p-21-activated kinase 6 (PAK6) is a serine-threonine protein kinase originally identified as an Androgen Receptor (AR) interacting protein. In current study, we determined the subcellular localization of PAK6 through mutational analysis. We have found that the N-terminal CRIB domain is partly responsible for plasma membrane targeting, the region between amino acid residues #292 to #368 is functionally relevant to plasma membrane localization and that amino acid residues #119 through #190 are responsible for nuclear targeting of PAK6, in addition to a stretch of positively charged N-terminal residues (#2-#11) since mutants lacking this sequence mis-localizes to cytoplasm. In junction forming epithelial cells, PAK6 is demonstrated to co-localize with B-catenin at adherens junctions, suggesting that PAK6 is an activation-dependent event and that PAK6 translocates from plasma membrane to the cytoplasm in response activation via the PKA signal pathway. / by Ciny John. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

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.

A existência de uma família gênica diversa de serino proteinases em Lepidóptera sugere que essas proteinases desempenham um papel importante na adaptação desses insetos à presença de inibidores de proteinases vegetais. Essas enzimas têm se revelado estarem envolvidas no processo digestivo de larvas de insetos. Larvas de Spodoptera frugiperda foram alimentadas com uma dieta suplementada com inibidor de proteinase de soja (IPS) e a expressão gênica de proteinases intestinais foi avaliada através de PCR em tempo real. Análises de transcrição anteriores mostraram a existência de dois grupos de serino proteinases: um grupo de genes constitutivamente expressos em larvas controle que é induzido pela dieta contendo IPS e um segundo grupo que está ausente no controle, mas que é também induzido por uma dieta rica em IPS. No presente trabalho foi observado um terceiro grupo de proteinases que não são nem induzidas nem reprimidas pela presença do IPS na dieta. Essa observação sugere que a adaptação de S. frugiperda ao IPS envolve a síntese de novas proteinases, a indução de enzimas preexistentes e ainda um terceiro grupo insensível à presença dos inibidores. Proteinases dos intestinos de larvas crescidas em dieta com IPS mostraram insensibilidade ao inibidor. As proteinases também foram insensíveis quando a atividade foi verificada com um inibidor de proteinases de amplo espectrum. Os resultados aqui apresentados propõem que a adaptação de S. frugiperda ao IPS segue uma estratégia generalizada, baseada na indução geral de um grande grupo de endoproteinases. / The existence of a diverse serine proteinase gene family in lepidopteran insects has suggested its significant role in the insect adaptation to plant proteinase inhibitors. These enzymes have been shown to be involved in the proteolytic digestion process of insect larvae. Spodoptera frugiperda larvae were fed on a diet supplemented with soybean proteinase inhibitor (SPI) and the gene expression of intestinal proteinases was evaluated by real time PCR. Previous transcription analyses found two groups of intestinal serine proteinases: one group of genes constitutively expressed in the control larvae that is induced by the SPI-containing diet during the experiment, and a second group that is absent in the control but also induced by the SPI rich diet. Herein was observed a third group of proteinases that are neither induced nor repressed by the presence of SPI in the diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis, up regulation of existing enzymes and that there is a third group insensitive to the presence of the inhibitors. Proteinases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteinases were also insensitive when the activity was checked with a broad-spectrum potato proteinase inhibitor. The results here presented propose that adaptation of S. frugiperda to SPI follows a \"shotgun\" approach, based on a general up regulation of a large set of endoproteinases.

Chu Ling Hon Matthew. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 201-223). / Abstracts in English and Chinese. / Declaration --- p.i / Thesis/Assessment Committee --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Acknowledgements --- p.viii / General abbreviations --- p.xi / Abbreviations of chemicals --- p.xv / Table of Contents --- p.xvi / List of Figures --- p.xxiii / List of tables --- p.xxviii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Severe Acute Respiratory Syndrome (SARS) - Three Years in Review --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Clinical presentation --- p.3 / Chapter 1.1.3 --- Diagnostic tests --- p.5 / Chapter 1.2 --- Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) --- p.7 / Chapter 1.2.1 --- SARS - Identification of the etiological agent --- p.7 / Chapter 1.2.2 --- The coronaviruses --- p.9 / Chapter 1.2.3 --- The genome organization of SARS-CoV --- p.11 / Chapter 1.2.4 --- The life cycle of SARS-CoV --- p.13 / Chapter 1.3 --- Spike Glycoprotein (S protein) of SARS-CoV --- p.15 / Chapter 1.3.1 --- SARS-CoV S protein --- p.15 / Chapter 1.3.2 --- S protein-driven infection --- p.17 / Chapter 1.4 --- SARS-CoV S Protein Fusion Core --- p.22 / Chapter 1.4.1 --- Heptad repeat and coiled coil --- p.22 / Chapter 1.4.2 --- The six-helix coiled coil bundle structure --- p.25 / Chapter 1.5 --- 3C-like Protease (3CLpro) of SARS-CoV --- p.28 / Chapter 1.5.1 --- Extensive proteolytic processing of replicase polyproteins --- p.28 / Chapter 1.5.2 --- SARS-CoV 3CLpro --- p.30 / Chapter 1.5.3 --- Substrate Specificity of SARS-CoV 3CLpro --- p.31 / Chapter 1.6 --- SARS Drug Development --- p.32 / Chapter 1.6.1 --- Drug targets of SARS-CoV --- p.32 / Chapter 1.6.2 --- Current anti-SARS drugs --- p.36 / Chapter 1.7 --- Project Objectives --- p.39 / Chapter 1.7.1 --- Characterization of SARS-CoV S protein fusion core --- p.39 / Chapter 1.7.2 --- Characterization of SARS-CoV 3CLpr0 substrate specificity --- p.40 / Chapter 2 --- Materials and Methods --- p.42 / Chapter 2.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.42 / Chapter 2.1.1 --- Bioinformatics analyses of heptad repeat regions of SARS- CoV S protein --- p.42 / Chapter 2.1.2 --- Recombinant protein approach --- p.43 / Chapter 2.1.2.1 --- Plasmids construction --- p.43 / Chapter 2.1.2.2 --- Protein expression and purification --- p.52 / Chapter 2.1.2.3 --- Amino acid analysis --- p.57 / Chapter 2.1.2.4 --- GST-pulldown experiment --- p.58 / Chapter 2.1.2.5 --- Laser light scattering --- p.61 / Chapter 2.1.2.6 --- Size-exclusion chromatography --- p.62 / Chapter 2.1.2.7 --- Circular dichroism spectroscopy --- p.62 / Chapter 2.1.3 --- Synthetic peptide approach --- p.64 / Chapter 2.1.3.1 --- Peptide synthesis --- p.64 / Chapter 2.1.3.2 --- Native polyacrylamide gel electrophoresis --- p.65 / Chapter 2.1.3.3 --- Size-exclusion high-performance liquid chromato-graphy --- p.66 / Chapter 2.1.3.4 --- Laser light scattering --- p.66 / Chapter 2.1.3.5 --- Circular dichroism spectroscopy --- p.67 / Chapter 2.2 --- Identification of SARS-CoV Entry Inhibitors --- p.70 / Chapter 2.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.70 / Chapter 2.2.2 --- Recombinant protein- and synthetic peptide-based biophysical assays --- p.74 / Chapter 2.2.3 --- Molecular modeling --- p.75 / Chapter 2.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.79 / Chapter 2.3.1 --- Protein expression and purification --- p.79 / Chapter 2.3.2 --- """Cartridge replacement"" solid-phase peptide synthesis" --- p.80 / Chapter 2.3.3 --- Peptide cleavage assay and mass spectrometric analysis --- p.83 / Chapter 3 --- Results --- p.84 / Chapter 3.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.84 / Chapter 3.1.1 --- Bioinformatics analyses of heptad repeat regions of SARS- CoV S protein --- p.84 / Chapter 3.1.2 --- Recombinant protein approach --- p.87 / Chapter 3.1.2.1 --- "Plasmids construction of pET-28a-His6-HRl, pGEX-6P-l-HR2 and pGEX-6P-l-2-Helix" --- p.87 / Chapter 3.1.2.2 --- Protein expression and purification --- p.92 / Chapter 3.1.2.3 --- GST-pulldown experiment --- p.101 / Chapter 3.1.2.4 --- Laser light scattering --- p.103 / Chapter 3.1.2.5 --- Size-exclusion chromatography --- p.105 / Chapter 3.1.2.6 --- Circular dichroism spectroscopy --- p.107 / Chapter 3.1.3 --- Synthetic peptide approach --- p.112 / Chapter 3.1.3.1 --- Peptide synthesis --- p.112 / Chapter 3.1.3.2 --- Native polyacrylamide gel electrophoresis --- p.116 / Chapter 3.1.3.3 --- Size-exclusion high-performance liquid chromatography --- p.117 / Chapter 3.1.3.4 --- Laser light scattering --- p.122 / Chapter 3.1.3.5 --- Circular dichroism spectroscopy --- p.124 / Chapter 3.2 --- Identification of SARS-CoV Entry Inhibitors --- p.129 / Chapter 3.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.129 / Chapter 3.2.2 --- Recombinant protein- and synthetic peptide-based biophysical assays --- p.131 / Chapter 3.2.3 --- Molecular modeling --- p.135 / Chapter 3.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.141 / Chapter 3.3.1 --- Protein expression and purification --- p.141 / Chapter 3.3.2 --- Substrate specificity preference of SARS-CoV 3CLpr0 --- p.142 / Chapter 3.3.3 --- "Primary and secondary screening using the ""cartridge replacement strategy""" --- p.142 / Chapter 4 --- Discussion --- p.149 / Chapter 4.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.149 / Chapter 4.1.1 --- Design of recombinant proteins and synthetic peptides of HR regions --- p.149 / Chapter 4.1.2 --- Recombinant protein approach --- p.151 / Chapter 4.1.3 --- Synthetic peptide approach --- p.153 / Chapter 4.1.4 --- Summary of the present and previous studies in the SARS-CoV S protein fusion core --- p.157 / Chapter 4.2 --- Identification of SARS-CoV Entry Inhibitors --- p.167 / Chapter 4.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.167 / Chapter 4.2.2 --- Identification of peptide inhibitors --- p.168 / Chapter 4.2.3 --- Identification of small molecule inhibitors --- p.172 / Chapter 4.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.183 / Chapter 4.3.1 --- A comprehensive overview of the substrate specificity of SARS-CoV 3CLpro --- p.184 / Chapter 4.3.2 --- The development of the rapid and high-throughput screening strategy for protease substrate specificity --- p.188 / Appendix --- p.191 / Chapter I. --- Nucleotide Sequence of S protein of SARS-CoV --- p.191 / Chapter II. --- Protein Sequence of S protein of SARS-CoV --- p.194 / Chapter III. --- Protein Sequence of 3CLpro of SARS-CoV --- p.195 / Chapter IV. --- Vector maps --- p.196 / Chapter 1. --- Vector map and MCS of pET-28a --- p.196 / Chapter 2. --- Vector map and MCS of pGEX-6P-l --- p.197 / Chapter V. --- Electrophoresis markers --- p.198 / Chapter 1. --- GeneRuler´ёØ 1 kb DNA Ladder --- p.198 / Chapter 2. --- GeneRuler´ёØ 100bp DNA Ladder --- p.198 / Chapter 3. --- High-range Rainbow Molecular Weight Markers --- p.199 / Chapter 4. --- Low-range Rainbow Molecular Weight Markers --- p.199 / Chapter VI. --- SDS-PAGE gel preparation protocol --- p.200 / References --- p.201

Chong Lin-Tat. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 76-78). / Abstracts in English and Chinese. / Chapter Chapter 1 --- introduction / Chapter 1.1 --- Severe acute respiratory syndrome Coronavirus (SARS CoV) --- p.13 / Figure 1.1 Genome organization and putative functional ORFs of SARS CoV --- p.14 / Chapter 1.2 --- SARS main protease / Chapter 1.2.1 --- Three dimensional structure --- p.15 / Figure 1.2 Ribbon illustration of the SARS-coronavirus main protease --- p.17 / Figure 1.3 Surface representations of P1 and P2 substrate-binding pocket of main protease --- p.18 / Chapter 1.2.2 --- Substrate specificities --- p.19 / Table 1.1. Eleven predicted cleavage sites of SARS CoV main protease --- p.21 / Chapter 1.3 --- Protein-based FRET assay system --- p.22 / Figure 1.4. The principle of fluorescent resonance energy transfer (FRET) --- p.24 / Chapter 1.4 --- Objectives --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- General Techniques / Chapter 2.1.1 --- Preparation and transformation of competent E. coli DH5a and23 BL21 (DE3)pLysS --- p.26 / Chapter 2.1.2 --- Minipreparation of plasmid DNA (Invitrogen) --- p.27 / Chapter 2.1.3 --- Spectrophotometric quantitation DNA --- p.28 / Chapter 2.1.4 --- Agarose gel electrophoresis / Chapter 2.1.5 --- Purification of DNA from agarose gel (Invitrogen) / Chapter 2.1.6 --- Restriction digestion of DNA fragments --- p.29 / Chapter 2.1.7 --- Ligation of DNA fragments into vector / Table 2.1. Standard recipe of ligation reaction --- p.30 / Chapter 2.1.8 --- SDS-PAGE electrophoresis --- p.31 / Table 2.2. Standard recipe of separating gel for SDS-PAGE --- p.32 / Table 2.3. Standard recipe of stacking gel for SDS-PAGE --- p.33 / Chapter 2.2 --- Sub-cloning and site-directed mutagenesis / Chapter 2.2.1 --- Sub-cloning of SARS Co V main protease --- p.34 / Chapter 2.2.2 --- Sub-cloning of Substrate / Chapter 2.2.3 --- Site-directed mutagenesis of substrate variant --- p.35 / Table 2.4 Primer sequence for generating substrate variants --- p.36 / Table 2.5. Standard recipe of Polymerase Chain Reaction (PCR) --- p.40 / Table 2.6. Polymerase Chain Reaction (PCR) profile --- p.41 / Chapter 2.3 --- Sample preparation / Chapter 2.3.1 --- Expression of recombinant proteins --- p.42 / SARS CoV main protease / Substrate and substrate variants / Chapter 2.3.2 --- Purification of recombinant proteins / SARS CoV main protease / Substrate and substrate variants / Chapter 2.4 --- Protein-based FRET kinetic analysis --- p.45 / Chapter 2.5 --- A model for substrate-enzyme binding by docking simulation --- p.46 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Preparation of SARS CoV main protease and substrate / Chapter 3.1.1 --- Expression and purification of SARS main protease --- p.48 / Figure 3.1. Purification profile of SARS CoV main protease --- p.49 / Chapter 3.1.2 --- Expression and purification of substrate and substrate variants --- p.50 / Figure 3.2. Purification profile of substrate and substrate variants --- p.51 / Chapter 3.2 --- A novel protein-based FRET assay system was established / Chapter 3.2.1 --- "With the cleavage of active main protease, absorbance at 528nm dropped while signal at 485nm were slightly increased" --- p.52 / Figure 3.3. Absorbance at 528nm dropped and 485nm increased with the substrate hydrolysis --- p.53 / Chapter 3.2.2 --- FRET efficiency ratio (528/485) decreased over time --- p.54 / Figure 3.4. FRET efficiency ratio (528/485) decreased over time --- p.55 / Chapter 3.2.3 --- Comparable kcat/Km value of SARS CoV main protease was obtained --- p.56 / Figure 3.5. Catalytic parameter (kcat/ Km) was determined from the slope of straight Line --- p.57 / Chapter 3.3 --- Main protease activity towards substrate variants at different substrate-binding sites (S2'-S2) --- p.58 / Table 3.1. Kinetic parameterrs of 76 substrate variants in descending order --- p.59 / Chapter 3.3.1 --- S2'substrate-binding site --- p.60 / Chapter 3.3.2 --- S1' substrate-b inding site / Chapter 3.3.3 --- S1 substrate-binding site / Chapter 3.3.4 --- S2 substrate-binding site / Figure 3.6. Kinetic analysis of some typical substrate variants against main protease --- p.62 / Figure 3.7. SDS-PAGE analysis of some typical substrate variants against main protease --- p.63 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Quantitative and high-throughput analysis by protein-based FRET assay system --- p.64 / Chapter 4.2 --- Substrate specificities of SARS CoV main protease at S2'-S2 subsites / Chapter 4.2.1 --- β-strand conformation was preferred at S2，subsite / Chapter 4.2.2 --- Residues with small aliphatic side chain were preferred at S1 ´ة subsite --- p.65 / Chapter 4.2.3 --- "Glutamine at S1 subsite was absolutely conserved, but alternatives were disclosed" --- p.66 / Figure 4.1. Glutamine was not absolutely conserved in S1 subsite --- p.67 / Chapter 4.2.4 --- Hydrophilic residues were tolerated at S2 subsite --- p.68 / Figure 4.2. Hydrophilic residues were tolerated at S2 subsite --- p.70 / Table 4.1. Summary of types of residues preferred at individual subsites --- p.71 / Chapter 4.3 --- Predicted conformation of substrate towards SARS CoV main protease at S2' and S1' subsites --- p.72 / Figure 4.3. Small residues were preferred at S1´ة subsite and Val at S2' subsite was more favoured than the native one --- p.73 / Chapter Chapter 5 --- Summary --- p.74 / Chapter Chapter 6 --- Future work --- p.75 / References --- p.76

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.

La cardosine A est une protéase aspartique identifiée il y a plus de 20 ans dans les cellules du chardon Cynara cardunculus. Sa distribution dans tous les tissus de la plante et ses caractéristiques enzymatiques ont été caractérisées par approches biochimiques. La cardosine A a des fonctions essentielles dans la reproduction, la mobilisation de réserves protéiques, et le remaniement de membranes. Pour assumer ces différentes fonctions, la cardosine A doit pouvoir transiter et s’accumuler dans différents compartiments intracellulaires : vacuole de stockage, vacuoles lytiques, ou autres compartiments membranaires. Il n’y a cependant que très peu de données disponibles sur les mécanismes de biosynthèse, de tri, de transport et d’adressage aux différents compartiments cellulaires. De récents travaux suggèrent que l’expression en modèle hétérologue pourrait être utilisée pour une meilleure compréhension de la biologie intracellulaire de la cardosine A. Les résultats de cette étude montrent que l’expression transitoire de la cardosine A dans les feuilles de Nicotiana tabacum est un bon modèle expérimental pour explorer le transport de la cardosine A dans la cellule. En effet dans ce système les mécanismes de maturation et de transport de la protéine à la vacuole sont conservés. De plus, une lignée stable d’Arabidopsis thaliana exprimant la cardosine A sous promoteur inductible s’est également avérée un bon modèle d’étude du transport intracellulaire de la cardosine A. L’utilisation de ces systèmes hétérologues a permis de combiner l’expression de formes mutées de la cardosine A (dans lesquelles des séquences spécifiques ou des acides aminés avaient été tronqués ou modifiés) avec des approches de biochimie et d’imagerie cellulaire pour identifier des signatures moléculaires responsables de l’adressage vacuolaire de la protéine. Nos résultats montrent que la cardosine A a deux déterminants vacuolaires dans sa séquence protéique : le domaine “PSI”, qui définit un déterminant d’adressage vacuolaire original et propre à certaines protéases aspartiques, et un peptide C-terminal appartenant à la classe bien définie des ctVSD. De plus, les résultats montrent que la présence de ces deux déterminants illustre la capacité d’emprunter deux routes distinctes pour atteindre la vacuole : le domaine PSI peut permettre d’attendre la vacuole sans passer par le Golgi, tandis que le domaine C-ter négocie un transport classique Reticulum, Golgi, Prévacuole, Vacuole. Cette capacité à choisir deux routes différentes n’est pas observée pour la cardosine B, autre protéase aspartique du chardon présentant une haute homologie de séquence avec la cardosine A. Pour expliquer cette capacité de la cardosine A à emprunter deux routes vacuolaires différentes, l’hypothèse d’un rôle possible de la glycosylation dans le tri des protéines entre les deux routes vacuolaires est alors étudiée. L’expression de la cardosine A dans les graines en germination d’Arabidopsis thaliana révèle que la protéine peut s’accumuler d’une manière différentielle dans les graines en absence de germination ou pendant la germination, tout au long du système endomembranaire jusqu’à la vacuole de réserve ou dans les vacuoles lytiques en formation. Les expériences de blocage de transport du Reticulum au Golgi n’ont pas permis de conclure d’une manière certaine si les accumulations vacuolaires dérivaient d’un transport pouvant court-circuiter le Golgi comme dans les feuilles de Nicotiana. Au total, la cardosine A constitue une protéine modèle pour étudier les transports vacuolaires chez Nicotiana tabacum and Arabidopsis thaliana, deux systèmes hétérologues qui permettent de développer des méthodes complémentaires pour une exploration fonctionnelle des mécanismes impliqués. Cette étude permet de contribuer à une meilleure connaissance de la biologie des cardosines en particulier, et des protéases aspartiques en général. / The aspartic proteinase cardosin A is a vacuolar enzyme found to accumulate in protein storage vacuoles and lytic vacuoles in the flowers and in protein bodies in seeds of the native plant cardoon. Cardosin A has been first isolated almost two decades ago and has been extensively characterized since, both in terms of distribution within the tissues and of enzyme biochemistry. In the native system, several roles have been addressed to cardosin A in reproduction, mobilization of reserves and membrane remodeling. To participate in such diverse events, cardosin A must accumulate and travel to different compartments inside the cell: protein storage vacuoles, lytic vacuoles, cytoplasmic membrane (and eventually outside the cell). However, not much information is available regarding cardosin A biogenesis, sorting or trafficking to the different compartments. Recent studies have approached the expression of cardosin A in Arabidopsis thaliana and Nicotiana tabacum. These preliminary observations were the starting point of a detailed study of cardosin A expression, localisation, sorting and trafficking routes, resourcing to several and very different methods. It has been showed that transient expression of cardosin A in Nicotiana tabacum leaf is a good system to explore cardosin A trafficking inside the cell, as the protein is processed in a similar manner as the control and accumulates in the vacuole. Furthermore, an Arabidopsis thaliana line expressing cardosin A under an inducible promoter was explored to understand cardosin A dynamics in terms of vacuolar accumulation during seed germination events. Similarly to the Nicotiana tabacum one, this system was also validated for cardosin A expression and it allowed to conclude that the protein’s expression did not retrieved any phenotype to the cells or individuals. However, experiments conducted in BY-2 cells revealed to be inconclusive since cardosin A expression in this system is not predictable. The data obtained along this work using several cardosin A mutated forms, lacking specific domains or point-mutated, allowed to determine that cardosin A has two Vacuolar Sorting Determinants in its protein sequence: the PSI, an unconventional sorting determinant, and the C-terminal peptide, a C-terminus sorting determinant by definition. Furthermore, it was also demonstrated that each domain represents a different route to the vacuole: the PSI bypasses the Golgi Apparatus and the C-terminal peptide follows a classic Endoplasmic Reticulum-Golgi Apparatus-Prevacuole route to the vacuole. This difference in the trafficking routes is not observed for cardosin B sorting determinants as both the PSI and C-terminal peptide from cardosin B needs to pass the Golgi Apparatus to reach the vacuole. A putative role for glycosylation in the trafficking routes is further discussed as cardosin A PSI, contrary to cardosin B, is not glycosylated. The production of mutants affecting cardosin A glycosylation sites supported this idea. Moreover, cardosin A expression in germinating Arabidopsis thaliana seeds revealed a differential accumulation in non-germinated and germinated seedlings. Cardosin A was detected along the secretory pathway to the Protein Storage Vacuole in association with the Endoplasmic Reticulum, Golgi Apparatus, Prevacuole and newly formed Lytic Vacuoles. The drug Brefeldin A caused the protein to be retained in the Golgi Apparatus, despite some amount being still detected in the vacuole, not being clear if the Golgi Apparatus bypass observed in Nicotiana tabacum leaves occurs in this system. As a whole, cardosin A confirmed to be a good model to study vacuolar sorting in these two systems that complement each other in terms of approaches available. This study provided good results in order to understand in more detail cardosin A biology in particular and vacuolar trafficking of plant Aspartic Proteinases as a group.

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

Graduation date: 2003