Dissertations / Theses on the topic 'Porirua'

Submitted by Diogo Misoguti (diogo.misoguti@gmail.com) on 2016-06-30T13:02:59Z No. of bitstreams: 1 cp108652.pdf: 3211869 bytes, checksum: 99fdf86f3617cb57359337d87c540797 (MD5)
Approved for entry into archive by Diogo Misoguti (diogo.misoguti@gmail.com) on 2016-06-30T13:03:49Z (GMT) No. of bitstreams: 1 cp108652.pdf: 3211869 bytes, checksum: 99fdf86f3617cb57359337d87c540797 (MD5)
Made available in DSpace on 2016-06-30T13:03:49Z (GMT). No. of bitstreams: 1 cp108652.pdf: 3211869 bytes, checksum: 99fdf86f3617cb57359337d87c540797 (MD5) Previous issue date: 2009
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
FAPESP: 04/14434-3

Orientadores: Marcelo de Carvalho Ramos, Maria Cecilia Barisson
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-13T01:15:13Z (GMT). No. of bitstreams: 1 Milan_Arlete_M.pdf: 2380396 bytes, checksum: 8b3168431347ab625bac1135be8ebb1c (MD5) Previous issue date: 2007
Resumo: Introdução: A Pseudomonas aeruginosa é um importante agente de infecção nosocomial e motivo de grande preocupação por apresentar, com freqüência, resistência a diversos antimicrobianos. Os principais mecanismos de resistência encontrados são: inativação por enzimas, alterações na permeabilidade da membrana celular e promoção de efluxo. O conhecimento dos mecanismos de resistência que operam nesses microrganismos, bem como o estudo epidemiológico-molecular desses isolados, pode contribuir para a melhor compreensão e conseqüente controle dessas infecções no ambiente hospitalar. Objetivos: Determinar a Concentração Inibitória Mínima do Imipenem de Pseudomonas aeruginosa resistentes a esse antimicrobiano, isoladas de pacientes internados em hospital; Determinar a frequência de produção de metalo-beta-lactamases e da expressão da porina Opr D2 de Pseudomonas aeruginosa resistentes ao Imipenem, isoladas de pacientes internados em hospital; Genotipar e estudar a relação genética de Pseudomonas aeruginosa resistentes ao Imipenem, isoladas de pacientes internados em hospital. Materiais e Métodos: Foram estudadas 138 cepas isoladas de diversas amostras clínicas, identificadas bioquimicamente por técnicas de rotina e por automação. A triagem para a detecção de amostras produtoras de MBL's foi realizada pela fita de Etest combinada com EDTA. A extração e eletroforese de proteínas da parede celular foi executada para avaliar a expressão de porina Opr D2. Para a genotipagem foi utilizada a técnica de PFGE. Resultados e Discussões: Foram estudados 138 isolados, encaminhados como resistentes ao Imipenem. Em 128 desses isolados a resistência foi confirmada através do teste de difusão com disco e com a determinação do MIC (n = 98), através do Etest. A pesquisa da porina Opr D2 foi realizada em todos os isolados resistentes, ou seja, 128 casos. Em 68 deles não havia a expressão dessa proteína. Quando combinados os resultados da pesquisa de metalo-beta-lactamases e da deleção da porina Opr D2, encontramos 24 isolados que exibiam ambos os mecanismos de resistência. Da análise genotípica de 117 isolados, mostrou um acentuado polimorfismo, mesmo em isolados obtidos em um mesmo paciente. Não foram caracterizados surtos. Conclusões: A maioria das cepas de Pseudomonas aeruginosa resistentes ao Imipenem, isoladas em ambiente hospitalar, possui elevada resistência; A produção de meta-lobeta-lactamases constitui um mecanismo de resistência importante nesses isolados; A deleção da porina Opr D2 de membrana celular foi observada na maioria dos isolados. Em cerca de um terço deles havia também produção de metalo-beta-lactamase; A conglomeração desses isolados foi baixa, não caracterizando surtos em todos os casos; A variabilidade genotípica dos isolados foi intensa, mesmo em um determinado paciente, indicando que as ações relativas ao controle da disseminação desse patógeno deve ser de natureza rotineira, com as precauções de contato de universais e com o controle do uso de antimicrobianos
Abstract: Objective: Metallo-ß-lactamase production and Opr D2 channels expression was investigated in Imipenem resistant Pseudomonas aeruginosa recovered from hospital acquired infections. Design: Descriptive study in a convenient sample of organisms. Setting: Two 400-bed general teaching hospital, both tertiary care teaching hospitals, in Campinas, Brazil. All Imipenem resistant P. aeruginosa, recovered from March, 2000 through December 2004 from hospitalized patients were collected. Methods: Disk diffusion tests were used to confirm Imipenem resistance. Etest MBL was done to check for MBL production, and. Imipenem MIC's. Opr D2 expression was checked using cellular membrane proteins electrophoresis PFGE-genotyping was done in all isolates. Results: A sample of 128 Imipenem resistant Pseudomonas aeruginosa isolates was collected during the study period. Most isolates exhibited Imipenem MIC's = 256 µg / mL. Macrorestriction analysis (SpeI) using pulsed field gel electrophoresis (PFGE) showed a substantial polymorphism. Only 15 strains could be allocated to seven clusters, six with two isolates and one with three isolates. Ninety-nine Imipenem resistant isolates were screened for MBL production, and 87 were screened for both MBL, and porin Opr D2 expression. Sixty four isolates showed MBL production. Conclusion: Dissemination of MBL producing-genotypically heterogenous Pseudomonas aeruginosa strains was documented in the hospitals studied. Lack of Opr D2 combined with MBL production contributed to the high imipenem-resistance rates observed
Mestrado
Ciencias Basicas
Mestre em Clinica Medica

Au cours de ce travail, différents glucosylamines et aminodésoxyglucoses ont été synthétisés et caractérisés par différentes méthodes spectroscopiques dont l’IRTF, la RMN 1H, 13C et MALDI-Tof MS. L’étude des propriétés biologiques de ces molécules réalisée, d’une part, avec deux champignons du bois, Coriolus versicolor et Poria placenta, et d’autre part, avec trois microorganismes potentiellement rencontrés dans des aliments, Listeria innocua, Salmonella typhimurium et Fusarium proliferatum ont indiqué une contribution positive de la N-alkylation, du degré de N-substitution et de la quaternisation sur l’inhibition de leur croissance. Par ailleurs, l’impact sur la bioactivité, de la position du groupe amine sur le sucre, a été étudié. Il a été montré que la position du groupe amine sur le C-1 du glucose conduisait à une activité antifongique contre C. versicolor et P. placenta plus prononcée alors que la position C-3 du glucose était favorable à une activité antimicrobienne contre L. innocua et S. typhimurium
In this study different glucosylamines and amino desoxyglucoses were synthesized and characterised using various spectroscopic methods including IRFT, both 1H and 13C NMR spectroscopy and MALDI-Tof MS. Biological assessment of these compounds realised with two wood decay fungi, Coriolus versicolor and Poria placenta on one hand, and with three food microorganisms Listeria innocua, Salmonella typhimurium and Fusarium proliferatum on other hand, indicated a positive impact of both N-alkylation and degree of N-substitution and quaternisation on their growth inhibition. Furthermore, a biological impact of the amine position on sugar was studied. It was found that amine function attached to the C-1 of glucose conducted to the best antifungal activity against both C. versicolor and P. placenta while that fixed on the C-3 of glucose was indicated for antibacterial activity against L. innocua and S. typhimurium

Made available in DSpace on 2014-06-11T19:29:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2003-05-15Bitstream added on 2014-06-13T21:00:07Z : No. of bitstreams: 1 andrade_mcn_me_ilha.pdf: 261991 bytes, checksum: 006fec132b03beb5f46f5030a336fa9b (MD5)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A ocorrência de fungos contaminantes e competidores é comum durante o cultivo de shiitake em toros. No Brasil, poucos são os trabalhos que identificam e analisam os efeitos destes microrganismos na produção de shiitake. No entanto, sabe-se que a incidência destes fungos em grandes proporções pode levar à improdutividade dos toros. Portanto, o presente trabalho teve como objetivo testar o efeito da cal hidratada e do fungicida benomyl no controle de fungos contaminantes e sua resposta na produção de shiitake em toros. Para tanto, testou-se anteriormente in vitro o efeito do benomyl nas concentrações 0,5; 1,0; 2,0; 4,0; 8,0 e 16,0 mg/mL no crescimento micelial das linhagens de shiitake JAB-L; JAB-K; LE-96/17; LE-95/01 e LE-96/22, de modo a selecionar a linhagem mais tolerante as concentrações de benomyl propostas, comparando com o crescimento das mesmas sem a presença deste fungicida (testemunha). O delineamento deste experimento foi inteiramente casualizado em fatorial 5X7, tendo no total trinta e cinco tratamentos, cada qual com três repetições, sendo cada repetição correspondente a uma placa de Petri. Observou-se que a única linhagem de shiitake a não sofrer qualquer efeito do benomyl nas concentrações propostas foi a LE-96/17, sendo a escolhida para ser utilizada no experimento em toros, a qual foi submetida a maior concentração de benomyl (16,0 mg/mL). O delineamento do experimento em toros foi inteiramente casualizado, com três tratamentos: testemunha; cal (8 kg de cal/ 60 litros de água) aplicados nos toros de produção logo após a inoculação e após cada choque de indução; e benomyl (16,0 mg/mL) aplicados quinzenalmente a partir da inoculação dos toros. Cada tratamento conteve 60 repetições, sendo a unidade experimental correspondente a um toro. Os toros utilizados foram de Eucalyptus urophylla, os quais foram inoculados... .

Orientador: Luiz Antônio Graciolli
Banca: Luzia Doretto Paccola Meirelles
Banca: Marli de Fátima Stradioto Papa
Abstract: The occurrence of contaminants fungus and competitors are common during the shiitake cultivation in logs. In Brazil, few are the works that identify and they analyze the effects of these microorganisms in the shiitake production. However, it is known that the incidence of these fungus in great proportions can take to the unproductiveness the logs. Therefore, the present work had as objective to test the effect of the moisturized whitewash and of the fungicide benomyl in the control of contaminant fungus and his answer in the shiitake production in logs. For so much, it was tested in vitro previously the effect of the benomyl in different concentrations (0.5; 1.0; 2.0; 4.0; 8.0 and 16.0 mg/mL) in the mycelial growth of five shiitake lineages (JAB-L; JAB-K; LE-96/17; LE-95/01 and, LE-96/22), in way to select the most tolerant lineage to the concentrations of proposed benomyl, comparing with the growth of the same ones without the presence of this fungicide (control). A completely randomized design in a factorial scheme 5X7, tends in the total 35 treatments, each one with three repetitions, corresponding to a plate of Petri. It was observed that only shiitake lineage did not suffer any effect of the benomyl in the proposed concentrations was the LE-96/17, being the chosen to be used in the experiment in logs, when the largest benomyl concentration was submitted (16.0 mg/mL). The other study was conducted in logs in an interely randomized design, with three treatments: control; whitewash (8 kg of whitewash / 60 liters of water) applied in the production logs soon after the inoculation and after each induction shock; and benomyl (16.0 mg/mL) applied biweekly starting from the inoculation of the logs. Each treatment contained 60 repetitions, being the experimental unit corresponding to a log. The used logs were of Eucalyptus urophylla, which were... (Complete abstract, click electronic address below).
Resumo: A ocorrência de fungos contaminantes e competidores é comum durante o cultivo de shiitake em toros. No Brasil, poucos são os trabalhos que identificam e analisam os efeitos destes microrganismos na produção de shiitake. No entanto, sabe-se que a incidência destes fungos em grandes proporções pode levar à improdutividade dos toros. Portanto, o presente trabalho teve como objetivo testar o efeito da cal hidratada e do fungicida benomyl no controle de fungos contaminantes e sua resposta na produção de shiitake em toros. Para tanto, testou-se anteriormente in vitro o efeito do benomyl nas concentrações 0,5; 1,0; 2,0; 4,0; 8,0 e 16,0 mg/mL no crescimento micelial das linhagens de shiitake JAB-L; JAB-K; LE-96/17; LE-95/01 e LE-96/22, de modo a selecionar a linhagem mais tolerante as concentrações de benomyl propostas, comparando com o crescimento das mesmas sem a presença deste fungicida (testemunha). O delineamento deste experimento foi inteiramente casualizado em fatorial 5X7, tendo no total trinta e cinco tratamentos, cada qual com três repetições, sendo cada repetição correspondente a uma placa de Petri. Observou-se que a única linhagem de shiitake a não sofrer qualquer efeito do benomyl nas concentrações propostas foi a LE-96/17, sendo a escolhida para ser utilizada no experimento em toros, a qual foi submetida a maior concentração de benomyl (16,0 mg/mL). O delineamento do experimento em toros foi inteiramente casualizado, com três tratamentos: testemunha; cal (8 kg de cal/ 60 litros de água) aplicados nos toros de produção logo após a inoculação e após cada choque de indução; e benomyl (16,0 mg/mL) aplicados quinzenalmente a partir da inoculação dos toros. Cada tratamento conteve 60 repetições, sendo a unidade experimental correspondente a um toro. Os toros utilizados foram de Eucalyptus urophylla, os quais foram inoculados... (Resumo completo, clicar acesso eletrônico abaixo).
Mestre

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-04T14:54:05Z No. of bitstreams: 1 vanessacordeirodias.pdf: 2034556 bytes, checksum: 89059620f1b31c4177f6ae487a15c672 (MD5)
Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-07T15:33:46Z (GMT) No. of bitstreams: 1 vanessacordeirodias.pdf: 2034556 bytes, checksum: 89059620f1b31c4177f6ae487a15c672 (MD5)
Made available in DSpace on 2016-06-07T15:33:46Z (GMT). No. of bitstreams: 1 vanessacordeirodias.pdf: 2034556 bytes, checksum: 89059620f1b31c4177f6ae487a15c672 (MD5) Previous issue date: 2015-12-18
FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais
As bactérias Gram-negativas não fermentadoras, como Pseudomonas aeruginosa e Acinetobacter baumannii, estão amplamente disseminadas no ambiente e estão cada vez mais associados a infecções nosocomiais graves. O uso extensivo e indiscriminado de antibióticos tem contribuído para um aumento do número de infecções causadas por A. baumannii e P. aeruginosa resistentes a uma grande variedade de agentes antimicrobianos, incluindo β-lactâmicos. O objetivo deste trabalho foi avaliar características fisiológicas e moleculares da resistência aos carbapenêmicos em P. aeruginosa e A. baumannii isolados em um hospital terciário. A partir de estudos com amostras clínicas de pacientes admitidos num hospital terciário foram isolados, em 2012, A. baumannii (n=44) e P. aeruginosa (n=28) resistentes aos carbapenêmicos e em 2013, P. aeruginosa (n=19) e A. baumannii (n=44) com igual fenótipo. As amostras bacterianas recuperadas em 2012 foram submetidas a testes de susceptibilidade aos antimicrobianos. Marcadores genéticos relacionados com a síntese de β-lactamases blaOXA-23, blaOXA-24, blaOXA-51, blaOXA-58 e blaOXA-143 foram testados por PCR. A partir das linhagens isoladas no estudo de 2013, testes fisiológicos foram realizados para avaliar a atividade hemolítica, estresse oxidativo, tolerância aos biocidas, formação de biofilme e determinação da resistência aos antimicrobianos. Marcadores genéticos relacionados com a síntese de β-lactamases (ampC, blaKPC, blaSIM, blaIMP, blaSPM-1, blaVIM, blaGIM, blaOXA e blaNDM-1), sistemas de efluxo (adeB, mexB, mexD, mexF e mexY) e perda de porina (oprD) foram pesquisados por PCR. Foram analisados dados epidemiológicos de todos os pacientes avaliados. No estudo de 2012, a polimixina B foi a única droga eficaz para todos os isolados. Os marcadores genéticos foram observados apenas em isolados de Acinetobacter. O genótipo mais frequente observado foi blaOXA-23+/blaOXA-51+ (45,5%), seguido por blaOXA-51+/blaOXA-143+ (41%). Os genes blaOXA-24 e blaOXA-58 não foram detectados. Uma elevada taxa de mortalidade foi observada (> 70%) entre os pacientes. No estudo de 2013, idade avançada, predomínio de indivíduos internados em UTI, uso de dispositivos médicos invasivos, como cateter venoso, tratamento anterior com fluoroquinolonas ou ß-lactâmicos em combinação com um inibidor da β-lactamase e estadia prolongada no hospital foram fatores predisponentes para infecção por estes microrganismos. Colistina demonstrou atividade, in vitro, contra todas as amostras bacterianas avaliadas. Tigeciclina foi também efetiva para linhagens de A. baumannii. P. aeruginosa resistente aos carbapenêmicos não foi capaz de produzir hemolisinas. Essas linhagens foram menos tolerantes ao estresse oxidativo e alguns biocidas, mas mostraram um aumento da capacidade de formação de biofilme em relação aos isolados sensíveis. Os principais mecanismos de resistência presentes em linhagens de A. baumannii resistentes aos carbapenêmicos foi síntese de oxacilinases (OXA-23, OXA-51 e OXA-143), ausência de oprD e presença de bomba de efluxo (AdeABC). Em P. aeruginosa foram encontrados genes para bombas de efluxo (MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM), blaSPM-1, além de ausência de oprD. Estes resultados confirmam a dificuldade de manejo terapêutico de pacientes com infecções associadas a microrganismos multirresistentes, com impacto direto na mortalidade e controle epidemiológico dessas linhagens nos centros de saúde.
The non-fermenting Gram-negative bacteria, such as Pseudomonas aeruginosa and Acinetobacter baumannii are widespread in the environment and are increasingly associated with severe nosocomial infections. Extensive and indiscriminate use of antibiotics has contributed to an increased number of infections caused by A. baumannii and P. aeruginosa that are resistant to a wide variety of antimicrobials, including β-lactams. This study aimed to evaluate physiological and molecular characteristics of carbapenem resistance in P. aeruginosa and A. baumannii. From studies with clinical samples from patients admitted to a tertiary hospital, were isolated in 2012 A. baumannii (n=44) and P. aeruginosa (n=28) resistant to carbapenems and in 2013, P. aeruginosa (n=19) and A. baumannii (n=44) with similar phenotype. The bacterial samples recovered in 2012 were submitted to antibiotic susceptibility testing. Genetic markers related to β-lactamases synthesis blaOXA-23, blaOXA-24, blaOXA-51, blaOXA-58 and blaOXA-143 were screened by PCR. From strains recovered in the 2013 study, physiological tests were performed to evaluate hemolytic activity, oxidative stress, biocides tolerance and biofilm formation, besides determination of antimicrobial resistance patterns. Genetic markers related to β-lactamases synthesis (ampC, blaKPC, blaSIM, blaIMP, blaSPM-1, blaVIM, blaGIM, blaOXA and blaNDM-1), efflux systems (adeB, mexB, mexD, mexF and mexY) and loss of porin (oprD) were screened by PCR. Epidemiological data about all of these patients were analyzed. In the 2012 study, polymyxin B was the only effective drug for all isolates. Genetic markers were observed only in Acinetobacter isolates. The most frequent genotype observed was blaOXA-23+/blaOXA-51+ (45,5%), followed by blaOXA-51+/blaOXA-143+ (41%). The genes blaOXA-24 and blaOXA-58 were not detected. High mortality rate (> 70%) between the pacients was observed. In a prospective study, advanced age, predominance of individuals hospitalized in ICU, use of invasive medical devices, such as venous catheter, previous treatment with fluoroquinolones or β-lactams in combination with β-lactamase inhibitor and prolonged stay in hospital were predisposing factors for infection by these microorganisms. Colistin has shown activity, in vitro, against all assessed bacterial samples. Tigecycline was also effective for strains of A. baumannii. Carbapenem-resistant P. aeruginosa was not able to produce hemolysin. These strains were less tolerant to oxidative stress and some biocides, but they showed increased ability of biofilm formation in relation to susceptible isolates. The major mechanisms of carbapenems resistance present in A. baumannii strains was oxacilinases synthesis (OXA-23, OXA-51 and OXA-143), oprD absence and efflux pump presence (AdeABC). In P. aeruginosa was found genes encoding efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM) and blaSPM-1; besides oprD absence. These results confirm the difficulty of therapeutic management of patients with infections associated with multidrug resistant microorganisms, with direct impact on mortality and epidemiological control of multidrug-resistant strains in health centers.

P. aeruginosa e espécies de Acinetobacter são causas comuns de diversas infecções em pacientes hospitalizados, principalmente nos internados em centros de tratamento intensivo. Além disso, esses microrganismos se destacam por apresentarem resistência, intrínseca e adquirida, a várias classes de antibióticos, conferindo à bactéria fenótipos de multirresistência e panresistência. O objetivo deste estudo foi avaliar a participação de integrons (elementos genéticos que carreiam genes de resistência), de genes codificadores de metalo--lactamases, da perda de porinas (canais protéicos da membrana externa), e da atividade de efluxo aumentada, como determinantes do fenótipo de multirresistência e panresistência. Foram estudadas 147 P. aeruginosa e 57 Acinetobacter spp. isolados de pacientes hospitalizados no Hospital Universitário da Universidade Federal de Juiz de Fora, no período de 2003 a 2006. O perfil de sensibilidade destes isolados foi determinado por disco de difusão e utilizado para classificá-las como multirresistentes (MDR) e não multirresistentes (n-MDR). A variabilidade clonal dos isolados foi investigada por PFGE. Os isolados pertencentes aos grupos MDR e n-MDR foram investigados quanto a presença de integrons de classe 1, 2 e 3, por PCR e análise de RFLP. Os cassetes gênicos contidos nestes integrons, assim como genes codificadores de carbapenemases (ex. IMP, VIM e SPM), foram detectados por PCR e identificados por seqüenciamento. Avaliação da expressão gênica de bombas de efluxo (mexB, mexY, mexD e adeB) e de porina (OprD) foi conduzida por real-time RT-PCR. Os dados apresentados para os isolados do grupo MDR foram comparados àqueles do grupo n-MDR e a associação entre os determinantes de resistência e o fenótipo MDR foi calculada estatisticamente. Fenótipo de multiresistência foi observado em 42,2% e 84,2% das P. aeruginosa e Acinetobacter spp. estudadas. Nenhum isolado bacteriano apresentou fenótipo panresistente. Em 65 (44,2%) dos isolados de P. aeruginosa, foram detectados integrons de classe 1. Esses elementos apresentaram relação estatisticamente significativa com fenótipos MDR em P. aeruginosa. Entretanto, a maioria desses integrons não carreava nenhum cassete gênico (43/65) ou continham apenas cassetes gênicos de resistência a aminoglicosídeos (19/65). Entre os isolados de Acinetobacter spp., 11 (17,5%) apresentaram integrons de classe 1 e 30 (47,6%) integrons de classe 2. Apenas os últimos foram estatisticamente associados com fenótipos MDR. A pesquisa de metalo--lactamase (MBL) revelou a produção de enzimas SPM em 24 isolados de P. aeruginosa. Os estudos de expresão gênica demonstraram que, entre os sistemas de efluxo mais relatados para P. aeruginosa, MexXY-OprM foi o que mostrou maior diferença entre o nível de expressão dos grupos MDR e n-MDR, sugerindo que este sistema de efluxo desempenha importante papel no fenótipo MDR. Diminuição, em média de 66,4%, da produçãode OprD também foi um padrão encontrado nos isolados MDRem relação aos n-MDR. Dois grupos clonais de P. aeruginosa e dois de Acinetobacter spp. foram predominantes e tiveram relação com presença de integrons, produção de SPM-1 e com fenótipo MDR. Portanto, esse fenótipo pode ser consequência de acúmulo de determinantes de resistência em clones específicos.
The non-fermenting pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter spp. are important causes of nosocomial infections. Theses species are often associated with a multidrug resistance (MDR) phenotype, due to intrinsic and acquired resistance genes. Some determinants of resistance, such as integrons, carbapenemases, overexpression of efflux systems and porins loss may be associated with the MDR phenotype. The aim of this study was to evaluate the association of non-MDR and MDR phenotypes in P. aeruginosa and Acinetobacter spp. to the presence of integrons and carbapenemases encoding genes, the overexpression of mexY, mexB, mexD and adeB genes and loss of the outer membrane protein, OprD. These resistance determinants were evaluated in 147 P. aeruginosa and 57 Acinetobacter spp., isolated from in-patients of University Hospital of UFJF. Isolates with different PFGE and non-susceptibility profiles were grouped according to MDR or non-MDR phenotypes. PCR and real-time RT-PCR were used to investigate the presence of class 1, 2 and 3 integrons and carbapenemase encoding genes and the expression of mexY, mexB, mexD and adeB efflux pumps and OprD porin, respectively. Class 1 integrons were one of the most common genetic elements present in MDR P. aeruginosa (44,2%), but the phenotype could not be attributed to these elements, since they showed empty (43/65) or only aminoglycoside gene cassettes (19/65). Class 2 integrons were the most common genetic elements in MDR Acinetobacter spp., and this association was statistically significant. SPM encoding gene was the only carbapenemase gene found in P. aeruginosa and, predominantly, in the PFGE cluster A. Expression of MexXY-OprM determined by real-time RT-PCR was the highest variable between MDR and non-MDR P. aeruginosa isolates (almost 10-fold). Reduction of 66.4% in OprD expression was observed in MDR P. aeruginosa, in comparison with non-MDR ones. It is concluded that the most important genetic determinants in the MDR phenotype of P. aeruginosa were SPM-1 production, followed by MexXY-OprM over expression and diminished production of OprD, while class 2 integrons was the most important genetic determinant of MDR phenotype in A. baumannii.

Os micro-organismos podem infectar seu hospedeiro por diferentes vias, sendo a principal o trato respiratório. O reconhecimento pela mucosa dessas vias pode desencadear inibição da proliferação e bloqueio da entrada microbiana, assim como estimular resposta direcionada a memória imunológica para prevenir posteriores infecções. Alguns micro-organismo, como as bactérias Neisseria meningitidis e Neisseria lactamica, são capazes de modular a resposta imune de mucosa diretamente, ou por meio das células epiteliais respiratórias. Este trabalho propôs, então, a avaliação das porinas B provenientes destas bactérias como moduladoras da produção de IL-8 nas linhagens BEAS-2B e Detroit 562. Também foi avaliada a dependência deste estímulo ao receptor TLR2. Ambas as porinas se ligaram a TLR2 e por este receptor estimularam a produção de IL-8. O perfil de produção foi dependente da expressão de TLR2 pelas células. A porina lactâmica induziu menos IL-8 por regular negativamente a expressão de TLR2, mas sua afinidade pelo receptor se mostrou maior que a da porina meningocócica. As porinas são então moduladoras das células de mucosa, fato que somado a atividade adjuvante destas proteínas por via parenteral estimulou a avaliação destas como adjuvantes de mucosa. O modelo escolhido para a avaliação foi o de inoculação intranasal de camundongos, utilizando como antígeno o lipopolissacarídio pouco imunogênico de Franciscella tularensis atenuada (Ft-LPS). A análise foi baseada no título de anticorpos IgG e IgM séricos. A porina meningocócica se mostrou a mais imunogênica, mas por ser originária de patógeno acarreta maior risco biológico em sua produção. Para viabilizar a porina meningocócica como adjuvante, a mesma foi substituída por porina homóloga produzida de modo recombinante em Escherichia coli não patogênica. A porina recombinante foi avaliada pelo mesmo sistema in vivo e comparada a adjuvantes experimentais de ação conhecida (rCTB, QS-21 e ODN 1826). A porina apresentou o melhor desempenho entre todos os adjuvantes, principalmente dois meses após o fim do esquema vacinal. O mesmo adjuvante foi adicionado ao vírus da raiva para caracterizar a amplitude de antígenos para sua aplicação e o efeito biológico dos anticorpos induzidos. Os resultados obtidos por via intranasal com antígeno da raiva confirmaram a propriedade de adjuvante de mucosa da porina recombinante, aumentando os títulos de IgG séricos. O ensaio biológico dos anticorpos por RFFIT comprovaram a funcionalidade dos anticorpos gerados, neutralizando a infectividade viral em células BHK-21. O uso da porina por via subcutânea não aumentou o nível de anticorpos neutralizantes, mas aumentou o de IgG. Não foi detectada imunidade celular específica de linfócitos do baço ao vírus da raiva nos parâmetros avaliados, independente da adição de adjuvantes. Em resumo, as porinas foram caracterizadas como relevantes na imunomodulação de células da mucosa respiratória por infecção meningocócica. A modulação também foi relevante para o aumento de resposta humoral frente a diferentes antígenos, por diferentes vias de administração, o que demonstra a eficiência e versatilidade da porina recombinante como adjuvante imunológico.
Microorganisms can invade the host through many routes, specially the respiratory tract. The respiratory mucosa is responsible for recognition, inhibition, proliferation and entry blockade of microorganisms, besides incitation of immunological memory to prevent further infections. Some microorganisms, such as Neisseria meningitidis and Neisseria lactamica, can modulate the mucosa immune response directly or through stimulation of respiratory epithelial cells. The present work proposed the evaluation of porin B proteins, derived from these microorganisms, as modulators of IL-8 production on respiratory epithelial cell strains BEAS-2B and Detroit 562. TLR2 receptor dependency for the modulation was also evaluated. Both porins bounded to TLR2 and through this receptor were able to stimulate IL-8 production, whereas this profile was correlated with TLR2 expression. Lactamica porin (Nlac PorB) induced less IL-8 and TLR2 expression, also for a shorter period of time. The effect caused by Nlac PorB was attributed to TLR2 down regulated expression, since its binding affinity to the receptor is greater than meningococcal porin (Nmen PorB). Porins were therefore able to immune modulate mucosal cells, fact that allied with their parenteral adjuvant activity incited evaluation of porins as potential mucosal adjuvants. The model chosen for the evaluation was intranasal immunization of mice, using as the antigen a low immunogenic lipopolysaccharide extracted from attenuated Franciscella tularensis (Ft-LPS). The evaluation was based on IgG and IgM serum titers. After the immunization scheme, Nmen PorB induced higher IgG and IgM titers than Nlac PorB. Although Nmen PorB was more efficient, it comes from a pathogen. To overcome the risk of its production, it was replaced by recombinant porin (rPorB) produced by Escherichia coli. rPorB was evaluated by the same model and compared with well known experimental adjuvants (rCTB, QS-21 e ODN 1826). rPoB had the highest IgM and IgG titers among all adjuvants tested, specially two months after vaccination. The same adjuvant was also combined with a viral antigen to characterize its application wideness and biological function of incited antibodies. Results obtained with rabies antigen by intranasal route confirmed the mucosal adjuvant properties of rPorB, increasing IgG titers induced by the antigen. These antibodies were also capable of virus neutralization, as demonstrated in RFFIT assays. rPoB didn´t raise neutralizing antibody titers by subcutaneous route, but increased IgG titers. Cellular immunity was undetectable in spleen lymphocytes with the screening method used, regardless of adjuvant addition. In conclusion, porins were characterized as revelant for immunomodulation of the respiratory mucosal cells, caused by infection with meningococcus. The immunomodulation was also revelant for increase of humoral reponse to different antigens and by different routes, pointing out recombinant porin B as an efficient and versatile immunological adjuvant.

Introdução: Isolados clínicos de Pseudomonas aeruginosa multirresistentes estão associados a elevadas taxas de mortalidade. A resistência ao imipenem é uma urgência global, uma vez que é considerado o tratamento de escolha para infecções associadas a bactérias Gram negativas multirresistentes. Assim, elucidar os mecanismos de resistência é de vital importância para realizar um controle epidemiológico efetivo da disseminação deste tipo de isolado. Objetivos: Caracterizar os principais mecanismos de resistência ao imipenem em 76 isolados clínicos brasileiros de Pseudomonas aeruginosa, recuperados em 2004/2007, de 4 centros hospitalares do Estado de São Paulo. Material e métodos: Foram investigados: i) o perfil de resistência com determinação da CIM do imipenem; ii) a detecção de metalo-betalactamases (MBL) através de métodos fenotípicos e genotípicos; iii) a sensibilidade e especificidade do método de dupla difusão do disco na detecção de MBL; iv) a presença de genes codificadores de metilases 16S RNAr e sua associação com fenótipos aminoglicosídeo resistentes; v) alterações da permeabilidade por perda da porina OprD; vi) a presença ou ausência do gene oprD por PCR; vii) triagem fenotípica para expressão de bombas de efluxo através da determinação da CIM de quinolonas, cefalosporinas e carbapenêmicos na presença/ausência de inibidores específicos, realizando uma análise comparativa com o método de disco combinado; viii) os genes associados às bombas de efluxo mexA e mexE, através de PCR; ix) caracterizar a expressão das bombas de efluxo MexAB-OprM e MexEF-OprN, x) a clonalidade dos isolados por tipagem genotípica, através de ERIC-PCR, avaliando a relação genética (dendrograma) e sua associação com o predomínio de um determinado mecanismo de resistência. Resultados: Dentre os isolados de P. aeruginosa resistentes ao imipenem estudados (n=76, CIM50 e CIM90 = 32 µg/mL e > 512 µg/mL, respectivamente) 82% apresentaram um fenótipo de multirresistência. O principal mecanismo de resistência ao imipenem foi a produção de MBL, detectada em 74% dos isolados, e destes, 62% carregavam o gene blaSPM-1 e 12% carregavam o gene blaVIM-like. O método de dupla difusão do disco identificou a produção de MBL em 61% dos isolados. A combinação CAZ/MAA apresentou maior sensibilidade na detecção de MBL associada à SPM-1 (89%), mostrando uma especificidade de 86%. A presença do gene rmtD foi confirmada em 66% das amostras resistentes aos aminoglicosídeos, sendo que a presença concomitante do gene rmtD e do gene blaSPM-1 foi confirmada em 61% dos isolados. A deleção da porina OprD foi observada em 71% dos isolados. Dentre os isolados MBL positivos, 66% apresentaram ausência desta porina e, dentre as amostras MBL negativas, 85% não apresentaram OprD. Assim, para a resistência ao imipenem foi confirmada a contribuição de dois mecanismos, mediados pela presença de MBL e ausência de porina OprD. Em 13% (10/76) isolados, a deleção da porina OprD esteve associada à presença de seqüências de inserção (SI) em uma região anterior ao gene oprD. Por outro lado, a ausência de amplificação da região 736/1394 do gene oprD, em 11% (9/76) dos isolados, sugeriu a presença de polimorfismos. O gene mexA esteve presente em 92% dos isolados, enquanto que o gene mexE esteve presente em 82% dos isolados. A triagem de bombas de efluxo por disco combinado e análise da CIM na presença de reserpina, CCCP e PAβN, utilizando levofloxacina, meropenem, aztreonam, imipenem ou levofloxacina, não teve correlação com a superexpressão dos sistemas MexAB-OprM e MexEF-OprN. Ambos os métodos careceram de especificidade e sensibilidade quando comparados ao PCR em tempo real. A superexpressão dos sistemas mexA e mexE foi confirmada em 35% (7/20) isolados MBL negativos, enquanto que 11% (6/56) isolados MBL positivos apresentaram superexpressão do gene mexA ou mexE, sendo que 7% (4/56) isolados MBL positivos superexpressaram ambos os genes. A superexpressão dos sistemas MexAB-OprM e MexEFOprN como único mecanismo de resistência ao meropenem e imipenem foi confirmada em 10% (2/20) dos isolados MBL negativos. Nos 76 isolados, a tipagem genotípica por ERIC-PCR, identificou a presença de 24 clusters (considerando 90% de similaridade na análise do dendrograma). Conclusão: A convergência de múltiplos mecanismos de resistência em P. aeruginosa parece ser um evento favorável para a seleção de clones endêmicos multirresistentes disseminados na região Sudeste do Brasil.
Introduction: Clinical isolates of Pseudomonas aeruginosa are associated with high mortality rates. Resistance to imipenem is a global concern, since it is a drug of choice for the treatment of infections produced by multidrug-resistant Gram-negative bacteria. Thus, research on resistance mechanisms is crucial to carry out an effective program for infection control and epidemiology of imipenem-resistant strains. Objective: to characterize the major mechanisms of imipenem resistance in 76 clinical isolates of P. aeruginosa recovered from clinical samples collected, from 2004 to 2007, in four hospitals in the State of São Paulo, Brazil. Material and methods: Isolates were screened for: i) resistance profile to antibacterial agents, determining the MIC of imipenem; ii) the detection of metallo-beta-lactamase (MBL) by phenotypic and genotypic methods, iii) MBL detection by using a double-disk diffusion test (D-test), determining the sensitivity and specificity of the assay; iv) the presence of genes encoding 16S rRNA methylases and their association with aminoglycoside-resistant phenotypes, v) changes in the bacterial permeability due to porin (OprD) loss; vi) the presence or absence of the oprD gene by using PCR; vii) phenotypic expression of efflux pumps by determining the MIC of quinolones, cephalosporins and carbapenems in the presence/absence of specific inhibitors, performing a comparative analysis with a combined-disk method, viii) genes encoding efflux pumps proteins (mexC and mexX) by PCR; ix) MexAB-OprM and MexEF efflux pumps expression; x) clonal relatedness, by ERIC-PCR genotyping, regarding the predominance of major resistance genotypes. Results: Among imipenem-resistant P. aeruginosa strains (n=76, MIC50 e MIC90 = 32 µg/mL e > 512 µg/mL, respectively) 82% showed a multidrug-resistant phenotype. The main mechanism of imipenem resistance was the MBL production detected in 74% strains, of which 62% harbored the blaSPM-1 gene, and 12% harbored the blaVIM-like gene. The D-test identified MBL production in 61% strains. In this regard, CAZ/MAA was the most sensitive combination for MBL detection associated to SPM-1 enzyme (89%), exhibiting 86% specificity. The presence of the rmtD 16S rRNA methylase gene was confirmed in 66% aminoglycoside-resistant strains. Moreover, presence of both rmtD and blaSPM-1 genes was identified in 61% strains. Loss of OprD porins was observed in 71% strains. In this regard, 66% MBL positive strains and 85% MBL negative strains showed OprD loss. Thus, MBL production and OprD loss contributed to imipenem resistance in P. aeruginosa. Most likely, in 13% (10/76) strains the porin loss was associated to insertion sequences (SI) inserted upstream of the oprD gene. On the other hand, in 11% (9/76) strains the absence of a PCR product targeting the 736/1394 region of the oprD gene, suggested the presence of polymorphisms. The mexA gene was identified in 92% strains, whereas the mexE gene was identified in 82% strains. Results obtained from efflux pump screening by using a combined-disk assay and MIC determination in the presence of reserpine, CCCP e PABN (using levofloxacin, meropenem, aztreonam or imipenem) was not correlated with results obtained from MexAB-OprM and MexEF-OprN overexpression analysis by RT-PCR. In this regard, both combined-disk and MIC assay showed lack of specificity and sensitivity in comparison to RT-PCR. Overexpression of mexA and mexE genes was confirmed in 35% (7/20) MBL-negative and 11% (6/56) MBL-positive strains, respectively, being 7% (4/56) MBL-positive strains overexpressed both genes. The overexpression of MexAB-OprM and MexEF-OprN efflux pumps, as only mechanism of resistance to meropenem and imipenem was observed in 10% (2/20) MBL-negative strains. ERICPCR typing revealed the presence of 24 clusters among 76 imipenem-resistant P. aeruginosa strains (≥ 90% similarity). Conclusion: The convergence of multiple mechanisms of resistance in Pseudomonas aeruginosa seems to be a favorable event for the selection of multiresistant clones endemic in the southeastern region of Brazil.

Memoria para optar al Título Profesional de Ingeniero de la Madera
Nothofagus obliqua (Mirb.) Oerst (roble o pellín), es una especie que se distribuye desde la Región de Valparaíso hasta la Región de Los Lagos; también habita en Argentina. Esta especie se caracteriza por la alta durabilidad natural que presenta su madera en individuos adultos. Sin embargo, para la madera proveniente de renovales, con diferentes características de color y densidad, no existen antecedentes sobre su comportamiento frente al ataque de hongos de pudrición u otros agentes de biodeterioro. Actualmente, la madera de renovales de roble es sometida a un tratamiento de “vaporizado”, el que tiene como objetivo igualar el color rosado a rojizo del duramen de la madera de renoval, con el rojo oscuro del duramen de roble pellín, mucho más apreciado en el mercado. Con el objetivo de proporcionar antecedentes sobre la durabilidad natural de la madera de renoval de roble y de la influencia del tratamiento de cambio de color (vaporizado), se evaluó la resistencia de estas maderas frente a la acción de hongos de pudrición mediante la pérdida de peso, solubilidad en soda y el % de lignina. Para realizar el estudio se utilizó madera de renoval de roble, empleándose 40 probetas de albura y 40 de duramen. Se aplicó el proceso de vaporizado a 20 probetas de cada grupo, de manera de compararlas con el comportamiento de la madera sin vaporizar frente al ataque del hongo de pudrición blanca Polystictus versicolor y de pudrición café Poria monticola, utilizando además, 20 probetas de roble adulto como testigo. La mitad de las probetas de cada conjunto fue evaluada a los 2 meses (T1) y el resto a los 4 meses (T2) desde el inicio del ataque fúngico. Los resultados indicaron que el vaporizado no tuvo influencia sobre la resistencia de la madera de renoval frente al ataque de P. versicolor, que generó el %PP más alto para todos los tipos de madera a los 2 meses de acción fúngica. Así mismo, el % de solubilidad en soda indicó que el hongo de pudricion blanca provocó el mayor biodeterioro en comparación con el hongo de pudricion café. Para el caso de P. monticola, se observó que el vaporizado tampoco tuvo efecto sobre la resistencia al ataque de pudrición, generándose el mayor biodeterioro a los 4 meses (T2) que a los 2 meses (T1). Esto señaló finalmente, que la madera de renoval de roble no es una madera durable, considerando su resistencia frente al ataque de hongos de pudrición.
Nothofagus obliqua (Mirb.) Oerst (roble o pellín), is a specie allocated from Valparaiso Region to Los Lagos Region, Chile, also it can be found in Argentina. This kind is characterised by its high natural durability of its wood in adults members. Nevertheless, there are not history, for wood from secondary forests, with different characteristics of color and density, about its behaviour against the fungal attack or other biodeterioration agents. Nowadays, the roble wood from secondary forests is subjected to a “steaming” treatment, which aims to equalize pink to reddish heartwood color of the wood from secondary forests, with the dark red of the roble pellin heartwood, much more appreciated in the market. In order to provide history about the natural durability of the roble secondary forest wood, and of the influence of the color change treatment (steaming), the resistance of these woods against the action of fungal through weightloss, soda solubility and the lignin percentage was evaluated. Roble wood (renoval) was used to carry out the research, using 40 test tubes for sapwood and 40 test tubes for heartwood. Steaming process was tested in 20 test tubes of each group to do the comparison with those samples that were not under the process but under the white rot fungi attack Polystictus versicolor and the brown fungi Poria monticola. Also 20 test tubes of old roble were used as reference sample. Half of the samples of each group was checked at two months (T1) and the remaining samples at four months (T2) since the attacks of fungi started. The results shown that the steaming did not affect the wood resistance against the attack of P. versicolor produced the highest %PP for all the samples at 2 months of the test. Moreover, the %S indicated that the white fungi produced a bigger biodeterioration than the brown one. In the case of P. Monticola, it was observed that the steaming had no effect neither about the resistance of fungal attack, generating the greatest deterioration at 4 months (T2) than 2 months (T1). This finally pointed that the roble secondary forest wood is not a durable one, considering its resistance against the fungal attack

This essay looks at memorials and monuments raised in Finland dedicated to the civil war 1918 from both the red and the white side. The earliest memorials are from 1918 and the newest one from 1964. The difference between the two sides and the change over time is looked at through a few chosen monuments and memorials analyzed using semiotics and reception aesthetics. The use of symbols is extensive throughout the period, but the form they take, as well as their connotation, change over time, depending on the political context they are created in. They are created with different aims.

碩士
國立臺灣大學
化學工程學研究所
95
Abstract In this research, porous chitosan microparticles were prepared by using a emulsification/freeze-gelation method. The effects of various processes parameters were investigated. The BSA-loading capability of the porous chitosan microparticles and that of a commercial powder were compared. First, the optimum HLB value of the surfactant utilized in this method was found to be in the range of 8.6-11.0. The size of the porous chitosan microparticles became smaller as the HLB values of the different surfactants approached the optimum HLB value. These microparticles prepared by using different surfactants had different structures；some were dense porous and some were loose porous. Second, Span 20/Tween 21 was found to be the optimum combination of surfactants with the HLB value of 10. The microparticles had homogeneous and loose porous structure, and the particle size was 153.4 μm and C.V. was 33.9%. Third, in the emulsification system when the proportion of petroleumbenzin (mL) / 1 wt% chitosan solution (mL) / surfactants (g) was adjusted to 100/20/1.5, the particle yield was increased by 3.2 folds with size of 163.3 μm (C.V. = 37.3%). Fourth, this research also investigated the influence of freezing temperature. The water uptake capability of microparticles increased obviously when the freezing temperature decreased. The water uptake capability was about 2300% when the freezing temperature was -25℃, which was 5.5 folds of that of commercial powder. Finally, the application of the porous chitosan microparticles in BSA loading indicated that BSA loading was complete at low BSA concentration (＜0.8 mg/mL) and was 190 mg BSA/g chitosan at high BSA concentration (＞2.0 mg/mL). The loading capacity of these microparticles was 4 folds of that of commercial powder. This research demonstrated that the properties of the porous chitosan microparticles could be modulated by varying the process parameters for application in various fields.

碩士
國防醫學院
生物化學研究所
93
Poria cocos, an important Chinese herb medicine, is widely used for a variety of distresses with no apparent adverse effects for long term usage. Despite a highly valued traditional medicine, its mechanism of action remains to be elucidated. The main ingredients of this herb medicine are a group of triterpenoid compounds, which are generally attributed for most of its pharmacological effects. Since triterpenoids have a common steroid-like structure, they are expected to have certain steroid-related pharmacological activities. In this study, a series of triterpenoids from poria cocos, compound K1, K2, K3, and K4, were investigated for their effect on nuclear receptors for estrogen, glucocorticoid, progesterone, mineralcorticoid, and androgen. Among these compounds, K1 activated the transcription of the estrogen-responsive luciferase reporters in human MCF-7 cells at a concentration between 10-50 μM. The effect was specifically inhibited by ER antagonist ICI 182,780, suggest that the effect of K1 is ER-dependent. The expression of several estrogen-regulated genes, including estrogen receptor (ER), progesterone receptor (PR), pS2, c-fos, and cathepsin D were also shown to be regulated by compound K1 in an identical pattern as that of estradiol. Competitive binding studies, however, failed to demonstrate detectable binding between compound K1 and ER. These results suggest that compound K1 may bind to ER at very low affinity or may not bind ER directly but activated ER in other signaling pathways. Although failed to show direct binding to ER in our assay system, our data indicated that poco-K1 acts as a weak, but genuine, phytoestrogen in eliciting estrogen responsiveness in human cells. Compound K3, on the other hand, potently activated luciferase activity from the glucocorticoid responsive MMTV-luciferase reporter in human HEK293 cells. Treatment HEK293 cells with K3 enhanced nucleus translocation of glucocorticoid receptor (GR) as does dexamethasone (Dex), though in a much lower scale. Similar to the action of Dex, compound K3 enhanced the expression of IκB and down regulated expression of GR. Competitive GR binding assay indicated that K3, at a concentration of 10~50 uM may weakly compete for binding to GR. In spite of the above glucocorticoid response, compound K3 exhibited other activities which may not related to GR. Western blot analysis indicated that K3 facilitated nuclear translocation of NF-κB factor. In the presence of GR and PR antagonist RU486, the glucocorticoid response of compound K3 was greatly enhanced rather then abolished. These results indicate that, in addition to function as a weak ligand to elicit GR responsiveness through binding to GR, compound K3 also exhibited evident GR-independent activities. Taken together, results shown in this study demonstrated that triterpenoids from poria cocos exhibited steroid-like activities to modulate the action of steroid hormone receptors. Our study thus provide molecular basis for understanding of the action mechanism of this traditional herb medicine and shed light on potential therapeutic applications of these compounds.

碩士
國立臺灣大學
園藝暨景觀學系
102
Poria cocos protein (PCP), which is purified from dried sclerotium of Poria cocos, is a 35.6 kDa heterodimer protein including 14.3 and 21.3 kDa glycosyl subunits. In our previous results, PCP could active RAW264.7 cells and mouse peritoneal macrophages. This protein could also up-regulate murine T cells to secrete IFN-γ in the presence of anti-CD3/CD28 mAbs and inhibit TH2 immune-response in patch test for atopic dermatitis. However, PCP could not directly activate the differentiation of CD90.2+ T cells to TH1 cells, suggesting antigen-presenting cells as dendritic cells (DCs) could be responsible for the activation of PCP on TH1 cells. In this study, we first found that PCP could up-regulate the expression of CD40, CD80, and CD86 and induce secretion of IL-6 and IL-12p70 on murine DCs. In the results of PCP incubated with DCs of TLR2-/- and TLR4-/- mice, we found that PCP might induce DCs activation through neither TLR2 nor TLR4 pathway. Second, PCP significantly increased the antigen-presentimg ability of murine DCs to promote cell proliferation and IFN-γ secretion of OVA-specific DO11.10 CD90.2+ T cells. Meanwhile, these DCs could inhibit the OVA-specific IL-4 secretion by DO11.10 CD90.2+ T cells. According to the results, we suggested that PCP can help DCs presenting antigen and skewing immune-response to TH1. Finally, in OVA-induced asthma mouse model, oral administration of PCP (100 μg/day) could increase the production of IFN-γ in bronchoalveolar lavage fluid (BALF) and OVA-specific IgG2a in serum. Oral administration of PCP could also down-regulate the amount of eosinophils and TH2 cytokines (IL-4, IL-5, and IL-13) in BALF, OVA-specific IgE in serum, and cell infiltration in mouse lung sections. In conclusion, PCP can activate DCs to promote differentiation of TH1 cells and suppress TH2 immune-response in mouse model of asthma.

碩士
輔仁大學
營養科學系
98
Poria cocos (SchW.) wolf Poria is a well-known herb among traditional Chinese medicine. It contains both edible and medical characteristics. Many studies demonstrated that poria cocos play many functions such as anti-tumor, antioxidant and promote antibody secretion, etc. According to our Lab’s previous study showed that the OVA sensitized airway inflammatory mice administrated with low dose extract of poria cocos, could increase TH1 type cytokine secretion, however, high dose extract of poria cocos represented decrease TH1 and TH2 responses. Therefore, the optimal effects of the TA need to be clarify in vivo. In this study, we fed mice with low (LPC), medium (MPC) or high (HPC) dosages of extract of poria cocos in order to elucidate its role on immune response. Firstly, mice looks normal on the appearance and physiology on all groups. The levels of serum antibody and phagocytosis of blood cells were not changed among three groups. Intestinal immune response shown that poria cocos-extract fed groups shown no differences on total numbers of lymphocyte (isolated from PP and NLM), cell proliferative activity, and cytokine secretion. Then, the percentage of subpopulation of IgA+ B cell in the peyer’s patch of 3 poria cocos fed groups (L, M, and HPC) all elevated as compared to the control group significantly. Thus, extract of poria cocos might modulate the isotype switching of naïve B cell to IgA+ B cell. Meanwhile, the effects of splenocytes immune functions, such as cell population and proliferative response, were no change among group. However, the weight of spleen and total cell numbers of splenocyte in HPC group represent significantly lower when compared to the control group. Accumulated data suggested that extract of poria cocos might promote intestinal IgA+ B cell population and reduce the splenocytes numbers upon HPC administration. Key words: extract of poria cocos, intestinal immune response.

碩士
國防醫學院
藥學研究所
92
Fu-Ling, the dried sclerotium of Poria cocos (Schw.) Wolf, is one the most important and common in Chinese traditional medicine used to treatment of edema with oliguria, dizziness, restlessness and insomnia, and diminished function of the spleen marked by anorexia, loose stools or diarrhea, and also a popular food in China. Previous studies demonstrated it contained lanostane type triterpenes exhibited almost biological activities, e.g. immuodulatory activity. Being interested in exploring of the quality of plant materials and bioactivity of Fu-Ling, the reinvestigation of this plant was undertaken in this study. Being compared the quality of two mainly cultivated area, Hubei and Yunnan, in the production of Fu-Ling, the EtOH extract of Fu-Ling of Hubei (10 Kg) was separated on silica gel and preparative HPLC to give four lanostane type compounds, pachymic acid (1), tumulosic acid (2), polyporenic acid C (3), and 3-epidehydrotumulosic acid (4), as same as the isolation of Yunnan’s. The quantity of total triterpene and polyporenic acid (3) of Hubei’s is more than that of Yunnan’s, but the quantity of pachymic acid (1) Hubei’s is less than that of Yunnan’s. In addition, a new 4,5-secolanostane triterpenoid, named fulin C, isolated from Yunnan’s Fu-Ling. Their structures were determined on the basis of the spectral data comparision with the literature data and an authentic sample. In bioactivity studies, poco-K2 and poco-K4 at dose of 0.001~1μM exhibited significantly enhance glucose absorption in Caco-2 transwell system common used as nutrimental facilitation and drug absorption test.

Chen, Jialun.
Thesis Ph.D. Chinese University of Hong Kong 2015.
Includes bibliographical references (leaves 97-112).
Abstracts also in Chinese.
Title from PDF title page (viewed on 29, September, 2016).

碩士
國立臺灣大學
園藝學研究所
93
A new immunomodulatory protein (PCP) is purified from the dried sclerotium of an therapeutic fungi, Poria cocos (Schw.) Wolf, by extraction using 5 % cold acetic acid in the presence of 0.1 % 2-mercaptoethanol, followed by ammonium sulfate fractionation, DE-52 anion-exchange and FPLC chromatography. Gel filtration chromatography shows that PCP has a molecular mass of 35.6 kDa. SDS-PAGE electrophoresis reveals that PCP contains two subunits with molecular mass of 14.3 and 21.3 kDa, respectively. Based on these results, we suggest that PCP is a heterodimer protein. Additionally, isoelectric focusing electrophoresis shows that the pI value of PCP is around 5.2. The amino acid composition and N-terminal amino acid sequences of these two subunits of PCP are also obtained. Three hybridoma clones, which secrete monoclonal antibodies recognizing PCP, are obtained. The specificity and isotype of these monoclonal antibodies are also confirmed. Immunocytochemical analysis suggests that PCP mainly expressed on cell wall, cell membrane, and nucleus of Poria cocos sclerotium. PCP directly activates RAW 264.7 macrophages and enhances the production of both nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) by LPS-induced cells, and increases the mRNA expression of iNOS, TNF-a, IL-1b, IL-6, IL-12, and IL-18 of the cells. Polymyxin B (PMB) and LAL tests show that the capability of PCP activating macrophages is not due to LPS contamination. Furthermore, PCP activates murine splenocytes, markedly enhances cell proliferation and metabolization of the cells, and also increases their secretion of gamma-interferon (IFN-g) in vitro. Flow cytometry analysis reveals that PCP significantly promotes cell proliferation in both T cells and B cells by increasing S and G2/M phases of DNA content. Additionally, PCP up-regulates the mRNA expression of IL-2, IFN-g, IL-4, and IL-5. However, PCP stimulates IFN-g but not IL-4 secretion in murine splenocytes. Taken together, these findings suggest that PCP can activate murine splenocytes and drive their Th1 development. This study suggests that PCP is an immune stimulant and can strengthen the immune response of its host and having medicinal capability.

碩士
輔仁大學
營養科學系
96
Asthma is a chronic inflammatory disorder of the airway. The symptoms of asthma include recurrent episodes of wheezing, cough, chest tightness and breathlessness. Our previous study showed that BALB/c mice administrated with the extract of poria cocos (PC) resulted reduced IL-5 secretion, but enhanced IFN-γ secretion on Con A-stimulated splenocyte. According to this, we proposed that there may exist some ingredients in the extracts of poria cocos (PC) which have beneficial effect of immunoregulation on murine airway inflammation. This study tried to clarify the potential mechanisms of attenuating asthma on mice administered extracts of PC. The BALB/c mice were sensitized with OVA (ovalbumin), and administered with different dosages of FL (1.488 mg/ kg-BW), A (0.440 mg/ kg-BW), B (0.416 mg/ kg-BW) and C (0.348 mg/ kg-BW), or the corticosteroid, prednisolone (5 mg/ kg-BW; as a positive control for this study), or the asthma group that received no treatment. The effects of extracts of PC on the hallmarks of bronchial asthma on mice have been examed. Our data showed that B and C treatment could eliminate the AHR response, IL-5 and eotaxin secretion in BALF, but there had no effects on the percentage of eosinophilas in BALF, total IgE and antigen-specific IgE production in sera. The airway inflammatory mice administered with B or C compound isolated from PC, can significant increase IFN-γ and IL-2 production of Con A, PMA/ ionomysin, or OVA-stimulated splenocytes, but was no effect on IL-4 and IL-5 production. According to these data, it suggested that the ingredient of B or C compound may have beneficial effects on attenuating airway inflammation in this early-feeding study. We also processed the acute oral toxicity test in ICR mice for investigating the safety test of B and C compound. Our results revealed that no abnormality symptoms found after given B (25 mg/ kg-BW) or C (10 mg/ kg-BW) during 14 day’s experimental period. Hematological related parameters were around the normal range and no obvious genotoxicity in micronucleus test of mice fed with extra-high dosages of B or C compounds. Besides, there was no significant difference of the weights of heart, liver, lung and kidney, but B or C feeding resulted in decreasing the weight of immune organs, such us spleen and thymus. Our results indicated that there was no toxicity effect on murin of short term and extra high dosage fed with B or C compound. For study the therapeutic effect on establish asthmatic mice, we fed mice with high dose of B’ (2.5 mg/ kg-BW), C’ (1 mg/ kg-BW) or Pred to established asthmatic mice for continued five days after those mice reciving their final OVA immunizatoion. In this late stage feeding study of asthmatic mice, our data showed that B’ and C’ could both decrease AHR response, and eosinophil infiltrated in BALF, however, it did not affect the secretion of IL-5 and eotaxin in BALF. The pathologic histopathological assay of lung section revealed that the B’ and C’ treated group had less inflammatory cells infiltrated in peribrochial and peribronchiolar regions as compared to the asthma group. The B’ and C’ administrated could also decrease OVA-specific IFN-γ and IL-4 secretion. In conclusion, our data suggested that the extracts of PC have beneficial roles in attenuating asthma. The ingredients of B and C isolated from PC provide a possible therapeutic application in asthma according to the late stage feeding study. We also demonstrated that there were no toxicity effects in B and C compound. According to these data, we predict that the extracts of PC on relief asthma may through regulating T cell function. It will need more studies to demonstrate the effect of B and C on regulating T cell signaling pathway in future.

碩士
明新科技大學
化學工程與材料科技系碩士班
103
Poria cocos (Schw.) Wolf is an important oriental edible and medical fungus. The main active component isolated from Poria cocos is triterpenes and pachymaran. Pharmacology studies have proven that its active ingredients possess anti-inflammatory, anti-tumor, anti-emetic, ant-inflammation, anti-nephritis, anti-diabetes, anti-aging, immunity, sedative, diuretic and antioxidant properties. Recently, although many researchers focus on activity components and function of fermentation product as well as development in health-food, little literature was reported concerning effects of culture conditions on production and antioxidant activity of polysaccharides of Poria cocos by submerged culture. Thus, the project was proposed to investigate the optimization of culture conditions (including pH, temperature and aeration rate) on the production of polysaccharide in submerged culture of Poria cocos by one-factor-at-a-time design methodology and their antioxidant properties. The results showed that the optimal operation conditions are pH 3.0, 36℃and 0.6 vvm in 5 L stirred tank bioreactor submerged cultures. Besides, In 20 L stirred tank bioreactor culture on day 7, the optimal operation conditions also can obtain the maximum biomass and exopolysaccharide (EPS) production with 5.614 gL-1 and 236.32 mgL-1, respectively. The crude EPS from submerged cultures of Poria cocos was purified by using Sepharose CL-4B column chromatography with four EPS peaks (EPS-A, EPS-B, EPS-C and EPS-D) eluted. Antioxidant activity results point out that EPS-C has a high antioxidant activities and scavenging effects on superoxide anions. Corresponding EC50 values are 4.03 mgmL-1 and 0.25 mgmL-1. The high EC50 values of scavenging effects on 1,1-diphenol-2-picrylhydrazyl radicals of EPS-A are 0.04 mgmL-1. EPS-A showed high reducing powers with EC50 values of 0.018 mgmL-1. Crude EPS from 20 L stirred tank bioreactor culture by Poria cocos. Besides, the protein/polysaccharide ratios of EPS-A, EPS-B, EPS-C, and EPS-D are 31.46%, 32.16%, 31.81%, and 31.46% (w/w), respectively. The protein/polysaccharide ratio of crude EPS from 20 L stirred tank bioreactor culture by Poria cocos is 34.2% (w/w). Molecular weight analyses of EPS-A, EPS-B, EPS-C, and EPS-D by using Molecular Sieve chromatography tell that molecular weights of four fractions are 4.13×106Da for EPS-A, 2.481×104Da for EPS-B, 2.48×104Da for EPS-C, and 2.81×105 Da for EPS-D. In addition, molecular weight of crude EPS from 20 L stirred tank bioreactor culture by Poria cocos is 7.23×103 Da. Keywords: Poria cocos; antioxidant properties; polysaccharide; submerged cultur

碩士
國立宜蘭大學
食品科學系碩士班
102
Poria cocos is an edible and medicinal fungi, the main bioactive compounds are polysaccharides and triterpenoids. The pharmacological studies confirmed that it has several effects such as anti-inflammatory, antioxidant, immunomodulatory, anti-tumor, hypoglycemic, sedative, diuretic, antiemetic etc. The objectives of this study were (1) to explore the microwave power, extraction time of the crude polysaccharides and triterpenoids content in the Poria cocos solid-state fermented products, and to compare its extraction efficiency with hot water extraction and ultrasonic extraction, and (2) to study hypoglycemic function of the crude Poria cocos extracts. The results showed that the highest crude polysaccharides and triterpenoids contents were in Poria cocos solid-state fermented adlay and soybean, respectively. The solvents used for extracting crude polysaccharides and triterpenoids were water and 95% ethanol, respectively. The solid-liquid ratio was 1:20. The optimal microwave extraction was controlled the microwave output power at 300 W heating for 5 min. Both STZ-induced diabetic mice (type I diabetes) and STZ with NA-induced diabetic mice (similar to type II diabetes) by tube-feeding 50 mg/kg ethanol extracted from Poria cocos solid-state fermented soybean product had good hypoglycemic effec.

博士
國立臺灣大學
食品科技研究所
92
Abstract Wolfiporia cocos gilbertson (WCG) [the sclerotium of Poria cocos (Schw.) Wolf], an oriental fungus termed as Fu-Ling in Chinese, has been widely used as a Chinese traditional herbal medicine, such as a tonic food for health promotion, for centuries. In the present study, a neutral polysaccharide fraction from WCG (WCGPS) was isolated by cool-water extraction and the subsequent ethanol precipitation. The effect of WCGPS on the proliferation and differentiation of human leukemic cells, U937 and HL-60, was investigated in vitro. Results showed that the conditioned medium prepared with WCGPS of 15 mg/mL- stimulated human blood mononuclear cells (WCGPS-MNC-CM) exhibited an potent activity in suppressing the proliferation of U937 and HL-60 cells by up to 87.3% and 74.7%, respectively. Furthermore, WCGPS-MNC-CM treatment induced about 66.6% of the U937 cells and 49.4% of the HL-60 cells to differentiate into mature monocytes/macrophages, which also markedly express surface antigens of CD11b, CD14, and CD 68. The differentiated U937 and HL-60 cells displayed physiological functions such as phagocytosis and respiratory burst. However, neither WCG-PS alone nor normal MNC-CM showed similar effects. When consider both of the results from DNA ladder test and MTT test together, it demonstrated that the minor part of leukemic cells without differentiation underwent the apoptosis procedure. Interestingly, the levels of interferon (IFN)-g and tumor necrosis factor alpha (Geneva-Popova and Murdjeva 1999) in MNC-CM prepared without WCGPS treatment were very low, however, they were substantially increased up to about 41 and 10 folds, respectively, in WCGPS-MNC-CM. Antibody neutralization experiments of the WCGPS-MNC-CM were performed and the results revealed that the growth-inhibiting and differentiation-inducing activities of PSPS-MNC-CM were mainly due to the elevated cytokines, especially IFN-g and TNF-a. Therefore, these two cytokines acted synergistically on inhibiting tumor cell growth and inducing differentiation of the targeted U937 and HL-60 cells. The molecular weight of WCGPS was approximately 160 kDa, as estimated by gel permeation chromatography. Results revealed that WCGPS was capable of inhibiting proliferation and inducing differentiation of human leukemic U937 and HL-60 cells by stimulating cytokine production from human peripheral blood mononuclear cells. It suggests that WCGPS is a biological response modifier, instead of a cytotoxic reagent, and may be potential to be an adjuvant in cancer therapy.

碩士
東吳大學
微生物學系
85
The major goal of this study is to design pairs of specific PCR primers, based on the sequences of ITS region, for the detection of Poria cocos and Polyporus umbellatus in powdered chinese medicine. The internal transcribed spacers (ITS) of nuclear ribosomal DNA were sequenced and analyzed. The length of ITS region in Poria cocos ranges from 1419 in standard strains CCRC36022, 1529 in CCRC36023, to 1699 bases in Poria cocos herbs; and in Polyporus umbellatus, it ranges from 625 in standard strains CCRC36435 and Polyporus umbellatus herbs, to 663 bases in CCRC36407. In order to develop unique PCR primers to detect Poria cocos and Polyporus umbellatus were cloned in T- vector. The resulting clone was confirmed by sequencing. The cloned DNA fragment contained the ITS1, ITS2 and 5.8S rRNA gene. PCR primers specific for Poria cocos and Polyporus umbellatus was obtained, basedon the ITS region sequence. The specificity of these pairs of primers was tested with 4 kind of powered chinese medicine. All these powered chinese medicine showed the specific positive band reaction with these pairs of primers. The other kind of chinese herbs (especially the sort of fumgi) were tested and none showed the specific positive band. The sensitivity of the detection level was 0.1ng of genomic DNA extractedfrom pure Poria cocos and Polyporus umbellatus, or powered chinese medicine. At the end of this study, we use RFLP to reconfirm the PCR product amplified by these specific primers. These PCR product have the same restriction site as the pure Poria cocos and Polyporus umbellatus.

碩士
輔仁大學
營養科學系碩士班
101
SLE (systemic lupus erythematosus) is an autoimmune disease and lupus nephritis (LN) is the major complication of SLE. In our previous study, we found that fed NZB/W F1 mice (SLE prone mice) with PC ( a triterpenoid purified from poria cocos) could attenuated LN. Many documents demonstrated that over-proliferated mesangial cells (MC) in glomerulus concerned about the pathological mechanism of LN. MC is a kind of specialized macrophage in kidney, and activated MC will secrete many pro-inflammatory substances such as MCP-1, IL-6 and so on. In order to study the beneficial role of PC on inhibiting LPS (lipopolysaccharide) induced pro- inflammation, firstly, we prepared peritoneal exudated cells (PEC, rich of macrophages) isolated from NZB/ W F1 or BALB/ c mice， and co-cultured with 80 uM PC in vitro. The data suggested that PC could inhibited the MCP-1 and IL-6 mRNA and protein expression in NZB/ W F1 mice, but not in BALB/c mice. In part two study, we established stable-primary culture system of MCs derived from NZB/ W F1 mice and DBA/W F1 mice, MCs were stimulated with LPS then co-cultured with 80 uM PC to investigate the effects of PC on expression of inflammatory mRNA and protein in MCs. Real-time qPCR showed PC could significantly decreased level of MyD88, IL-6 and MCP-1 mRNA expression. By the way, we also performed the mice inflammation protein array , and we found that PC could decrease many pro-inflammatory protein expression, such as IL-6, MCP-1, RANTES and KC. Collected data suggested that PC may attenuated LN through TLR-4 downstream MyD88- NF-κB signaling and inhibit pro-inflammatory related cytokines. PC may play a beneficial role on further developing it as a therapeutic agent to LN disease.

碩士
國立中山大學
生物科學系研究所
95
PCWE has been used widely in oriental traditional medicine in treating edema and diarrhea. Recent studies have shown that PCWE may also have anti-tumor and anti-inflammation acts. However, few studies have been conducted to reveal the mechanisms of these effects. In the present study, we tried to elucidate the mechanisms by investigating the effects on PCWE on the regulation of ion transport across the epithelial membranes of colon, which is also useful in explaining the anti-diarrhea and anti-edema effects. Alternation in membrane potential and short-circuit current (Isc) were examined using the Ussing chamber technique. Our results showed that PCWE decreased Isc upon application to the apical side. Amiloride inhibited this Isc induced by PCWE indicating that PCWE acted on amiloride-sensitive sodium channel of the epithelium. However, when PCWE was applied to the serosal side, the Isc was not changed, indicating a minimal influence of this substance on ATP-driving Na+/K+ counter transporters. Our data also showed that the Isc decreased by PCWE could be inhibited by bumetanide and chlorothiazide (Cl‾ channel inhibitors). We therefore concluded that PCWE could both enhance sodium transport and stimulate the secretion of Cl‾ in colon epithelium.

碩士
輔仁大學
營養科學系
100
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that affects various organs. It has been associated with interactions between the endocrine system , the environment, and genetic factors, but the pathogenesis is not yet fully understood. Conventionally, immunosuppressive agents such as steroids have been used to treat SLE patients. However, due to severe adverse effects of immunosuppressive agents, alternative treatments instead of traditional medication are now under development. According to our preliminary studies, the extracts of Poria cocos (PC) play beneficial roles in attenuating the progression of asthma on mouse models, and it may through its anti-inflammatory effects, probably. Both pathological mechanisms of asthma and SLE were related of shifting the immune status from Th1-dominant to Th2-dominant immune responses, therefore, we would like to explore whether PC contributes to the remission of SLE. Spontaneous SLE prone female NZB/W F1 mice were divided into the control group and the other three experimental groups (N = 8/group), which been fed with 0.1, 0.5, or 2.5 mg PC per kg body weight per day, respectively, for five weeks. Collected data showed that the experimental groups had showed significantly decreased levels of sera IgA and IgG, and fewer CD5+ B lymphocytes in Peyer's patches than those of the control group. Besides, the experimental group with the highest dosage of PC treatment represented the highest total cell number of B and T lymphocytes, as well as total leukocytes in the splenocytes among the experimental groups on two-week-fed's interval. In summary, PC may modulate the generation of white blood cells and B lymphocytes in lupus-like mice. In our study, when we fed NZB/W F1 mice with the highest dosage of PC (2.5 mg PC per kg body weight per day) for 5 weeks, accumulated data suggested that PC had no disadvantageous response to the innate immunity. The role of PC in immunoregulation still needs further investigation.

碩士
國立宜蘭大學
食品科學系碩士班
106
Soybean residue and rice bran are rich in nutrients and active components, but high moisture content of extracted residue or high lipase activity could cause rapidly decaying and hard to be application except for animal feed or fertilizer. Radio frequency (RF) can rapidly heat up water molecules or ions in food by rapid conversion of electric field; therefore, RF drying can overcome the heat and mass transfer resistances to accelerate drying processing. The objectives of this study were to dry the soybean residue and to inactive lipase of rice bran by RF heating. Then the soybean residue and rice bran were the media for Poria cocos solid-state fermentation to increase bioactive components and improve antioxidant activities. The results showed that they required 14, 22 and 30 min for reducing moisture content from 80% to 15% to dry 1, 1.5 and 2 kg of soybean residue by RF, respectively. RF drying rate was 25 times of 45℃ cold air drying, and the total energy consumption was reduced to 1/9. The total polyphenols content and DPPH free radical scavenging ability in RF soybean residue were higher than cold air dried soybean residue, but there was no significant difference in color between two different drying methods of soybean residue. RF dried soy residue contains 54.5% of carbohydrates, 27.6% of crude protein and 10.6% of crude fat. On the other hand, 1 kg rice bran was stabilized by RF with gap of 6 cm heating 2 min to inhibit lipase activity without affecting color and antioxidant activity. RF stabilized rice bran contains 48.3% of carbohydrates, 15.4% of crude protein and 19.1% of crude fat. RF really stabilized rice bran during 8 weeks storage at 4, 25, and 37℃. Finally, Poria cocos solid-state fermentation was carried out with different fermentation time periods (0, 30 and 60 days) with 500 g mixed medium (40% moisture content) with different ratios of soybean residue and rice bran (1:0, 2:1, 1:1, 1:2 and 0:1). All the contents of crude polysaccharide, crude triterpenoids, total polyphenols and flavonoids in Poria cocos solid-state fermentation product increased with the increase of the proportion of rice bran in the mixed media and fermentation time. However, considering the time cost and other factors, the suitable Poria cocos solid-state fermentation conditions: 40% of the moisture content, soybean residue and rice bran = 1:1, at 25℃ for 30 days. It took only 30, 200 sec at the RF electrode gap of 15 cm to pasteurize and reduce the moisture content below 15%; therefore, it could replace the traditional sterilization 60 min in an autoclave and cold air drying 100 min at 45℃. RF heating greatly shorten pasteurization and drying time of Poria cocos solid-state fermentation product in 3 min to complete, but also retain the appearance of Poria cocos white mycelium color, to avoid browning. The Poria cocos fermentation product after RF treatment contained 5.03% of mycelium, 9.83% of crude polysaccharide, 4.43% of crude triterpenoids, 3.54 mg gallic acid equivalent/g DW of total polyphenols, 0.38 mg quercetin equivalent/g DW of flavonoid content and good antioxidant capacity. Zebrafish animal experiments confirmed the 200 mg/L extracts from the Poria cocos solid-state fermentation product to have antioxidant activity.

碩士
國立臺灣海洋大學
食品科學系
92
The cellulase was selected among various commercial enzymes cellulase, α-amlyase, and β-glucosidase, to hydrolyze Fu-lin to produce the hydrolysate with immunoactivity. The optimal temperature and pH for cellulase action on Fu-lin were 45℃ and pH 5.5, respectively, with 3% Fu-lin being the maximal working concentration. The hydrolysate obtained from cellulase degradation of Fu-lin at pH 5.5 for 24 hr had the highest increasing effects on the cell proliferation and IgM secretion of a human hybridoma cell HB4C5, and therefore was further fractioned by Sephacryl S-200 HR gel filtration chromatography. At 1,000 ppm both the hydrolysate and gel filtration fraction increased the IgM secretion of HB4C5 by 199% and 189%, respectively. Both samples could not increase the cell proliferation of mouse macrophage RAW 264.7, but they did stimulate the NO secretion. In addition, the sample of gel filtration fraction significantly inhibited the cell proliferation of human acute promyelocytic leukemia HL-60 and human ailing promyelocytic leukemia K562. In vitro both the hydrolysate and its gel filtration fraction significantly increased the cell proliferation of mouse peritoneal exudates cells. In the presence of concanavalin A, the hydrolysate and the gel filtration fraction at 70 ppm increased the cell proliferation of mouse spleen lymphocyte by 126% and 130%, respectively. However, they did not affect the IgG and IgM secretion of mouse speen lymphocytes. In vivo the sample of gel filtration fraction significantly accelerate the IgG and IgM production in mice. After peritoneal injection of mice for 2 weeks, the contents of serum IgG and IgM for group administrated with the gel filtration fraction were significantly higher them those of control; while only till 4 weeks of injection, the contents of serum IgM and IgA for the groups administrated with water extract or the hydrolysate of Fu-lin were significantly higher then those of control. The cell counts, cell proliferation rate and NO secretion of peritoneal exudates cell were significantly higher for the groups injected with samples of hydrolysate and gel filtration fraction. In the presence of Con A the cell proliferation of mouse spleen lymphocyte were increased by 125% for the group injected with sample of gel filtration fraction, compared with that of control. In the presence of lipopolysaccharide, the cell proliferation rates of mouse spleen lymphocyte were increased by 146%, 156% and 165% for the groups injected with samples of PBS extract, hydrolysate and gel filtration fraction, respectively.

碩士
國立臺灣大學
食品科技研究所
93
Polysaccharide-rich fraction (PRF) from dried fruiting body of Fu-Ling [Poria cocos (Schew.) Wolf] was prepared from cool-water extracts by ethanol precipitation to study its immunomodulatory and anti-tumor effects in vivo. In non-specific immune response, oral administration of PRF for 33 days did not affect the growth and relative organ weight of Balb/c mice. Relative proliferation ratio of PHA-stimulated spleonocytes in mice was significantly increased, while natural killer activity, splenocyte surface markers of T, Th, Tc, and B cells were similar to the corresponding result of the control group. Mice were immunized with ovalbumin (OVA) to conduct the specific immune response experiment. It was found that oral administration of PRF induced the reduction of anti-OVA IgG and IgM levels. Oral administration of 100 mg/kg bw PRF significantly reduced the percentage of CD8(+) Tc splenocytes, while higher dose of 200 mg/kg bw significantly increased CD4(+) Th and CD8(+) Tc percentage. In anti-tumor test of CT26-implanted Balb/c mice, about 32.1% of tumor was suppressed at dose of 400 mg/kg bw PRF. The content of serum IgG and proliferation of PHA-stimulated splenocytes for mice orally administrated with PRF were significantly higher than that of control group. Ratio of lymphocyte of CT26-implanted Balb/c mice decreased in splenocytes, but oral administration of PRF increased the cell numbers of Th, Tc, and B cells.

碩士
國立臺灣大學
食品科技研究所
105
Depression is a chronic illness that significantly affects a person’s moods, behaviour and general health. Predicted to be the first contribution of global burden disease in 2030 according to WHO. Inflammation is a protective response of the human body to various harmful stimuli including physical trauma, noxious chemicals, and microbiologic agents. However, chronic inflammation can lead to serious tissue damage and inflammation-associated diseases, like depression. At present, there are several classes of classical antidepressants in clinical practice. However, most of these drugs are undesirable, due to their side effects and efficacy for only a certain proportion of patients. Thus, there is an unmet need for safe, well‐tolerated and powerful antidepressants. Poria cocos Wolf, commonly known as ‘Fu-Ling’, is a medicinal fungus and it is widely distributed in Korea, China, and East Asian countries. This fungus has long been utilized as a traditional medicine for its sedative, diuretic, and tonic effects. Recently, the sclerotia of Poria cocos were shown to have many biological activities including anticancer, anti-inflammatory, antioxidant and stabilize the mind. Therefore the main purpose of the current study was to verify whether water extract of Poria cocos conferred an antidepressant-like effect on the depressive mouse model established by forced swim test and unpredictable chronic mild stress, then explore its possible mechanism. The results shown that Poria cocos may decrease the immobility time. And the depression-related behaviors including sucrose preference test and open field test, indicated that water extract of Poria cocos 300 mg/kg can improve the depression symptoms. Western blot analysis displayed down-regulated expressions of p38 mitogen-activated protein kinase (MAPK) and decreased the expressions of tumor necrosis factor-alpha (TNF-α). Thus, it was supposed that water extract of Poria cocos might be useful for the treatment of chronic depression possibly through p38 MAPK pathway. In conclusion, we found that water extract of Poria cocos exhibited anti-inflammatory effects on depression prevention.

碩士
國立臺灣海洋大學
食品科學系
93
The purpose of this thesis are to explore the efficacy of an anti-aging and whitening cosmetics that containing different extracts of Chinese herbal remedies composed of Ligusticum chuanxiong, Ocimum basilicum L., Poria cocos, Angelica dahurica and Chyueh laau chyuh jouh fang with different solvents. The study includes： 1. To evaluate the efficacy of anti-aging and whitening of the extracts and optimize the combination of Chinese herbal remedies by observation the chelating ferrous ions capacities, tyrosinase inhibition and the scavenging capacity of DPPH radical, hydrogen peroxide and superoxide radical also determining cells viability of the cultured fibroblast and melanin inhibition of the cultured melanocyte, 2. To prepare and characterize the cream products containing different extracts of Chinese herbal remedies extracts. The characteristics of cream products studied including storage stability, water holding capacity, skin elasticity, skin whitening, skin roughness and safety test. The results obtained are: The Chelating ferrous ions capacities, the scavenging effects for DPPH radical and superoxide radical, increased with increasing the concentration of the extracts of Chinese herbal remedies. The 95% ethanol extracts of Ligusticum chuanxiong, Ocimum basilicum L., Angelica dahurica and the 50% ethanol extracts of Poria cocos and Chyueh laau chyuh jouh fang had the highest scavenging effect for DPPH radical and superoxide radical than the other two extracts of Chinese herbal remedies. Cell viability results showed the extracts concentration between 1.95- 500 μg/ml with different solvents showed no significant different on CDD-966-SK and A 375 viability. Chinese herbal remedies extracts revealed 80% inhibition on melanin content of A 375 at ≦250 μg/ml and 50- 70% inhibition on melanin content of A 375 at 250-1,000 μg/ml. The stabilities of cosmetic product containing with or without Chinese herbal remedies extracts were stable for sonication (135 W, 42KHz, 5 min), for centrifuging (3000 rpm, 30 min), and for more than two months at high temperature (50�aC) storage. The pH of cream products did not change significantly. These results showed that cream product had good stability. pH of cream products containing different concentrations of Chinese herbal remedies extracts are between 5.6-5.9. The safety test resulted in no erythema based on the Daize score test. Water holding capacity in terms of electrical capacitance ratio were higher for those containing Chinese herbal remedies extracts than that of the control. Skin eleasticity (R2, R8) for those whom have applied the cream containing Chinese herbal remedies extracts increased over time during four weeks test period especial for those applying cream product containing the 50% ethanol extracts of Chyueh laau chyuh jouh fang extracts showed significant better results. Skin whitening effect increased over time during four weeks test period and did show a significant difference for melanin index. skin roughness decreased over time during four weeks test period and did show a significant difference for skin roughness index.

博士
國立臺灣大學
園藝學研究所
97
Poria cocos (Schw.) Wolf is an important Oriental medical fungus with multiple functionalities, yet its bioactive substances and mechanisms involved have not been fully characterized. The objective of the present study was to investigate the bioactive substance from P. cocos and its molecular mechanism involved in immune modulation focused on macrophage and lymphocyte activation. A novel immunomodulatory protein (P. cocos immunomodulatory protein; PCP) was purified from the dried sclerotium of P. cocos (Schw.) Wolf using DE-52 cellulose and gel filtration chromatography. Chromatography and electrophoresis results indicated that the native PCP (35.6 kDa) is a disulfide-linked heterodimeric glycoprotein consisting of 14.3 and 21.3 kDa subunits with N- and O-glycosylation. PCP was capable of stimulating RAW 264.7 macrophages in vitro through the induction of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) as well as the regulation of nuclear factor-kappa B (NF-κB)-related gene expression. In primary mouse macrophages, we observed an increase in the expression of major histocompatibility complex (MHC) class II and CD86 molecules on peritoneal cavity macrophages. PCP directly activated macrophages to induce Toll-like receptor (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent signaling. We demonstrated the cell surface interactions of PCP with TLR4 and the capacity of PCP for TLR4 tyrosine phosphorylation. Results obtained with peritoneal macrophages from TLR4-deficient C57BL/10ScN mice revealed that PCP-induced activation and PCP cell surface binding were significantly attenuated. Moreover, enzymatic deglycosylation decreased PCP-mediated responses, indicating that the glycosylated portion of PCP was a key factor in PCP signaling through TLR4 in peritoneal macrophages. Further investigation on lymphocyte activation indicated that PCP directly activated mouse splenocytes, markedly increased cell proliferation and the levels of interferon-gamma (IFN-γ) secretion but not IL-5 production. Similarly, the selectively enhanced transcriptional expression of IL-2 and IFN-γ by PCP was demonstrated using quantitative real-time PCR. Although there were slight increases in the total cell population of CD4+ and CD8+ T cells in PCP-stimulated splenocytes, PCP significantly increased expression of the activation marker CD69 on both splenic CD4+ and CD8+ T cells. The potent CD4+ and CD8+ T cell-activating capability of PCP was demonstrated by the enhancement of cell proliferation, cytokine secretion, activation marker CD44 and CD69 expression upon anti-CD3/CD28 costimulation. The expression of T-bet, tyrosine phosphorylation of STAT4, IFN-γ and IL-2 secretion during PCP-induced CD4+ T cell activation were upregulated. In contrast to the functional deficiency of deglycosylated PCP on macrophage activation, the core protein of PCP was shown to be involved strongly in induction of T cell activation, as demonstrated by inhibition of T cell response using deproteinized PCP. In vivo experiments indicated that oral administration of PCP (50 mg/kg body weight) suppressed the level of serum IgG1, and enhanced amounts of serum IgG2a and T helper 1 (Th1)-associated cytokine secretion in BALB/c mouse spleen cell cultures. Oral administration of PCP upon immunization with ovalbumin (OVA) exhibited that OVA specific IgG2a levels were also significantly increased compared with those of PBS-treated mice, suggesting that PCP could suppressed OVA-induced Th2 response to drive Th1 development. Taken together, these studies characterize a new potential immune stimulator, PCP, which induces TLR4-dependent activation within murine macrophages and triggers a Th1-dominant immune response. These observations provide strong support for further studies of PCP and P. cocos to explore their overall modulatory nature toward mammalian cells and reveal their pharmaceutical potential and industrial value.