M. Tech. (Chemistry, Department of Chemistry), Vaal University of Technology / Infectious diseases, a group of illnesses caused by specific pathogens or its toxins is a leading cause of death globally. Treatment with antibiotics is a key intervention in the control and management of many infectious disease. However, the increasing incidence of antibiotics failure, due to the emergence of drug resistant pathogens, is rendering the use of antibiotics chemotherapy ineffective. A possible solution is to synthesize new compounds with broad spectrum characteristics and superior drug performances as alternative to conventional antibiotics. Schiff Bases are biologically active ligands. They form metal complexes with superior biological activities. This research aims to synthesize some Schiff Base metal complexes and investigate their biological effects on Staphylococcus aureus and Enterococcus faecalis. Metal acetylacetonates of Vanadium, Copper, Cobalt, Zinc, Magnesium, Manganese, Cadmium, Nickel and Iron were synthesized and characterized by Fourier transform infrared spectroscopy. Four Schiff bases, LI, L2, L3 and L4 were also synthesized by the condensation of 4- (diethylamino)-2-hydroxybenzaldehyde with 4-nitrobenzohydrazide and 4-methoxybenzohydrazide to form L1 and L2. 4-(dimethylamino) benzaldehyde was reacted with 4-nitrobenzohydrazide and 4-methoxybenzohydrazide to form L3 and L4 respectively. The Schiff base ligands were then reacted with synthesized Vanadium, manganese, cobalt and magnesium acetylacetonates to form Schiff base complexes (SBC 1A to 4D). Schiff bases ligands and complexes were characterized by FT-IR, 1H-NMR, 13C-NMR, TGA and DTA. Fourier Transform infrared spectroscopy (FTIR) of the acetylacetonates showed the formation of metal acetylacetonates as characterized by the absence of the carbonyl stretching n(C=O) vibration in metal acetylacetonate spectra as compared to pure acetylacetone. Metal acetylacetonates also showed the presence of metal oxygen vibration frequency, n(M-O-C), in the spectra obtained. Thermogravimetric analysis (TGA) and Derivative or Differential Thermogravimetric analysis (DTA) of the Schiff base ligands showed the presence of a single decomposition product in L1, L2, L3 and L4 indicating the formation of a single reaction product while those of Schiff base complexes showed the formation of several decomposition products. Proton and carbon thirteen Nuclear Magnetic Resonance (1H- and 13C-NMR) spectroscopy of the Schiff base ligands indicated the presence of hydrogen and carbon-13 in different environments. The chemical shifts of the hydrogens and carbon-13 provided evidence that Schiff base ligands were formed. The strongest evidence is the presence of the azomethine hydrogen and carbon in the spectra of the Schiff base ligands. The presence of aromatic hydrogens and carbon at chemical shift environments found in literature also confirmed the formation of Schiff base ligand. The NMR spectra of Schiff base complexes showed the presence of azomethine (HC=N) and aromatic hydrogens at expected chemical shifts. The synthesized Schiff bases and their corresponding metal complexes were screened for their invitro antibacterial activities against two Gram-positive (Staphylococcus aureues and Enterococcus feacalis) bacterial strains by the Agar-well diffusion methods.The ligands and complexes were tested against confirmed S. aureus and E. faecalis strains and only 4 exhibited antimicrobial activities. The ligands and complexes were effective against the S. aureusand E. faecalis isolates. / VUT

No presente estudo, a proposta foi avaliar a ação antimicrobiana sobre o biofilme de Candida albicans (SC 5314) e determinar a mínima concentração inibitória (MCI) de cinco fármacos utilizados no tratamento de estomatite protética (nistatina, miconazol, cetoconazol, itraconazol e diacetato de clorexidina), quando incorporados em reembasadores resilientes temporários à base de resina acrílica (Trusoft e Softone) bem como o efeito dessa modificação sobre as propriedades de dureza Shore A e rugosidade superficial dos materiais. Para determinação das MCIs, o biofilme fúngico foi formado sobre corpos de prova circulares (10 mm x 1 mm) dos materiais (n = 6) modificados (experimentais) ou não (controle) pela adição dos fármacos. Diferentes concentrações dos antimicrobianos foram testadas e a viabilidade celular determinada espectrofotometricamente pelo ensaio de redução de sais de tetrazólio- XTT, nos períodos de 24 h, 48 h, 7 e 14 dias. As medidas espectrofotométricas foram convertidas em porcentagens de redução fúngica e as MCIs determinadas como aquelas suficientes para inibir 90% ou mais do crescimento de C. albicans. Para os ensaios de dureza e rugosidade, corpos de prova retangulares (36 mm x 7 mm x 6 mm) dos materiais resilientes (n= 8) foram confeccionados sem (controle) ou com incorporação dos fármacos nas MCIs previamente definidas. Após armazenamento em água destilada a 37°C por 24 h, 7 e 14 dias, foram realizados os testes de dureza em durômetro Shore A (Woltest, GSD-709A) e de rugosidade superficial em rugosímetro (Surftest SJ-301). Os resultados foram analisados estatisticamente por ANOVA 3 fatores, seguida pelo teste de Tukey (=0,05). De acordo com os resultados, as MCIs determinadas para fármacos incorporados aos materiais resilientes foram: 0,032, 0,256, 0,128, 0,256 e 0,064 g/mL para nistatina, miconazol, cetoconazol, itraconazol e clorexidina, respectivamente. A adição dos antimicrobianos em ambos os materiais não alterou os valores de dureza, ou resultou na sua diminuição em relação ao controle, exceto para a incorporação de miconazol ao Softone, que demonstrou maiores médias após 14 dias (P=0,0035). A incorporação de nistatina aos dois materiais, de clorexidina ao Trusoft e de cetoconazol ao Softone não alterou os valores de rugosidade em relação ao controle após 7 e 14 dias (P>0,05). Nesses períodos, o itraconazol aumentou a rugosidade dos materiais (P<0,0001). Em relação às 24 h iniciais, o período de 14 dias mostrou que a rugosidade com a adição de nistatina, miconazol e cetoconazol foi reduzida para o Trusoft (P<0,05) e não apresentou alteração para o Softone (P>0,05). Foi possível concluir que a incorporação dos antimicrobianos testados inibiu o crescimento de C. albicans nos materiais ao longo de 14 dias de avaliação. A adição de todos os fármacos testados, exceto o miconazol no Softone, não causou alterações deletérias à dureza dos materiais resilientes no período avaliado. No período final, a adição de nistatina, miconazol e cetoconazol em ambos os reembasadores e de clorexidina no Trusoft não resultou em efeitos adversos na rugosidade. / The purpose of this study was to evaluate the antimicrobial action on Candida albicans biofilm (SC5314) and determine the minimum inhibitory concentration (MIC) of five drugs for denture stomatitis\' treatment (nystatin, miconazole, ketoconazole, itraconazole, and chlorhexidine diacetate) incorporated into temporary denture relines (Trusoft e Softone) as well the effect of this addition on the Shore A hardness and surface roughness of the materials. For MIC determination, the fungal biofilm was formed on disc specimens (10mm x 1 mm) of the materials (n= 6) modified (experimental) or not (control) by the addition of drugs. Different dosages of the antimicrobials were tested and cellular viability was determined by spectrophotometric tetrazolium salt XTT reduction assay at 24 h, 48 h, 7 and 14 days of incubation. The spectrophotometric measurements were converted to percentage reduction in candidal growth and the MICs were determined as the concentrations necessary to inhibit 90% or more of fungal viability. For hardness and surface tests, rectangular specimens (36 mm X 7 mm X 6 mm) of the resilient materials (n= 8) were made without (control) or with incorporation of the MIC drugs. After storage in distilled water at 37°C for 24h, 7 and 14 days, the hardness tests were performed using a Shore A hardness tester (Woltest, GSD-709A) and the roughness assay was conducted in a surface roughness tester (Surftest SJ-301). Data were statistically analyzed by 3-way ANOVA followed by Tukeys test (=.05). According to the results, MICs of the drugs incorporated into the material were: 0.032, 0.256, 0.128, 0.256 e 0.064 g/mL for nystatin, miconazole, ketoconazole, itraconazole and chlorhexidine, respectively. The addition of the tested antimicrobial agents in both materials demonstrated no evident hardness change or resulted in its decrease compared to the control, except for miconazole incorporation into Softone, which increased the hardness values after 14 days (P = .0035). The addition of nystatin into the two materials, chlorhexidine into Trusoft and ketoconazole into Softone resulted in no significant changes of roughness values compared to the control after 7 and 14 days (P>.05). In these periods, itraconazole promoted increase of the roughness for both materials (P<.0001). Compare to the 24- h period, the roughness at 14- day time with the addition of nystatin, miconazole and ketoconazole was reduced for Trusoft (P<.05) and remained unaffected for Softone (P> .05). It can be concluded that the incorporation of antimicrobial agents inhibited the C. albicans growth on the materials up to 14 days. The addition of all tested drugs, except for the miconazole into Softone, resulted in no deleterious effects on hardness of the resilient materials over the evaluation time. At the end period, the incorporation of nystatin, miconazole and ketoconazole into both denture relines and chlorhexidine into Trusoft resulted in no detrimental changes on the roughness.

A resina acrílica para base de próteses removíveis é capaz de acumular biofilme e assim favorecer o aparecimento de diversos problemas na cavidade bucal dos usuários de próteses. A imobilização de um agente antisséptico na matriz polimérica tem potencial preventivo frente a esse acúmulo, mas necessita de maior investigação. O objetivo deste estudo foi avaliar as propriedades biológicas do brometo de metacriloiloxiundecilpiridínio (MUPB), um composto antisséptico capaz de copolimerizar-se com as resinas acrílicas. Foram determinadas as concentrações inibitória e fungicida/bactericida mínimas (CIM, CFM/CBM) do MUPB frente às espécies Candida albicans, Candida dubliniensis, Candida glabrata, Lactobacillus casei, Staphylococcus aureus e Streptococcus mutans, em comparação ao cloreto de cetilpiridínio (CCP). A seguir, investigou-se a citotoxicidade do MUPB em fibroblastos, comparando-o com o metil metacrilato (MMA). A avaliação da atividade antimicrobiana de diferentes concentrações do MUPB em massa (0, 0,3% e 0,6%) incorporado em uma resina acrílica termopolimerizável para base de próteses foi realizada por meio de testes de difusão em disco e quantificação de unidades formadoras de colônia (UFC) aderidas à resina após contato com suspensões de cada micro-organismo. A adesão microbiana também foi avaliada por microscopia eletrônica de varredura. Comparações entre o MUPB e as demais substâncias, bem como entre as concentrações incorporadas na resina, foram realizadas com &alpha;=0,05. O MUPB apresentou CIM inferior ao CCP para C. dublinienses e S. mutans (P=0,046 e 0,043, respectivamente). Para as demais espécies, as diferenças não foram significantes. Para a CFM/CBM, só foi encontrada diferença significante para C. albicans (P=0,046). O MUPB não polimerizado mostrou-se em torno de 20 vezes mais citotóxico que o MMA. Independente da concentração incorporada e da espécie, não houve formação de halo de inibição em torno dos espécimes. A incorporação do MUPB só influenciou na adesão de C. albicans (P=0,003), com menores contagens de UFC para o grupo com 0,6%. Conclui-se que o MUPB não polimerizado tem capacidade antimicrobiana próxima à do CCP, e alta citotoxicidade, se comparado ao MMA. A atividade antimicrobiana, após incorporação em uma resina acrílica para base de prótese, não depende de sua eluição, porém apresentou-se restrita à C. albicans. / Acrylic resins for removable denture base are capable of accumulating biofilm and thus favor the appearance of various problems in the edentulous oral cavity. An antiseptic agent immobilized within the polymeric matrix has a preventive potential against this accumulation, but requires further investigation. The aim of this study was to evaluate the biological properties of methacryloyloxyundecylpyridinium bromide (MUPB), an antiseptic monomer capable of copolymerizing with acrylic resins. The minimum inhibitory and fungicidal/bactericidal concentrations (MIC, MFC/MBC) of MUPB were determined against the species Candida albicans, Candida dubliniensis, Candida glabrata, Lactobacillus casei, Staphylococcus aureus and Streptococcus mutans, in comparison with cetylpyridinium chloride (CCP). After this, the cytotoxicity of MUPB was investigated in fibroblasts, compared with methyl methacrylate (MMA). The antimicrobial activity of different concentrations of MUPB (0, 0.3% and 0.6% w/w) incorporated into a heat polymerized denture base acrylic resin was evaluated by means of disk diffusion tests, and the CFUs adhered to the resin after contact with suspensions of each microorganism were quantified. Microbial adhesion was also evaluated by scanning electron microscopy. Comparisons were made between MUPB and the other substances, as well as between the concentrations incorporated into the resin (&alpha;=.05). MUPB presented a lower MIC than CCP for C. dubliniensis and S. mutans (P=.046 and .043, respectively). For the other species, the differences were not significant. For MFC or MBC, significant difference was found only for C. albicans (P=.046). Non polymerized MUPB was shown to be 20 times more cytotoxic than MMA. Irrespective of the concentration incorporated and the species, there was no growth of inhibition halo around the specimens. The incorporation of MUPB only influenced the adhesion of C. albicans (P=.003), with lower CFU counts for the group with the concentration of 0.6%. It was concluded that non polymerized MUPB has an antimicrobial capacity close to that of CCP, and high cytotoxicity when compared with MMA. The antimicrobial activity after incorporation within a denture base acrylic resin did not depend on its elution, but was shown to be restricted to C. albicans.

Neste trabalho, foi realizado um estudo crítico dos modelos metodológicos in vivo e in vitro sobre a avaliação microbiológica da clorexidina empregada como agente irrigante e medicação intracanal na terapia endodôntica. Para tanto, foi realizada uma busca sistemática nas bases de dados: Medline, Lilacs e BBO nos últimos dez anos sobre o assunto em questão. Foram selecionados os trabalhos que avaliaram a clorexidina de uma maneira não combinada com outras substâncias ou fármacos, abordando sua utilização tanto como irrigante endodôntico, e como medicação intracanal. Para uma melhor análise, os trabalhos foram agrupados de acordo com a forma de uso e metodologia com seus dados tabulados, respectivamente: Clorexidina como irrigante in vitro, Clorexidina como irrigante in vivo, Clorexidina como medicação intracanal in vitro e Clorexidina como medicação intracanal in vivo. Na análise, pode-se observar a efetividade antimicrobiana da clorexidina como irrigante nos ensaios laboratoriais e clínicos, sendo nas formas de gel e líquida com variação na concentração de 0.2 a 2%. Quanto o uso da clorexidina como medicação intracanal, pode ser constatada sua atividade antimicrobiana em ambos ambientes experimentais, embora com poucos trabalhos in vivo, seu desempenho foi observado apresentando largo espectro de ação quando utilizada a 2% em gel por um período de sete dias. / In this essay, it was realized a critical study of the methodological models, in vivo and in vitro, about the microbiological assessment of the chlorhexidine employed as root canal irrigants and dressing in the Endodontics. A systematic search was carried out on the databases: Medline, Lilacs and BBO from the last ten years concerning the respective subject. There were selected the papers which evaluated the chlorhexedine as an individual group, without joined to another substances or drugs, broaching its application as root canal irrigants as much as dressing. For improved analyzed, the investigations were divided according its way of use and methodology, with its data tabulated in the respective way: Chlorhexidine as irrigant in vitro, Chlorhexidine as irrigant in vivo, Chlorhexidine as intracanal dressing in vitro and Chlorhexidine as intracanal dressing in vivo. At the analysis, it can be observed the antimicrobial effectiveness of the chlorhexidine as irrigant in the clinical and laboratories tests, being the gel and liquid form with concentration changed from 0.2 to 2%. About the chlorhexidine as root canal dressing, it can be evidenced its antimicrobial activity in both experimental methods, although a few trials in vivo, its performance was observed showing a large spectral action when used in 2% gel for a period of seven days.

O objetivo deste estudo preliminar foi obter padrões metodológicos para viabilizar a utilização de um dispositivo acrílico intra-oral adaptável à mucosa palatina de ratos e passível de reembasamento com material resiliente temporário (Trusoft) modificado com antimicrobianos em suas mínimas concentrações inibitórias (MCIs) para biofilme de Candida albicans. Para delinear essa adequação, foram determinados parâmetros em relação à dieta e alojamento dos animais enquanto usuários desses dispositivos. A amostra total dos ratos (n=115) foi dividida em seis grupos: Controle Negativo (CN): sem dispositivo acrílico intra-oral; Controle Geral (CG): dispositivo acrílico sem reembasamento; Controle Positivo (CP): dispositivo reembasado com Trusoft sem fármaco; Diacetato de Clorexidina (CLO): dispositivo reembasado com Trusoft contendo clorexidina (0,064 g/mL); Cetoconazol (CET): dispositivo reembasado com Trusoft contendo cetoconazol (0,128 g/mL) e Nistatina (NIS): dispositivo reembasado com Trusoft contendo nistatina (0,032 g/mL). Os animais foram eutanasiados após 7 ou 14 dias da instalação dos dispositivos intraorais. As adaptações para a obtenção dos dispositivos incluíram a técnica de moldagem, método de reembasamento e formas de retenção à mucosa palatina dos animais. Para proporcionar a análise histopatológica descritiva padronizada, foram estabelecidos critérios em relação à obtenção das amostras histológicas, à seleção da região de interesse (RI) da análise e aos determinantes utilizados na descrição. Foi observado que a dieta pastosa e o alojamento em gaiolas com piso aramado sobre base de maravalha e papel craft possibilitaram melhores condições de sobrevivência aos animais. Os dispositivos intra-orais confeccionados individualmente e retidos via amarrilhos com fios ortodônticos nos incisivos e molares se mantiveram satisfatoriamente em posição durante todo o experimento para a maioria dos animais (72,6%), sobretudo no período inicial de 7 dias. O procedimento para o reembasamento dos dispositivos foi considerado apropriado em ambos os períodos testados. Houve perda de alguns animais por desnutrição (9,5%) ou durante a anestesia (4,1%). As amostras histológicas obtidas da mucosa palatina (tecido mole) foram descartadas por serem adequadas para a determinação da RI, sendo utilizadas apenas aquelas provenientes de tecidos mole e duro (n=12). A RI mais satisfatória para análise histopatológica correspondeu à área entre os primeiros molares, de um feixe neurovascular ao outro. A simples comparação visual das fotomicrografias obtidas na análise histopatológica descritiva sugeriu a ausência de infiltrado inflamatório em todos os grupos testados para ambos os períodos de avaliação. Após 7 dias, a espessura da camada córnea, estratificação das camadas do epitélio e disposição das fibras colágenas, dos vasos e das células da lâmina própria dos grupos de estudo se apresentaram similares ao observado para o grupo CN, havendo sinais de reabsorção óssea palatina apenas no grupo CG. Aos 14 dias, foi observado aumento da espessura de ortoqueratina e compressão das células epiteliais e do tecido conjuntivo da lâmina própria de todos os grupos em relação ao CN. Neste período, a reabsorção óssea palatina foi presente em todos os grupos, exceto CN e CP, com maior intensidade no grupo CG. Foi possível sugerir que os dispositivos acrílicos intra-orais podem ser considerados funcionais e viáveis para os testes de materiais para base de próteses e avaliação de tratamento para estomatite protética em ratos, sobretudo em períodos de até 7 dias. / The purpose of this study was to produce preliminary methodological standards to allow the use of an intraoral acrylic device adjusted to the palatal mucosa of rats and capable of relining with temporary resilient material (Trusoft) modified with antimicrobial agents in the minimum inhibitory concentrations (MICs) for Candida albicans biofilm. To design this adjustment, parameters were determined in relation to the diet and housing of animals as users of these devices. The total sample (n=115) was divided into six groups: Negative control (NC): without an acrylic intraoral device; Overall Control (OC): device without relining; Positive Control (PC): device relined with Trusoft without the addition of drugs; Chlorhexidine diacetate (CHL): device relined with Trusoft containing chlorhexidine diacetate (0.064 g/mL); Ketoconazole (KET): device relined with Trusoft containing ketoconazole (0.128g/mL); and Nystatin (NYT): acrylic device relined with Trusoft containing nystatin (0.032 g/mL). The animals were sacrificed after 7 or 14 days from the installation ofthe intra-oral devices. The adaptations for obtaining the devices included the impression technique, the reline method and the means of retaining in the palatal mucosa of animals. To obtain the standard descriptive histopathological analysis, criteria had been established in relation to obtaining the histological samples,selecting the region of interest (RI) of analysis and the criteria utilized in the description. It was observed that a paste diet and housing in cages with a wired fence floor with an additional base of wood shavings and craft paper enabled the best possible survival conditions for the animals. The individually made intra-oral devices retained with stainless steel wires at the incisors and molars remained satisfactorily in position throughout the experiment for most animals (72.6%), mainly in the initial period of 7 days. The procedure for the relining of the devices was deemed appropriate for both test periods. Some animals were lost from malnutrition (9.5%) or failure to recover from the anaesthetic (4.1%). The obtained histological samples of the palatine mucosa (soft tissue) were discarded as inappropriate for the determination of the RI, having utilized only those derived from hard and soft tissues (n = 12). The RI most satisfactory for the histopathological analysis corresponded to the area between the first molars from a palatal neurovascular bundle. A simple visual comparison of the photomicrographs obtained from the descriptive histopathological analysis suggested the absence of an inflammatory infiltrate in all the groups tested for both periods. After 7 days, the thickness of the keratin, the layers of the stratified epithelium and arrangement of the collagen fibers, cells and vessels of the lamina from the mucosa of the study groups presented similar findings to those observed for the NC group, having signals of palatine bone resorption only in the OC group. At 14 days, there was an increasing thickness of the keratin and compression of the epithelial cells and connective tissue of the lamina from the mucosa of all groups in relation to the NC group. In this period, the palatal bone resorption was present in all groups except the NC and PC groups, with a greater intensity in the OC group. It was possible to suggest that the intraoral acrylic devices may be considered as functional and viable for the testing of denture base materials and the evaluation of treatment for denture stomatitis in rats, especially in periods of up to 7 days.

A resina acrílica para base de próteses removíveis é capaz de acumular biofilme e assim favorecer o aparecimento de diversos problemas na cavidade bucal dos usuários de próteses. A imobilização de um agente antisséptico na matriz polimérica tem potencial preventivo frente a esse acúmulo, mas necessita de maior investigação. O objetivo deste estudo foi avaliar as propriedades biológicas do brometo de metacriloiloxiundecilpiridínio (MUPB), um composto antisséptico capaz de copolimerizar-se com as resinas acrílicas. Foram determinadas as concentrações inibitória e fungicida/bactericida mínimas (CIM, CFM/CBM) do MUPB frente às espécies Candida albicans, Candida dubliniensis, Candida glabrata, Lactobacillus casei, Staphylococcus aureus e Streptococcus mutans, em comparação ao cloreto de cetilpiridínio (CCP). A seguir, investigou-se a citotoxicidade do MUPB em fibroblastos, comparando-o com o metil metacrilato (MMA). A avaliação da atividade antimicrobiana de diferentes concentrações do MUPB em massa (0, 0,3% e 0,6%) incorporado em uma resina acrílica termopolimerizável para base de próteses foi realizada por meio de testes de difusão em disco e quantificação de unidades formadoras de colônia (UFC) aderidas à resina após contato com suspensões de cada micro-organismo. A adesão microbiana também foi avaliada por microscopia eletrônica de varredura. Comparações entre o MUPB e as demais substâncias, bem como entre as concentrações incorporadas na resina, foram realizadas com &alpha;=0,05. O MUPB apresentou CIM inferior ao CCP para C. dublinienses e S. mutans (P=0,046 e 0,043, respectivamente). Para as demais espécies, as diferenças não foram significantes. Para a CFM/CBM, só foi encontrada diferença significante para C. albicans (P=0,046). O MUPB não polimerizado mostrou-se em torno de 20 vezes mais citotóxico que o MMA. Independente da concentração incorporada e da espécie, não houve formação de halo de inibição em torno dos espécimes. A incorporação do MUPB só influenciou na adesão de C. albicans (P=0,003), com menores contagens de UFC para o grupo com 0,6%. Conclui-se que o MUPB não polimerizado tem capacidade antimicrobiana próxima à do CCP, e alta citotoxicidade, se comparado ao MMA. A atividade antimicrobiana, após incorporação em uma resina acrílica para base de prótese, não depende de sua eluição, porém apresentou-se restrita à C. albicans. / Acrylic resins for removable denture base are capable of accumulating biofilm and thus favor the appearance of various problems in the edentulous oral cavity. An antiseptic agent immobilized within the polymeric matrix has a preventive potential against this accumulation, but requires further investigation. The aim of this study was to evaluate the biological properties of methacryloyloxyundecylpyridinium bromide (MUPB), an antiseptic monomer capable of copolymerizing with acrylic resins. The minimum inhibitory and fungicidal/bactericidal concentrations (MIC, MFC/MBC) of MUPB were determined against the species Candida albicans, Candida dubliniensis, Candida glabrata, Lactobacillus casei, Staphylococcus aureus and Streptococcus mutans, in comparison with cetylpyridinium chloride (CCP). After this, the cytotoxicity of MUPB was investigated in fibroblasts, compared with methyl methacrylate (MMA). The antimicrobial activity of different concentrations of MUPB (0, 0.3% and 0.6% w/w) incorporated into a heat polymerized denture base acrylic resin was evaluated by means of disk diffusion tests, and the CFUs adhered to the resin after contact with suspensions of each microorganism were quantified. Microbial adhesion was also evaluated by scanning electron microscopy. Comparisons were made between MUPB and the other substances, as well as between the concentrations incorporated into the resin (&alpha;=.05). MUPB presented a lower MIC than CCP for C. dubliniensis and S. mutans (P=.046 and .043, respectively). For the other species, the differences were not significant. For MFC or MBC, significant difference was found only for C. albicans (P=.046). Non polymerized MUPB was shown to be 20 times more cytotoxic than MMA. Irrespective of the concentration incorporated and the species, there was no growth of inhibition halo around the specimens. The incorporation of MUPB only influenced the adhesion of C. albicans (P=.003), with lower CFU counts for the group with the concentration of 0.6%. It was concluded that non polymerized MUPB has an antimicrobial capacity close to that of CCP, and high cytotoxicity when compared with MMA. The antimicrobial activity after incorporation within a denture base acrylic resin did not depend on its elution, but was shown to be restricted to C. albicans.

病原體感染是包括植物的高等生物的主要健康危害之一。為抵禦入侵者，大多數植物會製造防禦蛋白，包括凝集素、蛋白酶抑製劑、抗真菌蛋白、核糖核酸酶和核糖體失活蛋白，並分佈在不同的器官，如葉、根、種子和塊莖。一些植物防禦蛋白被發現能表現出多種生物活性，如抗腫瘤活性、抗細菌活性和抗病毒活性，能抵抗多種植物病原菌和人類病原體。因此，一些植物防禦蛋白可能有潛力用於治療人類疾病，或保護農作物免受感染。 / 我們在研究中從不同的植物來源成功純化出各種防禦蛋白，包括：小芋頭塊莖中的血凝素、日本長芋中的凝集素、東北紅豆中的血凝素和抗真菌多肽、棕色芸豆中的凝集素、抗真菌多肽和胰蛋白酶抑製劑，玉豆一號中的凝集素以及小斑豆中的胰蛋白酶抑製劑。小芋頭血凝素被發現能誘導脾細胞的有絲分裂反應。日本長芋凝集素和東北紅豆血凝素被發現能對一些腫瘤細胞株（如乳腺癌MCF7細胞及鼻咽癌CNE2細胞）發揮抗增殖的作用。棕色芸豆凝集素能誘導脾臟細胞的有絲分裂反應以及抑制腫瘤細胞株（如乳腺癌MCF7細胞、肝癌HepG2及鼻咽癌CNE1和 CNE2細胞）的生長，而棕色芸豆抗真菌蛋白能抑制數種病原真菌物種的生長。研究這些防禦蛋白的生物活性有助找出其潛在應用價值，如藥用前景。 / Infection from pathogens is one of the major health hazards in higher organisms including plants. To defend against harmful invaders, most plants produce a variety of defense proteins including lectins, protease inhibitors, antifungal proteins, ribonucleases and ribosome-inactivating proteins. They may be present in different organs of the plants, such as leaves, roots, seeds and tubers. Some of the plant defense proteins were found to exhibit a variety of biological activities such as anti-tumor activity, anti-bacterial activity and anti-viral activity that act against various plant pathogens and also some human pathogens. Therefore, some plant defense proteins may have potential for therapeutic applications in human diseases, or protecting the crops from infections. / This study involved purification of defense proteins from different plant sources. The proteins that were successfully isolated included a hemagglutinin from small taro tubers, a lectin from Japanese yam tubers, a lectin and an antifungal peptide from northeast red beans, a lectin, an antifungal peptide and a trypsin inhibitor from brown kidney beans, a lectin from French bean cultivar no. 1 and a trypsin inhibitor from mini pinto beans. The small taro hemagglutinin was found to induce mitogenic response in splenocytes. The Japanese yam lectin and northeast red bean hemagglutinin were found to exert anti-proliferative activity toward some tumor cell lines including MCF7 and CNE2 cells. The brown kidney bean lectin induced a mitogenic response from murine splenocytes as well as inhibited the growth of tumor cell lines including MCF7, HepG2, CNE1 and CNE2 cells, while the brown kidney bean antifungal protein inhibited the growth of several pathogenic fungal species including M. arachidicola, S. turcica and B. maydis. Studying the biological activities of these defense proteins helps to find out their potential applications like therapeutic uses. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chan, Yau Sang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves i-xvii). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i-ii / 論文摘要 --- p.iii / Acknowledgements --- p.iv / List of Publications --- p.v / Table of Contents --- p.vi-vii / List of Figures --- p.viii-ix / List of Tables --- p.x / List of Abbreviations --- p.xi / Chapter Chapter 1 --- Introduction on plant defense proteins / Chapter 1.1 --- General introduction to plant defense proteins --- p.1-2 / Chapter 1.2 --- An overview on lectins --- p.3-18 / Chapter 1.2.1 --- History of lectins --- p.3-6 / Chapter 1.2.2 --- Classification of lectins --- p.7-11 / Chapter 1.2.3 --- Biological activities of lectins --- p.12-16 / Chapter 1.2.4 --- Applications of plant lectins --- p.16-18 / Chapter 1.3 --- An overview on defensins --- p.18-25 / Chapter 1.3.1 --- Types of defensins --- p.18-21 / Chapter 1.3.2 --- Mechanism of anti-microbial activity of defensins --- p.22-23 / Chapter 1.3.3 --- Application of defensins --- p.23-25 / Chapter 1.4 --- An overview on trypsin inhibitors --- p.25-38 / Chapter 1.4.1 --- Serpins --- p.26-28 / Chapter 1.4.2 --- Kunitz-type protease inhibitors --- p.29-31 / Chapter 1.4.3 --- Bowman-Birk protease inhibitors --- p.32-34 / Chapter 1.4.4 --- Physiological functions of protease inhibitors --- p.35-38 / Chapter 1.5 --- Aim of study --- p.38-41 / Chapter Chapter 2 --- Isolation and characterization of a hemagglutinin from small taros and a lectin from yam tubers / Chapter 2.1 --- Introduction --- p.42-45 / Chapter 2.2 --- Materials and Methods --- p.46-55 / Chapter 2.3 --- Results --- p.56-78 / Chapter 2.4 --- Discussion --- p.79-84 / Chapter Chapter 3 --- Isolation and characterization of two defense proteins from seeds of Phaseolus vulgaris cv. “northeast red bean“ / Chapter 3.1 --- Introduction --- p.85-86 / Chapter 3.2 --- Materials and Methods --- p.87-93 / Chapter 3.3 --- Results --- p.93-119 / Chapter 3.4 --- Discussion --- p.120-129 / Chapter Chapter 4 --- Isolation and characterization of three defense proteins from seeds of Phaseolus vulgaris cv. “brown kidney bean“ / Chapter 4.1 --- Introduction --- p.130-131 / Chapter 4.2 --- Materials and Methods --- p.131-136 / Chapter 4.3 --- Results --- p.136-175 / Chapter 4.4 --- Discussion --- p.176-189 / Chapter Chapter 5 --- Isolation and characterization of a lectin from French bean cultivar no. 1 beans and a trypsin inhibitor from mini pinto beans / Chapter 5.1 --- Introduction --- p.190-191 / Chapter 5.2 --- Materials and Methods --- p.191-194 / Chapter 5.3 --- Results --- p.195-212 / Chapter 5.4 --- Discussion --- p.213-221 / Chapter Chapter 6 --- General discussion / Chapter 6.1 --- Summary on purification protocols of the defense proteins in the study --- p.222-228 / Chapter 6.2 --- Chemical properties of the defense proteins in the study --- p.228-232 / Chapter 6.3 --- Biological activities of the defense proteins in the study --- p.232-238 / Chapter 6.4 --- Potential application of these defense proteins and future perspectives --- p.238-242 / References --- p.i-xvi

Reembasadores resilientes temporários contendo fármacos antifúngicos foram sugeridos como um tratamento adjunto para estomatite protética. No entanto, antes de utilizar clinicamente estes reembasadores modificados em humanos, é importante avaliar a sua biocompatibilidade em modelos animais. Este estudo avaliou a biocompatibilidade in vivo de um reembasador resiliente temporário para base de prótese (Trusoft) modificado por agentes antimicrobianos em suas mínimas concentrações inibitórias (MCIs) para biofilme de Candida albicans. Dispositivos acrílicos intra-orais (DIOs) foram confeccionados individualmente para 60 ratos Wistar. Os ratos foram divididos em 6 grupos (n=5): 3 grupos controle (Negativo: sem DIO; Geral: DIO sem reembasamento; Positivo: DIO reembasado com Trusoft sem fármacos) e 3 grupos experimentais (DIOs reembasados com Trusoft modificados por fármacos em suas respectivas MCIs: 0,032 g de nistatina, 0,064 g de diacetato de clorexidina e 0,128 g de cetoconazol). Os ratos com ou sem os DIOs foram eutanasiados após 7 e 14 dias de avaliação. A análise histopatológica qualitativa foi realizada comparando-se fotomicrografias de secções histológicas, que foram obtidas utilizando um microscópio óptico que abrangeu transversalmente a região intermolares. As alterações morfológicas no epitélio e queratina foram analisadas quantitativamente através da realização de planimetria computadorizada. Os dados quantitativos foram analisados utilizando ANOVA 2-fatores e teste de Tukey (=0,05). A análise quantitativa mostrou que apenas o grupo com DIO contendo cetoconazol diminuiu significativamente a espessura e a área do estrato córneo em comparação com os outros grupos (p<0,05), que não apresentaram diferenças significativas entre si (p>0,05). Estes resultados estiveram de acordo com os obtidos para análise qualitativa. A incorporação de MCIs de nistatina e diacetato de clorexidina no Trusoft não induziram alterações histopatológicas na mucosa palatina de ratos, sugerindo a biocompatibilidade in vivo deste protocolo para o tratamento de estomatite protética. / Temporary resilient denture liners containing antifungal drugs have been suggested as an adjunct treatment for denture stomatitis. However, before clinically using these modified liners in humans, it is important to assess their biocompatibility in animal models. This study evaluated the in vivo biocompatibility of a temporary soft denture liner (Trusoft) modified by antimicrobial agents at their minimum inhibitory concentrations (MICs) for biofilm formation by Candida albicans. Methods: Acrylic intraoral devices (IODs) were individually made for 60 Wistar rats. The rats were divided into the following 6 groups (n=5): 3 control groups (Negative: without IOD; General: IOD without relining; Positive: IOD relined with Trusoft without drugs) and 3 experimental groups (IOD relined with Trusoft modified by drugs at MICs: 0.032 g for nystatin, 0.064 g for chlorhexidine diacetate, and 0.128 g for ketoconazole). The rats with or without the IODs were sacrificed after 7 or 14 days of evaluation. Histopathological qualitative analysis was performed by comparing photomicrographs of histological sections, which were obtained using an optical microscope that transversely covered the inter-molar region. Morphological changes in the epithelium and keratin were quantitatively analyzed by performing computerized planimetry. Quantitative data were analyzed using 2-way ANOVA and Tukey\'s test (=0.05). Quantitative analysis showed that only the group with IOD containing ketoconazole significantly decreased the thickness and area of the stratum corneum compared with the other groups (p<0.05), which showed no significant differences between each other (p>0.05). These results were in accordance with those obtained for qualitative analysis. Incorporation of MICs of nystatin and chlorhexidine diacetate in Trusoft did not induce histopathological changes in the palatal mucosa of rats, suggesting the in vivo biocompatibility of this protocol for treating denture stomatitis.

Chong, Kam Lin. / Thesis submitted in: December 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 165-174). / Abstracts in English and Chinese. / ABSTRACT --- p.ii / ACKNOWLEDGEMENTS --- p.vii / TABLE OF CONTENTS --- p.viii / LIST OF ABBREVIATIONS --- p.xi / Chapter / Chapter 1. --- Introduction --- p.1 / Chapter 2. --- Literature review --- p.10 / Chapter 2.1 --- Prescribing patterns of antibiotics in hospital --- p.12 / Chapter 2.2 --- Worldwide problem of misuse of antibiotics --- p.15 / Chapter 2.2.1 --- Misuse of antibiotics in developed countries --- p.15 / Chapter 2.2.1.1 --- The United States of America --- p.16 / Chapter 2.2.1.2 --- The United Kingdom --- p.19 / Chapter 2.2.1.3 --- Australia --- p.23 / Chapter 2.2.1.4 --- Canada --- p.25 / Chapter 2.2.2 --- Misuse of antibiotics in African countries --- p.27 / Chapter 2.2.3 --- Misuse of antibiotics in Asian countries --- p.30 / Chapter 3. --- Objectives --- p.37 / Chapter 4. --- Methods --- p.39 / Chapter 4.1 --- Subjects --- p.39 / Chapter 4.2 --- Data --- p.41 / Chapter 4.3 --- Definition of terms --- p.44 / Chapter 4.3.1 --- Name of antibiotic --- p.44 / Chapter 4.3.2 --- Antibiotic course and antibiotic therapy --- p.44 / Chapter 4.3.3 --- Indications and types of antibiotic therapy --- p.47 / Chapter 4.3.4 --- Switch therapy --- p.50 / Chapter 4.3.5 --- Types of change --- p.50 / Chapter 4.3.6 --- Causes of change --- p.51 / Chapter 4.3.7 --- Clinical outcome of treatment --- p.53 / Chapter 4.3.8 --- Length of stay --- p.54 / Chapter 4.4 --- Determination of pattern of use --- p.54 / Chapter 4.5 --- Assessment of antibiotic therapies --- p.55 / Chapter 4.5.1 --- Assessment of indication and choice of agent --- p.59 / Chapter 4.5.2 --- Assessment of dosage --- p.60 / Chapter 4.5.3 --- Assessment of route of administration --- p.62 / Chapter 4.5.4 --- Assessment of duration of therapy --- p.62 / Chapter 4.6 --- Features of the guideline developed for the present study --- p.63 / Chapter 4.6.1 --- Ceftriaxone and cefotaxime are appropriate for treating serious infections --- p.72 / Chapter 4.6.2 --- Cefuroxime is not a first line agent --- p.72 / Chapter 4.6.3 --- Regimen for Helicobacter pylori eradication --- p.73 / Chapter 4.7 --- Statistical analysis --- p.73 / Chapter 5. --- Results --- p.74 / Chapter 5.1 --- Antibiotic courses and patients --- p.74 / Chapter 5.1.1 --- Inclusion and exclusion of antibiotic courses --- p.74 / Chapter 5.1.2 --- Patient sex and age --- p.76 / Chapter 5.1.3 --- Chronic disease/past medical history --- p.76 / Chapter 5.1.4 --- Length of stay --- p.78 / Chapter 5.2 --- Pattern of use --- p.79 / Chapter 5.2.1 --- Indications and sites of infection --- p.79 / Chapter 5.2.2 --- Types of antibiotic therapy --- p.83 / Chapter 5.2.3 --- Antibiotics prescribed in initial therapy --- p.84 / Chapter 5.2.4 --- Number of antibiotics in initial therapy --- p.87 / Chapter 5.2.5 --- Prescribing pattern of antibiotics in initial therapy --- p.88 / Chapter 5.2.5.1 --- Prescribing pattern of antibiotics in empirical treatment of lower respiratory tract infections --- p.89 / Chapter 5.2.5.2 --- Prescribing pattern of antibiotics in empirical treatment of sepsis --- p.90 / Chapter 5.2.6 --- Types of change --- p.92 / Chapter 5.2.7 --- Causes of change --- p.92 / Chapter 5.2.8 --- The relationship between causes of change and types of change --- p.93 / Chapter 5.2.9 --- Antibiotics prescribed in switch therapy --- p.96 / Chapter 5.2.10 --- Number of antibiotics in switch therapy --- p.99 / Chapter 5.3 --- Appropriateness of antibiotic therapy --- p.100 / Chapter 5.3.1 --- Appropriateness of empirical therapies --- p.101 / Chapter 5.3.2 --- Appropriateness of directed therapies --- p.102 / Chapter 5.3.3 --- Appropriateness of prophylactic therapies --- p.103 / Chapter 5.3.4 --- Appropriateness of unclassified therapies --- p.104 / Chapter 5.4 --- Clinical outcomes of treatment --- p.105 / Chapter 6. --- Discussion --- p.106 / Chapter 6.1 --- Limitations --- p.106 / Chapter 6.2 --- Method --- p.110 / Chapter 6.2.1 --- Symptom improved and not responded --- p.110 / Chapter 6.2.2 --- Grand round --- p.111 / Chapter 6.2.3 --- Susceptibility test result --- p.111 / Chapter 6.3 --- Results --- p.112 / Chapter 6.3.1 --- Patients --- p.112 / Chapter 6.3.2 --- Pattern of use --- p.113 / Chapter 6.3.2.1 --- Types of therapy --- p.113 / Chapter 6.3.2.2 --- Site of infection --- p.113 / Chapter 6.3.2.3 --- Prescribing pattern of antibiotics in initial therapy --- p.114 / Chapter 6.3.2.4 --- Relationship between types and causes of change --- p.115 / Chapter 6.3.3 --- Appropriateness of antibiotic therapies --- p.117 / Chapter 6.3.3.1 --- Misuse of empirical therapy --- p.118 / INAPPROPRIATE USE OF ANTIBIOTICS IN LOWER RESPIRATORY TRACT INFECTION --- p.118 / INAPPROPRIATE USE OF ANTIBIOTICS IN SEPSIS --- p.120 / MISUSE OF CEFUROXIME --- p.122 / MISUSE OF THIRD GENERATION CEPHALOSPORIN --- p.127 / Chapter 6.3.3.2 --- Appropriate use of directed therapy --- p.129 / Chapter 6.3.3.3 --- Appropriate use of prophylactic therapy --- p.130 / Chapter 6.3.3.4 --- Excessive use of unclassified therapy --- p.131 / Chapter 6.3.4 --- Clinical outcome of treatment --- p.133 / Chapter 6.4 --- Conclusion --- p.134 / Chapter 7. --- Summary --- p.136 / Chapter 8. --- Recommendations --- p.140 / APPENDICES --- p.143 / Chapter 1. --- Usual adult daily dose range of antibiotics --- p.144 / Chapter 2. --- Assessment of indications and choices of agent of initial therapies in an audit of 324 antibiotic courses in a medical ward --- p.146 / Chapter 3. --- Culture test results in an audit of use of antibiotics in a medical ward --- p.160 / Chapter 4. --- Generic to trade name conversion of antibiotics --- p.163 / REFERENCES --- p.165

Neste trabalho, foi realizado um estudo crítico dos modelos metodológicos in vivo e in vitro sobre a avaliação microbiológica da clorexidina empregada como agente irrigante e medicação intracanal na terapia endodôntica. Para tanto, foi realizada uma busca sistemática nas bases de dados: Medline, Lilacs e BBO nos últimos dez anos sobre o assunto em questão. Foram selecionados os trabalhos que avaliaram a clorexidina de uma maneira não combinada com outras substâncias ou fármacos, abordando sua utilização tanto como irrigante endodôntico, e como medicação intracanal. Para uma melhor análise, os trabalhos foram agrupados de acordo com a forma de uso e metodologia com seus dados tabulados, respectivamente: Clorexidina como irrigante in vitro, Clorexidina como irrigante in vivo, Clorexidina como medicação intracanal in vitro e Clorexidina como medicação intracanal in vivo. Na análise, pode-se observar a efetividade antimicrobiana da clorexidina como irrigante nos ensaios laboratoriais e clínicos, sendo nas formas de gel e líquida com variação na concentração de 0.2 a 2%. Quanto o uso da clorexidina como medicação intracanal, pode ser constatada sua atividade antimicrobiana em ambos ambientes experimentais, embora com poucos trabalhos in vivo, seu desempenho foi observado apresentando largo espectro de ação quando utilizada a 2% em gel por um período de sete dias. / In this essay, it was realized a critical study of the methodological models, in vivo and in vitro, about the microbiological assessment of the chlorhexidine employed as root canal irrigants and dressing in the Endodontics. A systematic search was carried out on the databases: Medline, Lilacs and BBO from the last ten years concerning the respective subject. There were selected the papers which evaluated the chlorhexedine as an individual group, without joined to another substances or drugs, broaching its application as root canal irrigants as much as dressing. For improved analyzed, the investigations were divided according its way of use and methodology, with its data tabulated in the respective way: Chlorhexidine as irrigant in vitro, Chlorhexidine as irrigant in vivo, Chlorhexidine as intracanal dressing in vitro and Chlorhexidine as intracanal dressing in vivo. At the analysis, it can be observed the antimicrobial effectiveness of the chlorhexidine as irrigant in the clinical and laboratories tests, being the gel and liquid form with concentration changed from 0.2 to 2%. About the chlorhexidine as root canal dressing, it can be evidenced its antimicrobial activity in both experimental methods, although a few trials in vivo, its performance was observed showing a large spectral action when used in 2% gel for a period of seven days.