Master of Science / Department of Entomology / Marcelo Ramalho-Ortigao / Sand flies (Diptera:Psychodidae) are vectors of parasites of the genus Leishmania transmitted to suitable vertebrate host during blood feeding. For blood feeding arthropods, including sand flies, blood meal digestion requires the secretion of inhibitory molecules, such as Kazal-type serine proteinase inhibitors that are involved in preventing the blood from coagulating within the mouthparts and the midgut. Previous studies have identified such molecules in mosquitoes, ticks, and triatomine bugs. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). We are interested in the role of these proteins as inhibitors of coagulation cascades, in addition to their potential effects on blood digestion in P. papatasi. Ppkzl1 is similar to thrombin and trypsin inhibitors in triatomines and mosquitoes and Ppkzl2 is similar to Kazal-type inhibitors in mosquitoes with unknown function. Analyses of expression profiles indicated that although both transcripts are expressed prior to blood feeding in the midgut of P. papatasi they are tightly regulated by the blood meal. Reverse genetics studies using RNAi-targeted knockdown of PpKzl1 and PpKzl2 by dsRNA injection did not result in a detectable effect on mRNA expression levels. Thus, we expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess activity against various serine proteinases. Recombinant PpKzl2 inhibited chymotrypsin at nanomolar levels and also inhibited thrombin and trypsin at micromolar levels, suggesting that native PpKzl2 is an active serine proteinase inhibitor and may regulate digestive enzymes and thrombin in the midgut. Leishmania development within the sand fly midgut is faced with several barriers that can severely impact the parasites. For transmission to occur, parasites must be able to overcome these barriers including digestive proteinases, escape from the peritrophic matrix, and midgut attachment. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sand fly midgut. Thus, targeting serine proteinase inhibitors may provide a new strategy to prevent transmission of Leishmania.

A Estomatite protética associada a Candida (EPC) acomete a mucosa bucal em contato com próteses removíveis e, clinicamente, caracteriza-se por eritema com diferentes graus de inflamação. Esta doença é considerada de etiologia multifatorial, isto é, uma associação de fatores como trauma, falta de higienização, uso contínuo da prótese, hipersensibilidade ao material usado na dentadura, diabetes, terapia imunossupressora e infecção por fungo, como diferentes espécies de Candida. Os principais fatores de virulência deste fungo são a formação de hifas, dimorfismo, alterações fenotípicas, aderência, persistência, sinergismo com as bactérias, interferências com o sistema de defesa do hospedeiro e a produção de enzimas hidrolíticas. Dentre as enzimas hidrolíticas, a proteinase aspartil secretada (SAP) é uma das mais importantes para a patogenia de C. albicans, sendo nociva para o tecido epitelial e para o sistema imune do hospedeiro. Não está totalmente compreendida a real penetração do fungo nos tecidos e sua correlação com a presença da SAP, na doença estomatite protética. Essa dificuldade de avaliação pode ser justificada pelas divergências intrínsecas e extrínsecas observadas em muitos aspectos, como diferentes costumes, hábitos sociais, estado emocional e fisiológico. A utilização de um modelo experimental em animais poderá minimizar essas divergências e fornecer condições mais padronizadas para o experimento. Neste trabalho, foram avaliadas, quantitativamente, a expressão gênica das enzimas SAP-2, SAP-5 e SAP-9, presentes no biofilme formado na superfície interna das placas acrílicas superiores de ratos e, microscopicamente, a invasão do fungo no tecido epitelial do palato. Para isso, foram selecionados 49 ratos Wistar, com 90 dias de vida, pesando em média 300g, os quais foram divididos em 3 grupos: Controle, Placa/Candida e Placa, acompanhados durante 2, 4 e 6 dias. Os resultados demostraram que, em 4 dias de uso da placa acrílica contaminada, houve, em alguns ratos, sinais clínicos de inflamação no palato duro; microscopicamente, hiperplasia epitelial, hiperqueratinização, invasão fúngica nas camadas superficiais do revestimento epitelial, microabscessos de Munro e hiperplasia papilar; e maior percentual de neutrófilos no Grupo Placa/Candida em relação aos Grupos Controle e Placa. Também no quarto dia de uso da placa acrílica superior, no Grupo Placa/Candida, o biofilme formado na sua superfície interna apresentou a mais alta expressão gênica relativa das enzimas SAP-2, SAP-5 e SAP-9 que os períodos de 2 e 6 dias de uso. Assim, a invasão fúngica no revestimento epitelial do palato duro pode estar correlacionada com a alta expressão de RNAm das SAPs -2, -5 e -9. / Denture stomatitis (D.S.) affects the oral mucosa in contact with removable dentures, and clinically characterized by erythema with varying degrees of inflammation. This disease is considered a multifactorial etiology, with a combination of factors such as trauma, lack of hygiene, continuous use of stents, hypersensitivity to the material used for dentures, diabetes, immunosuppressive therapy and fungal infection, such as different species of Candida. The main virulence factors of the fungus are the formation of hyphae, dimorphism, phenotypic changes, adherence, persistence, synergism with bacteria, interference with the host defense system and the production of hydrolytic proteins. Among the hydrolytic proteins, the secreted aspartyl proteinase (SAP) is one of the most important in the pathogenesis of C. albicans. SAP is harmful to both the epithelial tissue and to the host\'s immune system. It is not fully understood the real penetration of the fungus in the epithelium tissue and its correlation with the presence of SAP, in denture stomatitis. This difficulty in evaluation can be justified by the intrinsic and extrinsic differences observed in many aspects, different customs, social`s habits, emotional and physiological state. Using an experimental animal model may minimize these differences and provide more standardized conditions for the experiment. In the present work, the aim was to evaluate quantitatively the gene expression of enzymes SAP-2, SAP-5 and SAP-9 of the biofilm formed in internal surface of rat`s upper acrylic plates, and to analysis microscopically, the fungal invasion in palatal epithelial tissue. 49 Wistar rats were selected, 90 days old, weighing on average 300g. They were divided into three groups: Control Group, Plate/Candida and Plate, follow by 2, 4 and 6 days of the use of the plates. The results demonstrated, in four days of use of the acrylic plate, clinical signs of inflammation in the hard palate; microscopically epithelial hyperplasia, hyperkeratinization, fungal invasion in the superficial layers of the epithelial lining, Munro`s microabscess and papillary hyperplasia; and higher percentage of neutrophils in the Plate/Candida Group, compared to Control and Plate Groups. In the period of 4 days, the relative gene expression of the SAPs-2, -5 and -9 in biofilm also showed to be higher in Plate/Candida Group, compared with the periods of 2 and 6 days. Thus, the fungal invasion in the epithelial lining of the hard palate may be correlated with high mRNA expressionn of SAPs -2, -5 e -9.

Os insetos da ordem Hemiptera apresentam membranas lipoproteicas que revestem as microvilosidades das células intestinais, como se fossem dedos de luva, e formam expansões para o lúmen do intestino que parecem terminar em fundo cego. A presença das duas membranas no ápice dos enterócitos gera questões intrigantes de como se dá a formação da membrana perimicrovilar, como ocorre a absorção de nutrientes e como se dá o encaminhamento de enzimas digestivas para o lúmen intestinal. A digestão de proteínas baseada em enzimas originalmente lisossômicas é uma característica marcante nos Hemiptera, em especial os Heteroptera que evolutivamente voltaram a uma alimentação de polímeros. O presente estudo indica que os genes das proteinases tipicamente lisossômicas sofrem uma série de duplicações, havendo a manutenção de um gene para função puramente lisossômica e a divergência funcional dos demais genes para a função de digestão extracelular. O gene que mantém a função lisossômica não é modulado pela alimentação, além de ser expresso nos mais diversos tecidos. Já os genes que se especializaram na digestão extracelular têm a expressão aumentada com a ingestão de alimento, indicando sua função. Parece não haver diferença nas características relacionadas ao encaminhamento celular das proteínas produzidas por esses genes, indicando que o direcionamento para a rota secretória é devido à superexpressão dos genes relacionados à digestão. Enzimas como alfa-glicosidases, alfa-manosidases e aminopeptidases que participam da digestão extracelular seguem a rota secretória que envolve a formação de vesículas de dupla membrana. Foi possível ampliar o modelo de digestão incluindo a participação das catepsinas D, de uma alfa-glicosidase solúvel e a possível participação de uma tiolredutase além do definir o local de atuação das lipases. Temos agora uma visão global da participação das enzimas digestivas que atuam na digestão em D. peruvianus. / The insects of the order Hemiptera have lipoprotein membranes lining the microvilli of midgut cells, like glove fingers, and form expansions into the lumen of the intestine. The presence of two membranes on the apex of enterocytes thus generates intriguing questions about the formation of perimicrovillar membrane, the absorption of nutrients, and the targeting of digestive enzymes into the intestinal lumen. The digestion of proteins based on originally lysosomal enzymes is an important feature in Hemiptera, especially in Heteroptera that evolutionary returned to feed on polymers. The genes of typical lysosomal proteinases undergo a series of duplications followed by the maintenance of a gene for purely lysosomal function and functional divergence of other genes for extracellular digestion function. The gene that maintains the lysosomal function is not modulated by feeding, in addition to being expressed in diverse tissues. By the other hand, genes specialized in extracellular digestion are up regulated by food intake, indicating its function. No difference was found in the targeting in the proteins produced by these genes, which indicates that targeting to the secretory route is due to overexpression of digestion-related genes. Enzymes involved in extracellular digestion as alpha-glucosidase, alpha-mannosidase and aminopeptidase follow the secretory route that includes the formation of double membrane vesicles. In this study we increased the digestion model adding the participation of cathepsin D, a soluble alpha-glucosidase, and the possible participation of a tiolredutase, and also to defining the place of lipases operation. We now have a global view of the participation of digestive enzymes involved in digestion D. peruvianus.

by Ricardo Wong Chi Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 128-138). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview of Serine Protease Inhibitors --- p.1 / Chapter 1.2 --- Classification of Serine Protease Inhibitors --- p.2 / Chapter 1.2.1 --- Kunitz Type Serine Protease Inhibitors --- p.7 / Chapter 1.2.2 --- Bowman-Birk Type Serine Protease Inhibitors --- p.11 / Chapter 1.2.3 --- Squash Type Serine Protease Inhibitors --- p.16 / Chapter 1.3 --- Role of Serine Protease Inhibitors in Plants --- p.20 / Chapter 1.4 --- Nutritional Fact of Serine Protease Inhibitors --- p.22 / Chapter 1.5 --- Possible Applications of Serine Protease Inhibitors --- p.25 / Chapter 1.5.1 --- Medical Applications --- p.25 / Chapter 1.5.2 --- Agricultural Applications --- p.29 / Chapter 1.6 --- Rationale of the Present Study --- p.31 / Chapter Chapter 2 --- Screening of Seeds for Inhibitory Activities Against Serine Proteases --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and Methods --- p.37 / Chapter 2.2.1 --- Materials --- p.37 / Chapter 2.2.2 --- Extraction Method --- p.37 / Chapter 2.2.3 --- Assays for Proteases Inhibitory Activities --- p.38 / Chapter 2.2.3.1 --- Assay for Chymotrypsin Activity --- p.38 / Chapter 2.2.3.2 --- Assay for Trypsin Activity --- p.38 / Chapter 2.2.3.3 --- Assay for Elastase Activity --- p.39 / Chapter 2.2.3.4 --- Assay for Subtilisin Activity --- p.39 / Chapter 2.2.3.5 --- Assays for Protease Inhibitory Activities --- p.40 / Chapter 2.2.4 --- Determination of Protein Concentration --- p.41 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Extraction --- p.42 / Chapter 2.3.2 --- Serine Proteases Inhibitory Activities --- p.42 / Chapter 2.4 --- Discussion --- p.47 / Chapter Chapter 3 --- Isolation of Chymotrypsin Inhibitor and Trypsin Inhibitors from Momordica cochinchinensis Seeds --- p.49 / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Materials --- p.56 / Chapter 3.2.2 --- Protein Extraction --- p.57 / Chapter 3.2.3 --- SP-Sepharose Chromatography --- p.57 / Chapter 3.2.4 --- Reversed Phase High Pressure Liquid Chromatography --- p.58 / Chapter 3.2.5 --- Assays for Chymotrypsin and Trypsin Inhibitory Activities --- p.60 / Chapter 3.2.6 --- Titration of Chymotrypsin --- p.61 / Chapter 3.2.7 --- Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.62 / Chapter 3.2.8 --- Coupling of Trypsin-Sepharose 4B Affinity Column --- p.63 / Chapter 3.2.9 --- Affinity Chromatography on Trypsin-Sepharose 4B --- p.64 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- SP-Sepharose Chromatography --- p.65 / Chapter 3.3.2 --- Reversed Phase High Pressure Liquid Chromatography --- p.67 / Chapter 3.3.3 --- Summary of Purification --- p.71 / Chapter 3.3.4 --- Titration of Chymotrypsin --- p.74 / Chapter 3.3.5 --- Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.74 / Chapter 3.3.6 --- Affinity Chromatography on Trypsin-Sepharose 4B --- p.78 / Chapter 3.4 --- Discussion --- p.81 / Chapter Chapter 4 --- Characterization of Chymotrypsin Inhibitor and Trypsin Inhibitors --- p.88 / Chapter 4.1 --- Introduction --- p.88 / Chapter 4.2 --- Materials and Methods --- p.90 / Chapter 4.2.1 --- Materials --- p.90 / Chapter 4.2.2 --- Determination of Molecular Weight --- p.90 / Chapter 4.2.3 --- Amino Acid Sequence Analysis --- p.91 / Chapter 4.2.4 --- Surface Plasmon Resonance Measurement --- p.92 / Chapter 4.2.4.1 --- Immobilization of Ligands on the Surface of Optical Biosensors --- p.92 / Chapter 4.2.4.2 --- Determination of Kinetics Constants --- p.93 / Chapter 4.2.4.3 --- pH Dependence of the Inhibition by Chymotrypsin Inhibitor --- p.93 / Chapter 4.2.4.4 --- Data Analysis --- p.94 / Chapter 4.2.5 --- Effect of Chymotrypsin Inhibitor on the Estereolytic Activity and Proteolytic Activity of Chymotrypsin --- p.95 / Chapter 4.2.6 --- Specificities of the Inhibitors % --- p.96 / Chapter 4.2.7 --- Binding Ratio of CI to Different Proteases --- p.97 / Chapter 4.2.8 --- Effects of the Proteases on Their Corresponding Inhibitors --- p.97 / Chapter 4.2.8.1 --- Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.97 / Chapter 4.2.8.2 --- Assay for Chymotrypsin Inhibitory Activity --- p.98 / Chapter 4.3 --- Results --- p.99 / Chapter 4.3.1 --- Molecular Weight of the Inhibitors --- p.99 / Chapter 4.3.2 --- N-terminal Amino Acid Sequence --- p.99 / Chapter 4.3.3 --- Surface Plasmon Resonance Measurement --- p.102 / Chapter 4.3.3.1 --- Kinetics of Chymotrypsin Inhibitor --- p.102 / Chapter 4.3.3.2 --- Kinetics of Trypsin Inhibitors --- p.106 / Chapter 4.3.3.3 --- pH Dependence of the Inhibition by Chymotrypsin Inhibitor --- p.106 / Chapter 4.3.4 --- Effect of Chymotrypsin Inhibitor on the Estereolytic Activity and Proteolytic Activity of Chymotrypsin --- p.106 / Chapter 4.3.5 --- Specificities of the Inhibitors --- p.110 / Chapter 4.3.6 --- Binding Ratio of CI to Different Proteases --- p.112 / Chapter 4.3.7 --- Effects of the Proteases on Their Corresponding Inhibitors --- p.112 / Chapter 4.4 --- Discussion --- p.119 / Chapter Chapter 5 --- Conclusion --- p.125 / References --- p.128

Úlceras indolentes são úlceras corneais superficiais, espontâneas, que apresentam curso prolongado e que tendem a recidivar. Comumente observadas em cães de meia idade, da raça Boxer, provoca dor de início agudo e necessita de tratamento específico, já que este, quando não realizado de forma correta, pode prolongar o curso da lesão por semanas a meses. A doença é explicada por diversas alterações da superfície ocular. Com o objetivo de avaliar a eficácia dos tratamentos ambulatoriais preconizados no Serviço de Oftalmologia do Hospital Veterinário da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo (HOVET-FMVZ-USP), e as principais considerações observadas no levantamento dos prontuários, realizou-se estudo retrospectivo dos casos atendidos entre os anos de 1997 e 2008. Segundo os resultados, observou-se que a maioria dos cães da raça Boxer apresentaram úlcera indolente, distrofia corneal e catarata; que as úlceras indolentes foram mais frequentemente observadas em fêmeas de meia idade e que a maioria dos proprietários demoraram mais de 15 dias para levar seus animais ao HOVET-FMVZ-USP; que as alterações oculares mais frequentemente referidas pelos proprietários foram o blefarospasmo, olho vermelho e a secreção; que as principais características das lesões observadas após o exame oftalmológico foram que a maioria das úlceras eram transparentes, apresentando epitélio não aderido ou com algum grau de vascularização; unilaterais, mais frequentemente observadas no olho direito; de aparecimento espontâneo e localizadas no centro da córnea. Quanto ao tratamento, observou-se que os inibidores das proteinases foram as medicações mais frequentemente prescritas e que sua administração não interferiu no tempo de cicatrização corneal ou na formação de granuloma. Vitamina C, apesar de ter prolongado de maneira significante o tempo de cicatrização corneal, reduziu a inflamação, consideração observada pela diminuição da presença de granuloma. Debridamento/cauterização corneal, além de não interferir na formação de granuloma, acelerou, significativamente, o processo de cicatrização. A antibioticoterapia e a administração de Atropina 1 % não interferiu no tempo de cicatrização, mas se relacionaram diretamente, de forma estatisticamente significante, à presença de granuloma. O uso de anti-inflamatórios tópicos e sistêmicos também não interferiu no tempo de cicatrização, mas diminuíram, de maneira significante, a presença de granuloma nos cães em que foram administrados. Observou-se também que a não administração de atropina 1 %, antibióticos e anti-inflamatórios não interferiu no tempo de cicatrização, nem na formação de granuloma; que o tempo de alteração ocular, antes da primeira consulta e as características das lesões não interferiram, de maneira relevante, no tempo de cicatrização corneal. Portanto, conhecer os diversos tipos de tratamento se mostra fundamental para o sucesso da resolução da doença, já que este deve ser específico, realizado de forma cautelosa e por tempo indeterminado, cuidando para que a lesão não progrida e promovendo o retorno da transparência corneal. / Indolent ulcers are superficial corneal ulcers that occurs spontaneously, presents prolonged course and tend to relapse. Commonly observed in middle-aged Boxer dogs, causes pain of acute onset and requires appropriate treatment. The disease is explained by several changes on the corneal surface. Aiming to assess the effectiveness of clinical treatments, recommended by the Ophthalmology Service of the Veterinary Hospital, of the Veterinary College, of the University of São Paulo (HOVET-FMVZ-USP) and to evaluate major considerations registered on its medical records, a retrospective study was conducted (1997 2008). Results demonstrated that, during studied period: most Boxer dogs presented indolent ulcers, corneal dystrophy and cataracts; indolent ulcers were frequently observed in middle-aged female Boxers and most owners took more than 15 days to bring their animals to the hospital; blepharospasm, red eye and ocular discharge were the most owner´s referred ocular alterations at the primary consultation; main features of examined lesions were transparent ulcers presenting non adherent epithelium and/or some degree of vascularization; unilateral, often observed at the right eye, of spontaneous onset and located at the center of the cornea. Regarding treatment, proteinase inhibitors were the most often prescribed medications; its administration did not affect corneal healing or granuloma formation. Vitamin C prolonged, significantly, the corneal healing time, although, its administration reduced its inflammation, observed by the decrement on the granuloma frequency. Corneal debridement / cauterization, did not interfere on granuloma formation and was capable to accelerate, significantly, the healing process. Antibiotics and 1 % atropine did not affect the healing time, but were statistically related to the presence of granuloma. Topical and systemic antiinflammatories did not interfere at the healing time, but decreased, significantly, the presence of granuloma. Not to administer atropine 1%, antibiotics and antiinflammatories, did not interfere at the corneal healing time nor the formation of granuloma. Duration period of ocular alterations before the first consultation and characteristics of the lesions did not interfere at the corneal healing time. Therefore, to know the various types of treatments seems to be fundamental to the resolution of the indolent ulcer, as treatment must be specific, performed cautiously and for indefinitely period, preventing the progression of the lesion, and promoting the return of corneal transparency.

Orientador: Carlos Eduardo Vergani / Banca: Fernanda Lourenção Brighenti / Banca: Ewerton Garcia de Oliveira Mima / Banca: Karin Hermaa Neppelonbroek / Banca: Cláudia Helena Lovato da Silva / Resumo: O objetivo do presente estudo "in vitro" foi avaliar a interação entre os microorganismos C. albicans, S. aureus sensível (MSSA) e resistente (MRSA) à meticilina com relação a alguns dos seus principais fatores de virulência. Estes fatores foram analisados considerando os micro-organismos isoladamente e em culturas mistas. Foi verificada a interação entre esses micro-organismos através da contagem do número de colônias viáveis (UFC/mL), análise da atividade metabólica por meio do ensaio XTT e quantificação da biomassa total por meio do corante cristal violeta nos tempos de 90 minutos, 12, 24 e 48 horas. Para análise da produção de proteinase (SAP) e fosfolipase (PL-C), kits fluorimétricos foram utilizados. A estrutura dos biofilmes foi avaliada por microscopia eletrônica de varredura (MEV). Culturas simples e mistas dos micro-organismos também foram utilizadas para infectar um epitélio oral humano reconstituído (EOHR). A capacidade de colonização e invasão foi avaliada após 24 horas utilizando a técnica de hibridização "in situ" (PNA-FISH). A avaliação do dano causado pelos micro-organismos ao tecido foi realizada por quantificação da liberação da enzima lactato desidrogenase (LDH). Técnicas moleculares (RT-qPCR) foram empregadas para quantificação do número de micro-organismos colonizando o epitélio e também para avaliação da expressão de genes de virulência. A expressão gênica foi avaliada em superfície biótica (EOHR) e abiótica (placa de poliestireno de 24 poços) para culturas simples e mistas dos micro-organismos. Os resultados dos ensaios de quantificação foram analisados utilizando ANOVA a dois critérios com teste "post-hoc" de Tukey (α = 0,05), enquanto os dados de atividade enzimática foram analisados por ANOVA a um critério com correção de Welch seguido pelo teste de Games-Howell. Os resultados da expressão gênica foram... (Resumo completo clicar acesso eletrônico) / Abstract: The aim of this study was to evaluate, "in vitro", the interaction between C. albicans and methicillinsusceptible (MSSA) and resistant (MRSA) S. aureus in relation to some of their major virulence factors. These factors were analyzed considering the three microorganisms in single and dual cultures. The interaction between these microorganisms was evaluated by counting colony-forming units (CFU/mL), analysis of metabolic activity by the XTT assay and quantification of the total biomass by crystal violet dye at 90 minutes, 12, 24, and 48 hours. To analyze the production of proteinase (SAP) and phospholipase (PL-C), fluorometric kits were used. The structure of the biofilms was acessed by scanning electron microscopy (SEM). Further, single and dual cultures of microorganisms were used to infect a reconstituted human oral epithelium (RHOE). The pattern of colonization and invasion of the RHOE was evaluated after 24 hours using the in situ hybridization technique (PNA-FISH). The damage caused by microorganisms in the tissue was evaluated by quantification of the lactate dehydrogenase enzyme (LDH) released. Molecular techniques (RT-qPCR) were also used to quantify the number of microorganisms colonizing the epithelium and to evaluate the expression of virulence genes. Gene expression was evaluated in biotic (RHOE) and abiotic (24-well polystyrene microplate) surfaces for single and dual cultures. The results from quantification assays were compared using two-way ANOVA with Tukey post-hoc test, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test. The results of gene expression were analyzed by one-way ANOVA with Tukey for data satisfying the assumptions of normality and homoscedasticity or one-way Welch-ANOVA followed by Games-Howell for heteroscedastic data. LDH results were also...(Complete abstract electronic access below) / Doutor

A Estomatite protética associada a Candida (EPC) acomete a mucosa bucal em contato com próteses removíveis e, clinicamente, caracteriza-se por eritema com diferentes graus de inflamação. Esta doença é considerada de etiologia multifatorial, isto é, uma associação de fatores como trauma, falta de higienização, uso contínuo da prótese, hipersensibilidade ao material usado na dentadura, diabetes, terapia imunossupressora e infecção por fungo, como diferentes espécies de Candida. Os principais fatores de virulência deste fungo são a formação de hifas, dimorfismo, alterações fenotípicas, aderência, persistência, sinergismo com as bactérias, interferências com o sistema de defesa do hospedeiro e a produção de enzimas hidrolíticas. Dentre as enzimas hidrolíticas, a proteinase aspartil secretada (SAP) é uma das mais importantes para a patogenia de C. albicans, sendo nociva para o tecido epitelial e para o sistema imune do hospedeiro. Não está totalmente compreendida a real penetração do fungo nos tecidos e sua correlação com a presença da SAP, na doença estomatite protética. Essa dificuldade de avaliação pode ser justificada pelas divergências intrínsecas e extrínsecas observadas em muitos aspectos, como diferentes costumes, hábitos sociais, estado emocional e fisiológico. A utilização de um modelo experimental em animais poderá minimizar essas divergências e fornecer condições mais padronizadas para o experimento. Neste trabalho, foram avaliadas, quantitativamente, a expressão gênica das enzimas SAP-2, SAP-5 e SAP-9, presentes no biofilme formado na superfície interna das placas acrílicas superiores de ratos e, microscopicamente, a invasão do fungo no tecido epitelial do palato. Para isso, foram selecionados 49 ratos Wistar, com 90 dias de vida, pesando em média 300g, os quais foram divididos em 3 grupos: Controle, Placa/Candida e Placa, acompanhados durante 2, 4 e 6 dias. Os resultados demostraram que, em 4 dias de uso da placa acrílica contaminada, houve, em alguns ratos, sinais clínicos de inflamação no palato duro; microscopicamente, hiperplasia epitelial, hiperqueratinização, invasão fúngica nas camadas superficiais do revestimento epitelial, microabscessos de Munro e hiperplasia papilar; e maior percentual de neutrófilos no Grupo Placa/Candida em relação aos Grupos Controle e Placa. Também no quarto dia de uso da placa acrílica superior, no Grupo Placa/Candida, o biofilme formado na sua superfície interna apresentou a mais alta expressão gênica relativa das enzimas SAP-2, SAP-5 e SAP-9 que os períodos de 2 e 6 dias de uso. Assim, a invasão fúngica no revestimento epitelial do palato duro pode estar correlacionada com a alta expressão de RNAm das SAPs -2, -5 e -9. / Denture stomatitis (D.S.) affects the oral mucosa in contact with removable dentures, and clinically characterized by erythema with varying degrees of inflammation. This disease is considered a multifactorial etiology, with a combination of factors such as trauma, lack of hygiene, continuous use of stents, hypersensitivity to the material used for dentures, diabetes, immunosuppressive therapy and fungal infection, such as different species of Candida. The main virulence factors of the fungus are the formation of hyphae, dimorphism, phenotypic changes, adherence, persistence, synergism with bacteria, interference with the host defense system and the production of hydrolytic proteins. Among the hydrolytic proteins, the secreted aspartyl proteinase (SAP) is one of the most important in the pathogenesis of C. albicans. SAP is harmful to both the epithelial tissue and to the host\'s immune system. It is not fully understood the real penetration of the fungus in the epithelium tissue and its correlation with the presence of SAP, in denture stomatitis. This difficulty in evaluation can be justified by the intrinsic and extrinsic differences observed in many aspects, different customs, social`s habits, emotional and physiological state. Using an experimental animal model may minimize these differences and provide more standardized conditions for the experiment. In the present work, the aim was to evaluate quantitatively the gene expression of enzymes SAP-2, SAP-5 and SAP-9 of the biofilm formed in internal surface of rat`s upper acrylic plates, and to analysis microscopically, the fungal invasion in palatal epithelial tissue. 49 Wistar rats were selected, 90 days old, weighing on average 300g. They were divided into three groups: Control Group, Plate/Candida and Plate, follow by 2, 4 and 6 days of the use of the plates. The results demonstrated, in four days of use of the acrylic plate, clinical signs of inflammation in the hard palate; microscopically epithelial hyperplasia, hyperkeratinization, fungal invasion in the superficial layers of the epithelial lining, Munro`s microabscess and papillary hyperplasia; and higher percentage of neutrophils in the Plate/Candida Group, compared to Control and Plate Groups. In the period of 4 days, the relative gene expression of the SAPs-2, -5 and -9 in biofilm also showed to be higher in Plate/Candida Group, compared with the periods of 2 and 6 days. Thus, the fungal invasion in the epithelial lining of the hard palate may be correlated with high mRNA expressionn of SAPs -2, -5 e -9.

Abstract not available

Three protease inhibitor-like proteins (PILPs) identified from Bungarus multicinctus genome are structurally homologous with Kunitz-type proteinase inhibitor. The goal of the present study is to explore whether PILPs exhibit an inhibitory action on matrix metalloproteinase 2 (MMP-2) activity. Unlike PILP-1 and PILP-2, PILP-3 was found to inhibit MMP-2 activity as evidenced by specific substrate assay. Moreover, in vitro migration and invasion assays, and wound-healing assay showed that PILP-3 suppressed the migration and invasion of human neuroblastoma SK-N-SH cells. Pull-down assay and dot blotting-binding assay proved an interaction between PILP-3 and MMP-2. Nevertheless, PILP-3 did not affect either expression or secretion of MMP-2 in SK-N-SH cells. In terms of highly structural similarity between PILP-2 and PILP-3, two chimeric mutants in which amino acids at N-terminus and C-terminus of PILP-3 were substituted by those of PILP-2 were prepared. In contrast to N-terminus chimera, C-terminus mutant of PILP-3 was unable to inhibit MMP-2 activity and showed a reduction in binding with MMP-2. Taken together, our data suggest that PILP-3 may be a useful template for rational designing pharmaceutical agent in inhibiting MMP-2 activity.

The family of human kallikrein-related peptidases (KLKs) numbers 15 serine proteinases implicated in tumour progression. Despite the wide tissue distribution of KLKs and the numerous reports of their differential expression in pathological settings, the signalling mechanism(s) whereby these enzymes regulate tissue function are not yet known. Further, knowledge of the levels of their activity, as well as of their potential endogenous targets, has only been extracted from in vitro studies and cell culture systems. We hypothesized that KLKs can trigger tumour signalling via proteinase-activated receptors (PARs), a family of G-protein-coupled receptors. To test our hypothesis, we evaluated the ability of KLKs 5, 6, and 14: to activate or prevent signalling via PARs 1, 2, and 4 in cells and tissues expressing these receptors. Further, we used a novel activity-based probe approach, coupled with conventional immunoassay (ELISA), to determine the abundance of active KLK6 relative to total immunoreactive KLK6 in cancer-related biological fluids. We concluded that KLKs can regulate multiple signalling pathways triggered by PARs 1, 2, and 4, resulting in calcium release, platelet aggregation and vascular relaxation, and they can cause murine inflammation. Further, our activity-based ELISA demonstrated the presence of active KLK6 in ovarian cancer ascites fluids and cancer cell supernatants. We, therefore, suggest that tumours can produce active KLKs, which can potentially control tumour behaviour by regulating PAR activity.