Annexin A1 (Anx-A1) is a 37kDa protein that is secreted by some cells in response to glucocorticoids (GCs) and which mediates several of their acute anti-inflammatory effects. In addition to GCs, ‘mast cell stabilising’ cromones such as nedocromil also mobilise Anx-A1 by promoting its phosphorylation by protein kinase C (PKC) and hence its secretion, which explains their acute efficacy as anti-allergic agents. This thesis addresses a fundamental aspect of Anx-A1 in the actions of anti-allergic drugs. In this study, anti-allergic drugs such as H1 antagonists, mast cell stabilisers and ‘dual action’ drugs were first tested for their ability to enhance Anx-A1 phosphorylation in a model system using U937 cells. Biochemical and immuno-fluorescent techniques were used to study the mechanisms by which these drugs suppress mediator release from cord blood derived mast cells (CDMCs) and murine bone-marrow derived mast cells (BMDMCs) from wild type and Anx-A1 null-mice. This thesis suggest that PKC activation is crucial for Anx-A1 export in mast cells and nedocromil in the presence of dexamethasone, prolongs the duration of PKC activation and subsequently phosphorylation, externalisation and release of Anx-A1 from CDMCs. The ability of nedocromil to inhibit β-hexosaminidase, tryptase, histamine and PGD2 release are dependent on Anx-A1 in CDMCs. Interestingly, ketotifen, a ‘dual action’ drug possesses a similar pharmacological profile to nedocromil, but not promethazine, which does not act through the Anx-A1 release. Strong evidence supports the notion that the mechanisms of action of nedocromil are modulated by Anx-A1, thus the possibility that FPR2 might be involved in the acute actions of nedocromil was tested. Nedocromil inhibits the release of PGD2 through the activation of FPR2 but not the inhibition of histamine release. A possible explanation for this finding could be that Anx-A1 might be interacting with other FPR family members to exert the histamine inhibitory effects. Although only a small subset of the downstream intracellular signaling pathway of MAPK was tested, the results indicate that Anx-A1 differentially regulates the activation of p38 and JNK in CDMCs treated with nedocromil. These findings indicate a novel model system in which Anx-A1 mediates the pharmacological actions of anti-allergic drugs and thus has an important role in preventing the mast cell degranulation.

As análises dos voláteis por SPME/CG-EM das partes aéreas, flores e frutos de Bidens sulphurea e Bidens gardneri permitiram-nos constatar diferenças em suas composições químicas. Porém, em todas as frações analisadas os compostos majoritários foram os sequiterpenos b-cariofileno, germacreno D e biciclogermacreno. Apesar dos constituintes majoritários coincidirem nas frações analisadas das duas espécies, foi possível constatar a presença exclusiva de determinados metabólitos em cada fração. A partir das frações hexânicas, oriundas dos extratos etanólicos de B. sulphurea (partes aéreas e flores) e B. gardneri (partes aéreas), foram identificadas trinta e cinco, dezenove e vinte substâncias por CG-EM, respectivamente. Na fração hexânica das partes aéreas de B. sulphurea (BsfcEt/Hx) foram identificados, como constituintes majoritários, o óxido de cariofileno, espatulenol e _- cariofileno, enquanto que na fração hexânica de suas flores (BsflorEt/Hx) os principais constituintes identificados foram _-amirina e _-sitosterol e na fração hexânica das partes aéreas de B. gardneri (BgfcEt/Hx) foram os metabólitos _-estigmasterol e o trans-fitol. O estudo químico da espécie B. sulphurea (partes aéreas e flores) conduziu ao isolamento de um sesquiterpeno (1), 5 flavonas (2, 10, 13-15), 8 flavonóis (3-8, 11, 16), 1 aurona (9) e 2 chalconas (12, 17). Já o estudo de B. gardneri (partes aéreas) conduziu ao isolamento de 4 ácidos clorogênicos (21, 22, 27, 28), 3 poliacetilenos (18-20), 2 flavonas (25, 27), 3 flavanonas (23, 24, 30) e 2 chalconas (29, 31). Os metabólitos 3-O-_-glicopiranosil-tetradeca- 6(E),12(E)-dieno-8,10-diino-1,14-diol (18), 1-O-_-glicopiranosil-14-hidróxi-tetradeca-6(E), 12(E)-dieno-8,10-diino-3-ona (19), 4-metóxi-7-O-_-glicopiranosil-8,3-diidróxi-flavanona (23), 4-metóxi-7-O-_-(6-acetil)-glicopiranosil-8,3-diidróxi-flavanona (24) e 7-O-_-(6- trans-p-cumaroil)-glicopiranosil-8,34-triidróxi-flavanona (30) estão sendo descritos pela primeira vez na literatura. Enquanto que os flavonóides 3-O-_-xilopiranosil-quercetina (5), 3-O-a-arabinofuranosil-kaempferol (8) e 3-O-b-(6-trans-cafeoil)-galactopiranosil-quercetina (16) estão sendo relatados pela primeira vez na família Asteraceae e as substâncias 4(15)- eudesmeno-1_,6_-diol (1), 6-C-_-glicopiranosil-apigenina (2), 3-O-_-arabinofuranosilquercetina (6), 8-C-_-glicopiranosil-apigenina (13), 6-C-_-glicopiranosil-luteolina (14), 8-C- _-glicopiranosil-luteolina (15), ácido 1-metil-5-O-E-cafeoilquínico (22) e 7-O-_- glicopiranosil-apigenina (25) estão sendo relatadas pela primeira vez no gênero Bidens. Além disso, convém destacar que a partir das substâncias 2-O-_-glicopiranosil-trideca-3(E),11(E)- dieno-5,7,9-triino-1,13-diol (20), 4-metóxi-4-O-_-glicopiranosil-okanina (29) e 4-O-_-(6- trans-p-cumaroil)-glicopiranosil-okanina (31) há poucos relatos na literatura e ainda não foram descritos dados de suas propriedades biológicas. Na avaliação da atividade antialérgica das substâncias isoladas, os flavonóides causaram inibição da liberação de _-hexosaminidase de forma dose-dependente, sendo que as substâncias 11 e 31 foram as mais ativas e apresentaram CI50 de 5,1 ± 1,3 M e 5,8 ± 1,2 M, respectivamente. Dentre os extratos e frações avaliados biologicamente, observou-se que BsfcEt/Hx (maior teor de sesquiterpenos) causou um estímulo da liberação de _-hexosaminidase, enquanto que BsfcEt/Ac foi a fração mais ativa (CI50 = 1,3 ± 1,1g/mL). As análises desta última fração e de BsfcEt/DCM por CLAE-DAD-EM e CLAE-DAD-EM/EM revelaram que seus constituintes majoritários são os flavonóis 3, 4, 6 e 7. / The SPME/GC-MS analyses of aerial parts, flowers and fruits of Bidens sulphurea and Bidens gardneri showed the differences in their chemical compositions. However the sesquiterpenes _-caryophyllene, germacrene D and bicyclogermacrene were identified in all fractions analyzed as major compounds. It was observed the exclusive presence of metabolites in each fraction, in spite of major constituents were equal in fractions analyzed of two species. Thirty-five, nineteen and twenty substances were identified in the hexane fractions from ethanol extracts of B. sulphurea (aerial parts and flowers) and B. gardneri (aerial parts) by GC-MS. The major compounds were caryophyllene oxide, spathulenol and _-caryophyllene in the hexane fraction from aerial parts of B. sulphurea (BsfcEt/Hx), while _-amyrin and _- sitosterol were identified in the hexane fraction of its flowers and _-stigmasterol and transphytol were main constituents identified in the hexane fraction from aerial parts of B. gardneri. The chemical study of species B. sulphurea (aerial parts and flowers) led to the isolation of one sesquiterpene (1), five flavones (2, 10, 13-15), eight flavonols (3-8, 11, 16), one aurone (9) and two chalcones (12, 17). From B. gardneri (aerial parts), four chlorogenic acids (21, 22, 27, 28), three polyacetylenes (18-20), two flavones (25, 27), three flavanones (23, 24, 30) and two chalcones (29, 31) were isolated. The substances 3-O-_-glucopyranosyltetradeca-6(E),12(E)-dien-8,10-diin-1,14-diol(18), 1-O-_-glucopyranosyl-14-hydroxytetradeca-6(E),12(E)-dien-8,10-diin-3-one(19),4-methoxy-7-O_glucopyranosyl-8,3-dihydroxyflavanone (23), 4-methoxy-7-O-_-(6-acetyl)-glucopyranosyl-8,3-dihydroxyflavanone(24) and 7-O-_-(6-trans-p-coumaroyl)-glucopyranosyl-8,34-trihydroxyflavanone(30) are described for the first time in the literature. Whereas the flavonols 3-O-_-xylopyranosyl quercetin (5), 3-O-a-arabinofuranosyl kaempferol (8) and 3-O--(6-transcaffeoyl)-galactopyranosyl quercetin (16) are described for the first time in the Asteraceaeand 4(15)-eudesmene-1_,6_-diol (1), 6-C-_-glucopyranosyl apigenin (2), 3-O-_-arabinofuranosyl quercetin (6), 8-C-_-glucopyranosyl apigenin (13), 6-C-_-glucopyranosylluteolin (14), 8-C-_-glucopyranosyl luteolin (15), 1-methyl-5-O-E-caffeoylquinic acid (22)and 7-O-_-glucopyranosyl apigenin (25) for the first time in the genus Bidens. Moreover,there are few reports of the isolation and there are not studies of biological activities from 2-O-_-glucopyranosyl-trideca-3(E),11(E)-dien-5,7,9-triin-1,13-diol (20), 4-methoxy-4-O-_-glucopyranosyl okanin (29) and 4-O-_-(6-trans-p-coumaroyl)-glucopyranosyl okanin (31).The flavonoids showed inhibition of _-hexosaminidase released with dependent-doseresponse and the substances 11 (IC50 = 5,1 ± 1,3 M) and 31 (IC50 = 5,8 ± 1,2 M) were themost active. The BsfcEt/Hx fraction (highest concentration of sesquiterpenes) induced _-hexosaminidase released, while the BsfcEt/Ac fraction exhibited the lower IC50 (1,3 ±1,1g/mL). The flavonoids 3, 4, 6 and 7 were identified, by HPLC-DAD-MS and HPLCDAD-MS/MS, as major constituents in BsfcEt/Ac and BsfcEt/DCM fractions.

As análises dos voláteis por SPME/CG-EM das partes aéreas, flores e frutos de Bidens sulphurea e Bidens gardneri permitiram-nos constatar diferenças em suas composições químicas. Porém, em todas as frações analisadas os compostos majoritários foram os sequiterpenos b-cariofileno, germacreno D e biciclogermacreno. Apesar dos constituintes majoritários coincidirem nas frações analisadas das duas espécies, foi possível constatar a presença exclusiva de determinados metabólitos em cada fração. A partir das frações hexânicas, oriundas dos extratos etanólicos de B. sulphurea (partes aéreas e flores) e B. gardneri (partes aéreas), foram identificadas trinta e cinco, dezenove e vinte substâncias por CG-EM, respectivamente. Na fração hexânica das partes aéreas de B. sulphurea (BsfcEt/Hx) foram identificados, como constituintes majoritários, o óxido de cariofileno, espatulenol e _- cariofileno, enquanto que na fração hexânica de suas flores (BsflorEt/Hx) os principais constituintes identificados foram _-amirina e _-sitosterol e na fração hexânica das partes aéreas de B. gardneri (BgfcEt/Hx) foram os metabólitos _-estigmasterol e o trans-fitol. O estudo químico da espécie B. sulphurea (partes aéreas e flores) conduziu ao isolamento de um sesquiterpeno (1), 5 flavonas (2, 10, 13-15), 8 flavonóis (3-8, 11, 16), 1 aurona (9) e 2 chalconas (12, 17). Já o estudo de B. gardneri (partes aéreas) conduziu ao isolamento de 4 ácidos clorogênicos (21, 22, 27, 28), 3 poliacetilenos (18-20), 2 flavonas (25, 27), 3 flavanonas (23, 24, 30) e 2 chalconas (29, 31). Os metabólitos 3-O-_-glicopiranosil-tetradeca- 6(E),12(E)-dieno-8,10-diino-1,14-diol (18), 1-O-_-glicopiranosil-14-hidróxi-tetradeca-6(E), 12(E)-dieno-8,10-diino-3-ona (19), 4-metóxi-7-O-_-glicopiranosil-8,3-diidróxi-flavanona (23), 4-metóxi-7-O-_-(6-acetil)-glicopiranosil-8,3-diidróxi-flavanona (24) e 7-O-_-(6- trans-p-cumaroil)-glicopiranosil-8,34-triidróxi-flavanona (30) estão sendo descritos pela primeira vez na literatura. Enquanto que os flavonóides 3-O-_-xilopiranosil-quercetina (5), 3-O-a-arabinofuranosil-kaempferol (8) e 3-O-b-(6-trans-cafeoil)-galactopiranosil-quercetina (16) estão sendo relatados pela primeira vez na família Asteraceae e as substâncias 4(15)- eudesmeno-1_,6_-diol (1), 6-C-_-glicopiranosil-apigenina (2), 3-O-_-arabinofuranosilquercetina (6), 8-C-_-glicopiranosil-apigenina (13), 6-C-_-glicopiranosil-luteolina (14), 8-C- _-glicopiranosil-luteolina (15), ácido 1-metil-5-O-E-cafeoilquínico (22) e 7-O-_- glicopiranosil-apigenina (25) estão sendo relatadas pela primeira vez no gênero Bidens. Além disso, convém destacar que a partir das substâncias 2-O-_-glicopiranosil-trideca-3(E),11(E)- dieno-5,7,9-triino-1,13-diol (20), 4-metóxi-4-O-_-glicopiranosil-okanina (29) e 4-O-_-(6- trans-p-cumaroil)-glicopiranosil-okanina (31) há poucos relatos na literatura e ainda não foram descritos dados de suas propriedades biológicas. Na avaliação da atividade antialérgica das substâncias isoladas, os flavonóides causaram inibição da liberação de _-hexosaminidase de forma dose-dependente, sendo que as substâncias 11 e 31 foram as mais ativas e apresentaram CI50 de 5,1 ± 1,3 M e 5,8 ± 1,2 M, respectivamente. Dentre os extratos e frações avaliados biologicamente, observou-se que BsfcEt/Hx (maior teor de sesquiterpenos) causou um estímulo da liberação de _-hexosaminidase, enquanto que BsfcEt/Ac foi a fração mais ativa (CI50 = 1,3 ± 1,1g/mL). As análises desta última fração e de BsfcEt/DCM por CLAE-DAD-EM e CLAE-DAD-EM/EM revelaram que seus constituintes majoritários são os flavonóis 3, 4, 6 e 7. / The SPME/GC-MS analyses of aerial parts, flowers and fruits of Bidens sulphurea and Bidens gardneri showed the differences in their chemical compositions. However the sesquiterpenes _-caryophyllene, germacrene D and bicyclogermacrene were identified in all fractions analyzed as major compounds. It was observed the exclusive presence of metabolites in each fraction, in spite of major constituents were equal in fractions analyzed of two species. Thirty-five, nineteen and twenty substances were identified in the hexane fractions from ethanol extracts of B. sulphurea (aerial parts and flowers) and B. gardneri (aerial parts) by GC-MS. The major compounds were caryophyllene oxide, spathulenol and _-caryophyllene in the hexane fraction from aerial parts of B. sulphurea (BsfcEt/Hx), while _-amyrin and _- sitosterol were identified in the hexane fraction of its flowers and _-stigmasterol and transphytol were main constituents identified in the hexane fraction from aerial parts of B. gardneri. The chemical study of species B. sulphurea (aerial parts and flowers) led to the isolation of one sesquiterpene (1), five flavones (2, 10, 13-15), eight flavonols (3-8, 11, 16), one aurone (9) and two chalcones (12, 17). From B. gardneri (aerial parts), four chlorogenic acids (21, 22, 27, 28), three polyacetylenes (18-20), two flavones (25, 27), three flavanones (23, 24, 30) and two chalcones (29, 31) were isolated. The substances 3-O-_-glucopyranosyltetradeca-6(E),12(E)-dien-8,10-diin-1,14-diol(18), 1-O-_-glucopyranosyl-14-hydroxytetradeca-6(E),12(E)-dien-8,10-diin-3-one(19),4-methoxy-7-O_glucopyranosyl-8,3-dihydroxyflavanone (23), 4-methoxy-7-O-_-(6-acetyl)-glucopyranosyl-8,3-dihydroxyflavanone(24) and 7-O-_-(6-trans-p-coumaroyl)-glucopyranosyl-8,34-trihydroxyflavanone(30) are described for the first time in the literature. Whereas the flavonols 3-O-_-xylopyranosyl quercetin (5), 3-O-a-arabinofuranosyl kaempferol (8) and 3-O--(6-transcaffeoyl)-galactopyranosyl quercetin (16) are described for the first time in the Asteraceaeand 4(15)-eudesmene-1_,6_-diol (1), 6-C-_-glucopyranosyl apigenin (2), 3-O-_-arabinofuranosyl quercetin (6), 8-C-_-glucopyranosyl apigenin (13), 6-C-_-glucopyranosylluteolin (14), 8-C-_-glucopyranosyl luteolin (15), 1-methyl-5-O-E-caffeoylquinic acid (22)and 7-O-_-glucopyranosyl apigenin (25) for the first time in the genus Bidens. Moreover,there are few reports of the isolation and there are not studies of biological activities from 2-O-_-glucopyranosyl-trideca-3(E),11(E)-dien-5,7,9-triin-1,13-diol (20), 4-methoxy-4-O-_-glucopyranosyl okanin (29) and 4-O-_-(6-trans-p-coumaroyl)-glucopyranosyl okanin (31).The flavonoids showed inhibition of _-hexosaminidase released with dependent-doseresponse and the substances 11 (IC50 = 5,1 ± 1,3 M) and 31 (IC50 = 5,8 ± 1,2 M) were themost active. The BsfcEt/Hx fraction (highest concentration of sesquiterpenes) induced _-hexosaminidase released, while the BsfcEt/Ac fraction exhibited the lower IC50 (1,3 ±1,1g/mL). The flavonoids 3, 4, 6 and 7 were identified, by HPLC-DAD-MS and HPLCDAD-MS/MS, as major constituents in BsfcEt/Ac and BsfcEt/DCM fractions.

L'huile d'olive est la principale source d’apports lipidique dans le régime méditerranéen. Elle est reconnue comme une source de nutriments et est à l’origine de plusieurs effets biologiques bénéfiques pour la santé. Plusieurs études montrent notamment que la consommation d'huile d'olive peut être efficace dans le traitement de maladies cardiovasculaires. Cependant, peu d'études mettent en évidence un effet antiinflammatoire, antiallergique et anti-Alzheimer de l’huile d’olive. De même, aucune étude n’a déterminé un lien entre ces effets et les variétés d’olives dont sont issues les huiles ainsi que les composés présents responsables de ces effets. Le but de ce travail est donc d'évaluer l'effet biologique, et plus particulièrement le potentiel antiinflammatoire et antiallergique des composés hydrophiles obtenus après extraction en phase solide (SPE-Diol), à partir de plusieurs variétés d’huiles d'olive extra vierge (EVOO) issues de trois années de récolte différentes. L'effet neuroprotecteur de la variété Picholine, contre la maladie d'Alzheimer a également été évalué. Les résultats montrent que l'effet anti-inflammatoire et l’effet anti-allergique varies de façon concentration dépendante et en fonction de la variété d’EVOO. De plus, nous avons mis en évidence que la variété de Picholine a un effet neuroprotecteur. L’ensemble de ce travail a établi une corrélation entre des activités anti-inflammatoire, anti-allergique et anti-Alzheimer des huiles d’olives issues de plusieurs variétés et les différents composés présents, ce qui a permis de mettre en avant la variété Picholine. / Olive oil is the primary source of fat in the Mediterranean diet. It is not only known as a distinctive source of nutrients but also as an origin of several biological effects that represent a very important factor for the health. Several studies demonstrate that olive oil consumption has to be effective against heart and cardiovascular diseases. Few studies show up the anti-inflammatory, the anti-allergic and the anti-Alzheimer effects of olive oil but there is no study about the cultivar effect and the interference that could occur between the different olive oil compounds. The aim of this work is to evaluate the biological effect and more particularly the potential anti-inflammatory and anti-allergic of hydrophilic components obtained by solid phase extraction (SPE-Diol) of different extra virgin olive oil (EVOO) cultivars and from three different harvest years. Moreover the neuroprotective effect of Picholine cultivar against Alzheimer’s disease was also studied. The results show that the anti-inflammatory and the anti-allergic activities differ depending on the EVOO cultivar and they are concentration-dependent. The results show also that Picholine cultivar has an anti-Alzheimer activity. All of these studies have established a correlation between the anti-inflammatory, the anti-allergic and the anti-Alzheimer effects of different EVOO cultivars and different compounds present, which enabled us to highlight the Picholine cultivar.

Neste trabalho foram investigadas a atividade antialérgica de extratos, ou substâncias isoladas, obtidos de fontes naturais. Para isso foi utilizado o sistema biossensor baseado em mastócitos os quais liberam a enzima beta-hexosaminidase usada como marcador da degranulação. Para algumas substâncias naturais da classe dos flavonóides (quercetina-Qc e rutina-Rt) e ácidos polifenólicos (ácido dimetoxicinâmico-Dm e ácido cafeico-Cf), os ensaios biológicos foram conduzidos na presença de beta-ciclodextrina (beta-CD) a fim de estudar a eficiência do ensaio biológico e o efeito de complexação na atividade antialérgica. Inicialmente, foram investigadas, as propriedades espectroscópicas destes flavonóides e ácidos polifenólicos, na ausência, e presença de beta-CD. As mudanças nos espectros de absorção e fluorescência, em presença de beta-CD, mostraram que ocorre a associação dos fármacos com a beta-CD. Assim, as constantes de incorporação (Kc) foram determinadas pelo método de Higuchi e Connors e os resultados mostraram maior incorporação da Qc (Kc = 172 /M) na cavidade da beta-CD quando comparada a Rt (Kc = 139 /M). No caso dos polifenóis, Dm mostrou incorporação maior em relação ao Cf, com valores de Kc iguais a 718 e 278 /M, respectivamente. Os valores de Kc foram considerados apropriados para a aplicação de compostos de inclusão como agentes terapêuticos. Assim, os complexos de inclusão sólidos, foram preparados por uma adaptação do método da co-evaporação e caracterizados por Análise Termogravimétrica (TGA), Análise Térmica Diferencial (DTA), Calorimetria Diferencial de Varredura (DSC), espectroscopia na região do Infravermelho (FTIR) e Ressonância Magnética Nuclear de Prótons (1H-RMN). O parâmetro físico-químico para interação hidrofóbica (log P) foi determinado para os flavonóides e acidos polifenólicos e os resultados indicaram que a hidrofobicidade seguiu a seguinte ordem: Dm > Cf > Qc > Rt. Os complexos de inclusão foram mais eficazes para inibir a liberação da beta-hexosaminidase do que os fármacos na forma livre. A atividade anti-alérgica da Qc livre (IC50= 5,1 µM) mostrou um aumento de oito vêzes quando complexada com a beta-CD (IC50= 0,62 µM). Um aumento da atividade foi observado, também, para os complexos Rt/CD, Cf/CD e Dm/CD. Este efeito foi mais forte para os compostos com maior hidrofobicidade. A atividade antialérgica das substâncais naturais livres provenientes de várias classes de plantas tais como flavonóides, ácidos polifenólicos, terpenos, alcalóides e iridóides foi, também, investigada. Os flavonóides tais como quercetina (IC50= 5,1 µM), 7-metil quercetina (IC50= 6,2 µM), caempferol-3-glicosideo (IC50= 6,7 µM) and 4-O-(6-trans-p-coumaroil)--D-glicopyranosil okanina (IC50= 5,8 µM) mostraram a maior atividade antialérgica comparados ao fumarato de cetotifeno (IC50= 15,1 µM). Os extratos provenientes de diversas espécies de plantas tais como Bidens sulphurea, Bidens gardneri, Bidens graveolens, Mikania parodii Cabrera e Mikania pilosa Baker foram, também, investigados. Os resultados mostraram maior atividade para o extrato de Bidens obtido de acetato de etila. Este extrato é rico em derivados metilados de quercetina os quais exibiram forte atividade antialérgica quando utilizados no ensaio biológico como substância isolada. / Anti-allergic activity of extracts and isolated compounds obtained from natural sources was investigated using the mast-cell based biosensor system. Mast cells release beta-hexosaminidase enzyme which is used as a marker of degranulation. Flavonoids (quercetin-Qc and rutin-Rt) and polyphenolic acids (caffeic acid-Cf and dimethoxy cinnamic acid-Dm) were used as inclusion compounds with beta-cyclodextrin (beta-CD) in order to compare the efficiency of the biological assay and the anti-allergic activity of the drugs free or associated with beta-CD. Spectroscopic properties of the flavonoids and polyphenolic acids were monitored in the absence or presence of beta-CD. The absorbance and fluorescence spectra showed drug association with beta-CD; subsequently the stability constants (kc) of the drugs were obtained in accordance with the method of the Higuchi-Connors. The results showed higher association of Qc with beta-CD (Kc= 172 /M) compared to Rt (Kc= 139 /M). For the polyphenolic acids, Dm exhibited the higher association with beta-CD compared to Cf (718 and 278 /M respectively). The Kc values felt within the range considered adequate for the formation of inclusion complex, and they can be used to improve the bioavailability of the flavonoids and polyphenolic acids. The solid inclusion compounds were obtained by an adaptation of the co-evaporation method and characterized by Thermo Gravimetric Analysis (TG), Differential Thermal Analysis (DTA), Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR) and Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR). Physico-chemical parameter for hydrophobic interaction (log P) was determined for the flavonoids and polyphenolic acids and the results indicated that the hydrophobicity followed the order: Dm > Cf > Qc > Rt. The inclusion complexes were more effective at inhibiting beta-hexosaminidase release than plain drugs. The anti-allergic activity of plain Qc (IC50= 5.05 M) showed eightfold improvement when included inside the beta-CD cavity (IC50= 0.62 M). Higher biological activity on the part of the complex was also observed for the complexes Rt/CD, Cf/CD and Dm/CD. This effect was stronger for the compounds with higher hydrophobicity. The anti-allergic activity of plain natural compounds from several classes of plants such as flavonoids, polyphenolic acids, terpenes, alkaloids and iridoids was investigated. Flavonoids such as quercetin (IC50= 5.1 µM), 7-methyl quercetin (IC50= 6.2 µM), kaempferol-3-glycoside (IC50= 6.7 µM) and 4-O-(6-trans-p-coumaroil)--D-glucopyranosyl okanin (IC50= 5.8 µM) showed the stronger anti-allergic activity compared with ketotifen fumarate, a reference drug (IC50= 15.1 µM). Extracts proceeding from different species of plants such as Bidens sulphurea, Bidens gardneri, Bidens graveolens, Mikania parodii Cabrera and Mikania pilosa Baker were also investigated. The results showed stronger anti-allergic activity for ethyl acetate extracts obtained from Bidens specie. This extracts are rich in methylated quercetin derivatives which showed strong anti-allergic activity when assayed as isolated substances

Neste trabalho foram investigadas a atividade antialérgica de extratos, ou substâncias isoladas, obtidos de fontes naturais. Para isso foi utilizado o sistema biossensor baseado em mastócitos os quais liberam a enzima beta-hexosaminidase usada como marcador da degranulação. Para algumas substâncias naturais da classe dos flavonóides (quercetina-Qc e rutina-Rt) e ácidos polifenólicos (ácido dimetoxicinâmico-Dm e ácido cafeico-Cf), os ensaios biológicos foram conduzidos na presença de beta-ciclodextrina (beta-CD) a fim de estudar a eficiência do ensaio biológico e o efeito de complexação na atividade antialérgica. Inicialmente, foram investigadas, as propriedades espectroscópicas destes flavonóides e ácidos polifenólicos, na ausência, e presença de beta-CD. As mudanças nos espectros de absorção e fluorescência, em presença de beta-CD, mostraram que ocorre a associação dos fármacos com a beta-CD. Assim, as constantes de incorporação (Kc) foram determinadas pelo método de Higuchi e Connors e os resultados mostraram maior incorporação da Qc (Kc = 172 /M) na cavidade da beta-CD quando comparada a Rt (Kc = 139 /M). No caso dos polifenóis, Dm mostrou incorporação maior em relação ao Cf, com valores de Kc iguais a 718 e 278 /M, respectivamente. Os valores de Kc foram considerados apropriados para a aplicação de compostos de inclusão como agentes terapêuticos. Assim, os complexos de inclusão sólidos, foram preparados por uma adaptação do método da co-evaporação e caracterizados por Análise Termogravimétrica (TGA), Análise Térmica Diferencial (DTA), Calorimetria Diferencial de Varredura (DSC), espectroscopia na região do Infravermelho (FTIR) e Ressonância Magnética Nuclear de Prótons (1H-RMN). O parâmetro físico-químico para interação hidrofóbica (log P) foi determinado para os flavonóides e acidos polifenólicos e os resultados indicaram que a hidrofobicidade seguiu a seguinte ordem: Dm > Cf > Qc > Rt. Os complexos de inclusão foram mais eficazes para inibir a liberação da beta-hexosaminidase do que os fármacos na forma livre. A atividade anti-alérgica da Qc livre (IC50= 5,1 µM) mostrou um aumento de oito vêzes quando complexada com a beta-CD (IC50= 0,62 µM). Um aumento da atividade foi observado, também, para os complexos Rt/CD, Cf/CD e Dm/CD. Este efeito foi mais forte para os compostos com maior hidrofobicidade. A atividade antialérgica das substâncais naturais livres provenientes de várias classes de plantas tais como flavonóides, ácidos polifenólicos, terpenos, alcalóides e iridóides foi, também, investigada. Os flavonóides tais como quercetina (IC50= 5,1 µM), 7-metil quercetina (IC50= 6,2 µM), caempferol-3-glicosideo (IC50= 6,7 µM) and 4-O-(6-trans-p-coumaroil)--D-glicopyranosil okanina (IC50= 5,8 µM) mostraram a maior atividade antialérgica comparados ao fumarato de cetotifeno (IC50= 15,1 µM). Os extratos provenientes de diversas espécies de plantas tais como Bidens sulphurea, Bidens gardneri, Bidens graveolens, Mikania parodii Cabrera e Mikania pilosa Baker foram, também, investigados. Os resultados mostraram maior atividade para o extrato de Bidens obtido de acetato de etila. Este extrato é rico em derivados metilados de quercetina os quais exibiram forte atividade antialérgica quando utilizados no ensaio biológico como substância isolada. / Anti-allergic activity of extracts and isolated compounds obtained from natural sources was investigated using the mast-cell based biosensor system. Mast cells release beta-hexosaminidase enzyme which is used as a marker of degranulation. Flavonoids (quercetin-Qc and rutin-Rt) and polyphenolic acids (caffeic acid-Cf and dimethoxy cinnamic acid-Dm) were used as inclusion compounds with beta-cyclodextrin (beta-CD) in order to compare the efficiency of the biological assay and the anti-allergic activity of the drugs free or associated with beta-CD. Spectroscopic properties of the flavonoids and polyphenolic acids were monitored in the absence or presence of beta-CD. The absorbance and fluorescence spectra showed drug association with beta-CD; subsequently the stability constants (kc) of the drugs were obtained in accordance with the method of the Higuchi-Connors. The results showed higher association of Qc with beta-CD (Kc= 172 /M) compared to Rt (Kc= 139 /M). For the polyphenolic acids, Dm exhibited the higher association with beta-CD compared to Cf (718 and 278 /M respectively). The Kc values felt within the range considered adequate for the formation of inclusion complex, and they can be used to improve the bioavailability of the flavonoids and polyphenolic acids. The solid inclusion compounds were obtained by an adaptation of the co-evaporation method and characterized by Thermo Gravimetric Analysis (TG), Differential Thermal Analysis (DTA), Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR) and Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR). Physico-chemical parameter for hydrophobic interaction (log P) was determined for the flavonoids and polyphenolic acids and the results indicated that the hydrophobicity followed the order: Dm > Cf > Qc > Rt. The inclusion complexes were more effective at inhibiting beta-hexosaminidase release than plain drugs. The anti-allergic activity of plain Qc (IC50= 5.05 M) showed eightfold improvement when included inside the beta-CD cavity (IC50= 0.62 M). Higher biological activity on the part of the complex was also observed for the complexes Rt/CD, Cf/CD and Dm/CD. This effect was stronger for the compounds with higher hydrophobicity. The anti-allergic activity of plain natural compounds from several classes of plants such as flavonoids, polyphenolic acids, terpenes, alkaloids and iridoids was investigated. Flavonoids such as quercetin (IC50= 5.1 µM), 7-methyl quercetin (IC50= 6.2 µM), kaempferol-3-glycoside (IC50= 6.7 µM) and 4-O-(6-trans-p-coumaroil)--D-glucopyranosyl okanin (IC50= 5.8 µM) showed the stronger anti-allergic activity compared with ketotifen fumarate, a reference drug (IC50= 15.1 µM). Extracts proceeding from different species of plants such as Bidens sulphurea, Bidens gardneri, Bidens graveolens, Mikania parodii Cabrera and Mikania pilosa Baker were also investigated. The results showed stronger anti-allergic activity for ethyl acetate extracts obtained from Bidens specie. This extracts are rich in methylated quercetin derivatives which showed strong anti-allergic activity when assayed as isolated substances

全球過敏性疾病的患病率逐漸增加。大約30 - 40的世界人口患有一個或多個過敏性疾病，它絶對是一個國際性的公共健康問題。在過去三十年，過敏性皮膚炎的發病率增加了2-3 倍，當中患病率最高的是嬰兒和兒童，而且現時並沒有明確的治療方法。然而，對過敏性疾病有效的治療方法仍然缺乏，大多數傳統的治療涉及臨床改善，但不針對促進過敏性炎症發病機制中的主要因素。這些傳統的治療方法都有不良副作用。因此，發展一個更安全和非類固醇的治療方式成為了新的趨勢。 / 從過往的臨床試驗中，患有中度至嚴重過敏性皮膚炎的兒童服用由五種中藥制成的Pentaherbs（PHF）膠囊藥丸後， 顯著地改善他們的生活質數，降低了過敏性皮膚炎指數（SCORAD）及減少使用傳統藥物類固醇的份量，更沒有出現任何不良藥物作用。實驗結果指出PHF 有潛力替代類固醇，成為治療過敏性皮膚炎的取代品。在本研究中，我們使用炎症相關的細胞因子IL-33，激活在有或沒有與皮膚成纖維細胞一起培植下的嗜鹼性細胞系KU812 細胞， 來探討了PHF，牡丹皮（DP，PHF 的五種草藥之一）和沒食子酸（GA，牡丹皮的主要成分之一）的抗炎和抗過敏特性。 / 在過敏性炎症中，嗜鹼性粒細胞是一個重要的效應細胞。我們利用細胞因子IL-33 激活嗜鹼性粒細胞系KU812 細胞，並從研究結果發現出PHF，DP 和GA 能有效及顯著地抑制細胞間粘附分子ICAM-1 的表達，炎症相關趨化因子CCL2，CCL5，CXCL8 和促炎細胞因子IL-6 的釋放。這證實出PHF, DP 和GA有抗炎和抗過敏的特性。在進一步的研究中，我們加入一種常用醫治過敏性炎症藥物的合成類固醇地塞米松, 與PHF, DP 和GA 結合使用。從各種組合的不同濃度地塞米松與PHF，DP 和GA 中，我們發現聯合使用低濃度為0.01 微克/毫升的地塞米松和10 微克/毫升GA 可進一步抑制ICAM-1 在KU812 的表達，趨化因子CCL2 和CCL5 釋放。此外，沒食子酸可顯著抑制細胞內信號分子p38 絲裂原活化蛋白激酶 (MAPK)，IκB-α和JNK 的表達。這表明了黏附分子的表達，趨化因子和細胞因子的釋放的抑製是經由p38 MAPK，IκB-α和JNK 訊息傳遞路徑所調節。實驗證實PHF，DP 和GA 具有抗炎和抗過敏的特性，與過往的臨床試驗結果一致。 / 為了更進一步研究沒食子酸和地塞米松在過敏性炎症的發病機制中扮演的角色， 我們建立了一個體外的模仿患者皮膚皮炎症的模型，共同培養嗜鹼性細系KU812 細胞和皮膚成纖維細胞 。我們發現，單沒食子酸的應用已經可以顯著地抑制在KU812 細胞和成纖維細胞表面粘附分子的表達，並減低釋放過敏性炎症相關的趨化因子CCL2，CCL5，CXCL8 和促炎細胞因子IL-6。此外，地塞米松和沒食子酸的結合使用能增強抑制KU812 細胞面上ICAM-1 的表達，和皮膚成纖維細胞面上ICAM-1 和VCAM-1 的表達，與及趨化因子CCL2 和CXCL8，促炎性細胞因子IL-6 的釋放。 / 上述調查結果表明，天然植物產品PHF，DP 和GA 是具有消炎和抗過敏的作用，抑制嗜鹼性粒細胞趨化遷移至發炎處和隨後釋放的過敏性炎症介質，如炎症相關的趨化因子和促炎細胞因子。結果表明，沒食子酸天然植物產品可能是一個潛在的過敏性皮膚炎的治療劑，而沒食子酸和地塞米松的結合使用，可以有效降低在患者治療皮膚炎症時使用地塞米松的劑量。總結，這項研究結果揭示使用天然植物衍生產品具有更安全,高效力和副作用少的一種新治療方式。 / The worldwide prevalence of allergic diseases has been increasing gradually. Around 30 - 40% of the world population suffers from one or more allergic conditions, it is definitely a national public health issue. The incidence of Atopic Dermatitis (AD) has increased by 2-3 folds in the past 3 decades with no definitive cure, where the case is the highest during the early infancy and childhood. However, effective treatments on allergic diseases are still lacking, with most of the traditional treatment involves clinical improvement but not targeting the primary factors promoting the pathogenesis of allergic inflammation. These traditional treatments have undesirable side effects. Therefore, there has been a rising interest in the development of a safer and nonsteroid immunomodulation formula to cure the disease. / From previous clinical trails, it is revealed that children with moderate-to-severe AD treated with traditional Chinese Medicine, Pentaherbs formula (PHF), have significantly improved their quality of life, lowered the Scoring of Atopic Dermatitis (SCORAD) index and the use of topical steroids without any adverse drug effect, suggesting that PHF can be an alternative potential adjunct therapy for AD. In this present studies, we elucidated the in vitro anti‐inflammatory and anti‐allergic activities of PHF, Cortex Moutan / Danpi (DP, one of the five herbs in PHF) and gallic acid (GA, one of the main ingredients in Danpi) using human basophilic KU812 cells, with or without human dermal fibroblast, upon the activation with alarmin inflammation-related cytokine IL-33. / Human basophilic KU812 cells activated by alarmin cytokine IL-33 were used as basophil cell model for study since basophils are a crucial effector cells in allergic inflammation. Our results showed that PHF, DP and GA exhibited the anti-inflammatory and anti‐allergic activities indicated by significant suppressive effects on the intercellular adhesion molecule (ICAM)‐1 expression, the release of inflammation‐related chemokines CCL2, CCL5, CXCL8 and proinflammatory cytokine IL-6 from IL-33‐activated KU812 basophilic cells. The studies were further investigated with the combined use of synthetic steroid dexamethasone which is a common drug for AD. Among various combinations with different concentrations of dexamethasone with PHF, DP and GA, we demonstrated that the combined use of a concentration as low as 0.01 μg/ml dexamethasone and 10 μg/ml GA could further suppress ICAM‐1 expression, chemokines CCL2 and CCL5 release in IL-33 activated KU812 cells. Furthermore, gallic acid could significantly suppress the intracellular signaling molecules p38 MAPK, IκB-α and JNK in KU812 cells, thereby suggesting the underlying mechanisms for the suppressive effect on adhesion molecules expression, and the chemokines and cytokines release. Both in vivo and in vitro experiments show that PHF, DP and GA exhibit the anti-inflammatory and anti‐allergic activities in concordance to the previous clinical trials using PHF on AD children. / In order to further study the involvement of gallic acid and dexamethasone in the pathogenesis of AD, we then established an in vitro skin inflammatory cell model by co‐culturing human basophilic KU812 cells and human dermal fibroblasts mimicking the skin lesions of the AD patients. We revealed that the application of gallic acid alone could already significantly suppress the adhesion molecules expression on KU812 cell and fibroblasts, and the release of AD-related chemokines CCL2, CCL5, CXCL8 and pro-inflammatory cytokine IL-6 from the co‐culture. In addition, the combined use of dexamethasone and gallic acid showed an enhanced suppressive effect on ICAM-1 on KU812, and ICAM-1 and VCAM-1 on fibroblasts, AD‐releated chemokines CCL2 and CXCL8, and pro-inflammatory cytokine IL-6. / The above findings suggest that PHF, DP and GA are anti-inflammatory and anti-allergic natural plant products by suppressing the transmigration of basophils into the inflamed sites and the subsequent release of allergic inflammation mediators e.g. inflammation-related chemokines and proinflammatory cytokines.The results suggest that natural plant product gallic acid could be a potential therapeutic agent in treating skin inflammation in AD, and the combined use of gallic acid with dexamethasone could lower the dosage of dexamethasone used in AD patients. Together, of the results of this study shed light for a novel therapeutic modality of AD using a safer natural plant derived product with high potency and less side effects to treat AD. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Yan Ping Kelly. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 107-122). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.III / 摘要 --- p.VI / PUBLICATIONS --- p.IX / ABBREVIATIONS --- p.XI / TABLES OF CONTENTS --- p.XIII / Chapter CHAPTER 1: --- General Introduction / Chapter 1.1 --- Allergy --- p.1 / Chapter 1.1.1 --- Definition of Allergy --- p.1 / Chapter 1.1.2 --- Allergic diseases and their prevalence --- p.2 / Chapter 1.1.3 --- Allergic inflammation and its characteristics --- p.2 / Chapter 1.1.4 --- Treatment of allergy --- p.4 / Chapter 1.1.5 --- Atopic Dermatitis --- p.7 / Chapter 1.2 --- Biology of basophils --- p.8 / Chapter 1.2.1 --- Development of basophils --- p.8 / Chapter 1.2.2 --- Morphology and phenotype --- p.9 / Chapter 1.2.3 --- Mast cells and basophils --- p.11 / Chapter 1.2.4 --- Basophils and allergic inflammation --- p.12 / Chapter 1.2.5 --- Human basophilic KU812 cell line --- p.13 / Chapter 1.3 --- Adhesion molecules in allergic inflammation --- p.14 / Chapter 1.3.1 --- Selectins --- p.14 / Chapter 1.3.2 --- Integrins --- p.16 / Chapter 1.3.3 --- Immunoglobulin gene super family --- p.16 / Chapter 1.4 --- Chemokines in allergic inflammation --- p.18 / Chapter 1.4.1 --- C chemokines --- p.18 / Chapter 1.4.2 --- CC chemokines --- p.18 / Chapter 1.4.3 --- CXC chemokines --- p.19 / Chapter 1.4.4 --- CX3C chemokines --- p.19 / Chapter 1.5 --- Cytokines in allergic inflammation --- p.20 / Chapter 1.5.1 --- Proinflammatory cytokines --- p.20 / Chapter 1.5.2 --- Anti-inflammatory cytokines --- p.23 / Chapter 1.6 --- Signal Transduction in allergic inflammation --- p.25 / Chapter 1.6.1 --- Intracellular signaling mechanisms --- p.25 / Chapter 1.6.2 --- RAS-RAF-MAPK pathway --- p.27 / Chapter 1.6.3 --- JAK/STAT pathway --- p.28 / Chapter 1.6.4 --- PI3K-Akt pathway --- p.28 / Chapter 1.6.5 --- NF-κB pathway --- p.28 / Chapter 1.7 --- Aim of Study --- p.29 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Cell Culture --- p.32 / Chapter 2.1.2 --- Serum Supplements --- p.33 / Chapter 2.1.3 --- Recombinant human cytokine --- p.33 / Chapter 2.1.4 --- Dexamethasone --- p.33 / Chapter 2.1.5 --- Phosphate-buffered saline --- p.34 / Chapter 2.1.6 --- Dimethyl sulfoxide --- p.34 / Chapter 2.1.7 --- Nucleotide-binding oligomerization domain ligands --- p.34 / Chapter 2.1.8 --- BAY 117082 --- p.34 / Chapter 2.1.9 --- Cell surface and intracellular immunofluorescence staining --- p.35 / Chapter 2.1.10 --- In vitro XTT based toxicology assay kit --- p.38 / Chapter 2.1.11 --- Quantitative analysis of inflammatory mediators release --- p.38 / Chapter 2.1.12 --- Natural Products --- p.39 / Chapter 2.1.13 --- Animal Experiment --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Cell Culture --- p.41 / Chapter 2.2.2 --- Preparation of plant extracts --- p.42 / Chapter 2.2.3 --- Cell toxicity of the natural products --- p.42 / Chapter 2.2.4 --- Flow cytometric analysis of cell surface expression of molecules --- p.43 / Chapter 2.2.5 --- CBA assay --- p.43 / Chapter 2.2.6 --- Flow cytometric analysis of activated intracellular molecules --- p.44 / Chapter 2.2.7 --- Allergic asthmatic mice model --- p.45 / Chapter 2.2.8 --- Statistical analysis --- p.45 / Chapter Chapter 3: --- Anti-inflammatory and anti-allergic properties of Pentaherbs formula, Danpi and Gallic acid / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.1.1 --- Basophils in inflammation --- p.46 / Chapter 3.1.2 --- IL-33 --- p.47 / Chapter 3.1.3 --- Natural plant products --- p.48 / Chapter 3.1.4 --- Dexamethasone --- p.51 / Chapter 3.1.5 --- Hypothesis and aim of study --- p.52 / Chapter 3.2 --- Results --- p.54 / Chapter 3.2.1 --- Cell cytotoxicity of PHF, DP and GA on human basophilic KU812 cells --- p.54 / Chapter 3.2.2 --- Effect of adhesion molecules expression on IL-33-activated KU812 cells treated with PHF, DP and GA --- p.56 / Chapter 3.2.3 --- Effect of PHF, DP and GA on inflammation-related chemokines CCL2,CCL5, CXCL-8 production from IL-33-activated KU812 cells --- p.59 / Chapter 3.2.4 --- Effect of PHF, DP and GA on pro-inflammatory cytokine IL-6 production from IL-33-activated KU812 cells --- p.64 / Chapter 3.2.5 --- Intracellular signaling pathways involved in GA treatment on IL33-activated KU812 cells --- p.67 / Chapter 3.2.6 --- Effect on the adhesion molecules expression, chemokines and cytokines release of IL-33-activated human basophilic KU812 cells upon the combined treatment of PHF/DP/GA with dexamethasone --- p.73 / Chapter 3.2.7 --- In vivo effect of PHF and DP on Th2 and inflammatory cytokines concentration in serum or BALF in allergic inflammatory mice models --- p.76 / Chapter 3.3 --- Discussion --- p.79 / Chapter Chapter 4: --- Gallic acid and Dexamethasone in Atopic Dermatitis / Chapter 4.1 --- Introductions --- p.84 / Chapter 4.1.1 --- Atopic Dermatitis --- p.84 / Chapter 4.1.2 --- Basophils in AD --- p.86 / Chapter 4.1.3 --- Dermal fibroblasts in AD --- p.86 / Chapter 4.1.4 --- Hypothesis --- p.87 / Chapter 4.2 --- Results --- p.88 / Chapter 4.2.1 --- Effect of the combined use of GA and dexamethasone on ICAM-1 expression on KU812 cells co-cultured with fibroblasts --- p.88 / Chapter 4.2.2 --- Effect of the combined use of GA and dexamethasone on ICAM-1 and VCAM-1 expression on fibroblasts co-cultured with KU812 cells --- p.90 / Chapter 4.2.3 --- Effect on chemokines release from the co-culture upon the treatment with GA and dexamethasone --- p.93 / Chapter 4.2.4 --- Effect on cytokine release from the co-culture treated with GA and dexamethasone --- p.96 / Chapter 4.3 --- Discussions --- p.98 / Chapter Chapter 5: --- Concluding Remarks and Future Prospective / Chapter 5.1 --- Concluding remarks --- p.100 / Chapter 5.2 --- Future prospective --- p.101 / References --- p.107

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