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La pathologie pleurale est fréquente dans la pratique médicale. Dans certaines séries, une effusion pleurale est retrouvée chez 10 % des patients hospitalisés en médecine interne. Les épanchements pleuraux peuvent refléter tant une pathologie pleurale princeps que les manifestations dun grand nombre de pathologies pulmonaires et extra-thoraciques. La recherche étiologique de ces pleurésies impose un diagnostic différentiel vaste sous-tendu par des processus physiopathologiques très différents. En particulier, il est de première importance didentifier une pathologie pleurale maligne eu égard à sa prise en charge spécifique et à son pronostic extrêmement réservé. Comme nous lavons rappelé dans une revue de littérature explicitant lexploration pleurale actuellement admise, limagerie conventionnelle ne présente pas de critère spécifique pour le diagnostic différentiel bénin vs malin dune pathologie pleurale. De même, la rentabilité de la thoracocentèse et de la biopsie pleurale percutanée demeure relativement faible et justifie fréquemment le recours à la thoracoscopie médicale. Dans notre travail, nous avons poursuivi trois objectifs. Notre premier objectif a été dévaluer lintérêt de la tomographie à émission de positrons (TEP) utilisant pour traceur le 18F-fluorodéoxyglucose (18FDG) dans le diagnostic étiologique de la pathologie pleurale et, en particulier, dans lidentification des effusions malignes. Dans ce cadre, nous avons aussi évalué la faisabilité dune mesure semi-quatitative de lhyperfixation du 18FDG dans le diagnostic différentiel bénin vs malin dune pleurésie, ainsi que dans la localisation thoracique vs extra-thoracique des pleurésies métastatiques. Nous avons pu établir deux valeurs seuils distinguant ces groupes respectifs. Notre deuxième objectif a été dévaluer la valeur pronostique de lintensité de lhyperfixation pleurale du 18FDG. Nous avons ainsi démontré que la quantification de lhyperfixation du 18FDG au niveau de la pleurésie présentait une valeur pronostique chez des patients présentant une néoplasie pulmonaire non à petites cellules avec pleurésie métastatique. Fort de ces données, nous avons positionné limagerie métabolique dans lapproche diagnostique de la pathologie pleurale et précisé ses indications cliniques. Enfin, notre troisième objectif a été dévaluer la valeur de lanalyse biochimique, en particulier de cytokines ou de facteurs de croissance dans le diagnostic étiologique des épanchements pleuraux. Nous avons étudié lintérêt du dosage pleural de 3 cytokines/facteurs de croissance (Interleukine-6 (IL-6)), Transforming growth factor-β1 (TGF-β1) et Vascular endothial growth factor (VEGF)) dans les pleurésies malignes. Nous avons démontré que, contrairement à lIL-6 et au TGF-β1, seul le VEGF présentait un taux significativement plus élevé dans les pleurésies malignes comparativement aux bénignes. Nous avons montré que le taux de VEGF pleural était corrélé à des marqueurs de lagressivité de la pleurésie (taille de lépanchement, nombre de globules rouges au sein de la pleurésie, taux pleural de glucose et de lactate déshydrogénase (LDH)). Toutefois, malgré ses taux pleuraux accru dans les pleurésies malignes, le dosage du VEGF dans le liquide pleural revêtait une rentabilité diagnostique modeste dans la distinction entre une pleurésie maligne et une bénigne. En outre, nous avons démontré que dans le diagnostic bénin vs malin dune pleurésie, la combinaison des trois critères classiques de Light, définissant un exsudat (rapport des taux pleuraux/sanguins des protéines, des LDH et la valeur absolue du taux de LDH pleuraux supérieur au 2/3 de la norme supérieur), présentait une sensibilité et une rentabilité diagnostiques similaires à celles du VEGF voisine de 60%. En conclusion, limagerie métabolique par TEP est discriminante pour affirmer la malignité dune pathologie pleurale et constitue un facteur pronostique indépendant de survie des patients atteints de pleurésies métastatiques de tumeur pulmonaire non à petite cellule. Dès lors la TEP sintègre dans lalgorithme décisionnel du diagnostic, et de la prise en charge dune pathologie pleurale et permet déviter de recourir systématiquement à une technique dinvestigation pleurale invasive. Quoique significativement plus élevés dans les pleurésies malignes, le taux pleural de VEGF présente un pouvoir discriminant modeste dans le diagnostic différentiel bénin vs malin dune pleurésie exsudative. Toutefois, ces observations aident à la compréhension physiopathologique des pleurésies tumorales et ouvrent la porte à de nouvelles thérapeutiques. Summary: Pleural involvements are common in various respiratory diseases including inflammatory, infectious, occupational or neoplastic pathological entities and pleural thickening and pleurisy are usual radiological presentation. Pleural pathology is found in 10 % of the patients hospitalized in a department of internal medicine. Pleural effusions can be induced both by primary pleural disease and by lung and extra-thoracic pathologies. Finding their etiology imposes a vast and sometimes difficult differential diagnosis as different physiopathological processes may be involved in thickening and effusions. In particular, its of first importance to identify malignant pleural disease in consideration of its specific treatment and its dire prognosis. Here we review the currently accepted pleural diseases investigation methods. There are no specific diagnosis criteria for malignancy with conventional pleural imaging by radiology, ultrasound, scanning and nuclear magnetic resonance. The analysis of chemistry, bacteriology and cytology of pleural fluid obtained by thoracocentesis makes a significant contribution to the diagnostic approach even though its diagnostic sensitivity does not exceed 62 %. Similarly, although allowing tissue examination, the sensitivity of closed-needle pleural biopsies does not exceed 51 %. Therefore the invasive thoracoscopy is often justified to find out the accurate diagnosis of pleural diseases, achieving a diagnostic sensitivity greater than 95%. Our goals in this thesis were threefold. Our first objective was to study the accuracy of positron emission tomography (TEP) using the 18F-fluorodeoxyglucose (18FDG) to distinguish between benign and malignant disease in exudative lymphocytic pleural effusions and pleural thickening. We evaluated the diagnostic performance of 18FDG TEP imaging with semi-quantitative analysis for differentiating benign from malignant pleural effusions and for guiding the search for the primary tumor (extra-thoracic vs thoracic location) of pleural metastases. We established two threshold values distinguishing benign from malignant pleural lesions on the one hand and extra-thoracic from thoracic malignant pleurisies on the other hand. Our second objective was to estimate the value of the pleural 18FDG uptake intensity as an independent survival prognosis factor in non small cell lung cancer with pleural extension (T4 NSCLC). We showed an inverse relationship between survival and pleural metabolic activity while there was no relationship between survival and the primary tumor metabolic activity. Finally, our third objective was to assess the value of biochemical analysis and in particular the value of cytokine (IL-6) and growth factors ((TGF-ß 1, VEGF) in differentiating benign from malignant pleural effusions. No significant difference was found between malignant and benign effusions with respect to IL-6 and TGF- pleural levels. In contrast to IL-6 and TGF-pleural VEGF concentrations were significantly higher in malignant than in benign effusions. Furthermore VEGF levels correlated with the effusion volume, pleural LDH and glucose levels and red cell counts. Nevertheless, although being greater in malignant pleural effusions, pleural VEGF levels are of a rather limited clinical interest in distinguishing between malignant and benign pleural effusions. VEGF levels did not perform better than the use the 3 combined Lights criteria in this attempt with a quite similar sensitivity around 60%. In conclusion, the metabolic imaging by TEP is an accurate method for differentiating benign from malignant pleural diseases and the intensity of the pleural 18FDG uptake seems to be an independent survival prognostic factor in non small cell lung cancer with pleural malignant effusion. Our results suggest TEP could be integrated in the investigation algorithm of pleural diseases and facilitate decision-making as to when begin invasive procedures. Raised pleural VEGF levels in malignant effusions are related to the aggressiveness of the tumors and, although of limited diagnostic value, should prompt the evaluation in the near future of new treatment strategy using anti-VEGF.

Tuberculosis is the second leading cause of morbidity and mortality worldwide due to an infectious disease. Development of a new tuberculosis (TB) vaccine would be facilitated by a better understanding of the mechanisms of protection induced by the current TB vaccine, Mycobacterium bovis BCG. Recombinant guinea pig (rgp)CCL5 and anti-rgpCCL5 were developed and characterized. The biological activity of rgpCCL5 was determined in a chemotaxis assay using T lymphocytes and pleural exudate cells. The specificity of rabbit anti-rgpCCL5 polyclonal antibody was confirmed by Western blot. RgpCCL5 was used to stimulate alveolar and peritoneal macrophages in vitro. and cytokine/chemokine gene expression was evaluated using real-time PCR. RgpCCL5 stimulated TNFα, IL-1β, CCL2, and CXCL8 mRNA expression and TNFα protein production (as assessed in the L929 cell bioassay) in macrophages. The effect of BCG-vaccination on CCL5 expression and production in leukocytes infected with M. tuberculosis was examined in vitro and in vivo. Polyclonal anti-rgpCCL5 was used to develop an ELISA assay to quantify gpCCL5 protein levels, and real-time PCR was used to detect CCL5 mRNA. Leukocytes isolated from BCG-vaccinated guinea pigs and infected in vitro with virulent M. tuberculosis demonstrated significantly elevated gpCCL5 mRNA and protein compared to cells from naive animals. The response of gpCCL5 to M. tuberculosis in vivo was studied in tuberculous pleural effusions, where peak levels of CCL5 mRNA and protein were reached at day 4 post-induction. Disease severity, cellular differentiation, histology, and cytokine/chemokine mRNA levels in pleural cells and granulomas were analyzed on day 4 in guinea pigs induced with tuberculous pleurisy and treated with either rgpCCL5 or anti-rgpCCL5 by direct intra-pleural injection. In these studies, neutralizing CCL5 resulted in reduced macrophage accumulation, diminished levels of IFNγ, TNFα, and CCL5 mRNA in pleural effusion cells, and reduced spontaneous lymphocyte proliferation. Together these studies suggest an important role for gpCCL5 in activating leukocytes during M. tuberculosis infection.

Baccharis trimera (Less.) DC. (Asteraceae), popularmente conhecida como carqueja, é nativa da América sendo amplamente empregada na medicina popular para problemas hepáticos, digestivos, e como anti-inflamatória. Em sua composição química destaca-se maior acúmulo de flavonas, flavonóis e de diterpenos labdanos e clerodanos. Diversos estudos vêm sendo realizados, em especial para as atividades anti-inflamatórias e antimicrobianas. Com isso, iniciou-se o fracionamento do extrato de B. trimera visando à identificação da fração ou compostos responsáveis pela atividade anti-inflamatória. Das partes aéreas de B. trimera foram obtidas as frações diclorometano, acetato de etila, n-butanol e aquosa, além das frações saponinas e flavonóides. Essas seis frações foram avaliadas para a atividade anti-inflamatória através do modelo de inflamação aguda da pleurisia induzida por carragenina e foram avaliados os seguintes parâmetros no líquido pleural: volume de exsudato, contagem de leucócitos totais, diferencial citológico, concentração protéica e determinação de NO no exsudato. Todas as frações testadas diminuíram significativamente os parâmetros analisados quando comparados ao grupo carragenina (ANOVA, P < 0,05), evidenciando assim potencial atividade antiinflamatória. A determinação de fenóis totais e a atividade antioxidante foram avaliadas através dos métodos de Folin-Ciocalteu e através do ensaio espectrofotométrico com o radical 2,2 difenil-1-picril-hidrazil (DPPH.), respectivamente. As frações flavonóides e acetato de etila (1 mg/ml) apresentaram alto potencial antioxidante, não se diferenciando estatisticamente (ANOVA, P < 0,05) das substâncias de referência vitamina C, quercetina e luteolina nas mesmas concentrações, sugerindo dessa forma que a ação antioxidante destas frações devese à presença de flavonóides. A continuidade dos estudos fitoquímicos e a identificação do potencial antioxidante e anti-inflamatório de B. trimera, além de contribuir para a diferenciação química das espécies do gênero Baccharis poderão credenciá-la para o desenvolvimento de um fármaco com atividade anti-inflamatória e antioxidante. / Baccharis trimera (Less.) DC. (Asteraceae), popularly known as "carqueja", is native to America and it is widely used in traditional medicine for gastrointestinal treatments, for liver diseases, as digestive and to anti-inflammatory purposes. It presented accumulation of flavones, flavonols and diterpenes. Several studies have been performed to study its anti-inflammatory and antimicrobial activities. Considering this, we initiated the fractionation of B. trimera extract in order to identify the fraction or compounds responsible for its anti-inflammatory activity. From the aerial parts of B. trimera it were obtained the fractions: dichloromethane, ethyl acetate, n-butanol, aqueous and also saponins and flavonoids fractions. All fractions were characterized by TLC. These six fractions were evaluated for the anti-inflammatory activity using the carrageenan-induced pleurisy, as a model of acute inflammation via i.p. The following parameters were determined in the pleural fluid: the exudate volume, total leukocyte count, cytological differences, protein concentration and determination of NO. All tested fractions decreased significantly the analyzed parameters when compared to the carrageenan group (ANOVA, P < 0.05), showing potential antiinflammatory activity. The determination of total phenols and antioxidant activity were evaluated by the Folin-Ciocalteu spectrophotometric assay and by the radical 2.2- diphenyl-1-picryl-hidrazil (DPPH.) method, respectively. The flavonoid and ethyl acetate fractions (1 mg / ml) showed high antioxidant potential that were similar (ANOVA, P < 0.05) from that of the reference substances: vitamin C, quercetin and luteolin in the same concentrations, thus suggesting that the antioxidant activity of these fractions is due to the presence of flavonoids. The phytochemical studies and the identification of potential antioxidant and anti-inflammatory activities of B. trimera, will contribute to the chemical differentiation of Baccharis species and also it should support the development of a drug with anti-inflammatory and antioxidant activity.

Baccharis trimera (Less.) DC. (Asteraceae), popularmente conhecida como carqueja, é nativa da América sendo amplamente empregada na medicina popular para problemas hepáticos, digestivos, e como anti-inflamatória. Em sua composição química destaca-se maior acúmulo de flavonas, flavonóis e de diterpenos labdanos e clerodanos. Diversos estudos vêm sendo realizados, em especial para as atividades anti-inflamatórias e antimicrobianas. Com isso, iniciou-se o fracionamento do extrato de B. trimera visando à identificação da fração ou compostos responsáveis pela atividade anti-inflamatória. Das partes aéreas de B. trimera foram obtidas as frações diclorometano, acetato de etila, n-butanol e aquosa, além das frações saponinas e flavonóides. Essas seis frações foram avaliadas para a atividade anti-inflamatória através do modelo de inflamação aguda da pleurisia induzida por carragenina e foram avaliados os seguintes parâmetros no líquido pleural: volume de exsudato, contagem de leucócitos totais, diferencial citológico, concentração protéica e determinação de NO no exsudato. Todas as frações testadas diminuíram significativamente os parâmetros analisados quando comparados ao grupo carragenina (ANOVA, P < 0,05), evidenciando assim potencial atividade antiinflamatória. A determinação de fenóis totais e a atividade antioxidante foram avaliadas através dos métodos de Folin-Ciocalteu e através do ensaio espectrofotométrico com o radical 2,2 difenil-1-picril-hidrazil (DPPH.), respectivamente. As frações flavonóides e acetato de etila (1 mg/ml) apresentaram alto potencial antioxidante, não se diferenciando estatisticamente (ANOVA, P < 0,05) das substâncias de referência vitamina C, quercetina e luteolina nas mesmas concentrações, sugerindo dessa forma que a ação antioxidante destas frações devese à presença de flavonóides. A continuidade dos estudos fitoquímicos e a identificação do potencial antioxidante e anti-inflamatório de B. trimera, além de contribuir para a diferenciação química das espécies do gênero Baccharis poderão credenciá-la para o desenvolvimento de um fármaco com atividade anti-inflamatória e antioxidante. / Baccharis trimera (Less.) DC. (Asteraceae), popularly known as "carqueja", is native to America and it is widely used in traditional medicine for gastrointestinal treatments, for liver diseases, as digestive and to anti-inflammatory purposes. It presented accumulation of flavones, flavonols and diterpenes. Several studies have been performed to study its anti-inflammatory and antimicrobial activities. Considering this, we initiated the fractionation of B. trimera extract in order to identify the fraction or compounds responsible for its anti-inflammatory activity. From the aerial parts of B. trimera it were obtained the fractions: dichloromethane, ethyl acetate, n-butanol, aqueous and also saponins and flavonoids fractions. All fractions were characterized by TLC. These six fractions were evaluated for the anti-inflammatory activity using the carrageenan-induced pleurisy, as a model of acute inflammation via i.p. The following parameters were determined in the pleural fluid: the exudate volume, total leukocyte count, cytological differences, protein concentration and determination of NO. All tested fractions decreased significantly the analyzed parameters when compared to the carrageenan group (ANOVA, P < 0.05), showing potential antiinflammatory activity. The determination of total phenols and antioxidant activity were evaluated by the Folin-Ciocalteu spectrophotometric assay and by the radical 2.2- diphenyl-1-picryl-hidrazil (DPPH.) method, respectively. The flavonoid and ethyl acetate fractions (1 mg / ml) showed high antioxidant potential that were similar (ANOVA, P < 0.05) from that of the reference substances: vitamin C, quercetin and luteolin in the same concentrations, thus suggesting that the antioxidant activity of these fractions is due to the presence of flavonoids. The phytochemical studies and the identification of potential antioxidant and anti-inflammatory activities of B. trimera, will contribute to the chemical differentiation of Baccharis species and also it should support the development of a drug with anti-inflammatory and antioxidant activity.

Baccharis trimera (Less.) DC. (Asteraceae), popularmente conhecida como carqueja, é nativa da América sendo amplamente empregada na medicina popular para problemas hepáticos, digestivos, e como anti-inflamatória. Em sua composição química destaca-se maior acúmulo de flavonas, flavonóis e de diterpenos labdanos e clerodanos. Diversos estudos vêm sendo realizados, em especial para as atividades anti-inflamatórias e antimicrobianas. Com isso, iniciou-se o fracionamento do extrato de B. trimera visando à identificação da fração ou compostos responsáveis pela atividade anti-inflamatória. Das partes aéreas de B. trimera foram obtidas as frações diclorometano, acetato de etila, n-butanol e aquosa, além das frações saponinas e flavonóides. Essas seis frações foram avaliadas para a atividade anti-inflamatória através do modelo de inflamação aguda da pleurisia induzida por carragenina e foram avaliados os seguintes parâmetros no líquido pleural: volume de exsudato, contagem de leucócitos totais, diferencial citológico, concentração protéica e determinação de NO no exsudato. Todas as frações testadas diminuíram significativamente os parâmetros analisados quando comparados ao grupo carragenina (ANOVA, P < 0,05), evidenciando assim potencial atividade antiinflamatória. A determinação de fenóis totais e a atividade antioxidante foram avaliadas através dos métodos de Folin-Ciocalteu e através do ensaio espectrofotométrico com o radical 2,2 difenil-1-picril-hidrazil (DPPH.), respectivamente. As frações flavonóides e acetato de etila (1 mg/ml) apresentaram alto potencial antioxidante, não se diferenciando estatisticamente (ANOVA, P < 0,05) das substâncias de referência vitamina C, quercetina e luteolina nas mesmas concentrações, sugerindo dessa forma que a ação antioxidante destas frações devese à presença de flavonóides. A continuidade dos estudos fitoquímicos e a identificação do potencial antioxidante e anti-inflamatório de B. trimera, além de contribuir para a diferenciação química das espécies do gênero Baccharis poderão credenciá-la para o desenvolvimento de um fármaco com atividade anti-inflamatória e antioxidante. / Baccharis trimera (Less.) DC. (Asteraceae), popularly known as "carqueja", is native to America and it is widely used in traditional medicine for gastrointestinal treatments, for liver diseases, as digestive and to anti-inflammatory purposes. It presented accumulation of flavones, flavonols and diterpenes. Several studies have been performed to study its anti-inflammatory and antimicrobial activities. Considering this, we initiated the fractionation of B. trimera extract in order to identify the fraction or compounds responsible for its anti-inflammatory activity. From the aerial parts of B. trimera it were obtained the fractions: dichloromethane, ethyl acetate, n-butanol, aqueous and also saponins and flavonoids fractions. All fractions were characterized by TLC. These six fractions were evaluated for the anti-inflammatory activity using the carrageenan-induced pleurisy, as a model of acute inflammation via i.p. The following parameters were determined in the pleural fluid: the exudate volume, total leukocyte count, cytological differences, protein concentration and determination of NO. All tested fractions decreased significantly the analyzed parameters when compared to the carrageenan group (ANOVA, P < 0.05), showing potential antiinflammatory activity. The determination of total phenols and antioxidant activity were evaluated by the Folin-Ciocalteu spectrophotometric assay and by the radical 2.2- diphenyl-1-picryl-hidrazil (DPPH.) method, respectively. The flavonoid and ethyl acetate fractions (1 mg / ml) showed high antioxidant potential that were similar (ANOVA, P < 0.05) from that of the reference substances: vitamin C, quercetin and luteolin in the same concentrations, thus suggesting that the antioxidant activity of these fractions is due to the presence of flavonoids. The phytochemical studies and the identification of potential antioxidant and anti-inflammatory activities of B. trimera, will contribute to the chemical differentiation of Baccharis species and also it should support the development of a drug with anti-inflammatory and antioxidant activity.

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Silva.pdf: 2397176 bytes, checksum: 10d2601e2d4c7bb5bd425131e63a0ebc (MD5) Previous issue date: 2016-04-29 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Inflammation is a physiological process that begins in response to infection or tissue damage. It is a mechanism focusing on tissue repair after injury and consists of a cascade of cellular and microvascular events that aims to remove damaged tissue and generation of new ones. In this cascade is observed an increasing in microvascular permeability, followed by adhesion and cell infiltration at the site of injury, apoptosis, and growth of new tissue and blood vessels. The nitrostyrene is a group of compounds consisting of an aromatic ring linked to a chain of the nitro group (NO2). It has been studied by having various biological activities such as anti-apoptotic, anti-platelet or anti-microbial, although few studies have been made for the anti-inflammatory activity of these compounds. The aim of this study was to evaluate anti-inflammatory activity of four new compounds from the class of nitrostyrene (NPA, 7B, 7D and 7E) in in vitro/in vivo assays. Initially, the compounds were submitted to in vitro testing in a murine macrophages model activated by lipopolysaccharide (LPS) and MRC5 human lung fibroblast in order to assess the cytotoxic potential of such compounds. Shortly thereafter, they were evaluated for inhibition of nitric oxide (NO·), for dosage of the cytokine TNF-α and evaluation of the expression of proteins involved in the inflammatory process. To evaluate the acute inflammatory process in vivo was performed edema induction by carrageenan tests on zebrafish model of LPS-induced pleurisy in mice. The four new compounds tested showed cytotoxic activity on J774A.1 and MRC5 cell when tested in the concentrations (3.12-25 μg/ml). For THP-1 cytotoxic activity was starting concentration of 10 μg/ml. All compounds inhibited nitric oxide production by macrophages activated by LPS but only the NPA showed a possibly significant concentration-response at the concentrations of 10 and 20 μg/ml. This same compound also reduced TNF-α, NF-B and ERK 1/2 levels in THP-1 cells. The NPA possibly also inhibited the carrageenan-induced edema in Zebrafish fish and leukocyte migration into the pleural cavity of mice by inhibiting the intracellular signaling pathways NF-B and ERK 1/2 in in vivo assays. In conclusion, compounds tested in this study not only presented significant cytotoxic activity, as also showed an excellent in vitro/in vivo anti-inflammatory potential, most likely by acting in the inhibition of NF-B and ERK 1/2 pathways. Data were important to explore the biological activity of these novel compounds and their possible application in the treatment of inflammatory diseases. / A inflamação é um processo fisiológico que se inicia em resposta a uma infecção ou dano aos tecidos. É um mecanismo direcionado ao reparo tecidual após a lesão e consiste em uma cascata de eventos celulares e microvasculares que tem como objetivo a remoção de tecidos danificados e a geração de outros novos. Nessa cascata é observado o aumento da permeabilidade microvascular, seguido pela adesão e infiltração de células no local da lesão, apoptose celular e crescimento de novos tecidos e vasos sanguíneos. Os nitroestirenos são um grupo de compostos constituído por anel aromático ligado a uma cadeia do grupo nitro (NO2). Eles têm sido estudados por apresentar diversas atividades biológicas tais como anti-apoptótica, antiplaquetária e antimicrobiana, entretanto poucas pesquisas têm sido realizadas quanto à atividade anti-inflamatória desses compostos. O objetivo deste estudo foi avaliar atividade anti-inflamatória de quatro novos compostos da classe dos nitroestirenos (NPA, 7B, 7D e 7E) em ensaios in vitro/in vivo. Inicialmente, os compostos foram submetidos a ensaio in vitro em modelo de macrófagos murino ativados por lipopolissacarídeo (LPS) e fibroblastos de pulmão humano MRC5, a fim de avaliar a capacidade citotóxica desses compostos. Logo em seguida, foram avaliados quanto à inibição de óxido nítrico (NO·), dosagem da citocina TNF-α e avaliação da expressão de proteínas envolvidas com o processo inflamatório. Para avaliar o processo anti-inflamatório agudo in vivo foi realizado ensaios de indução do edema por carragenina em peixe Zebrafish e o modelo de pleurisia induzido por LPS em camundongos. Os quatro novos compostos testados apresentaram atividade citotóxica em célula J774A.1 e MRC5 quando testados nas concentrações (3.12-25 μg/mL). Para THP-1 a atividade citotóxica foi apartir da concentração 10 μg/mL. Todos os compostos inibiram a produção de óxido nítrico em macrófago ativado por LPS, entretanto somente NPA apresentou uma possível concentração-resposta significativa nas concentrações de 10 e 20 μg/mL. Este mesmo composto também reduziu os níveis de TNF-α, NF-B e ERK 1/2 em células THP-1. O NPA também possivelmente inibiu o edema induzido por carragenina em peixe Zebrafish e a migração de leucócitos na cavidade pleural de camundongos através da inibição das vias de sinalização intracelular NF-B e ERK 1/2 em ensaios in vivo. Em conclusão, compostos testados neste estudo nao somente apresentaram atividade citotóxica significativa, como também demonstraram um excelente potencial anti-inflamatório in vitro e in vivo, provavelmente por atuação de inibição das vias NF-B e ERK 1/2. Os dados obtidos foram importantes para exploração da atividade biológica destes novos compostos e sua possível aplicação na terapêutica de doenças inflamatórias.

Euclea natalensis A.DC. belongs to the Ebenaceae family, and is extensively distributed along the eastern coast of southern Africa. Many Euclea species are widely gathered by indigenous people because of their medicinal properties. Roots of these plant species are frequently used to treat respiratory complications such as chest pains, bronchitis, pleurisy and asthma. Ground root powder is topically applied in cases of leprosy and is used by some ethnic groups to treat toothache and headache. The bioactivity encountered is attributable to naphthoquinones, which are common phenolic compounds in the Ebenaceae family. Naphthoquinones isolated from E. natalensis (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) have exhibited a broad spectrum of antimicrobial activity. The demand for these products will escalate due the amount of plant material required to further research. We need to explore techniques that can maximize their productivity. The present study was conducted on E. natalensis, in an attempt to establish if there exists any correlation between the accumulation of naphthoquinones and stages of seedling growth, seasonal fluctuations and application of fertilizers. A possible correlation between seedling growth stages and the accumulation of naphthoquinones (shinanolone, 7-methyljuglone and diospyrin) was investigated in seeds and seedlings of Euclea natalensis. In this study, the seeds represented the first stage, whereas the second seedling stage was defined as the stage when the radicles were about 6 cm long. The lengths of the seedlings at the third, fourth and fifth seedling stages were 9 cm, 12 cm and 16 cm respectively. Plant materials collected from the five seedling stages were separately extracted using chloroform and the naphthoquinones were then quantified by means of High Performance Liquid Chromatography (HPLC). Mobile phase of MeCN: H2O: AcOH (62.5: 32.5: 5) was used as an eluent in an isocratic mode and at a flow rate of 0.8 ml/min. Standard curves of each of the four compounds were obtained by making a series of dilutions in the concentration range of 22.5 µg/ml to 2.25 µg/ml. Ten microlitres of each dilution was injected three times into the HPLC, and the run time for each injection was 20 minutes. Calibration curves were then generated and used for the quantification of each compound. Shinanolone, which was the only naphthoquinone detectible in seeds, accumulated at variable rates (P<0.01) and no trend could be established between its synthesis and seedling growth. The content of shinanolone ranged from 87.5 mg/kg dry weight (dw) in seeds to a high mean value of 1047 mg/kg (dw) during the fourth seedling stage. A significant correlation (P<0.01) was found between the mean concentrations of 7-methyljuglone and seedling growth. 7-Methyljuglone was quantified at a high mean level of 5003 mg/kg during the third seedling stage and was not detected in the seed samples. A positive correlation (P<0.01) was established between the concentration of diospyrin and seedling stages. Diospyrin was detected at an elevated mean concentration of 6182 mg/kg during the fifth seedling stage, which was higher than the other quantified naphthoquinones. Seasonal variation of naphthoquinones (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) was investigated from eleven plants of E. natalensis subsp. natalensis growing in natural populations, over a period of four seasons. The roots were harvested, dried, extracted and analysed as in the previous study. The mean levels of shinanolone and 7-methyljuglone were found to be uniform in all the seasons and no statistically significant variation could be found between seasonal changes and their mean concentrations. Accumulation of isodiospyrin and neodiospyrin varied significantly with seasonal changes (P<0.05). These two bioactive naphthoquinones were detected only in summer and autumn respectively, and not in winter. A statistically significant variation (P<0.05) was established between the levels of diospyrin and seasonal fluctuations. Diospyrin was detected at a mean concentration of 3190 mg/kg (dw) during spring, which was higher than the other naphthoquinones quantified in all four seasons. The effect of NPK fertilizers on growth performance and accumulation of naphthoquinones (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) in seedlings of E. natalensis grown in shade and under field conditions was investigated. Each group was subdivided into four subgroups, which were then subjected to four respective treatments of water-soluble foliar feed (2:1:2 (44) NPK) at three different concentrations. Treatments tested were as follows: Treatment 1 at 40 g/l, Treatment 2 and Treatment 3 at 20 g/l and 10 g/l respectively. The control group received only supplemental water. The first harvest was conducted after 6 months of application of fertilizers and the second one was done after 12 months of treatment. Roots and shoots were harvested and analysed separately. The naphthoquinones were quantified as previously described. The bioactivity of root extracts from seedlings was tested against Mycobacterium smegmatis and extracts with lower MIC were further tested on M. tuberculosis. Growth parameters differed between the two groups, with seedlings from the shadehouse showing more plant vigour than the field grown plants. No significant interaction could be established between the measured growth factors and treatment. A significant interaction (P<0.001) was found between Treatment 2 and shadehouse seedlings. Treatment 2 enhanced vegetative performance with the mean values of fresh weight of shoots and roots being twice as much as their respective control mean values. A significantly positive correlation was established between the concentration of shinanolone (P<0.01), isodiospyrin (P<0.05) and neodiospyrin (P<0.05) with fertilization from field-grown seedlings. Application of NPK fertilizers significantly (P<0.05) increased the accumulation of neodiospyrin in seedlings subjected to shadehouse conditions. The most potent naphthoquinone, 7-methyljuglone, was found to be abundant in all the extracts and was quantified at a high mean concentration of 10200 mg/kg from shadehouse seedlings. Root extracts of E. natalensis seedlings grown under field conditions were generally more active against the bacterial strain of M. smegmatis as compared to extracts acquired from roots of seedlings maintained under a shadehouse setting. A lowest minimum inhibitory concentration (MIC) of 0.78mg/ml against M. smegmatis was observed from the second harvest of field-cultivated seedlings of the control and Treatment 1 subgroups. The MIC values for shadehouse seedlings ranged from 1.6 to 6.3 mg/ml. Minimum bactericidal concentration (MBC) values from all the extracts tested were relatively higher than their respective MIC’s. Root extracts of E. natalensis were more active against M. tuberculosis and their MIC values were lower than the tested concentrations. Extracts acquired from field-grown seedlings were more active against M. smegmatis with a lowest MIC value of 0.78 mg/ml. Extracts from the control group and Treatment 1, which had less application of fertilizers were more active against strains of M. tuberculosis with MIC value of 10 µg/ml. This shows the selectivity of E. natalensis against the mycobacterial strain of M. tuberculosis. Based on the findings, synthesis and accumulation of naphthoquinones in E. natalensis is highly variable within individuals of the species investigated. Naphthoquinones accumulate in relatively higher amounts in roots of E. natalensis than in the aboveground structures, which validate their harvest by indigenous people. The concentration of shinanolone varied slightly and its production increased with seedling growth. The synthesis of 7-methyljuglone is independent of fertilisation as its accumulation was enhanced in seedlings subjected to control treatment. Neodiospyrin and isodiospyrin were always present in every sample obtained from the seedlings but they were not detectible in every profile of samples from mature plants. Diospyrin is the only naphthoquinone that was detected in every sample analysed and also quantified in high concentrations from mature plants harvested in spring. The study showed that depending on the requirement of a particular naphthoquinone for research, one could target the seasons and seedling stages recommended from this study. This study also showed that field-cultivated seedlings produced more potent naphthoquinones than the ones subjected to controlled environments. / Dissertation (MSc (Plant Science))--University of Pretoria, 2008. / Plant Production and Soil Science / unrestricted

Über die Ätiologie und Epidemiologie der Erkrankungen des Respirationstraktes im Frühneolithikum Mitteleuropas gibt es bislang keine umfassenden Studien. Die Häufigkeit, mögliche Geschlechts- und Altersunterschiede, Populationsunterschiede und Erkenntnisse über mögliche Ursachen und auslösende Faktoren der Atemwegserkrankungen zur Zeit der Bandkeramik sind von besonderem Interesse. Zudem soll aufgezeigt werden wie wichtig es ist, alle Strukturen zu untersuchen, die den „knöchernen Respirationstrakt“ repräsentieren. Von den ersten Ackerbauern und Viehzüchtern aus Wandersleben (Thüringen, Kreis Gotha) lagen insgesamt 112 erwachsene Individuen zur Untersuchung vor. Rippen und Schädel wurden mit paläopathologischen Methoden untersucht. Diese waren neben einer makroskopischen Begutachtung der Knochen, röntgenologische, endoskopische, lupenmikroskopische, lichtmikroskopische und rasterelektronenmikroskopische Untersuchungen, um eine verlässliche Diagnosestellung zu gewährleisten. 100 % (n = 71/71) der befundbaren Individuen zeigten Spuren chronischer Erkrankungen im Bereich der knöchernen Strukturen, die die oberen Atemwege umgeben. Bei 76,8% (n = 53/69) der Individuen mit befundbaren Rippen konnten Spuren von chronischen Erkrankungen aufgezeigt werden. Signifikante Geschlechts- oder Altersunterschiede bestehen insgesamt nicht. Bei der linearbandkeramischen Population aus Wandersleben ist davon auszugehen, dass es sich in vielen Fällen um Chronifizierungen von Erkältungskrankheiten und um die Folgen einer vergleichbar schlechten Luftqualität hauptsächlich im Haus handelt. Dazu haben die damaligen Lebensumstände, vor allem aber die sesshafte Lebensweise und Wirtschaftszweige mit Ackerbau und Viehzucht, maßgeblich beigetragen.