Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O gênero Pterodon compreende algumas espécies largamente distribuídas sobre a região central do Brasil. Seus frutos são comercialmente disponíveis no mercado da flora medicinal sendo amplamente utilizados pelas suas propriedades farmacológicas como antirreumático, anti-inflamatório e analgésicas. O objetivo deste trabalho foi biomonitorar o fracionamento do extrato hexânico de <i>Pterodon polygalaeflorus Benth</i>. (ExPpg), utilizando modelos de inflamação aguda (edema de pata e bolha de ar). O fracionamento/sub-fracionamento foi realizado por cromatografia em coluna de sílica e as amostras analisadas por cromatografia em camada fina e cromatografia gasosa acoplada a espectrômetro de massas. O edema de pata foi induzido em camundongos SW por injeção (s.c.) de carragenina. Uma hora antes da inoculação de carragenina os animais foram tratados v.o. com veículo (EtOH 15%, 1,25% Tween-20), indometacina (10 mg/kg p.c.) ou ExHPpg/frações/sub-frações de Ppg. No modelo bolha de ar, a cavidade foi desenvolvida em camundongos SW através da injeção de ar estéril (s.c.) no dorso e a inflamação induzida por carragenina. Uma hora antes de inocular a carragenina os animais foram tratados (v.o) com o veículo, indometacina (10 mg/kg) ou ExHPpg/Fr2Ppg/sub-frações. Após 4 h o exsudato da bolha foi coletado para contagem total e diferencial de leucócitos (Panótico rápido) e dosagem de proteínas (biureto), e a pele referente à bolha foi removida para análises macroscópica e histológica (HE). Também foi realizado estudo de migração de neutrófilos pelo ensaio do <i>transwell</i>. Após demonstração do efeito antiedematogênico do ExHPpg, este foi fracionado em quatro frações. A fração Fr2Ppg, mais ativa no modelo de edema de pata, também inibiu os diferentes parâmetros inflamatórios avaliados no modelo de bolha de ar, com a menor dose testada, e foi sub-fracionada em cinco sub-frações. Destas, as SF2.1 e SF2.2 foram as que mostraram melhor efeito anti-inflamatório pelo modelo da bolha de ar. Em relação ao grupo controle com carragenina, o ExHppg, Fr2Ppg, SF 2.1 e SF 2.2, na dose 0,02 mg/kg, exerceram inibições de 70,6%, 62,8%, 54,7% e 79% no número total de células no exsudato, reduções de 76,8%, 76,9%, 71,1% e 73,3% na concentração de proteína, respectivamente. Com a dose de 0,2 mg/kg, foram observadas inibições apenas para ExHPpg e SF 2.1, com intensidades menores, na leucometria total (62,9% e 48,62%, respectivamente), e na concentração de proteína para SF 2.1 e SF 2.2 (reduções de 68,2% e 30,4%, respectivamente). As análises macroscópica e histológica mostraram redução importante da vasodilatação e do infiltrado inflamatório pelo tratamento com o ExHppg, Fr2Ppg, SF 2.1 e SF 2.2. No ensaio de <i>transwell</i> a Fr2Ppg exibiu 31,4% de inibição na migração de neutrófilos. As sub-frações SF2.1 e SF2.2 foram avaliadas por GC-MS, identificando-se de diversos compostos majoritários. Em resumo, este trabalho confirma o potencial anti-inflamatório da espécie <i>Pterodon polygalaeflorus</i> e mostra um fracionamento efetivo do ExHPpg quanto à composição e ação anti-inflamatória. / The genus Pterodon comprises some species widely distributed over the central region of Brazil. Its fruits are commercially available on the market of the medicinal flora, being widely used by their pharmacological properties as antipyretic, anti-inflammatory and analgesic. The aim of this work was to guide the fractionation of hexanic extract of <i>Pterodon polygalaeflorus</i> Benth. fruits (ExPpg), using acute inflammation models (paw edema and air pouch). The extract fractionation was done by column chromatography on silica and the samples were analyzed by thin layer chromatography and gas chromatography coupled with mass spectrometer. Paw edema was induced in SW mice by (s.c.) injection of carrageenan in the right paw. One hour before the carrageenan inoculation, animals were orally treated with vehicle (EtOH 15, 1.25 Tween-20), indomethacin (10 mg/kg bow.) or ExHPpg/fractions/sub-fractions of Ppg. In the air pouch model, the cavity was developed in SW mice through sterile air injection (s.c.) on the back and the inflammation induced by carrageenan inoculation in this cavity. An hour before carrageenan injection the animals were orally treated with the vehicle, indomethacin (10 mg/kg) or ExHPpg/Fr2Ppg/sub-fractions. After four hours the air pouch exudate was collected for the total and differential leukocyte counts (fast Panotic) and protein determination (biuret reaction), and the skin of the air pouch was removed to macroscopic and histological analyses (HE). In vitro neutrophil migration was performed by transwell assay. After demonstration of the anti-edematogenic effect of ExHPpg, this extract was fractionated into four fractions. The Fr2Ppg fraction, most active in paw edema model, also inhibited, in the lowest dose tested, the different inflammatory parameters evaluated in air pouch model. Then, it was sub-fractioned in five sub-fractions. The SF 2.1 and SF 2.2 were the sub-fractions with higher anti-inflammatory effects in the air pouch model. In relation to the carrageenan control group, ExHPpg, Fr2Ppg, SF 2.1 and SF 2.2, in 0.02 mg/kg dose, showed inhibitions of 70.6%, 62.8%, 54.7% and 79% in the total leukocyte counts in the exudate and reductions of 76.8%, 76.9%, 71.1% and 73.3% in the protein concentration, respectively. With the 0.2 mg/kg dose, reductions were observed only for total leukocyte counts after treatment with ExHPpg or SF 2.1 (62.9 and 48.62, respectively), and for protein concentration in animals treated with SF 2.1 and SF 2.2 (reductions of 68.2% and 30.4%, respectively). Macroscopic and histological analyses of air pouch tissues showed important reductions of vasodilation and inflammatory infiltrate by treatment with ExHPpg, Fr2Ppg, SF 2.1 and SF 2.2. In the transwell assay the Fr2Ppg exhibited inhibition of 31.4% of neutrophil migration. The SF 2.1 and SF 2.2 sub-fractions were analyzed by GC-MS, identifying some major compounds. Concluding, this work confirms the anti-inflammatory potential of <i>Pterodon polygalaeflorus</i> species and shows an effective ExHPpg fractionation in relation to the composition and anti-inflammatory effect.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O gênero Pterodon compreende algumas espécies largamente distribuídas sobre a região central do Brasil. Seus frutos são comercialmente disponíveis no mercado da flora medicinal sendo amplamente utilizados pelas suas propriedades farmacológicas como antirreumático, anti-inflamatório e analgésicas. O objetivo deste trabalho foi biomonitorar o fracionamento do extrato hexânico de <i>Pterodon polygalaeflorus Benth</i>. (ExPpg), utilizando modelos de inflamação aguda (edema de pata e bolha de ar). O fracionamento/sub-fracionamento foi realizado por cromatografia em coluna de sílica e as amostras analisadas por cromatografia em camada fina e cromatografia gasosa acoplada a espectrômetro de massas. O edema de pata foi induzido em camundongos SW por injeção (s.c.) de carragenina. Uma hora antes da inoculação de carragenina os animais foram tratados v.o. com veículo (EtOH 15%, 1,25% Tween-20), indometacina (10 mg/kg p.c.) ou ExHPpg/frações/sub-frações de Ppg. No modelo bolha de ar, a cavidade foi desenvolvida em camundongos SW através da injeção de ar estéril (s.c.) no dorso e a inflamação induzida por carragenina. Uma hora antes de inocular a carragenina os animais foram tratados (v.o) com o veículo, indometacina (10 mg/kg) ou ExHPpg/Fr2Ppg/sub-frações. Após 4 h o exsudato da bolha foi coletado para contagem total e diferencial de leucócitos (Panótico rápido) e dosagem de proteínas (biureto), e a pele referente à bolha foi removida para análises macroscópica e histológica (HE). Também foi realizado estudo de migração de neutrófilos pelo ensaio do <i>transwell</i>. Após demonstração do efeito antiedematogênico do ExHPpg, este foi fracionado em quatro frações. A fração Fr2Ppg, mais ativa no modelo de edema de pata, também inibiu os diferentes parâmetros inflamatórios avaliados no modelo de bolha de ar, com a menor dose testada, e foi sub-fracionada em cinco sub-frações. Destas, as SF2.1 e SF2.2 foram as que mostraram melhor efeito anti-inflamatório pelo modelo da bolha de ar. Em relação ao grupo controle com carragenina, o ExHppg, Fr2Ppg, SF 2.1 e SF 2.2, na dose 0,02 mg/kg, exerceram inibições de 70,6%, 62,8%, 54,7% e 79% no número total de células no exsudato, reduções de 76,8%, 76,9%, 71,1% e 73,3% na concentração de proteína, respectivamente. Com a dose de 0,2 mg/kg, foram observadas inibições apenas para ExHPpg e SF 2.1, com intensidades menores, na leucometria total (62,9% e 48,62%, respectivamente), e na concentração de proteína para SF 2.1 e SF 2.2 (reduções de 68,2% e 30,4%, respectivamente). As análises macroscópica e histológica mostraram redução importante da vasodilatação e do infiltrado inflamatório pelo tratamento com o ExHppg, Fr2Ppg, SF 2.1 e SF 2.2. No ensaio de <i>transwell</i> a Fr2Ppg exibiu 31,4% de inibição na migração de neutrófilos. As sub-frações SF2.1 e SF2.2 foram avaliadas por GC-MS, identificando-se de diversos compostos majoritários. Em resumo, este trabalho confirma o potencial anti-inflamatório da espécie <i>Pterodon polygalaeflorus</i> e mostra um fracionamento efetivo do ExHPpg quanto à composição e ação anti-inflamatória. / The genus Pterodon comprises some species widely distributed over the central region of Brazil. Its fruits are commercially available on the market of the medicinal flora, being widely used by their pharmacological properties as antipyretic, anti-inflammatory and analgesic. The aim of this work was to guide the fractionation of hexanic extract of <i>Pterodon polygalaeflorus</i> Benth. fruits (ExPpg), using acute inflammation models (paw edema and air pouch). The extract fractionation was done by column chromatography on silica and the samples were analyzed by thin layer chromatography and gas chromatography coupled with mass spectrometer. Paw edema was induced in SW mice by (s.c.) injection of carrageenan in the right paw. One hour before the carrageenan inoculation, animals were orally treated with vehicle (EtOH 15, 1.25 Tween-20), indomethacin (10 mg/kg bow.) or ExHPpg/fractions/sub-fractions of Ppg. In the air pouch model, the cavity was developed in SW mice through sterile air injection (s.c.) on the back and the inflammation induced by carrageenan inoculation in this cavity. An hour before carrageenan injection the animals were orally treated with the vehicle, indomethacin (10 mg/kg) or ExHPpg/Fr2Ppg/sub-fractions. After four hours the air pouch exudate was collected for the total and differential leukocyte counts (fast Panotic) and protein determination (biuret reaction), and the skin of the air pouch was removed to macroscopic and histological analyses (HE). In vitro neutrophil migration was performed by transwell assay. After demonstration of the anti-edematogenic effect of ExHPpg, this extract was fractionated into four fractions. The Fr2Ppg fraction, most active in paw edema model, also inhibited, in the lowest dose tested, the different inflammatory parameters evaluated in air pouch model. Then, it was sub-fractioned in five sub-fractions. The SF 2.1 and SF 2.2 were the sub-fractions with higher anti-inflammatory effects in the air pouch model. In relation to the carrageenan control group, ExHPpg, Fr2Ppg, SF 2.1 and SF 2.2, in 0.02 mg/kg dose, showed inhibitions of 70.6%, 62.8%, 54.7% and 79% in the total leukocyte counts in the exudate and reductions of 76.8%, 76.9%, 71.1% and 73.3% in the protein concentration, respectively. With the 0.2 mg/kg dose, reductions were observed only for total leukocyte counts after treatment with ExHPpg or SF 2.1 (62.9 and 48.62, respectively), and for protein concentration in animals treated with SF 2.1 and SF 2.2 (reductions of 68.2% and 30.4%, respectively). Macroscopic and histological analyses of air pouch tissues showed important reductions of vasodilation and inflammatory infiltrate by treatment with ExHPpg, Fr2Ppg, SF 2.1 and SF 2.2. In the transwell assay the Fr2Ppg exhibited inhibition of 31.4% of neutrophil migration. The SF 2.1 and SF 2.2 sub-fractions were analyzed by GC-MS, identifying some major compounds. Concluding, this work confirms the anti-inflammatory potential of <i>Pterodon polygalaeflorus</i> species and shows an effective ExHPpg fractionation in relation to the composition and anti-inflammatory effect.

As the principal pro-inflammatory prostanoid, prostaglandin E2 (PGE2) serves as mediator of pain and fever in inflammatory reactions. The biosynthesis of PGE2 starts from arachidonic acid (AA). Cyclooxygenase (COX)-1 and/or COX-2 converts AA to prostaglandin H2 (PGH2), and PGE2 synthases transform PGH2 to PGE2. Current mainstream approach for treating inflammation-related symptoms remains the application of traditional non-steroidal anti-inflammatory drugs (tNSAIDs) and selective COX-2 inhibitors (coxibs). As both categories shut down the biosynthesis of all downstream prostanoids, their application renders several deleterious effects including gastrointestinalulceration and cardiovascular risk. Microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors, specifically blocking the production of inflammation-related PGE2, are expected to reduce the adverse effects while retain the anti-inflammation activity. Although several compounds have been reported, only a few have entered clinical trials and none was on the market. Particularly, most of the reported human mPGES-1 inhibitors were not active for wild-type mouse/rat mPGES-1 enzymes, which prevents using the well-established mouse/rat models of inflammation in preclinical studies. Therefore, we expect our designed inhibitors to also be potent against mouse mPGES-1 and thus is suitable for preclinical testing in wild-type mice.

Orientador: Jose de Assis Fonseca Faria / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-15T08:14:09Z (GMT). No. of bitstreams: 1 Walter_EduardoHenriqueMiranda_D.pdf: 5634901 bytes, checksum: 43f060f6a152462fae06f4a9bd631c70 (MD5) Previous issue date: 2010 / Resumo: Os sistemas assepticos sao uma das tecnologias mais empregadas na conservacao de bebidas a temperatura ambiente, compondo um mercado dinamico e promissor de produtos, embalagens e equipamentos. O objetivo desse trabalho foi avaliar um sistema asseptico projetado para produzir 1.000 L/h de leite longa vida e bebidas de alta acidez (dois tipos de chas e quatro refrescos mistos de maca, pera e pessego), com uma taxa de defeitos de 0,1%. As embalagens consistiram em bolsas flexiveis com termossoldagem tipo almofada de polietileno de baixa densidade pigmentado de branco com dioxido de titanio, tradicionalmente utilizado para leite pasteurizado, e laminado multicamada com propriedades de barreira ao oxigenio e a luz. Foram realizadas 15 producoes de leite longa vida e quatro das bebidas de alta acidez. Os testes foram conduzidos num sistema composto por equipamentos comerciais (tanques, bombas, valvulas, tubulacoes e trocador de calor) e em desenvolvimento (tanque de produto e maquina de embalagem tipo forma, enche e fecha), todos destinados as industrias de pequena escala de producao. O sistema foi avaliado com base em testes de esterilizacao comercial e analises de aceitacao sensorial dos produtos. A taxa de defeitos nas producoes de leite longa vida ficou na ordem de 2%, enquanto para as bebidas de alta acidez de 0,4%. A aceitacao sensorial do leite longa vida nas embalagens de polietileno, estocadas no escuro, variou entre quatro e sete semanas, de acordo com a qualidade da materia-prima, enquanto a aceitacao sensorial do produto exposto a luz foi de alguns dias. Na embalagem laminada, a luz nao afetou a estabilidade do produto, que teve uma vida de prateleira entre 12 e 24 semanas, dependendo da materia-prima. Esses resultados comprovaram a viabilidade das embalagens para a conservacao do produto. Entretanto, o sistema devera passar por melhorias, alcancando desse modo o potencial para aplicacao em industrias de pequena escala de producao. A simplicidade da linha de processamento e embalagem contribuiu para quebrar o paradigma de que os sistemas assepticos tem de ser sofisticados e onerosos / Abstract: Aseptic systems are one of the main technologies employed for shelf stable milk and juices, creating a dynamic and promising market for products, packaging and equipments. The aim of this study was to evaluate an aseptic system designed to produce 1000 L/h long-life milk and high acidity soft drinks (two teas and four soft drinks, mixed with apple, pear and peach), with a defect rate of 0.1 %. The packages were pillow-style pouches of low density polyethylene pigmented white with titanium dioxide, traditionally used for packaging pasteurized milk in Brazil, and multilayer laminate with oxygen and light barrier properties. There were performed 15 trials of long-life milk and four of soft drinks. Tests were conducted in the system composed of commercial equipment (tanks, pumps, valves, piping and heat exchanger), product tank, and form/fill/seal packaging machine, all used to small-scale production. The system evaluation was based on tests for commercial sterility and sensory acceptability testing of the products. The defect rate in the production of long-life milk was around 2%, while in high acidity drinks of 0.4%. The sensory acceptability of long-life milk in polyethylene containers, stored in the dark, ranged from four to seven weeks, according to the quality of raw material, while for the products exposed to light, it was of a few days. In the laminated bag, light did not affect the stability of the product, which had a shelf life ranged from 12 to 24 weeks, depending on the raw material used. These results have proved the feasibility of both packs for the conservation of the product. However, the system must go through improvements, thereby achieving the potential for application in small-scale industries. The simplicity of the processing and packaging system helped to break the paradigm that aseptic systems have to be sophisticated and expensive / Doutorado / Doutor em Tecnologia de Alimentos

In light of the exponential growth of the nicotine pouch industry, major tobacco-industry players have shown increasing interest in newer nicotine and tobacco products, despite the lack of discernible differences them. Therefore, this study aims to investigate the differentiation strategies of companies within the Swedish nicotine pouch market, and further, the long-term competitiveness of these strategies. The study has applied an abductive and qualitative method to interpret non-numerical data of 14 companies through a document analysis, complemented by two semi-structured interviews. Empirical findings have been analysed using a conceptual model based on previous theories and research on product differentiation. The thesis identified five traditional, alongside three industry-specific modes of differentiation apparent within the market. Primarily, companies differentiate through branding and marketing, adaptiveness, health advocacy and sustainable branding. Interestingly, besides flavour innovation, technology and innovation-driven differentiation is less common within the industry. Most strategies identified within the market are, however, easily imitable, and therefore not competitive long- term. Ultimately, findings suggest the industry is at a crossroads: Consumers currently hold a significant amount of power with a variety of options and little to nothing swaying them in one direction or the other.

Achyrocline satureoides (Lam) D.C., popularmente conhecida como \"marcela\", é utilizada popularmente para tratar diversas doenças, como mal estar gástrico e intestinal, inflamações, diabetes e outros. Como os mecanismos de ação do extrato de A. satureoides ainda não são conhecidos, o presente trabalho visou esclarecer os mecanismos antiinflamatórios do extrato bruto hidroalcoólico das inflorescências de A. satureoides, focando na migração e função fagocítica e microbicida de neutrófilos. Para tanto, o extrato hidroalcoólico foi administrado por gavage (50, 100 e 250mg/kg) em ratos Wistar, machos, adultos, e a inflamação foi induzida pela injeção do lipopolisacarídeo de E. coli (LPS; 2mL de solução; 500 &#181;g/mL em PBS) no tecido subcutâneo dorsal (modelo da bolsa de ar). Animais controles receberam volumes equivalentes de veículo do extrato e indometacina (30mg/kg). Foram quantificados o número de neutrófilos (câmara de Neubauer e esfregaços corados por Panótico) e a concentração de Leucotrieno B4 e CINC-1 (ELISA) no foco de lesão; a interação leucócito-endotélio em vênulas da microcirculação mesentérica após estímulo in situ pelo lipopolisacarídeo de E.coli (LPS; 30&#181;g/40&#181;L; por microscopia intravital); a expressão de moléculas de adesão e do toll-like receptor (TLR-4), além da quantificação do burst oxidativo (induzido pelo miristato-acetato de forbol - PMA) e fagocitose em neutrófilos circulantes (citometria de fluxo); análise histológica e quantificação dos marcadores hepáticos (AST, ALT e Gama-GT) e renais (uréia e creatinina) no plasma por espectrofotometria. Os resultados obtidos mostraram que o tratamento com o extrato reduziu o número de neutrófilos e a concentração de LTB4 e CINC-1 na bolsa de ar; reduziu a expressão de TLR4 pelos neutrófilos circulantes e a porcentagem de neutrófilos positivos para L-selectina e &#946;2-integrina; inibiu a adesão e o comportamento rolling de leucócitos ao endotélio microvascular; reduziu o burst induzido por PMA; aumentou o potencial de fagocitose, sem alterar o burst induzido por Staphylococcus aureus, não alterou a morfologia tecidual e a concentração sérica dos marcadores de atividade hepática e renal. Em conjunto, os dados obtidos mostram que dose, aparentemente, não tóxica do extrato de A. satureoides exerce efeito antiinflamatório in vivo frente ao LPS, quantificados pela redução da migração e pelas interferências nas atividades fagocítica e microbicida dos neutrófilos. / Achyrocline satureoides (Lam) D.C., popularly known as \"marcela\", is popularly used to treat several diseases, such as gastric and intestinal disorders, inflammation, diabetes and others. As the mechanisms of the extract of A. satureoides have not been elucidated, this study aimed to investigate the anti-inflammatory mechanisms of the crude hydroalcoholic extract of the flowers of A. satureoides, focusing on migration and phagocytic and microbicidal neutrophils function. The hydroalcoholic extract was administered by gavage (50, 100 and 250mg/kg) into male Wistar rats, adult, and inflammation was induced by injection of lipopolysaccharide E. coli (LPS; 2 mL of solution; 500&#181;g/mL in PBS) into the dorsal subcutaneous tissue (air pouch model). Control animals received equivalent volume of extract vehicle or indomethacin solution (30mg/kg). It was quantified the numbers of neutrophils (Neubauer chamber and stained smears by Panoptic) and concentrations of leukotriene B4 (LTB4) and CINC-1 (ELISA) in the focus of the injury; the leukocyte-endothelium interactions in mesenteric venules of the microcirculation after stimulation by LPS (30&#181;g/40&#181;L; intravital microscopy); the adhesion molecules and toll-like receptor (TLR-4) expressions and quantification of oxidative burst and phagocytosis in circulating neutrophils (flow cytometry); histological analysis and the hepatic markers (AST, ALT and gamma-GT) and kidney (urea and creatinine) concentrations in plasma by spectrophotometry. Data obtained showed that the treatment reduced the numbers of neutrophils and concentrations of LTB4 and CINC-1 in the subcutaneous tissue; reduced the TLR4 expression by circulating neutrophils and the number of &#946;2-integrin- and L-selectin-positive neutrophils; inhibited the leukocyte adhesion and the rolling behavior to vascular endothelium; reduced the burst evoked by PMA; increased the phagocytosis without changing the burst induced by Staphylococcus aureus; and did not alter the tissue morphology and concentration of hepatic and renal enzymes in the serum. Together, these data suggest that dose, apparently non-toxic, of extract of A. satureoides exerts anti-inflammatory effect in vivo, quantified by the reduced migration and by interference in the phagocytic and microbicidal activities of neutrophils.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Echinodorus macrophyllus (Alismataceae), conhecida como chapéu de couro no Brasil, é usada popularmente para tratar doenças reumáticas e inflamatórias. Neste trabalho, foram avaliados os efeitos antiinflamatórios do extrato aquoso de E. macrophyllus (EAEm) e suas frações etanólicas no modelo murino de air pouch. Para a obtenção das frações, 7 g do EAEm foram aplicadas em uma coluna cromatográfica aberta de sílica gel eluída com diferentes concentrações de etanol. Os cromatogramas do EAEm/frações foram obtidos usando um sistema de HPLC. Foram obtidas quatro frações, duas delas com maior rendimento. Resumidamente, a bolha de ar foi induzida pela injeção de 5 mL de ar estéril (s.c) no dorso de camundongos SW machos (25-35 g). Após 3 dias, mas 3 mL de ar estéril foram injetados para manter a bolha. No sexto dia, cada grupo (n = 4) foi tratado intraperitoneal (ip) ou oralmente (v.o) com EAEm (25 ou 250 mg/kg), Fr20 ou Fr40 (2,5, 25, 50 ou 100 mg/kg) e os controles com indometacina (10 mg/kg, v.o.) ou veículo (salina). Uma hora depois, 1 mL de salina ou de carragenina 1% estéril foi injetada dentro da bolha. Após 4 h, a cavidade foi lavada com NaCl 0,9%, EDTA 2 mM (1 mL), para a determinação do número de leucócitos, volume do exsudato e concentração de proteínas. Células do exsudato foram preparadas em citocentrífuga e coradas pelo método do Panótico para a contagem diferencial dos leucócitos. Cortes histológicos coletados dos diferentes grupos foram fixados com formol tamponado 10% (pH 7,4) por 7 dias, corados com HE e analisados em MO. A análise da expressão da iNOS e da COX-2 foi realizada em células do exsudato por RT-PCR. O acúmulo de nitrito (NO2&#8722;) no sobrenadante do cultivo de células RAW 264.7 foi determinado usando um ensaio colorimétrico baseado na reação de Griess. Os resultados foram expressos como média EP e comparados usando ANOVA seguido de teste de Dunnet. Os experimentos foram realizados em triplicata. No modelo air pouch, a injeção de carragenina 1% aumentou tanto a migração celular quanto a concentração de proteína no exsudato. Contudo, enquanto o pré-tratamento com a Fr40 aumentou a resposta inflamatória, o pré-tratamento com o EAEm e a Fr20, sobretudo por via i.p., inibiu esta resposta quando comparado ao grupo controle tratado apenas com o veículo. Assim, foram observadas as seguintes razões de inibição da migração de células: EAEm, i.p. a 25 mg/kg (66,44%) e a 250 mg/kg (87,27%) e Fr20 a 2,5 mg/kg (26,89%), 25 mg/kg (60,06%), 50 mg/kg (63,13%) e a 100 mg/kg (77,47%). Em relação à contagem diferencial, o EAEm e a Fr20 afetaram principalmente o número de neutrófilos, inibindo sua migração no exsudato. O EAEm e a Fr20 também reduziram a concentração total de proteínas no exsudato principalmente no tratamento i.p.; EAEm a 25 e 250 mg/kg mostrou 3,33 0,55 e 2,05 0,51 mg/mL, respectivamente, quando comparado aos grupos controles (Indometacina 2.88 0.64 mg/mL; Veículo 5.48 0.88 mg/mL). A Fr20 a 2,5, 25, 50 e 100 mg/kg mostrou 4,788 0,444, 1,417 0,519, 2,474 0,529 e 2,215 0, 361 mg/mL. A análise histológica mostrou infiltrado celular, principalmente composto de leucócitos polimorfonucleares ao longo da derme inflamada de animais tratados com veículo. O tratamento com o EAEm ou Fr20 reduziu a infiltração de leucócitos no tecido inflamado. Além disso, o tratamento com o EAEm e a Fr20 mostrou atividade supressora sobre a expressão de iNOS e COX-2, e mostrou efeitos inibitórios na produção de NO induzida por LPS. Concluindo, todos estes resultados confirmam o potencial antiinflamatório sugerido para esta planta e fornecem uma base para a compreensão de seus mecanismos moleculares de ação. Contudo, outros estudos devem ser realizados para melhor elucidar as vias pelas quais o EAEm e a Fr20 exercem seus efeitos antiinflamatórios. Além disso, estudos fitoquímicos devem ser realizados para identificar os compostos ativos no EAEm e na Fr20. / Echinodorus macrophyllus (Alismataceae), known as "chapéu de couro" in Brazil, is used popularly to treat rheumatic and inflammatory diseases. In this work we have evaluated the anti-inflammatory effects of the aqueous extract of E. macrophyllus (AEEm) and of its ethanolic fractions in mice air pouch model. Fractions were obtained by applying 7 g AEEm on a silica gel chromatography open column eluted with different ethanol concentrations. The fractions so obtained were evaporated under vacuum and lyophilized. Representatives chromatograms of EAEm/fractions were obtained using a HPLC system. We obtained four fractions, two with higher-yielding. Briefly, the air pouch was induced by 5 mL of sterile air injection (s.c.) on the back of male SW mice (25-35 g). After 3 days, 3 mL of sterile air has been injected again to keep it. After six days each group (n = 4) received intraperitoneal (i.p.) or oral (p.o.) treatment with AEEm (25 or 250 mg/kg), Fr20 or Fr40 (2.5, 25, 50 or 100 mg/kg) or controls indomethacin (10 mg/kg, p.o.) and vehicle (saline). One hour later 1 mL saline or carrageenan 1% sterile was injected into the pouch. After 4 h, the cavity was washed with NaCl 0.9%, EDTA 2 mM (1 mL), for determination of leukocyte numbers, final exudate volume and protein concentration. Cytospin preparations of exudates were stained with Panotic method for differential leukocyte count. Histological sections of tissue collected from different groups were fixed with 10% buffered formalin (pH 7.4) for 7 days and stained with HE and analyzed by MO. The iNOS and COX-2 expression analyses were performed on the exudate cells by RT-PCR. Accumulated nitrite (NO2&#8722;) in the media obtained from the RAW 264.6 cell cultures was determined using a colorimetric assay based on the Griess reaction. Results were expressed as mean SEM and compared using ANOVA and Dunnet&#8223;s test. Experiments were performed in triplicate. In air pouch model, carrageenan 1% increased both the cell migration and the exudate protein level. However, while pretreatment with Fr40 increased inflammatory response, the pretreatment with AEEm and Fr20, mainly i.p. inhibited its when compared to the control group treated only the vehicle. So, the following rates of inhibition of cell migration were observed: AEEm, i.p. at 25 mg/kg (66.44%) and at 250 mg/kg (87.27%) and Fr20 at 2.5 mg/kg (26.89%), at 25 mg/kg (60.06%), at 50 mg/kg (63.13%) and at 100 mg/kg (77.47%). Regarding the differential count, the EAEm and Fr20 affected mainly the content of neutrophils, inhibiting the neutrophils migration in exudate. AEEm and Fr20 also reduced the total protein level in exudates mainly in the i.p. treatment. AEEm at 25 and 250 mg/kg showed 3.33 0.55 and 2.05 0.51 mg/mL, respectively, when compared to controls groups (Indomethacin 2.88 0.64 mg/mL; vehicle 5.48 0.88 mg/mL). Fr20 at 2.5, 25, 50 and 100 mg/kg showed 4.788 0.444, 1.417 0.519, 2.474 0.529 and 2.215 0.361 mg/mL. The histological analysis showed cellular infiltrate, mainly composed by polymorphonuclear leukocytes throughout the inflamed dermis of animals treated with vehicle. Treatment with AEEm or Fr20 reduced the leukocyte infiltrate on inflamed tissue. In addition, treatment with AEEm and Fr20 showed suppressive activity on iNOS and COX-2 expression, and showed inhibitory effects on LPS-induced nitric oxide production. In conclusion, all these findings support an anti-inflammatory potential suggested for this plant and provides a basis for understanding their action molecular mechanism. However, further studies should be undertaken to better elucidate the pathways by which AEEm and Fr20 exert their anti-inflammatory effects. In addition, phytochemical studies must be underway to identify active compounds in AEEm and Fr20.

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This diploma thesis is concerned with assessment of two methods of gauging the flow rate used in the sphere of official measurements on profiles with an unrestricted water level. This is the method of collecting liquid into a volumetric vessel and the method of collecting liquid into a weighing vessel (pouch). The main purpose of this work was to specify uncertainties determined using method A and B for these two methods of gauging flow rate within the terms of addressing the Metrology Development Plan of the Czech Office for Standards, Metrology and Testing, because these uncertainties were previously determined using older methodology and using less accurate flow rate benchmarks. The entire work is based on extensive experimental measurement of the flow rate, using the assessed methods, executed at the Laboratory of Water Management Research in Brno. The method of collecting liquids into a volumetric vessel was assessed using four various vessel volumes - 9 l, 15 l, 30 l and 50 l. Relative uncertainties determined using method A and B in relation to flow rate are determined for each vessel in the experimental section of this work. Within the scope of this thesis, these uncertainties were also determined for the method of collection of liquid into weighing vessel (pouch), which was assessed for flow rates of from 0,5 l/s to 10,0 l/s.

Homotrimeric P2X3 and heteromeric P2X2/3 receptors are present in sensory ganglia and participate in pain perception. In order to develop pharmacological antagonists for these receptors, it is important to clarify which amino acid (AA) residues constitute the agonist binding pouch as well as to learn the stoichiometry of the receptor subunits forming a heteromeric receptor. We expressed the homomeric human (h)P2X3 receptor or its mutants in HEK293 cells and measured the ATP-induced responses by the whole-cell patch-clamp method. For the binding-site mutants, all conserved and some non-conserved AAs in the four nucleotide binding segments (NBSs) of the P2X3 subunit were sequentially replaced by alanine. Especially the positively charged AAs Lys and Arg appeared to be of critical importance for the agonist effects. We concluded that groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the P2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor. We were also interested to find out, whether two heteromeric receptors (P2X2/3 and P2X2/6), where P2X2 combines with two different partners, have an obligatory subunit stoichiometry of 1:2 or whether the subunit stoichiometry may be variable. For this purpose we used non-functional P2X2, P2X3 and P2X6 subunit-mutants to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. The subunit stoichiometry of P2X2/3 and P2X2/6 was found to be 1:2 and 2:1, respectively. Thus, recognitions sites between P2X2 and its partners rather than random association may govern the subunit compositions of the receptor trimers.