A raspagem subgengival e o alisamento radicular constituem o "padrão ouro" e o tratamento de eleição para a periodontite; porém, é um procedimento difícil de ser executado, que requer um intenso treinamento e que pode expor a dentina, causando hipersensibilidade dentinária pela remoção excessiva de cemento, ou produzir defeitos, como sulcos e ranhuras, além de deixar cálculo residual e não conseguir atingir toda as superfície radicular. Recentemente, um gel a base de papaína e cloramina foi introduzido no mercado (Papacárie), utilizado no tratamento da remoção de dentina cariada. Este gel poderia auxiliar na remoção do cálculo subgengival com menor desgaste do cemento. O objetivo deste trabalho foi comparar a eficácia e analisar a superfície radicular na utilização de um gel à base de papaína e cloramina, associado ao alisamento radicular, na região subgengival. Após receberem instruções de higiene oral, raspagem supragengival e polimento coronário, 18 pacientes com periodontite crônica, 6 mulheres e 12 homens, com idade média de 51 anos (8) foram tratados num modelo de boca dividida. O tratamento-teste foi constituído pela aplicação do gel na área subgengival por 1 min., seguida pelo alisamento radicular; o tratamento-controle foi constituído pela raspagem subgengival e alisamento radiculares. A terapia foi executada por 3 operadoras e os exames inicial, de 28 dias e 3 meses, foram realizados por um único examinador. Quatro dentes nunca tratados de dois outros pacientes (2 incisivos centrais inferiores e 2 premolares), com indicação para extração, foram submetidos ao tratamento teste e controle e, após a exodontia, analisados em microscopia eletrônica de varredura (MEV). Ao longo dos 3 meses, os resultados demonstraram significativa melhora nos parâmetros clínicos: sangramento à sondagem, profundidade de bolsa e ganho de inserção, tanto no lado-teste, como no lado-controle, principalmente aos 28 dias; mas não foi observada significância estatística quando ambas as formas de terapia foram comparadas. O índice de placa médio permaneceu alto ao longo do estudo. A análise do MEV demonstrou que o tratamento-teste deixou uma maior quantidade de cálculo residual sobre a superfície radicular; porém, áreas livres de cálculo também foram observadas. No tratamento-controle, verificaram-se regiões mais profundas não atingidas pelas curetas, áreas livres de cálculo e um sulco produzido pela cureta. Concluiu-se que tanto o tratamento-teste, como o controle, foram eficazes no tratamento da periodontite crônica nos 3 meses observados. / Although subgingival scaling and root planing are the gold standard for elective treatment of periodontitis, they are difficult procedures to perform. As well as requiring intensive training, they can expose the dentin, causing dentin hypersensitivity by excessive removal of cement, or produce defects such as ridges and grooves, leaving residual calculus, whilst the whole root surface cannot be reached. A papain- and chloramine-based gel (Papacárie) has recently been introduced to remove carious dentin. This gel may help in the removal of subgingival calculus with reduced consumption of cement. The objective of this study was to compare the effectiveness of a papain- and chloramine-based gel and analyze the root surface in the region associated with subgingival root planing. After receiving oral hygiene instructions, supragingival scaling and coronary polishing, 18 chronic periodontitis patients (6 women and 12 men with a mean age of 51 years 8) were treated using a split-mouth model. The test treatment was established by applying the gel to the subgingival area for 1 minute, followed by root planing; whilst the control treatment was established by subgingival scaling and root planing. The therapy was performed by 3 operators, examinations initially and after 28 days and 3 months being performed by a single examiner. Four previously untreated teeth (2 lower central incisors and 2 premolars) with indication for extraction in two other patients were treated as test and control and analyzed by scanning electron microscopy (SEM) following extraction. Although over the 3 months the results showed marked improvement in clinical parameters: bleeding on probing, pocket depth and attachment gain on both test and control sides, especially after 28 days; the difference between the two forms of therapy was not found to be statistically significant. The mean plaque index remained high throughout the study. The SEM analysis showed that the test treatment left a larger amount of residual calculus on the root surface, but areas free of calculus were also observed. In the control treatment, deeper areas unaffected by the scaling and root planing, areas free of calculus and a groove produced by the curette were found. It was concluded that both test and control treatments were effective in the treatment of chronic periodontitis observed over 3 months.

Hidrogéis são membranas formadas pela reticulação de cadeias poliméricas, empregados na área farmacêutica como produtos biomédicos. Dentre os principais polímeros selecionados para a síntese de hidrogéis, destaca-se a poliacrilamida (PAAM) devido às suas propriedades como hidrofilicidade e alto grau de intumescimento. Proteínas terapêuticas e enzimas são veiculadas em hidrogéis como carreadores de fármaco ou como dispositivos para tratamento de feridas e escaras na pele. Este trabalho teve como objetivo a síntese de uma membrana à base de PAAM favorável para veiculação de proteínas. As proteínas empregadas foram papaína e albumina de soro bovino (BSA) e as etapas do processo englobaram síntese da membrana, adição das proteínas no sistema, irradiação em condições específicas e caracterização da membrana. Ao utilizar temperaturas criogênicas na síntese e na irradiação das amostras, houve predomínio de reticulação da cadeia polimérica, fato que não ocorria em temperatura ambiente. As membranas foram obtidas com incorporação dos ativos na concentração de 0,2 a 1% (p/p), obtendo-se concentração de PAAM entre 4% a 10% (p/p), as quais receberam irradiação com raios gama provenientes de uma fonte 60Co, na dose de 25 kGy. Nas condições realizadas, as membranas não apresentaram citotoxicidade nem adesão celular, o perfil de liberação das proteínas foi adequado, a papaína manteve sua bioatividade preservada apesar do decaimento biológico e, segundo estudos de carga das moléculas, a membrana possui maior afinidade com a papaína, liberando-a mais lentamente. Desta forma, o método proposto e as membranas obtidas foram apropriados para a obtenção de um biomaterial. / The use of hydrogels for biomedical purposes has been extensively investigated. Polyacrylamide (PAAM) is widely used due to properties such as hydrophilicity and swelling degree. Pharmaceutical proteins correspond to highly active substances which may be applied for distinct purposes. This work concerns the development of radio-synthesized hydrogel for protein release using papain and bovine serum albumin (BSA) as model proteins. The polymer was solubilized (1% w/v) in water and lyophilized. The proteins were incorporated into the lyophilized polymer and the hydrogels were produced by simultaneous crosslinking and sterilization using gamma radiation at 25 kGy under frozen conditions. The produced systems were characterized in terms of swelling degree, gel fraction, crosslinking density, fluid handling capacity, determination pH at point of polymer zero charge and evaluated according to protein release, bioactivity, cytotoxicity and cell adhesion. The hydrogels developed presented different properties as a function of polymer concentration and the optimized results were found for the samples containing 4-10% polyacrylamide. Protein release was controlled by the electrostatic affinity of acrylic moieties of polymer and proteins. This selection was based on the release of the proteins during the experiment period (up to 50 hours), maintenance of enzyme activity and the nanostructure developed. The system was suitable for protein loading and release and according to the cytotoxic assay and cell adhesion it was also adequate for biomedical purposes and this method was able to generate a matrix to protein release.

The flavivirus West Nile virus (WNV) encodes a single polyprotein that is processed into three structural and seven nonstructural proteins. Various WNV bicistronic replicons that direct cap-dependent translation of an N-terminal viral capsid or capsid/Renilla luciferase fusion protein as well as IRES-dependent translation of the nonstructural proteins were constructed. An original replicon consisting of the WNV 5' NCR, the 5' 198 nts of the capsid coding sequence, which included the 5' cyclization sequence (Cyc), and an EMCV IRES followed by the WNV nonstructural genes and 3' NCR was generated. Real time qRT-PCR analysis of intracellular levels of this replicon RNA showed a 4 fold increase by 96 hr after transfection of BHK cells. Increasing the distance between the 5' Cyc and IRES by insertion of a 5' IRES flanking sequence alone or together with a Renilla luciferase reporter did not increase RNA replication. Addition of only a reporter decreased RNA replication. The insertion of an extended capsid coding sequence also did not enhance RNA replication, but did enhance both cap- and IRES-dependent translation of replicon RNA, as indicated by immunofluorescence and Western blot analysis. These results suggest the presence of a translation enhancer in the 3' portion of the capsid coding region. Simian hemorrhagic fever virus (SHFV) is a member of the family Arteriviridae, order Nidovirales. SHFV is unique among Nidoviruses in having three instead of two papain-like cysteine protease (PCP) motifs designated alpha, beta, and gamma, within the N-terminal region of its ORF1a. Mutations of putative PCP cleavage sites showed that the most efficient cleavage was by PCP beta at its downstream cleavage site. A large deletion located between the two catalytic residues of PCP alpha was hypothesized to render this protease inactive. However, processing was observed at the cleavage site following PCP alpha. Mutational analyses confirmed that PCP alpha is an inactive protease, and that the cleavage sites downstream of PCP alpha are cleaved by PCP gamma. When the catalytic residues of PCP gamma were mutated, PCP beta was also able to back cleave at these sites. This "back" cleavage is a previously unreported activity for an arterivirus PCP.

This thesis investigates the design, synthesis and biological assay of cysteine protease inhibitors within the papain superfamily of cysteine proteases. This is achieved by examining the effect of inhibitor design, especially warheads, on IC₅₀ values and structureactivity relationships between cysteine protease inhibitors of the papain superfamily. The representative proteases used are m-calpain, μ-calpain, cathepsin B and papain. Chapter One is an introductory chapter; Chapters Two-Four describe the design and synthesis of cysteine protease inhibitors; Chapter Five discusses assay protocol; and Chapter Six contains the assay results and structure-activity relationships of the synthesised inhibitors. Chapter One introduces cysteine proteases of the papain family and examines the structure, physiology and role in disease of papain, cathepsin B, m-calpain and μ-calpain. The close structural homology that exists between these members of the papain superfamily is identified, as well characteristics unique to each protease. Covalent reversible, covalent irreversible and non-covalent warheads are defined. The generic inhibitor scaffold of address region, recognition and warhead, upon which the inhibitors synthesised in this thesis are based, is also introduced. Chapter Two introduces reversible cysteine protease inhibitors found in the literature and that little is known about the effect of inhibitor warhead on selectivity within the papain superfamily. Oxidation of the dipeptidyl alcohols 2.6, 2.26, 2.29, 2.30, 2.35 and 2.36 utilising the sulfur trioxide-pyridine complex gave the aldehydes 2.3, 2.27, 2.19, 2.2, 2.21 and 2.22. Semicarbazones 2.37-2.40 were synthesised by a condensation reaction between the alcohol 2.3 and four available semicarbazides. The amidoximes 2.48 and 2.49 separately underwent thermal intramolecular cyclodehydration to give the 3-methyl-1,2,4- oxadiazoles 2.41 and 2.50. The aldehydes 2.3 and 2.27 were reacted with potassium cyanide to give the cyanohydrins 2.51 and 2.52. The cyanohydrins 2.51 and 2.52 were separately reacted to give 1) the α-ketotetrazoles 2.43 and 2.55; 2) the α-ketooxazolines 2.42 and 2.58; 3) the esterified cyanohydrins 2.60 and 2.61. A two step SN2 displacement reaction of the alcohol 2.6 to give the azide 2.62, an example of a non-covalent cysteine protease inhibitor. Chapter Three introduces inhibitors with irreversible warheads. The well-known examples of epoxysuccinic acids 3.1 and 3.5 are discussed in detail, highlighting the lack of irreversible cysteine protease specific inhibitors. The aldehydes 2.3 and 2.27 were reacted under Wittig conditions to give the α,β-unsaturated carbonyls 3.14-3.18. Horner- Emmons-Wadsworth methodology was utilised for the synthesis of the vinyl sulfones 3.20- 3.23. The dipeptidyl acids 2.24 and 2.28 were separately reacted with diazomethane to give the diazoketones 3.25 and 3.26. The diazoketones 3.25 and 3.26 were separately reacted with hydrogen bromide in acetic acid (33%) to give the α-bromomethyl ketones 3.27 and 3.28, which were subsequently reduced to give the α-bromomethyl alcohols 3.29-3.32. Under basic conditions the α-bromomethyl alcohols 3.29-3.32 ring-closed to form the peptidyl epoxides 3.33-3.36. Chapter Four introduces the disadvantages of peptide-based inhibitors. A discussion is given on the benefits of constraining inhibitors into the extended bioactive conformation known as a β-strand. Ring closing metathesis is utilised in the synthesis of the macrocyclic aldehyde 4.4, macrocyclic semicarbazone 4.15, the macrocyclic cyanohydrin 4.16, the macrocyclic α-ketotetrazole 4.18 and the macrocyclic azide 4.19. Chapter Five introduces enzyme inhibition studies. The BODIPY-casein fluorogenic assay used for establishing inhibitor potency against m-calpain and μ-calpain is validated. Assay protocols are also established and validated for cathepsin B, papain, pepsin and α- chymotrypsin. A discussion of the effect of solvent on enzyme activity is also included as part of this study. Chapter Six presents the assay results for all the inhibitors synthesised throughout this thesis and an extensive structure-activity relationship study between inhibitors is included. The alcohols 2.26 and 2.30 are unprecedented examples of non-covalent, potent, cathepsin B inhibitors (IC₅₀ = 0.075 μM selectivity 80-fold and 1.1 μM, selectivity 18-fold). The macrocyclic semicarbazone 4.15 is an unprecedented example of a potent macrocyclic cysteine protease inhibitor (m-calpain: IC₅₀ = 0.16 μM, selectivity 8-fold). The cyanohydrin 2.51 contains an unprecedented cysteine protease warhead and is a potent and selective inhibitor of papain (IC₅₀ = 0.030 μM, selectivity 3-fold). The O-protected cyanohydrin 2.61 is a potent and selective inhibitor of pepsin (IC₅₀ = 1.6 μM, selectivity 1.5-fold). The top ten warheads for potent, selective cathepsin B inhibition are: carboxylic acid, methyl ester, diazoketone, esterified cyanohydrin, α-bromomethyl ketone, α,β- unsaturated aldehyde, vinyl sulfones, α-bromomethyl-C₃-S,R-alcohol, alcohol and α,β- unsaturated ethyl ester. The selectivity of these warheads was between 5- and 130-fold for cathepsin B. The best inhibitors for cathepsin B were the α-bromomethyl ketone 3.26 (IC₅₀ = 0.075 μM, selectivity 16-fold), the α,β-unsaturated aldehyde 3.18 (IC₅₀ = 0.13 μM, selectivity 13-fold) and the esterified cyanohydrin 3.59 (IC₅₀ = 0.35 μM, selectivity 22- fold). Chapter Seven outlines the experimental details and synthesis of the compounds prepared in this thesis.

This thesis investigates the design, synthesis and biological assay of cysteine protease inhibitors within the papain superfamily of cysteine proteases. This is achieved by examining the effect of inhibitor design, especially warheads, on IC₅₀ values and structureactivity relationships between cysteine protease inhibitors of the papain superfamily. The representative proteases used are m-calpain, μ-calpain, cathepsin B and papain. Chapter One is an introductory chapter; Chapters Two-Four describe the design and synthesis of cysteine protease inhibitors; Chapter Five discusses assay protocol; and Chapter Six contains the assay results and structure-activity relationships of the synthesised inhibitors. Chapter One introduces cysteine proteases of the papain family and examines the structure, physiology and role in disease of papain, cathepsin B, m-calpain and μ-calpain. The close structural homology that exists between these members of the papain superfamily is identified, as well characteristics unique to each protease. Covalent reversible, covalent irreversible and non-covalent warheads are defined. The generic inhibitor scaffold of address region, recognition and warhead, upon which the inhibitors synthesised in this thesis are based, is also introduced. Chapter Two introduces reversible cysteine protease inhibitors found in the literature and that little is known about the effect of inhibitor warhead on selectivity within the papain superfamily. Oxidation of the dipeptidyl alcohols 2.6, 2.26, 2.29, 2.30, 2.35 and 2.36 utilising the sulfur trioxide-pyridine complex gave the aldehydes 2.3, 2.27, 2.19, 2.2, 2.21 and 2.22. Semicarbazones 2.37-2.40 were synthesised by a condensation reaction between the alcohol 2.3 and four available semicarbazides. The amidoximes 2.48 and 2.49 separately underwent thermal intramolecular cyclodehydration to give the 3-methyl-1,2,4- oxadiazoles 2.41 and 2.50. The aldehydes 2.3 and 2.27 were reacted with potassium cyanide to give the cyanohydrins 2.51 and 2.52. The cyanohydrins 2.51 and 2.52 were separately reacted to give 1) the α-ketotetrazoles 2.43 and 2.55; 2) the α-ketooxazolines 2.42 and 2.58; 3) the esterified cyanohydrins 2.60 and 2.61. A two step SN2 displacement reaction of the alcohol 2.6 to give the azide 2.62, an example of a non-covalent cysteine protease inhibitor. Chapter Three introduces inhibitors with irreversible warheads. The well-known examples of epoxysuccinic acids 3.1 and 3.5 are discussed in detail, highlighting the lack of irreversible cysteine protease specific inhibitors. The aldehydes 2.3 and 2.27 were reacted under Wittig conditions to give the α,β-unsaturated carbonyls 3.14-3.18. Horner- Emmons-Wadsworth methodology was utilised for the synthesis of the vinyl sulfones 3.20- 3.23. The dipeptidyl acids 2.24 and 2.28 were separately reacted with diazomethane to give the diazoketones 3.25 and 3.26. The diazoketones 3.25 and 3.26 were separately reacted with hydrogen bromide in acetic acid (33%) to give the α-bromomethyl ketones 3.27 and 3.28, which were subsequently reduced to give the α-bromomethyl alcohols 3.29-3.32. Under basic conditions the α-bromomethyl alcohols 3.29-3.32 ring-closed to form the peptidyl epoxides 3.33-3.36. Chapter Four introduces the disadvantages of peptide-based inhibitors. A discussion is given on the benefits of constraining inhibitors into the extended bioactive conformation known as a β-strand. Ring closing metathesis is utilised in the synthesis of the macrocyclic aldehyde 4.4, macrocyclic semicarbazone 4.15, the macrocyclic cyanohydrin 4.16, the macrocyclic α-ketotetrazole 4.18 and the macrocyclic azide 4.19. Chapter Five introduces enzyme inhibition studies. The BODIPY-casein fluorogenic assay used for establishing inhibitor potency against m-calpain and μ-calpain is validated. Assay protocols are also established and validated for cathepsin B, papain, pepsin and α- chymotrypsin. A discussion of the effect of solvent on enzyme activity is also included as part of this study. Chapter Six presents the assay results for all the inhibitors synthesised throughout this thesis and an extensive structure-activity relationship study between inhibitors is included. The alcohols 2.26 and 2.30 are unprecedented examples of non-covalent, potent, cathepsin B inhibitors (IC₅₀ = 0.075 μM selectivity 80-fold and 1.1 μM, selectivity 18-fold). The macrocyclic semicarbazone 4.15 is an unprecedented example of a potent macrocyclic cysteine protease inhibitor (m-calpain: IC₅₀ = 0.16 μM, selectivity 8-fold). The cyanohydrin 2.51 contains an unprecedented cysteine protease warhead and is a potent and selective inhibitor of papain (IC₅₀ = 0.030 μM, selectivity 3-fold). The O-protected cyanohydrin 2.61 is a potent and selective inhibitor of pepsin (IC₅₀ = 1.6 μM, selectivity 1.5-fold). The top ten warheads for potent, selective cathepsin B inhibition are: carboxylic acid, methyl ester, diazoketone, esterified cyanohydrin, α-bromomethyl ketone, α,β- unsaturated aldehyde, vinyl sulfones, α-bromomethyl-C₃-S,R-alcohol, alcohol and α,β- unsaturated ethyl ester. The selectivity of these warheads was between 5- and 130-fold for cathepsin B. The best inhibitors for cathepsin B were the α-bromomethyl ketone 3.26 (IC₅₀ = 0.075 μM, selectivity 16-fold), the α,β-unsaturated aldehyde 3.18 (IC₅₀ = 0.13 μM, selectivity 13-fold) and the esterified cyanohydrin 3.59 (IC₅₀ = 0.35 μM, selectivity 22- fold). Chapter Seven outlines the experimental details and synthesis of the compounds prepared in this thesis.

Made available in DSpace on 2014-06-11T19:27:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-15Bitstream added on 2014-06-13T20:56:48Z : No. of bitstreams: 1 lima_dc_me_araca.pdf: 774525 bytes, checksum: 538ef364e5dbda059945006d226d6a66 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Na Odontologia atualmente têm-se enfatizado a prática de técnicas menos invasivas para a remoção de tecido cariado, que preservem ao máximo as estruturas dentárias. O interesse clínico e laboratorial por essas técnicas tem aumentado em função da realização de estudos que comprovaram a eficácia da remoção manual de dentina infectada, bem como as vantagens da técnica conservadora e indolor. O objetivo desse estudo foi analisar e comparar os tratamentos mecânico manual (ART convencional) e químico-mecânicos (CarisolvTM e Papa-cárie®) na remoção de dentina cariada, por meio da contagem de Streptococcus mutans e Lactobacillus e avaliação clínica de sintomatologia dolorosa durante o procedimento restaurador. Para a realização do presente estudo foram selecionadas 32 crianças, de ambos os sexos, com idade entre 6 e 10 anos, que apresentavam no mínimo dois molares decíduos, com exposição de dentina cariada. As crianças foram divididas aleatoriamente em dois grupos homogêneos, totalizando 64 dentes. O grupo I foi submetido à remoção mecânica em um elemento dental e em outro dente a químico-mecânica com o uso de Carisolv e o grupo II, a remoção mecânica e a químico-mecânica com o uso do Papacárie®. Durante a realização dos tratamentos foram feitas coletas de dentina, antes e após a remoção de tecido cariado e aplicação de um questionário para a avaliação clínica. Posteriormente, foram realizadas diluições seriadas e cultivo das amostras em meios específicos, Mitis salivarius bacitracina sacarose para a contagem de Streptococcus mutans e Rogosa, para contagem de Lactobacillus ssp. Após o período de incubação, a 37º C e 72 horas, foi realizada a contagem de Unidades Formadoras de Colônias (UFC) e obtidos os valores médios do número de bactérias. / In Dentistry, nowadays, it has been emphasizing the practice of techniques less invasive for the removal of decayed tissue that preserve the maximum the dental structure. The clinical laboratorial interest for those techniques have been increasing due to studies that proved the effectiveness of the manual removal of infected dentine, as well as the advantages of the conservative and painless technique. The objective of this study was to analyze and to compare the manual mechanic (conventional ART) and chemical-mechanic (Carisolv and Papa-carie®) treatments in the removal of decayed dentine, through the score of Streptococcus mutans and Lactobacillus and clinical evaluation of pain symptoms during the restorative procedure. For the performance of the present study 32 children were selected, of both genders, with age between 6 and 10 years that presented at least two deciduous molars, with exhibition of decayed dentine. The children were divided at random in two groups, summing 64 teeth. The group I was submitted to the mechanical and chemical-mechanical removal with the use of Carisolv and the group II, mechanical and chemical-mechanic removal with Papacárie®. During the performance of the treatments, dentine sample were collected, before and after the removal of decayed tissue and application of a questionnaire for the clinical evaluation. Later, it was carried out seriate dilutions and cultivation of the samples in Mitis- salivarius bacitracin sucrose for the score of Streptococcus mutans and Rogosa, for score of Lactobacillus ssp. After the incubation period, at 37° C and 72 hours, it was carried out the score of the ColonY Forming Units (CFU) and obtained the mean values of the bacteria number.

A raspagem subgengival e o alisamento radicular constituem o "padrão ouro" e o tratamento de eleição para a periodontite; porém, é um procedimento difícil de ser executado, que requer um intenso treinamento e que pode expor a dentina, causando hipersensibilidade dentinária pela remoção excessiva de cemento, ou produzir defeitos, como sulcos e ranhuras, além de deixar cálculo residual e não conseguir atingir toda as superfície radicular. Recentemente, um gel a base de papaína e cloramina foi introduzido no mercado (Papacárie), utilizado no tratamento da remoção de dentina cariada. Este gel poderia auxiliar na remoção do cálculo subgengival com menor desgaste do cemento. O objetivo deste trabalho foi comparar a eficácia e analisar a superfície radicular na utilização de um gel à base de papaína e cloramina, associado ao alisamento radicular, na região subgengival. Após receberem instruções de higiene oral, raspagem supragengival e polimento coronário, 18 pacientes com periodontite crônica, 6 mulheres e 12 homens, com idade média de 51 anos (8) foram tratados num modelo de boca dividida. O tratamento-teste foi constituído pela aplicação do gel na área subgengival por 1 min., seguida pelo alisamento radicular; o tratamento-controle foi constituído pela raspagem subgengival e alisamento radiculares. A terapia foi executada por 3 operadoras e os exames inicial, de 28 dias e 3 meses, foram realizados por um único examinador. Quatro dentes nunca tratados de dois outros pacientes (2 incisivos centrais inferiores e 2 premolares), com indicação para extração, foram submetidos ao tratamento teste e controle e, após a exodontia, analisados em microscopia eletrônica de varredura (MEV). Ao longo dos 3 meses, os resultados demonstraram significativa melhora nos parâmetros clínicos: sangramento à sondagem, profundidade de bolsa e ganho de inserção, tanto no lado-teste, como no lado-controle, principalmente aos 28 dias; mas não foi observada significância estatística quando ambas as formas de terapia foram comparadas. O índice de placa médio permaneceu alto ao longo do estudo. A análise do MEV demonstrou que o tratamento-teste deixou uma maior quantidade de cálculo residual sobre a superfície radicular; porém, áreas livres de cálculo também foram observadas. No tratamento-controle, verificaram-se regiões mais profundas não atingidas pelas curetas, áreas livres de cálculo e um sulco produzido pela cureta. Concluiu-se que tanto o tratamento-teste, como o controle, foram eficazes no tratamento da periodontite crônica nos 3 meses observados. / Although subgingival scaling and root planing are the gold standard for elective treatment of periodontitis, they are difficult procedures to perform. As well as requiring intensive training, they can expose the dentin, causing dentin hypersensitivity by excessive removal of cement, or produce defects such as ridges and grooves, leaving residual calculus, whilst the whole root surface cannot be reached. A papain- and chloramine-based gel (Papacárie) has recently been introduced to remove carious dentin. This gel may help in the removal of subgingival calculus with reduced consumption of cement. The objective of this study was to compare the effectiveness of a papain- and chloramine-based gel and analyze the root surface in the region associated with subgingival root planing. After receiving oral hygiene instructions, supragingival scaling and coronary polishing, 18 chronic periodontitis patients (6 women and 12 men with a mean age of 51 years 8) were treated using a split-mouth model. The test treatment was established by applying the gel to the subgingival area for 1 minute, followed by root planing; whilst the control treatment was established by subgingival scaling and root planing. The therapy was performed by 3 operators, examinations initially and after 28 days and 3 months being performed by a single examiner. Four previously untreated teeth (2 lower central incisors and 2 premolars) with indication for extraction in two other patients were treated as test and control and analyzed by scanning electron microscopy (SEM) following extraction. Although over the 3 months the results showed marked improvement in clinical parameters: bleeding on probing, pocket depth and attachment gain on both test and control sides, especially after 28 days; the difference between the two forms of therapy was not found to be statistically significant. The mean plaque index remained high throughout the study. The SEM analysis showed that the test treatment left a larger amount of residual calculus on the root surface, but areas free of calculus were also observed. In the control treatment, deeper areas unaffected by the scaling and root planing, areas free of calculus and a groove produced by the curette were found. It was concluded that both test and control treatments were effective in the treatment of chronic periodontitis observed over 3 months.

Hidrogéis são membranas formadas pela reticulação de cadeias poliméricas, empregados na área farmacêutica como produtos biomédicos. Dentre os principais polímeros selecionados para a síntese de hidrogéis, destaca-se a poliacrilamida (PAAM) devido às suas propriedades como hidrofilicidade e alto grau de intumescimento. Proteínas terapêuticas e enzimas são veiculadas em hidrogéis como carreadores de fármaco ou como dispositivos para tratamento de feridas e escaras na pele. Este trabalho teve como objetivo a síntese de uma membrana à base de PAAM favorável para veiculação de proteínas. As proteínas empregadas foram papaína e albumina de soro bovino (BSA) e as etapas do processo englobaram síntese da membrana, adição das proteínas no sistema, irradiação em condições específicas e caracterização da membrana. Ao utilizar temperaturas criogênicas na síntese e na irradiação das amostras, houve predomínio de reticulação da cadeia polimérica, fato que não ocorria em temperatura ambiente. As membranas foram obtidas com incorporação dos ativos na concentração de 0,2 a 1% (p/p), obtendo-se concentração de PAAM entre 4% a 10% (p/p), as quais receberam irradiação com raios gama provenientes de uma fonte 60Co, na dose de 25 kGy. Nas condições realizadas, as membranas não apresentaram citotoxicidade nem adesão celular, o perfil de liberação das proteínas foi adequado, a papaína manteve sua bioatividade preservada apesar do decaimento biológico e, segundo estudos de carga das moléculas, a membrana possui maior afinidade com a papaína, liberando-a mais lentamente. Desta forma, o método proposto e as membranas obtidas foram apropriados para a obtenção de um biomaterial. / The use of hydrogels for biomedical purposes has been extensively investigated. Polyacrylamide (PAAM) is widely used due to properties such as hydrophilicity and swelling degree. Pharmaceutical proteins correspond to highly active substances which may be applied for distinct purposes. This work concerns the development of radio-synthesized hydrogel for protein release using papain and bovine serum albumin (BSA) as model proteins. The polymer was solubilized (1% w/v) in water and lyophilized. The proteins were incorporated into the lyophilized polymer and the hydrogels were produced by simultaneous crosslinking and sterilization using gamma radiation at 25 kGy under frozen conditions. The produced systems were characterized in terms of swelling degree, gel fraction, crosslinking density, fluid handling capacity, determination pH at point of polymer zero charge and evaluated according to protein release, bioactivity, cytotoxicity and cell adhesion. The hydrogels developed presented different properties as a function of polymer concentration and the optimized results were found for the samples containing 4-10% polyacrylamide. Protein release was controlled by the electrostatic affinity of acrylic moieties of polymer and proteins. This selection was based on the release of the proteins during the experiment period (up to 50 hours), maintenance of enzyme activity and the nanostructure developed. The system was suitable for protein loading and release and according to the cytotoxic assay and cell adhesion it was also adequate for biomedical purposes and this method was able to generate a matrix to protein release.

Submitted by Fabiana Gonçalves Pinto (benf@ndc.uff.br) on 2015-12-03T12:47:21Z No. of bitstreams: 1 Andréa Pinto Leite.pdf: 3263744 bytes, checksum: a7457bd2eeb01032b2cdded51f89df5d (MD5) / Made available in DSpace on 2015-12-03T12:47:21Z (GMT). No. of bitstreams: 1 Andréa Pinto Leite.pdf: 3263744 bytes, checksum: a7457bd2eeb01032b2cdded51f89df5d (MD5) Previous issue date: 2012 / Mestrado Profissional em Enfermagem Assistencial / As úlceras venosas são consideradas um problema de saúde pública pela dificuldade de cicatrização, alta recorrência, elevado custo de tratamento e grande repercussão na qualidade de vida do paciente. A busca de terapias que contribuam para cicatrização tem sido uma constante. Um produto utilizado no tratamento dessas úlceras é a papaína. Objetivo geral: Analisar a efetividade do gel de papaína a 2% e 4% no reparo tecidual das úlceras venosas. Método: Pesquisa clínica experimental, prospectiva, com o uso do Gel de Papaína a 2% e 4% produzido pela Farmácia Universitária, com formulação, conservação e distribuição adaptada para manutenção da estabilidade enzimática. A amostra por conveniência foi composta por 16 pacientes atendidos no ambulatório com 30 úlceras venosas, maiores que 2 cm2, conforme protocolo. As variáveis de interesse foram analisadas por meio do teste de Wilcoxon e McNemar, sendo p < 0,05. O projeto foi aprovado pelo Comitê de Ética em Pesquisa, com o no 196/08. Resultados: Nove (56,25%) participantes eram do sexo feminino; com predomínio de idade entre 51 a 59 anos, com média de 62,31 anos, desvio padrão de ±10,2; 13 (81,25%) apresentavam Índice de Massa Corporal acima do normal; 12 (75%) possuíam Hipertensão Arterial Sistêmica e quatro (25%) Diabetes mellitus associado ou não a outras comorbidades. Quanto às úlceras, a amostra foi dividida em três subgrupos, com área inicial de 2 a 3,9 cm2; de 3,9 a 20 cm2; e > 20 cm2, sendo que no primeiro subgrupo houve redução média de 1,6 cm2 (60%); no segundo de 6,5 cm2 (66,6%) e no terceiro 15,7 cm2 (23,7%). A redução média na área das 30 úlceras foi de 7,9 cm2 (50%). Seis (20%) cicatrizaram completamente, com tempo médio de 56,67 dias ± 9,83, sendo todas menores ou iguais a 4 cm2. Houve redução significativa no tecido de esfacelo de todas as úlceras (p < 0,001) e aumento significativo no tecido de epitelização (p < 0,001) do início para o final do tratamento. Houve também melhora significativa na profundidade das úlceras venosas (p = 0,001), no tipo de exsudato (p = 0,0001), na quantidade de exsudato (p < 0,0001) e no edema (p < 0,0001). Ocorreu epitelização da borda de uma (33,33%) das três úlceras que se apresentavam com bordas maceradas. Em relação ao relato de dor, de 13 houve melhora em cinco (38,46%); em relação ao prurido, de 17 houve melhora em seis (35,29%). Em relação à dor após o uso do produto, houve relato de quatro (25%) pacientes, sendo de baixa intensidade. Conclusão: O gel de papaína a 2% e a 4% mostrou ser efetivo na diminuição do esfacelo, favorecendo o processo de epitelização e proporcionando a cicatrização total de úlceras venosas menores ou iguais a 4 cm2 no período de 90 dias. A pesquisa contribuiu com evidências científicas acerca do uso do gel de papaína a 2% e a 4% em úlceras venosas / The venous ulcers are considered a problem in public health for its healing difficulty, high recurrence, high cost treatment and big repercussion in the patient´s quality of life. The search for therapies which contribute for the healing has been constant. A product used in the treatment of these ulcers is the papain. General objective: Analyze the effectiveness of the papain gel at 2% and 4% in the tissue repair of the venous ulcers. Method: Experimental clinical research, prospective, with the use of papain gel at 2% and 4% produced by the University Pharmacy, with formulation, preservation and distribution adapted to maintain the enzymatic stability. The convenience sample was made of 16 outpatients with 30 venous ulcers, larger than 2 cm2, according to protocol. The variables of interest were analyzed through Wilcoxon and McNemar tests, with p < 0,05. The project was approved by the Ethics in Research Committee, no 196/08. Results: Nine (56,25%) participants were female, with predominance of age between 51 and 59 years old, average of 62,31 years old, standard deviation of ±10,2; 13 (81,25%) presented body mass index above the normal; 12 (75%) had systemic high pressure and four (25%) diabetes mellitus associated or not to other co-morbidities. Regarding the ulcers, the sample was divided in three subgroups, with initial area from 2 to 3.9 cm2; from 3.9 to 20 cm2; and > 20 cm2. There was an average reduction in the first subgroup of 1.6 cm2 (60%); in the second one of 6.5 cm2 (66.6%) and the third one of 15.7 cm2 (23.7%). The average reduction in the area of the 30 ulcers was of 7.9 cm2 (50%). Six (20%) presented complete healing, with an average time of 56.67 days ± 9.83, all of them smaller or equal to 4 cm2. There was a significant reduction of the slough of all the ulcers (p < 0,001) and significant increase of the tissue of epithelization. (p < 0,001) from the beginning to the end of the treatment. There was also a significant improvement in the depth of the venous ulcers (p = 0,001), exudate type (p = 0,0001), in the exudate amount (p < 0,0001) and in the edema (p < 0,0001). There was epithelization of the edge in one (33.33%) of the three ulcers which were macerated. Concerning pain report, out of 13, there was improvement in five (38.46%); related to itching, out of 17, there was improvement in six (35.29%). Regarding pain after using the product, there were four low intensity reports (25%) by the patients. Conclusion: The papain gel at 2% and 4% presented to be effective in the decrease of the slough, promoting the epithelization process and providing complete healing of the venous ulcers smaller or equal to 4 cm2 in the period of 90 days. The research contributes with scientific evidence about the use of papain gel at 2% and 4% in venous ulcers

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-13T18:50:12Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Duarte, Letícia de Souza Guimarães [Dissertação, 2016].pdf: 2499210 bytes, checksum: 961dab593ba2ea593fadba0a224c15cc (MD5) / Made available in DSpace on 2017-03-13T18:50:12Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Duarte, Letícia de Souza Guimarães [Dissertação, 2016].pdf: 2499210 bytes, checksum: 961dab593ba2ea593fadba0a224c15cc (MD5) / A papaína, que corresponde a um complexo de enzimas extraídas do vegetal Carica papaya L., tem sido utilizada pela sua propriedade proteolítica em desbridamento enzimático de feridas. Diversos estudos descrevem o uso da papaína na forma de pó, solução, gel e creme sobre feridas, porém a baixa estabilidade da enzima nesses meios limita sua utilização em larga escala. O objetivo principal do trabalho consiste em desenvolver e avaliar formulações de papaína 10%(p/p) em gel carbômero, contendo antioxidantes como acetato de alfa-tocoferol e metabissulfito de sódio, associados a outros adjuvantes que permitam melhor estabilidade da enzima. As formulações iniciais para o estudo foram planejadas segundo desenho experimental fatorial 23, sendo as variáveis independentes representadas pelas concentrações de cisteína, de polissorbato 80 e de antioxidante (acetato de alfa-tocoferol ou metabissulfito de sódio). As amostras, mantidas sob refrigeração (5°C ± 3) por 7 dias, foram submetidas a ensaios de espectrofluorimetria para avaliação da atividade enzimática. Os resultados iniciais revelaram que as amostras que continham metabissulfito de sódio associado às maiores concentrações de polissorbato 80, apresentaram as melhores condições para manutenção da atividade proteolítica, enquanto que as amostras contendo alfa-tocoferol não foram capazes de manter a atividade. Sendo assim, realizou-se estudo de estabilidade com preparações contendo concentrações fixas de papaína 10% (p/p), polissorbato 80 2,0% (p/p), com variações nas concentrações de cisteína de 0,10, 0,13 e 0,16% (p/p) e metabissulfito de sódio de 0,50, 0,75 e 1,00% (p/p). Durante o período estudado, as amostras apresentaram aspecto homogêneo, sem mudanças na coloração e no odor. Na avaliação de pH não houve variação significativa dos valores (p>0,05). A variação de potencial Zeta foi menor que ǀ10ǀmV, não havendo diferença estatisticamente significativa em função do tempo (p>0,05). A partir dos resultados obtidos, novo desenho fatorial 23 foi traçado considerando como variáveis independentes: o tempo, as concentrações de metabissulfito e de cisteína. A presença de metabissulfito 0,5% ou 1,0% (p/p) associado a cisteína 0,16%(p/p) e polissorbato 80 2,0% (p/p), proporcionaram os menores valores de decaimento na concentração de papaína ativa nas formulações testadas por 28 dias. / Papain, which corresponds to a complex of enzymes from the plant Carica papaya L., has been used for its proteolytic property in enzymatic wound debridement. Several studies describe the use of papain as powder, solution, gel and cream forms over wounds; however, the low stability of the enzyme in the media limits its use in large scale. The main objective of this work is to develop and evaluate formulations with papain 10% (w/w) at carbomer gel, containing antioxidants as alpha-tocopherol acetate and sodium metabisulphite, associated with other adjuvants that enable better stability of the enzymes. The early formulations for the study were planned in a factorial experimental design 23, with the independent variables represented by cysteine, polysorbate 80 and antioxidant (alpha-tocopherol or sodium metabisulphite) concentrations. The samples, kept under refrigeration (5°C ± 3) for 7 days, were submitted to spectrofluorimetry essays for enzyme activity evaluation. Initial findings showed that the samples containing sodium metabisulphite associated with higher concentrations of polysorbate 80, presented the best conditions for proteolytic activity maintenance, whereas the samples containing alpha-tocopherol were not able to maintain activity. Thus, stability study was carried out with preparations with fixed concentrations of papain 10% (w/w), polysorbate 80 2.0% (w/w), with variations in the concentrations of cystein 0.10, 0.13 and 0.16% (w/w) and sodium metabisulphite 0.50, 0.75 and 1.00% (w/w). During the studied period, the samples showed a homogeneous appearance, without color or odor changes. At pH evaluation there was no significant change in values (p> 0.05). Zeta potential have varied less than |10|mV, with no statistically significant difference over time (p> 0.05). From the results, a new factorial design 23 was traced, considering as independent variables: time, metabisulphite and cystein concentrations. The presence of metabisulphite 0.5 % or 1.0 % (w/w) associated with cysteine 0.16 % (w/w), and polysorbate 80 2.0% (w/w), showed the lowest decay values of active papain concentration in the formulations tested for 28 days.