No description available.

Rostliny rodu Echinacea jsou léčivé byliny stimulující imunitní systém. Používají se zvláště v boji proti chřipce a infekcím horních cest dýchacích. V této práci bylo analyzováno 7 komerčních výrobků obsahujících výtažky z rostliny Echinacea vysokoúčinnou kapalinovou chromatografií. Bylo provedeno kvantitativní stanovení derivátů kyseliny kávové, jež jsou látky zodpovědné za léčivé účinky rostliny. Zjištěné obsahy kyseliny chlorogenové, kávové, cichorové a echinakosidu byly porovnány v jednotlivých výrobcích. Dále byly připraveny extrakty z kořene, listů a květů třapatky nachové pomocí extrakce za zvýšeného tlaku extrakčního rozpouštědla při různých podmínkách, aby se z obsahu účinných látek v nich přítomných zjistilo, které extrakční podmínky jsou nejlepší. Z naměřených dat bylo zjištěno, že největší celková koncentrace obsahových látek je přítomna ve výrobku Imunit Echinaceové kapky firmy Simply You. Jako nejlepší extrakční podmínky se ukázaly teplota 80 °C v kombinaci s rozpouštědlem ve složení EtOH/voda = 40/60, a to jak při extrakci kořene, tak i listů a květů.

Embora a grande maioria dos pacientes com asma tenha controle com o padrão de tratamento com corticosteroides, 5 a 10% destes asmáticos ainda desenvolvem algumas formas graves da doença e são considerados pacientes difíceis de controlar. A ativação do NFkB contribui para a manutenção e desenvolvimento de inflamação pulmonar crônica na fisiopatologia da asma. O éster fenetilico do ácido cafeico (CAPE), um componente ativo de própolis das colmeias de abelhas, é conhecido por ter propriedades antiinflamatórias e imunomoduladoras. É conhecido como um potente e um inibidor específico da ativação de NFk B. Nós anteriormente mostramos que Y27632, um inibidor de Rho quinase, atenuou a resposta pulmonar das vias aéreas, inflamação e remodelamento. Para o nosso conhecimento, tratamentos com os inibidores de NFk B ou Rho-quinase em comparação com o uso de corticosteroides não foram previamente investigados em um modelo animal de inflamação pulmonar crônica alérgica. MÉTODOS: Trinta camundongos BALB/C machos (20 a 25g) foram divididos em cinco grupos (n=6 em cada grupo): SAL (instilado com solução salina), OVA (expostos à ovalbumina), OVA-CAPE (expostos à ovalbumina e tratados com CAPE), OVA-RHO (expostos à ovalbumina e tratados com inibidor de Rho), OVA-CORT (expostos à ovalbumina e tratados com corticosteróide). Os animais foram sensibilizados para ovalbumina durante 28 dias e os controles receberam solução salina. A administração de corticosteróide (1mg/Kg/dia) e CAPE (10mg/Kg/dia) foi feita por via intraperitoneal e Y-27632 (10 mg/kg) administrado por via intranasal, todos nos dias 22, 24, 26 e 28 do protocolo experimental. Vinte e quatro horas após a conclusão do protocolo experimental, os animais foram submetidos a uma avaliação da curva de resposta à dose à metacolina, consideramos as respostas máximas da Resistência ao Sistema Respiratório (Rrs) e Elastância (Ers) às 24 horas após o último desafio com ovalbumina (respostas basais e máximas após a metacolina desafio 3, 30 e 300mg/mL). Posteriormente, os pulmões foram removidos e a análise histológica foi realizada usando morfometria. RESULTADOS: não houve diferenças nos valores basais de todos os grupos. expostos à ovalbumina Houve um aumento em Rrs e Ers após o desafio da metacolina em comparação com o grupo SAL (p < 0,05). No grupo exposto ao OVA, os tratamentos com CAPE, CORT e RHO reduziram a resposta máxima de Ers em relação ao OVA (p < 0,05). Em relação ao tratamento com somente CAPE atenuou a resposta máxima de Rrs em relação ao OVA (p < 0,05). Houve diminuição nos eosinófilos, conteúdo de fibras de colágeno, células positivas para iNOS, MMP-9, TIMP-1 em grupos OVA-CAPE, OVA-CORT e OVA-RHO em comparação com os animais sensibilizados (grupo OVA) (p < 0,05). CONCLUSÃO: a inibição de NFkB e Rho-quinase contribuiu para o controle da hiperresponsividade, inflamação, processo de remodelamento da matriz extracelular e ativação do estresse oxidativo. Embora os inibidores de NFkB e Rho-quinase sejam uma alternativa ao tratamento da asma, são necessários mais estudos / Although the great majority of asthma patients obtain control with the gold standard treatment using corticosteroids, 5 to 10% of these asthmatics still develop some severe forms of the disease, called difficult to control patients. Activation of NFk B contributes to the maintenance and development of chronic lung inflammation in pathophysiology of asthma. Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have anti-inflammatory and immunomodulatory properties. It is known as a potent and a specific inhibitor of NFk B activation. We previously have shown that Y27632, a Rho-quinase inhibitor, attenuated airway lung responsiveness, inflammation and remodeling. To our knowledge, treatments with the NFk B or Rho-kinase inhibitors compared to the use of corticosteroids have not been previously investigated in an animal model of chronic allergic lung inflammation. METHODS: Thirty male BALB/c mice (20 to 25g) were divided into five groups (n=6 in each group): SAL (saline-instilled), OVA (exposed-ovalbumin), OVA-CAPE (exposedovalbumin and treated with CAPE), OVA-RHO (exposed-ovalbumin and treated with Rho inhibitor), OVA-CORT (exposed-ovalbumin and treated with corticosteroid). The BALB/c mice were sensitized to ovalbumin for 28 days and controls received saline. The administration of corticosteroid (1mg/Kg/day) and CAPE (10mg/Kg/day) was made intraperitoneally and Y-27632 (10mg/kg) was intranasally, all on days 22, 24, 26 and 28 of the experimental protocol. Twentyfour hours after completion of the experimental protocol, animals were subject to an evaluation of dose response curve to methacholine. We considered the maximal responses of respiratory system resistance (Rrs) and elastance (Ers) 24 hours after the last challenge with ovalbumin (baseline and maximal responses after metacholine challenge 3, 30 and 300mg/mL). Afterwards, lungs were removed and histological analysis was performed using morphometry. RESULTS: There were no differences on baseline values of all groups. Ovalbumin-exposed mice had an increase in Rrs and Ers after methacholine challenge compared to SAL group (p < 0.05). In OVA-exposed groups, the treatments with CAPE, CORT and RHO reduced maximal response of %Ers compared to OVA (p < 0.05). In relation to %Rrs only CAPE treatment attenuated the maximal response compared to OVA (p < 0.05). There was a decrease in eosinophils, collagen fibers content, iNOS, MMP-9, TIMP-1 positive cells in OVACAPE, OVA-CORT and OVA-RHO groups compared to only sensitized animals (OVA group) (p < 0.05). CONCLUSION: NFkB and Rho-quinase inhibition contributes to the control of hyperresponsiveness, inflammation, extracellular matrix remodeling process and oxidative stress activation. Although NFkB and Rho-quinase inhibitors were an alternative to asthma treatment, more studies are needed

Mit den vorgestellten Untersuchungen sollten das Potential der in Europa vorkommenden Echten Goldrute für den Anbau evaluiert und der Einfluß von Erntetermin, Schnitthöhe und Düngungsmaßnahmen sowie der abiotischen Umweltfaktoren Licht und Wasser auf den Ertrag und den Inhaltsstoffgehalt von Solidago virgaurea L. geprüft werden. In vier Feldversuchen wurden Inhaltsstoffgehalte, Ertragsparameter, Entwicklungsstadien sowie morphologische Merkmale untersucht. Die Gehalte an Flavonolglycosiden, Phenolglucosiden und Kaffeesäurederivaten wurden mittels HPLC und nachfolgender DAD-Detektion bestimmt. 45 Akzessionen aus dem europäischen Verbreitungsgebiet wurden anhand ihres Blühtermines gegliedert, den Unterarten virgaurea und minuta zugeordnet, und mit Hilfe von 33 morphologischen Merkmalen charakterisiert. Die Analyse von 452 Einzelpflanzen ergab Spannweiten von 0.4 bis 2.4 % Flavonolglycosiden, 0.7 bis 4.6 % Kaffeesäurederivaten und 0 bis 1.6 % Phenolglucosiden. Die phytochemische Zusammensetzung war innerhalb der einzelnen Akzessionen, insbesondere in der Unterart virgaurea hoch variabel. In einer anbaubezogenen Gesamtbewertung der Akzessionen erwiesen sich Herkünfte der Unterart virgaurea aufgrund hoher Drogenerträge von maximal 109 dt/ha und eines ausgewogenen Inhaltsstoffspektrums als die Wertvollsten. Im Lauf der Ontogenese stieg der Drogenertrag der Echten Goldrute an, während die Inhaltsstoffgehalte schwankten (Kaffeesäurederivate, Flavonolglycoside) oder relativ kontinuierlich absanken (Phenolglucoside). Eine gehaltsorientierte Ernte müßte daher zum relativ frühen Zeitpunkt des mittleren Knospenstadiums erfolgen. Hohe Masse- und Inhaltsstofferträge sind in einem Ernteverfahren mit niedrigen Schnitthöhen von 15 bis 30 cm erreichbar. In voll besonnten Pflanzen wurde im Vergleich zu schattierten eine höhere Anreicherung von Flavonolglycosiden und Kaffeesäurederivaten festgestellt, die durch die UV-Schutzfunktion dieser Stoffgruppen erklärbar ist. Daher muß angenommen werden, daß ontogenetisch bedingte Gehaltsveränderungen der Flavonolglycoside und Kaffeesäurederivate durch unterschiedliche Strahlungsverhältnisse überlagert werden können. Stickstoffdüngung verursachte neben der erwarteten ertragssteigernden Wirkung Unterschiede im Inhaltsstoffgehalt. In der ungedüngten Variante wurden jeweils die höchsten Flavonolglycosidgehalte bestimmt, gefolgt von der organischen Düngung sowie der Mehrnährstoff- und Stickstoffdüngung. Während die ermittelten Stickstoff- (maximal 96 kg/ha N) und Phosphorentzüge (maximal 39 kg/ha P2O5) vergleichsweise gering waren, wurden Kaliumentzüge (maximal 172 kg/ha K2O) festgestellt, die auch im Vergleich zu anderen Blatt- und Krautdrogen relativ hoch ausfielen. Im Gesamtüberblick aller Einflüsse wurde deutlich, daß Flavonolglycoside, gefolgt von Kaffeesäurederivaten am empfindlichsten auf Umwelteinflüsse wie Lichteinstrahlung und Nährstoffverfügbarkeit reagierten, während die Gruppe der Phenolglucoside nur geringe Schwankungen zeigte. Die Ergebnisse aller Versuchsfragen spiegeln das Spannungsfeld von landwirtschaftlichem Ertrag und pharmazeutisch geforderter Qualität wider, die häufig negativ korreliert sind. Bei der Produktion der Droge von Echter Goldrute muß daher eine Balance zwischen beiden Zielgrößen gefunden werden. / The aim of the presented studies was to evaluate the potential for cultivation of the European Goldenrod (Solidago virgaurea L.) and to examine the influences of harvesting date, cutting height, fertilization and of the abiotic environmental factors light and water on drug yield and compound content of the plant. In four field trials a fingerprint of 10 constituents, several parameters of drug yield, development stages and morphological traits were examined. The contents of flavonol glycosides, phenolic glucosides and caffeic acid derivatives were determined by HPLC and UV-detection by DAD. 45 european accession were sorted by their dates of anthesis and subdivided into the subspecies virgaurea and minuta and thereafter characterized by 33 morphological traits. The analysis of 452 individual plants showed ranges of 0.4 to 2.4 % flavonol glycosides, 0.7 to 4.6 % caffeic acid derivatives and 0.0 to 1.6 % phenolic glucosides. Within the examined accessions, especially subspecies virgaurea, a high variability of the phytochemical composition was observed. In an evaluation, accessions of subspecies virgaurea turned out to be most suitable for cultivation due to high drug yields up to 106 dt per ha and well-balanced spectra of constituents. During ontogenesis the drug yield of S. virgaurea increased, while compound contents varied (caffeic acid derivatives, flavonol glycosides) or decreased continuously (phenolic glucosides). Therefore a harvest aiming at high compound contents has to be carried out at a early development stage of mid budding. High mass and constituent yields can be obtained by a harvesting method with low cutting heights of 15 to 30 cm above ground. Plants in a sunny environment accumulated significantly higher amounts of flavonol glycosides and caffeic acid derivatives than shaded plants, which can be explained by the UV-protective function of these compound groups. Therefore it can be concluded that phytochemical variations during ontogenesis could be modified by different light conditions. Beside the expected drug yield increase, fertilization with nitrogen caused differences in the compound contents. The highest flavonol glycosides contents were observed in the unfertilized treatment, followed by treatments with organic fertilization, multinutrient fertilizer and calcium ammonium nitrate. While the nitrogen uptake by the plant (maximal 96 kg/ha N) and phosphorus (maximal 39 kg/ha P2O5) were relatively low, contrary potassium uptake (maximal 172 kg/ha K2O) was relatively high compared to other herbal drugs. In an overview of all influences it became obvious that the reaction of flavonol glycosides and caffeic acid derivatives were relatively sensitive to environmental influences such as light conditions and nutrient availability, while the group of phenolic glucosides showed low variations. The results of all field trials reflect the conflict between agricultural yield and pharmaceutical quality which are frequently negatively correlated. Therefore a balance between these two goals has to be found during drug production of S. virgaurea.

Die mittlere Lebenserwartung des Menschen ist über die letzten 200 Jahre kontinuierlich gestiegen. Da Langlebigkeit ohne Gesundheit wenig Wert besitzt, ist es ein zentrales Anliegen, das Auftreten altersbedingter Krankheiten zu mindern. Besonders pflanzliche Phytochemikalien, im speziellen Polyphenole (PPs), sollen erheblich an der Gesundheitsförderung mitwirken. Die exakten Mechanismen jedoch, welche die Wirkvielfalt erklären könnten, sind nicht im Detail bekannt. Diese Fragen können nur durch in vivo Studien an Modelorganismen beantwortet werden, die sowohl die Lebensdauer, sowie physiologische und genetische Parameter einschließen. In dieser Studie wurden drei PPs mit lebensverlängernden Eigenschaften in C. elegans identifiziert: Quercetin (Q), Kaffeesäure (CA) und Rosmarinsäure (RA). Für alle drei PPs wurden hormetische Konzentration-Wirkungs-Kurven gefunden, dennoch war die Hormetin-typische Aktivierung einer Stressantwort (gemessen als Geneexpressions-Level von Hitzeschock-Proteinen) auf Q und RA beschränkt. Eine Umverteilung von Ressourcen nach dem Prinzip der „Disposable Soma Theorie“ konnte anhand von Abweichungen in der Größe, verändertem Lipid-Metabolismus und verzögerter Reproduktion (bei gleichbleibender Anzahl der Nachkommen), für alle drei PPs gezeigt werden. Während direkte CR-Effekte ausgeschlossen wurden, ist dies nicht möglich für durch CA und RA ausgelöste indirekte CR-Effekte, da beide die Lebensspanne von sir-2.1 Mutanten nicht verlängern konnten. Alle drei PPs verlängerten die Lebensspanne von mev-1 Mutanten, jedoch wurde eine erhöhte TAC in vivo und eine reduzierte oxidative Schädigung, nur durch Q- und CA- Gabe erreicht. Die genetischen Wirkwege der PPs wurden durch Lebensdauer- und Thermotoleranztests mit in alters-relevanten Genen mutierten Nematoden definiert. Die gesundheitsfördernden Eigenschaften von CA und RA konnten so osr-1, sek-1, sir-2.1 and unc-43, sowie daf-16 im Falle von CA, zugeschrieben werden. Die Mechanismen von Q wurden in größerem Umfang, durch die Integration von durchgeführten Lebensdauertests und Microarray-Studien einerseits und einer umfassenden Meta-Analyse von veröffentlichten, alters-relevanten Genexpressions-Profilen andererseits, analysiert. Q wirkt vermutlich durch ein komplexes Zusammenspiel von konservierten genetischen Signalwegen, im Speziellen dem Insulin-ähnlichen (ILS), TGF-beta, p38 MAPK, CAMKII und möglicherweise auch über eine von der Keimbahn und somatischen Gonade ausgehenden Signalwirkung. Zusammenfassend lässt sich sagen, dass sowohl in vivo antioxidative und prooxidative Eigenschaften, die Modulation auf Genebene, sowie eine Umverteilung von Ressourcen zu gewissen Teilen (abhängig vom PP) zur Lebensverlängerung beitragen. / The mean life expectancy of humans has increased continuously over the last 200 years. Since longevity is of little value in the absence of health, it is a central request to prevent the increasing burden of age-related diseases. It is suggested that phytochemicals in plants, specifically the polyphenols (PPs), are important factors to support the overall well-being. However, the precise mechanisms that can explain, in full, the magnitude of impact remains elusive. This knowledge gap can only be plugged by in vivo model organism approaches that integrate lifespan assays with physiological, and genetic parameters following the ingestion of PPs. In this study, three PPs with life-extending properties in C. elegans were identified: Quercetin (Q), Caffeic acid (CA) and Rosmarinic acid (RA). The underlying mechanisms were systematically studied by a broad spectrum of functional and genetic investigations. For all three compounds, life extension was characterized by hormetic concentration-response curves, but stress-response induction, a hallmark of hormetin action, was restricted to Q and RA, at least when assessed at the level of gene expression of heat shock proteins. A reallocation of resources in a disposable soma-like pattern could be shown for all three PPs, because the exposure to Q, CA and RA resulted in variations in body size, altered lipid-metabolism and a tendency towards a delay in reproductive timing. However, the total number of offspring was unaltered. While direct CR effects arising from reduced food uptake could be rejected, an indirect CR effect cannot be excluded for CA and RA, as these PPs failed to provoke longevity in sir-2.1 mutants. Furthermore, the in vitro versus in vivo antioxidative properties were evaluated. While all three PPs could prolong mev-1 lifespan, only Q and CA were shown to increase the TAC in vivo and reduce oxidative damage in the nematodes. To define the genetic pathways of PP action, lifespan and thermotolerance assays were performed in mutant animals devoid of aging-relevant genetic players. These experiments revealed that the health gaining properties of CA and RA both rely on osr-1, sek-1, sir-2.1 and unc-43, plus daf-16 in the case of CA. The mechanisms of Q action are partly distinct and were analyzed in more detail by integrating own mutant lifespan assays and microarray studies with an extensive meta-analysis of published gene expression profiles obtained under aging-relevant conditions. Quercetin is proposed to act through a complex interplay of conserved genetic pathways, for example Insulin-like signaling (ILS), TGF-beta signaling, p38 MAPK, CaMKII, and possibly also due to germline and somatic gonad signaling. Taken together, hormesis, in vivo antioxidative/prooxidative properties, modulation of genetic players, as well as the re-allocation of resources all contribute (to some extent and dependent on the polyphenol) to life extension. Summary 1

Embora a grande maioria dos pacientes com asma tenha controle com o padrão de tratamento com corticosteroides, 5 a 10% destes asmáticos ainda desenvolvem algumas formas graves da doença e são considerados pacientes difíceis de controlar. A ativação do NFkB contribui para a manutenção e desenvolvimento de inflamação pulmonar crônica na fisiopatologia da asma. O éster fenetilico do ácido cafeico (CAPE), um componente ativo de própolis das colmeias de abelhas, é conhecido por ter propriedades antiinflamatórias e imunomoduladoras. É conhecido como um potente e um inibidor específico da ativação de NFk B. Nós anteriormente mostramos que Y27632, um inibidor de Rho quinase, atenuou a resposta pulmonar das vias aéreas, inflamação e remodelamento. Para o nosso conhecimento, tratamentos com os inibidores de NFk B ou Rho-quinase em comparação com o uso de corticosteroides não foram previamente investigados em um modelo animal de inflamação pulmonar crônica alérgica. MÉTODOS: Trinta camundongos BALB/C machos (20 a 25g) foram divididos em cinco grupos (n=6 em cada grupo): SAL (instilado com solução salina), OVA (expostos à ovalbumina), OVA-CAPE (expostos à ovalbumina e tratados com CAPE), OVA-RHO (expostos à ovalbumina e tratados com inibidor de Rho), OVA-CORT (expostos à ovalbumina e tratados com corticosteróide). Os animais foram sensibilizados para ovalbumina durante 28 dias e os controles receberam solução salina. A administração de corticosteróide (1mg/Kg/dia) e CAPE (10mg/Kg/dia) foi feita por via intraperitoneal e Y-27632 (10 mg/kg) administrado por via intranasal, todos nos dias 22, 24, 26 e 28 do protocolo experimental. Vinte e quatro horas após a conclusão do protocolo experimental, os animais foram submetidos a uma avaliação da curva de resposta à dose à metacolina, consideramos as respostas máximas da Resistência ao Sistema Respiratório (Rrs) e Elastância (Ers) às 24 horas após o último desafio com ovalbumina (respostas basais e máximas após a metacolina desafio 3, 30 e 300mg/mL). Posteriormente, os pulmões foram removidos e a análise histológica foi realizada usando morfometria. RESULTADOS: não houve diferenças nos valores basais de todos os grupos. expostos à ovalbumina Houve um aumento em Rrs e Ers após o desafio da metacolina em comparação com o grupo SAL (p < 0,05). No grupo exposto ao OVA, os tratamentos com CAPE, CORT e RHO reduziram a resposta máxima de Ers em relação ao OVA (p < 0,05). Em relação ao tratamento com somente CAPE atenuou a resposta máxima de Rrs em relação ao OVA (p < 0,05). Houve diminuição nos eosinófilos, conteúdo de fibras de colágeno, células positivas para iNOS, MMP-9, TIMP-1 em grupos OVA-CAPE, OVA-CORT e OVA-RHO em comparação com os animais sensibilizados (grupo OVA) (p < 0,05). CONCLUSÃO: a inibição de NFkB e Rho-quinase contribuiu para o controle da hiperresponsividade, inflamação, processo de remodelamento da matriz extracelular e ativação do estresse oxidativo. Embora os inibidores de NFkB e Rho-quinase sejam uma alternativa ao tratamento da asma, são necessários mais estudos / Although the great majority of asthma patients obtain control with the gold standard treatment using corticosteroids, 5 to 10% of these asthmatics still develop some severe forms of the disease, called difficult to control patients. Activation of NFk B contributes to the maintenance and development of chronic lung inflammation in pathophysiology of asthma. Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have anti-inflammatory and immunomodulatory properties. It is known as a potent and a specific inhibitor of NFk B activation. We previously have shown that Y27632, a Rho-quinase inhibitor, attenuated airway lung responsiveness, inflammation and remodeling. To our knowledge, treatments with the NFk B or Rho-kinase inhibitors compared to the use of corticosteroids have not been previously investigated in an animal model of chronic allergic lung inflammation. METHODS: Thirty male BALB/c mice (20 to 25g) were divided into five groups (n=6 in each group): SAL (saline-instilled), OVA (exposed-ovalbumin), OVA-CAPE (exposedovalbumin and treated with CAPE), OVA-RHO (exposed-ovalbumin and treated with Rho inhibitor), OVA-CORT (exposed-ovalbumin and treated with corticosteroid). The BALB/c mice were sensitized to ovalbumin for 28 days and controls received saline. The administration of corticosteroid (1mg/Kg/day) and CAPE (10mg/Kg/day) was made intraperitoneally and Y-27632 (10mg/kg) was intranasally, all on days 22, 24, 26 and 28 of the experimental protocol. Twentyfour hours after completion of the experimental protocol, animals were subject to an evaluation of dose response curve to methacholine. We considered the maximal responses of respiratory system resistance (Rrs) and elastance (Ers) 24 hours after the last challenge with ovalbumin (baseline and maximal responses after metacholine challenge 3, 30 and 300mg/mL). Afterwards, lungs were removed and histological analysis was performed using morphometry. RESULTS: There were no differences on baseline values of all groups. Ovalbumin-exposed mice had an increase in Rrs and Ers after methacholine challenge compared to SAL group (p < 0.05). In OVA-exposed groups, the treatments with CAPE, CORT and RHO reduced maximal response of %Ers compared to OVA (p < 0.05). In relation to %Rrs only CAPE treatment attenuated the maximal response compared to OVA (p < 0.05). There was a decrease in eosinophils, collagen fibers content, iNOS, MMP-9, TIMP-1 positive cells in OVACAPE, OVA-CORT and OVA-RHO groups compared to only sensitized animals (OVA group) (p < 0.05). CONCLUSION: NFkB and Rho-quinase inhibition contributes to the control of hyperresponsiveness, inflammation, extracellular matrix remodeling process and oxidative stress activation. Although NFkB and Rho-quinase inhibitors were an alternative to asthma treatment, more studies are needed

La récente définition de Brachypodium distachyon comme modèle des graminées en fait un organisme de choix pour l’étude de leur paroi cellulaire, en particulier dans le cadre de leur utilisation comme matière première renouvelable pour le bioéthanol de seconde génération. Les lignines, dont les trois unités (H, G et S) proviennent de la polymérisation des monolignols, sont associées aux acides hydroxycinnamiques dans la paroi des céréales et représentent l’obstacle majeur à l’exploitation industrielle de la biomasse lignocellulosique. L’acquisition de connaissances sur les mécanismes dirigeant leur mise en place et leur organisation permettrait d’identifier des facteurs modulant les rendements de production qui y sont associés. Quatre familles de gènes ont été étudiées et l’implication dans la voie de biosynthèse des monolignols de trois gènes a été montrée : BdF5H2 possède une activité férulate-5-hydroxylase permettant la synthèse des précurseurs des unités S des lignines, BdCOMT3 est l’isoforme principale des acide cafféique O-Méthyltransférases et sa perte partielle de fonction cause une diminution de la quantité de lignine, la modification du rapport S/G et une baisse de quantité d’acide p-coumarique dans deux lignées mutantes indépendantes. Enfin, BdCAD1 est l’isoforme principale des alcools cinnamylique déshydrogénases : sa perte de fonction dans deux lignées indépendantes cause la diminution de la quantité globale de lignine et d’acide p-coumarique, une baisse du rapport S/G ainsi que l’accumulation de sinapaldéhyde. Par ailleurs ces deux lignées présentent des rendements de saccharification augmentés de plus d’un quart par rapport au sauvage. / Brachypodium distachyon was recently adopted as an experimental model for grass species. As such, it is used to study grass cell wall, in particular in the context of their use as renewable feedstock for the production of second generation bioethanol. Lignins are polymers of three main units (H, G and S) originating from the polymerization of monolignols, and are linked to hydroxycinnamic acids in grasses. They constitute the main bottleneck to industrial processes targeting lignocellulosic biomass and improving the understanding of the mechanisms directing their structure and deposition could lead to the identification of the factors modulating associated production yields. Four gene families were studied and the involvement of three genes in the monolignols biosynthetic pathway was shown: BdF5H2 displays a ferulate-5-hydroxylase activity enabling the synthesis of the S lignin units, BdCOMT3 is the main caffeic acid O-methyltransferase and its partial loss of function in two independent mutant lines leads to the reduction of lignin content, the modification of the S/G units ratio and a decrease in p-coumaric acid accumulation. BdCAD1 is the main cinnamyl alcohol dehydrogenase isoform: its loss of function in two independent mutant lines results in a decrease in lignin content and of the S/G ratio and the accumulation of sinapaldehyde. Moreover, these two lines display significatively increased saccharification yields.

[ES] El mercado actual del vino tinto demanda vinos de gran potencia visual, elevada concentración aromática y que no resulten excesivamente astringentes ni amargos. Asimismo, el consumidor final demanda cada vez más vinos que se desarrollen en una viticultura más orgánica que garantice la sostenibilidad del cultivo. Para cumplir con estas expectativas, se está limitando la utilización de compuestos químicos en el viñedo, sustituyéndolos por otros naturales con menor impacto medioambiental, y se están utilizando en bodega técnicas como la maceración prefermentativa, que contribuye a la extracción de antocianos y aromas, o la microoxigenación, que permite estabilizar los compuestos polifenólicos en un tiempo reducido. El color de los vinos depende de su concentración en antocianos, pero también la forma y el estado en que éstos se encuentren en el vino, que depende de diferentes factores, siendo uno de ellos el fenómeno de la copigmentación como paso anterior a la polimerización y estabilización. Además, el oxígeno juega un papel crucial en la evolución del color, así como en la oxidación de los polifenoles y en la formación de complejos más estables. El aroma también es muy importante en los vinos, ya que es uno de los primeros aspectos que percibimos. Por ello, es de gran importancia la utilización de técnicas de vinificación que permitan extraer el mayor potencial aromático de los precursores que se encuentran en las uvas. Para conseguir incrementar la calidad de los vinos tintos elaborados con la variedad Monastrell, estableciendo una metodología de elaboración que permita mejorar la concentración polifenólica y aromática de estos vinos, se ha estudiado el efecto de la aplicación en la uva, 10 días antes de la vendimia, de sustancias copigmentantes, tales como, extractos vegetales (romero, trigo sarraceno y té verde) y copigmentos puros (ácido cafeico, rutina y catequina). Asimismo, los copigmentos puros también se aplicaron a la entrada en bodega. Se ensayaron dos técnicas enológicas diferentes: la vinificación tradicional y la maceración prefermentativa en frío (6-8ºC), seguida de vinificación tradicional. Por último, una vez concluida la fermentación maloláctica, los vinos obtenidos fueron sometidos a una adición controlada de acetaldehído durante 45 días, seguido de un proceso de conservación en botella de 12 meses. Los resultados mostraron que los tratamientos con copigmentos, especialmente con extractos vegetales ricos en copigmentos, incrementaron la concentración de antocianos polimerizados y totales, y de compuestos aromáticos favorables para la calidad, destacando el extracto de romero por su acción directa sobre el color de los vinos de Monastrell. Utilizar la maceración prefermentativa en la elaboración de los vinos procedentes de tratamientos de copigmentación, ha permitido incrementar la concentración de polifenoles totales y taninos condensados, así como la de ésteres y acetatos. La microoxigenación, mediante la aplicación de acetaldehído durante la conservación de los vinos tratados previamente con copigmentos, dio lugar a un incremento del porcentaje de antocianos polimerizados, de la concentración de antocianos, de taninos condensados y de polifenoles totales, no afectando a la composición aromática de los vinos. Por todo ello, se puede establecer que la combinación de tratamientos de copigmentación en el campo, especialmente con extractos vegetales, de maceración prefermentativa durante la elaboración, y de microoxigenación durante la conservación, permite elaborar vinos de Monastrell con un color más estable, mayor concentración de compuestos polifenólicos, y mayor concentración de ésteres y acetatos, compuestos relacionados con aromas positivos florales, frutales y especiados. / [CA] El mercat actual del vi negre demana vins de gran potència visual, elevada concentració aromàtica i que no siguen excessivament astringents ni amargs. Així mateix, el consumidor final demana cada vegada més vins que es desenvolupen en una viticultura més orgànica que garanteisca la sostenibilitat del cultiu. Per complir amb aquestes expectatives, s'està limitant la utilització de compostos químics a la vinya, substituint-los per altres naturals amb menor impacte mediambiental, i s'estan utilitzant al celler tècniques com la maceració prefermentativa, que contribueix a l'extracció d'antocians i aromes, o la microoxigenació, que permet estabilitzar els compostos polifenòlics en un temps reduït. El color dels vins depèn de la seua concentració en antocians, però també en la forma i l'estat en que aquests es troben en el vi, que depèn de diferents factors, siguent un d'ells el fenòmen de la copigmentació com pas anterior a la polimerització i l'estabilització. A més a més, l'oxígen juga un paper crucial en l'evolució del color, així com en l'oxidació dels polifenols i en la formació dels complexos més estables. L'aroma també és molt important en els vins, ja que és un dels primers aspectes que percebem. Per això, és de gran importància la utilització de tècniques de vinificació que permeten extraure el major potencial aromàtic dels precursors que es troben al raïm. Per aconseguir augmentar la qualitat dels vins negres elaborats amb la varietat Monastrell, establint una metodologia d'elaboració que permeta millorar la concentració polifenòlica i aromàtica d'aquests vins, s'ha estudiat l'efecte de l'aplicació en el raïm, 10 dies abans de la verema, de substàncies copigmentants, com ara, extractes vegetals (romer, blat negre i te verd) i copigments purs (àcid cafeic, rutina i catequina). Així mateix, els copigments purs també es van aplicar a l'entrada al celler. Es van assajar dos tècniques enològiques diferents: la vinificació tradicional i la maceració prefermentativa en fred (6-8ºC), seguida de vinificació tradicional. Finalment, una vegada conclosa la fermentació malolàctica, els vins obtinguts van ser sotmesos a una addició controlada d'acetaldehid durant 45 dies, seguit d'un procés de conservació en ampolla de 12 mesos. Els resultats van mostrar que els tractaments amb copigments, especialment amb extractes vegetals rics en copigments, van augmentar la concentració d'antocians polimeritzats i totals, i de compostos aromàtics favorables per a la qualitat, destacant l'extracte de romer per la seua acció directa sobre el color dels vins de Monastrell. Utilitzar la maceració prefermentativa en l'elaboració dels vins procedents de tractaments de copigmentació, ha permès augmentar la concentració de polifenols totals i tanins condensats, així com la d'èsters i acetats. La microoxigenació, mitjançant l'aplicació d'acetaldehid durant la conservació dels vins tractats prèviament amb copigments, va donar lloc a un augment del percentatge d'antocians polimeritzats, de la concentració d'antocians, de tanins condensats i de polifenols totals, no afectant a la composició aromàtica dels vins. Per tot això, es pot establir que la combinació de tractaments de copigmentació en el camp, especialment amb extractes vegetals de maceració prefermentativa durant l'elaboració, i de microoxigenació durant la conservació, permet elaborar vins de Monastrell amb un color més estable, més concentració de compostos polifenòlics, i major concentració d'èsters i acetats, compostos relacionats amb aromes positus florals, fruitals i especiats. / [EN] The current red wine market demands wines with high visual power, great aromatic concentration and that are not excessively astringent or bitter. Furthermore, the final consumer increasingly demands developed wines in a more organic viticulture in order to ensure the sustainability of the crop. To meet these expectations, the use of chemical compounds in the vineyard is being limited, replacing them by another natural ones with less environmental impact, and techniques such as pre-fermentation maceration are being used in the winery, which contributes to the extraction of anthocyanins and aromas, or micro-oxygenation, that allows polyphenolic compounds to be stabilized in a reduced time. The colour of wines depends on their concentration in anthocyanins, but also the form and state in which they are found in the wine, which depends on several factors, one of them being the phenomenon of copigmentation as a previous step to polymerization and stabilization. In addition, the oxygen has a crucial role in colour's evolution, as well as, in the oxidation of polyphenols and in the formation of more stable complexes. Aroma is also very important in wines, due to the fact that is one of the first aspects that we perceive. For this reason, the use of vinification techniques that allow extracting the greatest aromatic potential of the precursors found in the grapes, is of great importance. In order to increase the quality of red wines made with the Monastrell variety, establishing a production methodology that allows improving the polyphenolic and aromatic concentration of these wines, the effect of the application on the grape, 10 days before harvest, has been studied, of copigmenting substances such as plant extracts (rosemary, buckwheat and green tea) and pure copigments (caffeic acid, rutin and catechin). Likewise, pure copigments were also applied in the cellar entrance. Two different enological techniques were tested: traditional vinification and cold pre-fermentation maceration (6-8ºC), followed by traditional vinification. Finally, once the malolactic fermentation was concluded, the wines obtained were subjected to a controlled addition of acetaldehyde for 45 days, followed by a 12-month bottle preservation process. The results showed that treatments with copigments, especially with plant extracts rich in copigments, increased the concentration of polymerized and total anthocyanins, and aromatic compounds favorable for quality, highlighting rosemary extract for its direct action on the colour of wines of Monastrell. Using pre-fermentation maceration in the production of wines from copigmentation treatments has made it possible to increase the concentration of total polyphenols and condensed tannins, as well as that of esters and acetates. Micro-oxygenation through the application of acetaldehyde during the conservation of wines previously treated with copigments gave rise to an increase in the percentage of polymerized anthocyanins, in the concentration of anthocyanins, in condensed tannins and in total polyphenols, without affecting the aromatic composition of the wines. For all of these reasons, it can be established that the combination of copigmentation treatments in the field, particularly with plant extracts, pre-fermentation maceration during production and micro-oxygenation during storage, allows the production of Monastrell wines with a more stable colour, higher concentration of polyphenolics, and higher concentration of esters and acetates, compounds related to positive floral, fruity and spicy aromas. / Anaya Martínez, JA. (2022). Efecto de la aplicación de copigmentos vegetales sobre la composición fenólica y aromática de vinos tintos de Monastrell elaborados con diferentes técnicas de vinificación [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185126 / TESIS

Cardiovascular diseases are among the leading causes of death worldwide and particularly in the developed World. The search for new therapeutic approaches for improving the functions of the damaged heart is therefore a critical endeavour. Myocardial infarction, which can lead to heart failure, is associated with irreversible loss of functional cardiomyocytes. The loss of cardiomyocytes poses a major difficulty for treating the damaged heart since terminally differentiated cardiomyocytes have very limited regeneration potential. Currently, the only effective treatment for severe heart failure is heart transplantation but this option is limited by the acute shortage of donor hearts. The high incidence of heart diseases and the scarcity donor hearts underline the urgent need to find alternative therapeutic approaches for treating cardiovascular diseases. Pluripotent embryonic stem (ES) cells can differentiate into functional cardiomyocytes. Therefore the engraftment of ES cell-derived functional cardiomyocytes or cardiac progenitor cells into the damaged heart to regenerate healthy myocardial tissues may be used to treat damaged hearts. Stem cell-based therapy therefore holds a great potential as a very attractive alternative to heart transplant for treating heart failure and other cardiovascular diseases. A major obstacle to the realisation of stem cell-based therapy is the lack of donor cells and this in turn is due to the fact that, currently, the molecular mechanisms or the regulatory signal transduction mechanisms that are responsible for mediating ES cell differentiation into cardiomyocytes are not well understood. Overcoming this huge scientific challenge is absolutely necessary before the use of stem cell-derived cardiomyocytes to treat the damaged heart can become a reality. Therefore the aim of this thesis was to investigate the signal transduction pathways that are involved in the differentiation of stem cells into cardiomyocytes. The first objective was the establishment and use of cardiomyocyte differentiation models using H9c2 cells and P19 stem cells to accomplish the specific objectives of the thesis. The specific objectives of the thesis were, the investigation of the roles of (i) nitric oxide (ii) protein kinase C (PKC), (iii) p38 mitogen-activated protein kinase (p38 MAPK) (vi) phosphoinositide 3-kinase (PI3K) and (vi) nuclear factor-kappa B (NF-kB) signalling pathways in the differentiation of stem cells to cardiomyocytes and, more importantly, to identify where possible any points of convergence and potential cross-talk between pathways that may be critical for differentiation to occur. P19 cells were routinely cultured in alpha minimal essential medium (α-MEM) supplemented with 100 units/ml penicillin /100 μg/ml streptomycin and 10% foetal bovine serum (FBS). P19 cell differentiation was initiated by culturing the cells in microbiological plates in medium containing 0.8 % DMSO to form embryoid bodies (EB). This was followed by transfer of EBs to cell culture grade dishes after four days. H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Differentiation was initiated by incubating the cells in medium containing 1% FBS. In both models, when drugs were employed, they were added to cells for one hour prior to initiating differentiation. Cell monolayers were monitored daily over a period of 12 or 14 days. H9c2 cells were monitored for morphological changes and P19 cells were monitored for beating cardiomyocytes. Lysates were generated in parallel for western blot analysis of changes in cardiac myosin heavy chain (MHC), ventricular myosin chain light chain 1(MLC-1v) or troponin I (cTnI) using specific monoclonal antibodies. H9c2 cells cultured in 1% serum underwent differentiation as shown by the timedependent formation of myotubes, accompanied by a parallel increase in expression of both MHC and MLC-1v. These changes were however not apparent until 4 to 6 days after growth arrest and increased with time, reaching a peak at day 12 to 14. P19 stem cells cultured in DMSO containing medium differentiated as shown by the timedependent appearance of beating cardiomyocytes and this was accompanied by the expression of cTnI. The differentiation of both P19 stem cells and H9c2 into cardiomyocytes was blocked by the PI3K inhibitor LY294002, PKC inhibitor BIM-I and the p38 MAPK inhibitor SB2035800. However when LY294002, BIM-I or SB2035800 were added after the initiation of DMSO-induced P19 stem cell differentiation, each inhibitor failed to block the cell differentiation into beating cardiomyocytes. The NF-kB activation inhibitor, CAPE, blocked H9c2 cell differentiation into cardiomyocytes. Fast nitric oxide releasing donors (SIN-1 and NOC-5) markedly delayed the onset of differentiation of H9c2 cells into cardiomyocytes while slow nitric oxide releasing donors (SNAP and NOC-18) were less effective in delaying the onset of differentiation or long term differentiation of H9c2 cells into cardiomyocytes. Akt (protein kinase B) is the key downstream target of PI3K. Our cross-talk data also showed that PKC inhibition and p38 MAPK inhibition respectively enhanced and reduced the activation of Akt, as determined by the phosphorylation of Akt at serine residue 473. In conclusion, PKC, PI3K, p38 MAPK and NF-kB are relevant for the differentiation of stem cells into cardiomyocytes. Our data also show that the PKC, PI3K and p38 MAPK signalling pathways are activated as very early events during the differentiation of stem cells into cardiomyocytes. Our data also suggest that PKC may negatively regulate Akt activation while p38 MAPK inhibition inhibits Akt activation. Our fast NO releasing donor data suggest that nitric oxide may negatively regulate H9c2 cell differentiation.