of adhesion molecules, including von Willebrand factor (VWF). Increased release of high-molecular-weight VWF multimers is associated with higher risk for thrombotic events. In physiological conditions, the multimeric pattern of plasma VWF is essentially determined by the action of ADAMTS-13 (a desintegrin and metalloprotease with thrombospondin type 1 domains). The aim of this study was to investigate if ADAMTS-13 activity and VWF subunit fragments were altered by hypoxia in cyanotic congenital heart disease. Fiftysix patients (age 1 to 7 years) with cyanotic congenital heart disease admitted to the Heart Institute for heart surgery were included in this longitudinal study. Patients were evaluated before (baseline) corrective surgery, postoperative 48 hours and postoperative 30 days. Patients were classified in two groups (A and B) based on the peripheral oxygen saturation after 30 days surgery. VWF antigenic concentration, VWF subunit composition, ADAMTS-13 activity and presence of ADAMTS-13 inhibitors were determined. Peripheral oxygen saturation, VWF:Ag and ADAMTS-13 activity were all increased significantly in both groups, in postoperative 48 hours and postoperative 30 days in comparison with baseline moment. Normalized density of VWF main subunit (225 kDa) and proteolytic fragment with 176 kDa tended to increase in postoperative 48 hours and postoperative 30 days in both groups. The rate between ADAMTS-13 activity and VWF:Ag was lower than 1 in postoperative 48 hours, an indicating of enzyme consumption; however, in the postoperative 30 days the rate was 1:1 and VWF:Ag values were near those of reference. 29% of patients presented ADAMTS-13 inhibitors at the baseline moment. A study of correlation among variables as peripheral oxygen saturation, VWF:Ag, VWF subunit composition and ADAMTS-13 was done. It was observed that ADAMTS-13 correlated slightly positively with VWF:Ag and with VWF fragments 176 and 140 kDa, mainly in group B; in group A, the correlation at postoperative 48 hours tended to be negative. Most of the patients improved their peripheral oxygen saturation. The increased value of variables observed in postoperative 48 hours can be explained by the endothelial injury and inflammation caused by the surgery itself. This indicates an equilibrium between VWF:Ag and ADAMTS-13 in physiological conditions. However, this equilibrium could disappear when VWF is increased, probably by enzyme consumption. We conclude that ADAMTS-13 can act as a protective mechanism in these patients with thrombotic tendency

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Arthritis is a debilitating disease of the joints caused by the accelerated breakdown of cartilage, resulting in painful, swollen joints. Cartilage protects the joint by absorbing the shock that would otherwise be transferred directly to the underlying bone. One crucial component of cartilage is a specialised molecule known as aggrecan. Aggrecan consists of a core protein with three globular domains (G1, G2 and G3) and is modified with over one hundred highly sulphated glycosaminoglycan chains. Two types of glycosaminoglycans are substituted along the length of the protein, chondroitin sulphate and keratan sulphate. The glycosaminoglycans impart a highly negative charge to the tissue, giving it the ability to retain water and resist compressive forces. / Aggrecan is lost from cartilage following cleavage by aggrecanases. Too little aggrecan in cartilage destabilises the structural integrity of the tissue and is associated with arthritis. Of the five known aggrecanase cleavage sites, it is cleavage within the interglobular domain (IGD) between the G1 and G2 domains at NITEGE373 - 374ARGSVI that directly contributes to loss of aggrecan function. / The chondroitin sulphate and keratan sulphate located between the G2 and G3 domains is responsible for maintaining the biomechanical properties of aggrecan. The role of keratan sulphate within the G1-G2 domain is unknown, but it is not thought to be essential for aggrecan function. However the literature suggests a possible role of keratan sulphate in facilitating aggrecanase cleavage of NITEGE373 - 374ARGSVI in the IGD. The aim of my project was to examine the role of keratan sulphate in aggrecanase-mediated cleavage of aggrecan in the IGD. Three major goals have been accomplished in this thesis: 1) Identification of a cell type capable of sustained keratan sulphate synthesis. 2) Expression of a recombinant G1-G2 protein substituted with keratan sulphate (rG1-G2). 3) Demonstration that endogenous N-linked keratan sulphate is sufficient to potentiate aggrecanase cleavage of rG1-G2 in the IGD. / Cultured cells do not synthesise keratan sulphate. Therefore identifying a cell type, and culture conditions to maximise keratan sulphate synthesis, was a major undertaking. Conditions were identified which allowed for maximal keratan sulphate synthesis, albeit on a small scale, in primary bovine keratocytes. Using a Vaccinia virus expression system, recombinant G1-G2 was expressed in primary bovine keratocytes. / Analysis of the rG1-G2 revealed that it was substituted with 5 kDa of keratan sulphate. One important aspect of the study was that the keratan sulphate was all N-linked to the core protein. Subsequent aggrecanase digests, comparing substrates before and after removal of keratan sulphate, showed that aggrecanase cleavage was markedly more efficient when keratan sulphate was present. The results contained in this thesis add significantly to the established literature by providing a greater understanding of the mechanisms involved in aggrecanase-mediated cleavage of aggrecan and cartilage destruction. The results suggest that aggrecan substitution with N-linked keratan sulphate potentiates aggrecanase activity. The results from this study identify N-linked keratan sulphate as a possible target for the development of new drugs for the management of arthritis.

Equine laminitis is a devastating condition that results in the failure of the tissue responsible for suspending the skeleton within the hoof capsule. The digital laminae is composed of two interdigitated layers, the dermal lamellae surrounding the distal pedal bone, and the epidermal lamellae, which interfaces with the hoof wall. During laminitis, these layers separate, allowing for rotation and sinking of the pedal bone. While there are multiple diseases and physiological conditions associated with the development of laminitis, including sepsis, metabolic syndrome, and unequal weight bearing, the exact cause remains elusive. Prior work by our research group identified the metalloprotease ADAMTS-4 as a potential early instigator of disease. The data presented herein catalogs the distribution of the substrates of this enzyme, aggrecan and versican, the ramifications of ADAMTS-4-mediated versican loss in the laminae, and further expands into the repression of the canonical wnt signaling pathway and potential additional metalloprotease (MMP) involvement in disease, utilizing a model of acute, carbohydrate-induced laminitis. Additionally, samples from other models of laminitis induction and clinical samples were screened for differential expression of relevant gene markers, including versican, members of the canonical wnt signaling pathway, and MMP-1 and -13. Together, these data provide a characterization of laminar pathology in the carbohydrate-induced model, as well as highlighting key similarities and differences amongst multiple methods of disease development, and lay important groundwork for developing clinical therapeutic interventions.

A Síndrome Antifosfolípide (SAF) é uma doença autoimune na qual há presença de anticorpos antifosfolípides [anticoagulante lúpico (AL), anticardiolipina (ACL), anti-beta2glicoproteína I (a-beta2GPI)]. É caracterizada por eventos trombóticos e/ou perdas gestacionais de repetição. Existe um ambiente pró-coagulante na SAF, pois os antifosfolípides (AFL) são capazes de induzir disfunção endotelial aumentando a expressão de moléculas de adesão como o fator de von Willebrand (FVW). Entre os inúmeros elementos envolvidos neste processo, o FVW e a relação com: sua principal enzima proteolítica, a ADAMTS-13, e com o FVIII são relativamente menos conhecidos. Os objetivos deste estudo foram: 1-verificar alterações no endotélio de pacientes com SAF primária (SAFP), através do marcador de lesão endotelial, o FVW, da ADAMTS-13 e do FVIII, avaliando a concentração antigênica, a atividade e a correlação entre estas proteínas; 2- Verificar se alguma destas variáveis é capaz de diferenciar os tipos de eventos trombóticos ocorridos nestes pacientes e se a presença dos AFL influenciam estas proteínas. O estudo, do tipo transversal, envolveu 39 pacientes com SAF primária, com idade mediana de 43 anos, em tratamento no ambulatório de Reumatologia do Hospital das Clínicas, e 39 controles sadios doadores da Fundação Pró-Sangue Hemocentro de São Paulo, pareados por sexo e idade com os pacientes. Os títulos de ACL e a-beta2GPI, as determinações antigênicas e de atividade das proteínas FVW, ADAMTS-13, FVIII e PF4 foram realizados por ELISA, e a atividade do FVIII foi determinada pelo método cromogênico. A análise das subunidades do FVW foi realizada por Western immunoblotting. AL foi detectado utilizando ensaios de coagulação de acordo com as recomendações da Sociedade Internacional de Trombose e Hemostasia. Os resultados foram apresentados em: 1- pacientes SAFP e controles, e 2- pacientes SAFP agrupados em relação ao tipo de evento e ao tipo de AFL presente. Os pacientes apresentaram aumento da concentração antigênica do FVW (74±6 x 69±11 UI/dL; p=0,016), ADAMTS-13 (1,3±0,34 x 0,82±0,12 Ug/mL; p < 0,0001), FVIII (106±19 x 91±15 UI/dL; p=0,0003),da ligação FVW:FVIII (144±17 x 134±20%; p=0,082) e da atividade do FVIII (117±38 x 98±30%; p=0,0021) comparado aos controles. O PF4 apresentou-se diminuído nos pacientes em relação aos controles (96±12 x 101±8 UI/mL; p=0,014). Os pacientes com trombose arterial apresentaram correlação positiva e significante entre a atividade e o antígeno do FVW (r=0,468; p=0,028) e da ADAMTS-13 (r =0,635; p=0,001); os pacientes com trombose venosa apresentaram esta correlação positiva e significante na ADAMTS-13 (r=0,635; p=0,001). Quando os pacientes foram analisados pelo tipo de antifosfolípide, não se observou diferenças nas variáveis estudadas. Os pacientes com SAFP parecem apresentar disfunção endotelial. No entanto, aparentemente existe um mecanismo de equilíbrio para evitar a formação de um novo trombo. O papel do FVW e a sua relação com a ADAMTS-13, em diferentes doenças, ainda é relativamente pouco conhecido, mas está sendo apontado como importante na patogênese de estados pró-trombóticos como os presentes em pacientes com SAF / Antiphospholipid syndrome (APS) is an autoimmune disease, characterized by vascular thrombosis and /or pregnancy morbidity, in association with antiphospholipid antibodies (aPL) (lupus anticoagulant (LA), anticardiolipin (ACL), anti-beta2glicoprotein I (a-beta2GPI). Antiphospholipid (APL) seems to induce endothelial dysfunction by increasing the expression of adhesion molecules such as von Willebrand factor (VWF). This results in a prothrombotic state in APS. Among the several elements involved in this process, some are relatively less known, such as VWF and its relationship with ADAMTS-13, its main proteolytic enzyme. The aim of this study was to evaluate endothelial dysfunctions in patients with primary APS (PAPS), by examining correlation among the soluble endothelial marker, VWF, the enzyme ADAMTS-13, and FVIII protein. The relationship of these proteins and the presence of arterial and/or venous thrombosis, and presence of APL was also evaluated. This cross-sectional study involved 39 PAPS patients, with a median age of 43 years, who have been treated in the Outpatient Clinics, Department of Rheumatology, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, and 39 healthy subjects blood donors from the Fundação Pró-Sangue Hemocentro de São Paulo, matched for sex and age with patients. Levels of APL (ACL and a-beta2GPI), concentration and activities of VWF, ADAMTS-13, FVIII and PF4 proteins were measured with ELISA. LA was detected with coagulation assays according to updated guidelines from the International Society on Thrombosis and Haemostasis, and FVIII activity was measured by chromogenic method. Analysis of VWF subunits was performed by Western immunoblotting. The results were evaluated according to:1- PAPS patients and controls, and2- PAPS patients grouped in relation to type of event and the type of APL. Patients showed higher VWF antigen concentration (74±6 x 69±11 IU/dL, p=0.016), ADAMTS-13 (1.3±0.34 x 0.82±0.12 Ug/mL, p < 0.0001), FVIII (106±19 x 91 ± 15 IU/dL, p=0.0003), VWF binding to FVIII (144±17 x 134 ± 20%, p=0.082) and activity of FVIII (117±38 x 98±30%, p=0.0021) than controls. The PF4 was decreased in patients compared to controls (96±12 vs. 101±8 IU/mL, p=0.014). VWF antigen and activity correlated well (Pearson´s r =0.468; p=0.028) as well as ADAMTS-13(Pearson´s r=0.635; p=0.001) in patients with arterial thrombosis. However, in patients with venous thrombosis only ADAMTS-13 had a good correlation (Pearson´s r =0.492; p=0.045). When patients were analyzed by the type of aPL, no differences in the studied variables were observed. Patients with PAPS seem to present endothelial dysfunction. However, apparently there is an attempt to balance mechanism to prevent a new thrombus formation. The role of VWF and its relation with ADAMTS-13 in different diseases is still relatively unknown. However it has been considered as important in the pathogenesis of prothrombotic states such as those present in patients with APS

A Síndrome Antifosfolípide (SAF) é uma doença autoimune na qual há presença de anticorpos antifosfolípides [anticoagulante lúpico (AL), anticardiolipina (ACL), anti-beta2glicoproteína I (a-beta2GPI)]. É caracterizada por eventos trombóticos e/ou perdas gestacionais de repetição. Existe um ambiente pró-coagulante na SAF, pois os antifosfolípides (AFL) são capazes de induzir disfunção endotelial aumentando a expressão de moléculas de adesão como o fator de von Willebrand (FVW). Entre os inúmeros elementos envolvidos neste processo, o FVW e a relação com: sua principal enzima proteolítica, a ADAMTS-13, e com o FVIII são relativamente menos conhecidos. Os objetivos deste estudo foram: 1-verificar alterações no endotélio de pacientes com SAF primária (SAFP), através do marcador de lesão endotelial, o FVW, da ADAMTS-13 e do FVIII, avaliando a concentração antigênica, a atividade e a correlação entre estas proteínas; 2- Verificar se alguma destas variáveis é capaz de diferenciar os tipos de eventos trombóticos ocorridos nestes pacientes e se a presença dos AFL influenciam estas proteínas. O estudo, do tipo transversal, envolveu 39 pacientes com SAF primária, com idade mediana de 43 anos, em tratamento no ambulatório de Reumatologia do Hospital das Clínicas, e 39 controles sadios doadores da Fundação Pró-Sangue Hemocentro de São Paulo, pareados por sexo e idade com os pacientes. Os títulos de ACL e a-beta2GPI, as determinações antigênicas e de atividade das proteínas FVW, ADAMTS-13, FVIII e PF4 foram realizados por ELISA, e a atividade do FVIII foi determinada pelo método cromogênico. A análise das subunidades do FVW foi realizada por Western immunoblotting. AL foi detectado utilizando ensaios de coagulação de acordo com as recomendações da Sociedade Internacional de Trombose e Hemostasia. Os resultados foram apresentados em: 1- pacientes SAFP e controles, e 2- pacientes SAFP agrupados em relação ao tipo de evento e ao tipo de AFL presente. Os pacientes apresentaram aumento da concentração antigênica do FVW (74±6 x 69±11 UI/dL; p=0,016), ADAMTS-13 (1,3±0,34 x 0,82±0,12 Ug/mL; p < 0,0001), FVIII (106±19 x 91±15 UI/dL; p=0,0003),da ligação FVW:FVIII (144±17 x 134±20%; p=0,082) e da atividade do FVIII (117±38 x 98±30%; p=0,0021) comparado aos controles. O PF4 apresentou-se diminuído nos pacientes em relação aos controles (96±12 x 101±8 UI/mL; p=0,014). Os pacientes com trombose arterial apresentaram correlação positiva e significante entre a atividade e o antígeno do FVW (r=0,468; p=0,028) e da ADAMTS-13 (r =0,635; p=0,001); os pacientes com trombose venosa apresentaram esta correlação positiva e significante na ADAMTS-13 (r=0,635; p=0,001). Quando os pacientes foram analisados pelo tipo de antifosfolípide, não se observou diferenças nas variáveis estudadas. Os pacientes com SAFP parecem apresentar disfunção endotelial. No entanto, aparentemente existe um mecanismo de equilíbrio para evitar a formação de um novo trombo. O papel do FVW e a sua relação com a ADAMTS-13, em diferentes doenças, ainda é relativamente pouco conhecido, mas está sendo apontado como importante na patogênese de estados pró-trombóticos como os presentes em pacientes com SAF / Antiphospholipid syndrome (APS) is an autoimmune disease, characterized by vascular thrombosis and /or pregnancy morbidity, in association with antiphospholipid antibodies (aPL) (lupus anticoagulant (LA), anticardiolipin (ACL), anti-beta2glicoprotein I (a-beta2GPI). Antiphospholipid (APL) seems to induce endothelial dysfunction by increasing the expression of adhesion molecules such as von Willebrand factor (VWF). This results in a prothrombotic state in APS. Among the several elements involved in this process, some are relatively less known, such as VWF and its relationship with ADAMTS-13, its main proteolytic enzyme. The aim of this study was to evaluate endothelial dysfunctions in patients with primary APS (PAPS), by examining correlation among the soluble endothelial marker, VWF, the enzyme ADAMTS-13, and FVIII protein. The relationship of these proteins and the presence of arterial and/or venous thrombosis, and presence of APL was also evaluated. This cross-sectional study involved 39 PAPS patients, with a median age of 43 years, who have been treated in the Outpatient Clinics, Department of Rheumatology, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, and 39 healthy subjects blood donors from the Fundação Pró-Sangue Hemocentro de São Paulo, matched for sex and age with patients. Levels of APL (ACL and a-beta2GPI), concentration and activities of VWF, ADAMTS-13, FVIII and PF4 proteins were measured with ELISA. LA was detected with coagulation assays according to updated guidelines from the International Society on Thrombosis and Haemostasis, and FVIII activity was measured by chromogenic method. Analysis of VWF subunits was performed by Western immunoblotting. The results were evaluated according to:1- PAPS patients and controls, and2- PAPS patients grouped in relation to type of event and the type of APL. Patients showed higher VWF antigen concentration (74±6 x 69±11 IU/dL, p=0.016), ADAMTS-13 (1.3±0.34 x 0.82±0.12 Ug/mL, p < 0.0001), FVIII (106±19 x 91 ± 15 IU/dL, p=0.0003), VWF binding to FVIII (144±17 x 134 ± 20%, p=0.082) and activity of FVIII (117±38 x 98±30%, p=0.0021) than controls. The PF4 was decreased in patients compared to controls (96±12 vs. 101±8 IU/mL, p=0.014). VWF antigen and activity correlated well (Pearson´s r =0.468; p=0.028) as well as ADAMTS-13(Pearson´s r=0.635; p=0.001) in patients with arterial thrombosis. However, in patients with venous thrombosis only ADAMTS-13 had a good correlation (Pearson´s r =0.492; p=0.045). When patients were analyzed by the type of aPL, no differences in the studied variables were observed. Patients with PAPS seem to present endothelial dysfunction. However, apparently there is an attempt to balance mechanism to prevent a new thrombus formation. The role of VWF and its relation with ADAMTS-13 in different diseases is still relatively unknown. However it has been considered as important in the pathogenesis of prothrombotic states such as those present in patients with APS

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No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AnaliseImunohistoquímicaAdamts.pdf: 2182822 bytes, checksum: f614e4856b0e4ff30df45d31a8939a93 (MD5) Previous issue date: 2014 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O ameloblastoma e o tumor odontogênico cístico calcificante (TOCC) são tumores odontogênicos que tem origem do epitélio odontogênico, porém ainda não é conhecido o estímulo ou gatilho que leva à transformação neoplásica desses tumores. O comportamento biológico das lesões é distinto, pois o ameloblastoma é um tumor mais agressivo e com taxa de recorrência significativa. Já o TOCC é um tumor menos agressivo e raramente há recorrência e por esse motivo foi utilizado como controle no estudo. Portanto, a elucidação completa dos mecanismos pelos quais esses tumores odontogênicos apresentam tais comportamentos biológicos continua sendo um desafio para os pesquisadores. As c (A Disintegrin and Metalloproteinase with ThromboSpondin) são metaloendopeptidases que são dependentes de zinco em seu domínio catalítico. Essas enzimas possuem ampla atividade catalítica contra uma variedade de substratos como os proteoglicanos (agrecan, brevican e versican), que são proteínas presente na matriz extracelular (MEC). As ADAMTS exibem características estruturais que lhes conferem um grande potencial para exibir múltiplas funções. Exibem função crucial em vários processos como proliferação, adesão, invasão e sinalização celular. As alterações nessas enzimas estão presentes em diversos tumores, o que sugere que estas proteínas podem estar envolvidas no processo carcinogênico em diferentes caminhos. Especificamente a ADAMTS-1 tem sido correlacionada com a tumorigênese de algumas neoplasias como no câncer de mama, pulmão e pâncreas. Assim como a ADAMTS, agrecan, brevican e versican são expressos em vários tumores e a regulação alterada desses proteoglicanos pode contribuir para o desenvolvimento da carcinogênese. Neste trabalho foram estudadas ADAMTS-1, agrecan, brevican e versican no ameloblastoma e TOCC. Foram incluídos 20 casos de ameloblastoma e 6 casos de TOCC, utilizados como controle. A expressão de ADAMTS-1, agrecan, brevican e versican foi avaliada por imunohistoquímica e as áreas de marcação foram mensuradas e analisadas. Para análise de correlação entre as proteínas estudadas utilizou-se o teste de Spearman. Todas as amostras de ameloblastoma expressaram ADAMTS-1, agrecan, brevican e versican. Todas as amostras de TOCC também expressaram as mesmas proteínas, porém numa quantidade significativamente menor que no ameloblastoma. A diferença de expressão de ADAMTS-1 e brevican no epitélio do ameloblastoma e do TOCC foi significante estatisticamente (p<0,0105). Assim como a expressão de agrecan e versican, no epitélio do ameloblastoma e do TOCC, também foi estatisticamente significante (p<0,0067) e (p<0,0148), respectivamente. Não houve correlação entre as proteínas estudadas. / Ameloblastoma and calcifying cystic odontogenic tumor (CCOT) are odontogenic tumors with origin odontogenic epithelium, but it is not yet known stimulus or trigger that lead to neoplastic transformation of tumors. The biological behavior of the lesions is distinct because the ameloblastoma is more aggressive and significant rate of tumor recurrence. CCOT is a less aggressive tumor and recurrence rarely there and therefore was used as a control in the study. Therefore, the complete elucidation of the mechanisms by which these odontogenic tumors show such biological behavior remains a challenge for researchers. The ADAMTS (A Disintegrin and Metalloproteinase with thrombospondin) are metalloendopeptidases who are dependent on zinc in its catalytic domain. These enzymes have catalytic activity against a broad range of substrates including proteoglycans (aggrecan, brevican and versican), which are proteins present in the extracellular matrix (ECM). The ADAMTS exhibit structural features that confer great potential to display multiple functions. Exhibit crucial role in various processes such as proliferation, adhesion, invasion and cell signaling. Changes to these enzymes are present in various tumors, suggesting that these proteins may be involved in the carcinogenic process in different ways. Specifically, ADAMTS-1 has been correlated with tumorigenesis of some cancers such as in breast, lung and pancreatic cancer. Like ADAMTS, aggrecan, versican and brevican are expressed in various tumors and altered regulation of proteoglycans may contribute to the development of carcinogenesis. In this work ADAMTS-1, aggrecan, brevican and versican in ameloblastoma and CCOT were studied, 20 cases of ameloblastoma and 6 cases of TOCC, used as controls were included. We evaluated the expression of ADAMTS-1, aggrecan, brevican and versican by immunohistochemical study and the marking areas were measured and analyzed. To correlation analysis between the studied proteins used the Spearman test. All samples of ameloblastoma expressed ADAMTS-1, aggrecan, brevican and versican. All samples TOCC also expressed the same proteins, but in significantly less than the amount ameloblastoma. The difference in expression of ADAMTS-1 and brevican in the epithelium of ameloblastoma and of TOCC was statistically significant (p<0.0105). As the expression of aggrecan and versican, between ameloblastoma and TOCC, in the epithelium was also statistically significant (p<0.0067) and (p<0.0148), respectively. There was no correlation between the proteins studied.

smig-1(ev809) is a novel suppressor of multiple distal tip cell (DTC) migration mutants in the nematode Caenorhabditis elegans. In the C. elegans hermaphrodite, the two U-shaped gonad arms develop and form as a result of the migration of two DTCs. smig-1(ev809) suppresses DTC migration defects of mutants encoding components of intracellular glycosylation pathways as well as extracellular basement membrane glycoproteins. The smig-1(ev809) mutation bypasses the requirement for a fully functional chondroitin pathway and MIG-17 metalloprotease in DTC pathfinding. I found that i) suppression of the hypomorphic chondroitin mutant mig-22(k141) is not completely dependent on MIG-17 activity; ii) the smig-1(ev809) mutant is likely to be a loss- of-function suppressor; and iii) SMIG-1 does not visibly affect the localization of poorly glycosylated MIG-17 on the gonad surface. Understanding SMIG-1 function will shed light on the role of glycosylation, the extracellular matrix and basement membranes in cell migration during development.

smig-1(ev809) is a novel suppressor of multiple distal tip cell (DTC) migration mutants in the nematode Caenorhabditis elegans. In the C. elegans hermaphrodite, the two U-shaped gonad arms develop and form as a result of the migration of two DTCs. smig-1(ev809) suppresses DTC migration defects of mutants encoding components of intracellular glycosylation pathways as well as extracellular basement membrane glycoproteins. The smig-1(ev809) mutation bypasses the requirement for a fully functional chondroitin pathway and MIG-17 metalloprotease in DTC pathfinding. I found that i) suppression of the hypomorphic chondroitin mutant mig-22(k141) is not completely dependent on MIG-17 activity; ii) the smig-1(ev809) mutant is likely to be a loss- of-function suppressor; and iii) SMIG-1 does not visibly affect the localization of poorly glycosylated MIG-17 on the gonad surface. Understanding SMIG-1 function will shed light on the role of glycosylation, the extracellular matrix and basement membranes in cell migration during development.

Introduction: Durant la pathogenèse d’ostéoarthrose (OA), les cytokines pro-inflammatoires IL-1β (Interleukin-1 beta) et TNF-α (Tumor necrosis factor alpha) stimulent la dégradation des agrécanes par l’aggrécanase-1 ou ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motif). Ces cytokines peuvent stimuler plusieurs voies de signalisation conduisant ainsi à l’augmentation de l’expression des ADAMTS dans les chondrocytes humains. Les TIMPs (tissue inhibitor of metalloproteinases) présentent des inhibiteurs endogènes de l’ADAMTS. Nous avons démontré que la Rapamycine (un immunosuppresseur et un inhibiteur du mamalian target of Rapamycin (mTOR)) peut avoir des effets bénéfiques dans cette pathologie. Notre étude examine l’effet de la Rapamycine sur l’expression de l’ADAMTS-4 induit par les cytokines, son implication dans certaines voies de signalisation, et son effet sur l’expression du TIMP-3. Méthodes: Des chondrocytes normaux sont traités avec la Rapamycine seule ou stimulés aussi avec l’IL-1β et le TNF-α. Les effets de la Rapamycine sur l’expression de l’ADAMTS-4 et du TIMP-3 ont été étudiés par l’analyse RT-PCR et l’activité enzymatique a été étudiée par la technique d’ELISA. Les effets de la Rapamycine sur certaines voies de signalisation ont été étudiés par le Western blot. Résultats: Nous avons trouvé que la Rapamycine inhibe l’expression de l’ARNm de l’ADAMTS-4 induit par les cytokines pro-inflammatoires dans les chondrocytes humains. L’activité enzymatique de l’ADAMTS-4 induit par l’IL-1β a été légèrement diminuée par la Rapamycine. En plus, cette dernière a montré de différents effets sur plusieurs voies de signalisation stimulées par l’IL-1β et le TNF-α telles que les voies des MAPKs (Mitogen activated protein kinase), de l’AKT, et de la p70 S6 kinase. La Rapamycine a inhibé partiellement l’activation de la phosphorylation de l’ERK1/2 MAPK (extracellular signal-regulated protein kinase MAPK) en présence du TNF-α seulement. En outre, la Rapamycine a inhibé la phosphorylation des protéines p38 MAPK, JNK (c-Jun N-terminal kinase), et AKT activée par l’IL-1β seulement. En plus, la phosphorylation de la protéine p70 S6K stimulée par l’IL-1β et le TNF-α a été inhibée par la Rapamycine. D’autre part, nous avons démontré que le niveau du TIMP-3 a été augmenté en présence de la Rapamycine. Conclusion: Ces résultats suggèrent que la Rapamycine peut bloquer l’action de l’ADAMTS-4 via l’inhibition de l’activation des MAPKs, de l’AKT, et de la p70 S6K. La Rapamycine pourrait ainsi être considérée pour la prévention de la perte du cartilage chez les patients ostéoarthritiques. / Introduction: During the pathogenesis of osteoarthritis, the pro-inflammatory cytokines IL-1β (Interleukin-1 beta) and TNF-α (Tumor necrosis factor alpha) stimulate the degradation of aggrecans by aggrecanase-1 or ADAMTS-4 (A disintegrin and metalloproteinase with thrombospondin motif). These cytokines may stimulate several signaling pathways leading to an increased expression of ADAMTS-4 in human chondrocytes. The TIMPs (tissue inhibitor of metalloproteinases) are endogens inhibitors of ADAMTS-4. It has been shown that Rapamycin (an immunosuppressor and an inhibitor of the mammalian target of rapamycin or mTOR) may have beneficial effects in this pathology. Our study investigates the impact of Rapamycin on cytokine-induced expression of ADAMTS-4, his implication in certain signaling pathways, and his effects on the expression of TIMP-3. Methods: Human chondrocytes were pretreated with different doses of Rapamycin either alone or stimulated with IL-β and TNF-α. The effect on ADAMTS-4 expression was examined by RT-PCR analysis and enzyme activity by ELISA. Impact of Rapamycin on the activation of different signaling pathways was measured by Western blot analysis. Results: We have shown that Rapamycin down-regulated pro-inflammatory cytokines-induced ADAMTS-4 mRNA expression in human chondrocytes. The IL-1β-induced-enzymatic activity of ADAMTS-4 was slightly inhibited by Rapamycin. Furthermore, Rapamycin present different effects on pro-inflammatory cytokines-stimulated activation of certain signaling pathways such as MAPKs (Mitogen activated protein kinases), AKT, and P70 S6 kinase. Moreover, Rapamycin partially inhibits TNF-α-induced activation of phosphorylation of ERK1/2 MAPK (extracellular signal-regulated protein kinase MAPK). Thus, Rapamycin inhibit IL-1β-induced activation of phosphorylation of the proteins p38 MAPK, JNK (c-Jun N-terminal kinase), and AKT. Also, Rapamycin inhibit IL-1β and TNF-α-activation of phosphorylation of the protein p70 S6 kinase. In other way, we have shown that the level of TIMP-3 has been increased in the presence of Rapamycin. Conclusion: These results suggest that Rapamycin down-regulates the expression of ADAMTS-4 by inhibiting the cytokine activation of MAPKs, AKT, and p70 S6 kinase. Thus Rapamycin could be considered as potential therapeutic agent for prevention of cartilage loss in patient with osteoarthritis.