Tesi sul tema "Antitumoral properties"

The members of the epidermal growth factor (EGF) / ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this thesis work we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. The lack of EGFR-mediated growth-stimulatory activity prompted us to evaluate EGFt for targeted delivery of 111In, an Auger electron emitter, into EGFR-positive cancer cells. An 111In-DTPA-EGFt radioconjugate was developed and its properties were analyzed and compared to those of 111In-DTPA-hEGF. First we determined that 111In-DTPA-EGFt displays high specificity and affinity for EGFR. However, the cellular uptake of 111In-DTPA-EGFt resulted to be lower than that of 111In-DTPA-hEGF. Once internalized, 111In-DTPA-EGFt showed a high efficiency to accumulate into the cell nucleus, where the radioactivity emitted by 111In may damage the DNA. In accordance, 111In-DTPA-EGFt showed to be cytotoxic in vitro against breast cancer cells, although its cytotoxicity was lower compared to 111In-DTPA-hEGF. In vivo studies revealed a longer half-life in blood for 111In-DTPA-EGFt than for 111In-DTPA-hEGF and higher uptake in the kidney, with minor accumulation in other normal tissues. 111In-DTPA-EGFt accumulated in MDA-MB-468 tumors where, interestingly, 111In-DTPA-EGFt was detected in a great proportion in the cell nucleus. All the data obtained from this work indicate that EGFt may be a potential EGFR blocker for cancer therapy and also an attractive ligand for delivery of cytotoxic agents into the nucleus of EGFR-positive cancer cells.
Els receptors i lligands de la família del factor de creixement epidèrmic (EGF)/ErbB són dianes molt importants en el desenvolupament de teràpies contra el càncer. No obstant, l’eficàcia terapèutica dels fàrmacs dirigits a atacar aquesta via i que són utilitzats actualment en clínica és limitada. Per aquest motiu la recerca de noves molècules que inactivin els receptors d’aquesta família mitjançant noves estratègies és avui dia una de les vies més explorades. En aquesta tesi s’ha desenvolupat un pèptid idèntic al factor de creixement epidèrmic EGF que li manca la seva part C-terminal (EGFt) com a nou inhibidor de EGFR. El disseny d’aquest pèptid truncat s’ha basat en la superposició tridimensional de l’estructura de l’EGF i de l’inhibidor de la carboxipeptidasa de patata (PCI), un bloquejador de la via de l’EGFR descrit prèviament pel nostre grup. El pèptid ha estat produït en E.coli i s’ha aconseguit obtenir un alt rendiment de la proteïna i amb la seva conformació estructural correcta. Hem observat que l’EGFt in vitro té una capacitat molt menor per induir dímers del receptor i també la seva fosforilació si la comparem amb l’activitat que té l’hEGF natiu. Per altra banda, l’EGFt promou la internalització del receptor i la seva translocació al nucli cel·lular tal i com ho fa l’hEGF, tot i que no estimula el creixement cel·lular. A més, l’EGFt competeix amb els lligands natius de la família i inhibeix la proliferació cel·lular. La manca d’activitat estimuladora del creixement cel·lular d’aquest pèptid quan s’uneix a l’EGFR ens va portar a provar la utilització de l’EGFt com a vehicle de toxines dirigit a cèl·lules tumorals que sobreexpresessin EGFR. Concretament, es va produir un radioconjugat de EGFt amb l’isòtop radioactiu emissor d’electrons Auger Indi-111. Les propietats d’aquest radioconjugat es van analitzar i es van comparar amb el radioconjugat produït amb hEGF natiu. En primer lloc es va determinar que 111In-DTPA-EGFt té una alta especificitat i afinitat per EGFR. No obstant, la captació cel·lular de 111In-DTPA-EGFt va resultar ser menor que la de 111In-DTPA-hEGF. Un cop internalitzat, 111In-DTPA-EGFt va mostrar una alta eficiència per acumular-se en el nucli de la cèl·lula, on la radioactivitat emesa per 111In danya l'ADN. 111In-DTPA-EGFt va mostrar ser citotòxic in vitro contra cèl·lules de càncer de mama, encara que la seva citotoxicitat va ser menor en comparació amb 111In-DTPA-hEGF. Els estudis in vivo van revelar una vida mitjana més llarga en sang per 111In-DTPA-EGFt que per 111In-DTPA-hEGF, una major captació en el ronyó i una menor acumulació en altres teixits normals. 111In-DTPA-EGFt es va detectar en els tumors de cèl·lules MDA-MB-468 on el radiocompost es va acumular preferentment en el nucli de la cèl·lula. Les dades recollides en aquest treball indiquen que l’EGFt pot tenir un gran potencial com a bloquejador en teràpia pel càncer i a més pot ser un bon lligand per utilitzar com a vehicle d’agents citotòxics dirigits al nucli de cèl·lules tumorals positives en EGFR.

Nano-based delivery systems have attracted a great deal of attention in the past two decades as a strategy to overcome the low therapeutic index of conventional anticancer drugs. Nanoparticles enable the delivery of a great variety of drugs including analgesics, anti-Alzheimer's drugs, cardiovascular drugs, and several macromolecules into the brain after intravenous injection of animals. The mechanism of nanoparticle-mediated drug transport across the blood-brain barrier appears to be receptor-mediated endocytosis followed by drug release within the endothelial cells. Modification of the nanoparticle surface with covalently attached targeting ligands lead to the adsorption of specific proteins after injection is necessary for this receptor-mediated uptake. A very critical and important requirement for nanoparticulate brain delivery is that the employed nanoparticles are biocompatible and, moreover, rapidly biodegradable, i.e. over a time frame of a few days. In addition to enabling drug delivery to the brain, PLGA-based nanoparticles, with bisabolol inside, may importantly reduce the drug's toxicity. Because of the possibility to treat severe central nervous system diseases such as brain tumors and to even transport proteins and other macromolecules across the blood–brain barrier, this technology holds great promise for a non-invasive therapy of these diseases.

L'objectif de ce travail de thèse a été de préparer de nouveaux dendrimères ou dendrons phosphorés, qu'ils soient neutres, cationiques ou anioniques, dans le but d'élargir le panel des applications dans le domaine de la nanomédecine en tant que molécules actives par elles-mêmes ou en association avec certains médicaments. Au chapitre 1, la synthèse d'un nouveau dendrimère phosphoré polycationique comportant en surface 5 groupements pyrrolidinium et une amine protonnée a été menée à bien. Ce dendrimère associé au microRNA-30d permet d'obtenir des polyplexes qui sont transférés dans les cellules cancéreuses. Il a également un bon effet thérapeutique sur les cellules cancéreuses du sein. Au Chapitre 2, l'objectif était de développer un système de délivrance de médicaments capables de pénétrer les lésions cérébrales pour proposer un traitement de la maladie de Parkinson. Pour cela, il a été mis au point la formation d'un nanocomplexe formé d'un dendrimère phosphoré porteur de groupements hydroxyl en surface, combiné avec la fibronectine, qui règle la prolifération et la différenciation et la motilité cellulaire. Sur un modèle murin de la maladie de Parkinson, il a été mis en évidence une pénétration efficace au niveau de la BBB (blood brain barrier) de l'assemblage dendrimère phosphoré AK-123 et fibronectine exerçant une activité anti-inflammatoire et antioxydante permettant de soulager efficacement les symptômes observés et démontrant le grand potentiel pour un traitement clinique de la maladie de Parkinson mais aussi ouvrant un espoir de viser d'autres maladies neurodégénératives. Au Chapitre 3, les résultats obtenus dans le domaine de la maladie de Parkinson lors de l'action conjuguée d'un dendrimère phosphoré de génération 2 comportant 48 groupements hydroxy en surface et de la fibronectine nous ont incités à diversifier la nature et la structure de ces dendrimères afin d'avoir des premières indications de leur activité en général et d'aborder éventuellement une étude SAR (relation structure activité). Pour cela nous avons pu préparer tout un ensemble de nouveaux dendrimères phosphorés neutres de générations 1 et 2, caractérisés par des structures internes différentes de celles des dendrimères utilisés dans le chapitre précèdent, et de longues chaines en surface comportant également des groupements hydroxyle. Ces dendrimères ont l'avantage de posséder des groupements amines protonées permettant une solubilité en milieu aqueux. Nous avons pu également préparer des dendrons phosphorés neutres et chargés. Des résultats préliminaires concernant leurs propriétés ont pu être mis en évidence. Au Chapitre 4, Le but de notre étude a porté tout d'abord sur la synthèse de dendrimères phosphorés anioniques originaux et à l'utilisation d'un de ces dendrimères en l'occurrence le dendrimère AK 137 qui présente une activité anti-inflammatoire optimale et une grande aptitude pour la délivrance de protéines. Des nanocomplexes stables formés par ce dendrimère et diverses protéines en solution aqueuse (bovine serum albumin (BSA), ribonucleaseA (RNaA), ovalbumine (OVA) et fibronectine (FN) ont été obtenus. Nous avons pu démontrer que l'association AK-137@FN NCs bloque l'activation de certaines voies de signalisation (NF-kB et P13K/Akt), induit la polarisation de macrophages vers des phénotypes M2, inhibe la sécrétion de cytokines pro-inflammatoires (TNF-alpha, IL-1beta et IL6) et augmente les propriétés antioxydante de FN in vitro. Les effets thérapeutiques de l'association AK-137@FN ont été démontrés sur des modèles de souris ALI (acute lung injury) et AGA (acute gout arthritis) sans observation de toxicité systémique
The objective of this thesis work was to prepare new phosphorous dendrimers or phosphorous dendrons, either neutral, cationic or anionic, with the aim of broadening the range of applications in the field of nanomedicine as molecules active by per se. or in combination with certain medications. In Chapter 1, the synthesis of a new polycationic phosphorus dendrimer comprising 5 pyrrolidinium groups on the surface and a protonated amine was successfully completed. This dendrimer associated with microRNA-30d makes it possible to obtain polyplexes which are transferred into cancer cells in an optimal N/P ratio of 10. These polyplexes are cytocompatible and transfer miR-30d to suppress glycolysis associated with SLC2A1. The inhibition of the migration and invasion of murine cancer cells in vitro and in vivo were thus demonstrated. This dendrimer can be considered as an important component for therapy based on the use of miR-30d. In Chapter 2, we develop a drug delivery system capable of penetrating BBB (blood brain barrier) to provide treatment for degenerative disorders. For this, the formation of a nanocomplex was developed consisting of a phosphorus dendrimer carrying hydroxyl groups on the surface, combined with fibronectin, which regulates proliferation and differentiation and cell motility. In a mouse model of Parkinson's disease, effective penetration at the level of the BBB of the phosphorous dendrimer assembly AK-123 and fibronectin was demonstrated, exerting an anti-inflammatory and antioxidant activity allowing to decrease effectively the symptoms observed and demonstrating the great potential for clinical treatment of Parkinson's disease but also hold great promise to be used to tackle other neurodegenerative disorders. In Chapter 3, the results obtained in the field of Parkinson's disease during the combined action of a generation 2 phosphorous dendrimer comprising 48 hydroxyl groups on the surface and fibronectin have encouraged us to diversify the nature and structure of these dendrimers in order to have first indications of their activity in general and to possibly approach a SAR (structure activity relationship) study. According to these, we prepared a whole set of new neutral phosphorus dendrimers of generations 1 and 2, characterized by internal structures different from those of the dendrimers used in the previous chapter, incorporating long chains on the surface and also comprising hydroxyl groups. These dendrimers have the advantage of having protonated amine groups allowing solubility in an aqueous medium. We were also able to prepare neutral and charged phosphorous dendrons. Preliminary results concerning their properties have been demonstrated. In Chapter 4, the aim of this study was focused on the synthesis of original anionic phosphorus dendrimers and the application of these dendrimers, and particularly of the dendrimer AK 137 which presents optimal anti-inflammatory activity and great efficiency for the delivery of proteins. We demonstrate that the AK-137@FN NCs association blocks the activation of certain signalling pathways (NF-kB and P13K/Akt), induces the polarization of macrophages towards M2 phenotypes, inhibits the secretion of pro-cytokines. (TNF-alpha, IL-1beta and IL6) and increases the antioxidant properties of FN in vitro. The therapeutic effects of the AK-137@FN combination have been demonstrated in ALI (acute lung injury) and AGA (acute gout arthritis) mouse models without observation of systemic toxicity

Amanita ponderosa é uma espécie de cogumelos silvestres, comestível, característica de alguns microclimas mediterrânicos, existente na Península Ibérica. Neste estudo, avaliaram-se as propriedades biológicas e toxicológicas destes cogumelos e culturas, com vista a valorizar o seu potencial biotecnológico. Desenvolveu-se também uma metodologia de monitorização, utilizando técnicas de microanálise e imunológicas para screening e análise da especificidade de produção dos compostos bioativos. Os corpos de frutificação apresentaram um relevante conteúdo mineral e um perfil molecular correlacionável com o local de colheita. Tanto os cogumelos como as culturas e seus extratos revelaram baixa toxicidade, in vitro e in vivo, apresentaram propriedades antioxidantes, capacidade hepatoprotetora e efeito antiproliferativo em células MDA-MB-231. Estes resultados sugerem que A. ponderosa e/ou os seus extratos podem constituir uma importante fonte de compostos bioativos, com potencial valor nutracêutico e medicinal, podendo ser utilizados como suplementos alimentares, coadjuvantes no tratamento de doenças hepáticas e/ou tumorais; Characterisation of Amanita ponderosa wild mushrooms: production of metabolites with biological activity Abstract: Amanita ponderosa is a species of wild edible mushrooms that grows in some Mediterranean microclimates in the Iberian Peninsula. In this study, we evaluated the biological and toxicological properties of these mushrooms and cultures, in order to enhance their biotechnological potential. A monitoring methodology was also developed using microanalysis and immunological techniques, for screening and specificity evaluation of bioactive compounds production. The fruiting bodies presented a relevant mineral content and a characteristic molecular profile correlated with the geographical location. Either mushrooms or cultures and extracts have shown low toxicity in vitro and in vivo, and presented antioxidant properties, hepatoprotective effect and MDA-MB-231 antiproliferative activity. These results suggest that A. ponderosa and their extracts may constitute an important source of bioactive compounds with antioxidant benefits, nutraceutical potential and medicinal value, that can be used as dietetic supplements and as co-adjuvant of liver and cancer disease treatments.

Research on the putative health benefits has indicated that kefir, a traditional fermented milk, might have antimutagenic and antitumor properties. The major objective of the present thesis was to isolate and identify antitumor compounds in cow's milk kefir and investigate the possible mechanisms involved. High speed centrifugation (HSC), molecular weight cut-off filtration (MWCO), size exclusion high performance liquid chromatography (SEC-HPLC) and reverse phase-HPLC (RP-HPLC) were utilized for fractionation of kefir and a cell culture model was developed to screen for the antiproliferative effects of the kefir fractions. The antiproliferative effects of bacteria-free extracts from different fermentation stages of kefir production, as well as bacteria-free extracts from milk and yogurt were compared. The results showed that extracts from an early stage of fermentation (i.e., kefir mother culture) and the final commercial kefir product both exerted dose-dependent inhibition effects on human mammary tumor MCF-7 cells, yogurt extracts showed less potent antiproliferative effects, while pasteurized milk extracts showed no antiproliferative effects. No antiproliferative effects of the kefir extracts were observed on human mammary epithelial cells (HMEC) whereas the yogurt extracts showed antiproliferative action in HMEC cells at a high dose. A fraction of the kefir mother culture isolated by HSC, MWCO and RP-HPLC contained components that inhibited MCF-7 cell growth and had no effect on HMEC cells. Characterization of the bioactive fraction using mass spectrometry (MS) indicated that the main components in the fraction are likely fragments of kefiran and/or ceramide containing compounds such as gangliosides. The growth inhibitory effect may be mainly caused by the induction of TNF-alpha in MCF-7 cells. Whole extracts of kefir depleted glutathione (GSH) in MCF-7 cells, while the SEC-HPLC Fraction 7 and the RP-HPLC Fraction 30 induced GSH produc

Kyoto University (京都大学)
0048
新制・課程博士
博士(工学)
甲第11886号
工博第2579号
新制||工||1361(附属図書館)
23666
UT51-2005-N720
京都大学大学院工学研究科物質エネルギー化学専攻
(主査)教授 西本 清一, 教授 中條 善樹, 教授 木村 俊作
学位規則第4条第1項該当

Cancer is a growing health problem around the world and according to estimates from the International Agency for Research on Cancer (IARC), 14.1 million new cancer cases and 8.2 million cancer deaths worldwide have been reported in 2012 (Ferlay et al., 2015). By 2030, the global burden is expected to grow to 21.7 million new cancer cases and 13 million cancer deaths simply due to the growth and aging of the population. Indicaxanthin ((2S)-2,3-dihydro-4-[2-[(2S)-2a-carboxypyrrolidin-1- yl]ethenyl]pyridine-2a,6-dicarboxylic acid), a betalain pigment from cactus pear fruit, has been the object of sound experimental work over the latest years. As many phytochemicals, indicaxanthin is a redox-active compound and has been shown to act as antioxidant in a number of in vitro studies (Allegra et al., 2005; Turco Liveri et al., 2009). Interestingly, thanks to its charged portions, ionizable groups and lipophilic moieties, it is amphiphilic at physiological pH (Turco Liveri et al., 2009) and has been demonstrated to interact with cell membranes (Tesoriere et al., 2006; Turco Liveri et al., 2009). This feature is critical to allow bioactive compounds to interact with cells and to initiate signaling events. In this regard, indicaxanthin has been showed to modulate specific redox-dependent signaling pathways involved in macrophage activation and apoptosis, epithelial and endothelial dysfunction in vitro (Allegra et al., 2014; Tesoriere et al., 2015). Remarkably, and in contrast with the majority of dietary phytochemicals, indicaxanthin is highly bioavailable (Tesoriere et al., 2004). The molecule has been shown to cross unaltered intestinal epithelial cell in vitro being absorbed through paracellular junctions (Tesoriere et al., 2013). In line with that, indicaxanthin has been found in human plasma at a 7 μM peak concentration 3 h after the ingestion of four cactus pear fruits containing 28 mg of the pigment (Tesoriere et al., 2004). Moreover, its amphiphilicity allows it to cross the blood-brain-barrier and localize within the CNS (Allegra et al., 2015). Finally, thanks to its bioavailability and redox-modulating properties, indicaxanthin exerts significant pharmacological effects in vivo. Indeed, oral administration of the PhC at nutritionally-relevant doses (2 μmol/kg) generates, in rats, a plasma peak concentration of 0.2 μM able to exert strong anti-inflammatory effects in an in vivo model of acute inflammation (Allegra et al., 2014). The causative link between inflammation and melanoma has accurately been explored in the recent years (Bald et al., 2014; Meyer et al., 2011; Reinhardt et al., 2017; Soudja et al., 2010). Experiments in mice revealed that UV-induced skin inflammatory responses can cause the reactive proliferation and migration of melanocytes (Zaidi et al., 2011). More recently, it has been shown that reciprocal interactions between melanoma and immune cells in a pro-inflammatory microenvironment provide a source of phenotypic heterogeneity that drives therapy resistance and metastasis (Bald et al., 2014; Landsberg et al., 2012). In keeping this perspective, we decided to investigate the effects of Indicaxanthin against human melanoma cell proliferation and in a model of cutaneous melanoma. We here demonstrate that indicaxanthin induces apoptosis of human melanoma cells through the inhibition of the NF-κB pathway and the downstream anti-apoptotic signaling events in vitro and these effects were paralleled in vivo in a murine model of melanoma. Finally, preliminary data on six healthy volunteers, showed that indicaxanthin is able to modulate TNF- and Il-6 production in a whole blood ex vivo model. Furthermore, the phytochemical induces an increase in the phagocytosis of 5 different Gram-negative pathogens on whole blood assay, without exerting antimicrobial effects on them. Interestingly, preliminary data on 4 of the 6 volunteers showed that the observed effects maybe attributed to the modulation of LTB4 levels, strictly correlated to the activation of immune cells.

The interferon (IFN) regulatory factors (IRF) are transcriptional mediators of the cellular innate immune defense. Two of these factors, IRF-3 and IRF-7, are the principal inducers of type I IFN (IFN-alpha and -beta), which are key players in the antiviral response. Type I IFNs also exert strong antiproliferative and antiangiogenic effects and are widely used as adjuvants for therapy against cancer. When properly activated, macrophages can be tumoricidal making them attractive additions to standard cancer therapies. To this end, tolerance and activity of human autologous IFN-gamma-activated macrophages (MAK cells) have been assessed in pilot trials in cancer patients. In the present study, we tested the hypothesis that activation of IRF-3 and IRF-7 with subsequent type I IFN production may potentiate the antitumor functions of macrophages. Adenoviral vectors were generated to deliver constitutively active forms of IRF-3 (Ad-F3) or IRF-7 (Ad-F7) into human macrophages. Gene expression profiles of Ad-F3 or Ad-F7 transduced macrophages were assessed by microarray analysis. While IRF-3 strongly upregulated the expression of pro-apoptotic Noxa, IRF-7 poorly induced Noxa but upregulated Mcl-1- a Noxa-binding apoptosis inhibitor. Ad-F3 triggered rapid apoptosis and activation of the mitochondrial apoptotic pathway in macrophages. Active IRF-7 induced the expression of tumoricidal molecules (TRAIL, IFN-alpha and -beta), chemokines and genes involved in antigen processing/presentation. Ad-F7 macrophages exerted a cytostatic activity on different cancer cell lines and had an increased capacity to cross-present antigen. In conclusion, Ad-F3 or Ad-F7 appears to differentially regulate apoptosis and antitumor properties of macrophages, with active IRF-7 leading to the acquisition of antitumor effector functions.
Les facteurs régulateurs d'interferons (IRF) sont les médiateurs transcriptionnels de la réponse immunitaire innées. Deux de ces facteurs, IRF-3 et IRF-7, induisent l'expression des interferons de type I (IFN-alpha et -beta) importants pour la réponse antivirale et antitumorale. Les IFNs ont également des effets antiprolifératifs et antiangiogénics et sont couramment utilisés comme adjuvants en thérapie contre le cancer. Après activation, les macrophages presentment des activités antitumorales ce qui en font une alternative prometteuse au thérapie standard. À cet effet, la tolérance et l'activité des macrophages autologues humains activés à l'IFN-gamma (cellules MAK) ont été évaluées dans des essais pilotes chez des patients souffrants du cancer. Afin d'étudier le role d'IRF-3 et IRF-7 dans la regulation des fonctions antitumorales des macrophages, des vecteurs adénoviraux ont été produits pour livrer les formes constitutivement actives d'IRF-3 (Ad-F3) ou d'IRF-7 (Ad-F7) dans les macrophages humains. L' expression des gènes dans les macrophages transduits avec Ad-F3 ou Ad-F7 a été mesurée par microarray. IRF-3 induit fortement l'expression du gène pro-apoptotic Noxa. En revanche, IRF-7 induit faiblement Noxa mais induit Mcl-1 - un inhibiteur de l'apoptose qui interagit avec et inhibe Noxa. La transduction avec Ad-F3 induit l'activation rapide de la voie apoptotique mitochondriale dans les macrophages. La forme active d'IRF-7 induit l'expression des molecules tumoricidales (TRAIL et IFN-alpha et -beta) de chemokines et de genes impliqués dans la presentation d'antigènes. De plus, les macrophages transduits avec Ad-F7 exercent une activité cytostatique sur différentes lignées cancéreuses et une capacité de presentation-croisée accrue. En conclusion, Ad-F3 ou Ad-F7 semblent réguler l'apoptose et les propriétés anti-tumorales des macrophages de manière différente, l'expression d'IRF-7 menant à l'acquisition de fonctions e

The aim of the study was to evaluate the expression of immunosuppressive (FOXP3, NKG2A), and cytotoxic (perforin) or cytotoxic / immunomodulating (IFNγ) T-cell properties representing biomarkers in the peripheral blood CD8h T-cell population of patients with advanced renal cell carcinoma (RCC) or high risk cutaneous melanoma and healthy controls by multicolour flow cytometry. Determination of the percentage of functionally competing T-cell subsets (especially immunosuppressive) in the CD8hCD57+ T-cell subpopulation in future may serve as one of parameters enabling to assess the overall status of antitumor immune response and select cancer patients most suitable for antitumor immunotherapy while dismissing those to whom it would be ineffective or even harmful.
Darbo tikslas buvo įvertinti imunosupresines (FOXP3, NKG2A), citotoksines (perforin) bei citotoksines / imunomoduliuojančias (IFNγ) savybes atspindinčių žymenų raiškos skirtumus išplitusiu inkstų vėžiu ar didelės rizikos odos melanoma sergančių pacientų periferinio kraujo CD8h T limfocitų populiacijoje, lyginant su kontroline grupe. Skirtingas T limfocitų savybes atspindinčių žymenų raiška buvo tiriama tėkmės citometrijos būdu. Nustatyta, kad inkstų vėžiu ar odos melanoma sergančių pacientų periferiniame kraujyje Įvairių subpopuliacijų (ypač imunosupresinės) nuošimčio nustatymas CD8hCD57+ T limfocitų populiacijoje ateityje gali būti naudingas klinikinėje praktikoje, individualizuojant priešnavikinę imunoterapiją ir selektyviai parenkant tik tuos pacientus, kuriems imuninės sistemos aktyvinimas sukeltų navikinių ląstelių naikinimą, o ne dar labiau gilintų imunosupresiją.

Les préparations de Viscum album (VA), connu sous le nom vernaculaire de gui européen, sont fréquemment utilisées en support des traitements anticancéreux, principalement pour améliorer la qualité de vie des malades et réduire la croissance des tumeurs. Elles sont connues pour exercer des effets anti-tumoraux. Il existe de plus en plus de données scientifiques faisant état de liens étroits entre cancer et inflammation. Étant donné que la prostaglandine E2 (PGE2) induite par la cyclo-oxygénase 2 (COX-2) joue un rôle clef dans l’inflammation, j’ai exploré la régulation du système COX-2-PGE2 par VA et ses mécanismes sous-jacents. J’ai montré que VA exerce ses effets anti-inflammatoires en inhibant sélectivement l’expression de COX-2 et en diminuant la production de PGE2 qui en découle, par le biais d’une déstabilisation de l’ARNm de COX-2. En plus de leurs propriétés cytotoxiques, il a été montré que les préparations de VA ont également des effets immunostimulants. Les différentes préparations de VA sont hautement hétérogènes du fait de leurs compositions biochimiques qui varient selon la récolte, l’espèce de l’arbre hôte et les méthodes de préparation qui peuvent influer sur leur efficacité clinique. De ce fait, j’ai réalisé une étude comparative sur cinq préparations de VA dans le but d’analyser leurs capacités de maturation et d’activation des cellules dendritiques (DC) qui peuvent à leur tour présenter une réponse immunitaire anti-tumorale. Les résultats ont montré que parmi les cinq préparations,VA Qu Spez induit de manière significative l’activation des DC et la sécrétion de cytokines pro-inflammatoires telle que l’IL-6, l’IL-8 et le TNF-α qui induisent la production d’IFN-γ,orientant de ce fait la réponse immunitaire vers une réponse Th1. L’orchestration de la11fonction des cellules myélomonocytiques est un élément central à l’interface entre inflammation et cancer. Il constitue un paradigme expliquant la plasticité et la fonction des macrophages. Mon étude met en évidence l’influence de VA Qu Spez sur la polarisation des macrophages qui passent d’un état alternatif (M2) à un état dit classique (ou M1). Les macrophages M2 sont connus pour polariser les réponses immunitaires Th2, pour participer à l’élimination des parasites, pour diminuer l’inflammation, pour promouvoir le remodelage tissulaire et la progression des tumeurs et pour avoir des fonctions immunorégulatrices. Les macrophages M1 sont impliqués dans la réponse Th1, favorisent la résistance aux pathogènes intracellulaires et aux tumeurs et promeuvent des réactions de désagrégation tissulaires. L’ensemble de ces résultats permet de comprendre les propriétés anti-inflammatoires et immunostimulantes des préparations de VA. Des recherches complémentaires permettront d’améliorer les stratégies d’utilisation thérapeutique de VA et son utilisation dans les soins de support aux traitements anticancéreux
Viscum album (VA) preparations, commonly known as European mistletoe, are frequentlyused as complementary therapy in cancer, mainly to improve quality of life of the patients andto reduce the tumor growth. They are known to exert anti-tumoral effects. There is increasing evidence of the convoluted connection of cancer and inflammation. As cyclooxygenase-2(COX-2)-induced prostaglandin E2 (PGE2) plays a key role in the inflammation, I explored the regulation of COX-2-PGE2 axis by VA and underlying mechanisms. I found that VA exerts anti-inflammatory effects by selectively inhibiting COX-2 expression and ensuing PGE2 production. Inhibition of COX-2 expression implicates COX-2 mRNA destabilisation. In addition to their cytotoxic properties, they have also been shown to have immunostimulatory properties. Each VA preparations are highly heterogeneous because oftheir chemical composition which varies depending on the time of harvest, species of host treeand manufacturing methods, together which might influence clinical efficacy of VA.Therefore I performed a comparative study involving five different preparations of VA concerning maturation and activation of dendritic cells (DCs) which in turn may manifestanti-tumoral immune response. Results showed that among all five preparations, VA Qu Spez significantly induces DC activation, secretion of pro-inflammatory cytokines such as IL-6, Il-8 and TNF-α, enhancing IFN-γ production hence promoting Th1 immune response. The orchestration of myelomonocytic cell function is a key element that links inflammation and cancer and provides a paradigm for macrophage plasticity and function. My study reveals the effect of VA Qu Spez in switching the M2 macrophages which are known to participate inpolarizing Th2 responses, help with parasite clearance, dampen inflammation, promote tissue remodelling and tumor progression and have immunoregulatory functions, towards classicallyactivated M1 macrophages which are part of a polarized Th1 response and mediate resistance13to intracellular pathogens and tumors and elicit tissue-disruptive reactions. These results together should assist in understanding the anti-inflammatory and immunostimulatory properties of VA preparations and further research is warranted to improve the therapeutic strategies of use of VA and their role as complimentary therapy in cancer

Lectins are carbohydrate-binding proteins of non-immune origin, widely distributed in all the living organisms. They play a role in different biological processes and, due to their carbohydrate specific binding, some lectins have been found to possess antitumoral properties. A particular case is the recognition of the T-antigenic determinant (Galβ1-3GalNAcα) on the malignant cell surfaces, which is present in 90% of human carcinomas. Upon binding to the carbohydrate, some lectins can cause apoptosis, cytotoxicity, inhibition of tumour growth, thus preventing the proliferation of tumoral cells. Considering the fact that these carbohydrates are masked on healthy cells, the highly specific carbohydrate-lectin interaction can be exploited to target only malignant cells. Although the function and the biological properties of several lectins have been determined, there are still many lectins that remain to be structurally and functionally characterized. As reported in the literature, some Tremella fuciformis proteins have been investigated for their potential therapeutical properties and in the light of this, the crude extract of this fungus was examined to assess the presence of lectins. A lectin of 11 KDa, named TFL, was isolated and purified from the dried fruiting bodies and used for testing several crystal screening conditions. The best crystals were grown in 0.1 M TRIS pH 8.5, 1.5 potassium phosphate dibasic, 1 % DMSO and the final data sets were collected at the ESRF of Grenoble, revealing the three-dimensional structure of the protein. TFL belongs to the space group P1211 and the cell parameters of the crystal are the following: a = 61.62 Å, b = 61.83 Å, c = 67.84 Å, with β = 106.87 °. The protein is a monomer, composed of six β-sheets (from A to F) that arrange to form the so called “β-barrel”. In addition, two α-helixes, named H1 and H2 can be recognized in the structure, which is also stabilized by the presence of two disulphide bridges that connect Cys 54 to Cys 70 and Cys 97 to Cys 100, respectively. Thermal protein stability was analyzed by means of differential scanning calorimetry, revealing that TFL does not undergo a two-state unfolding process and its denaturation is reversible, a feature that has been rarely observed for lectins before. In addition, chemical and pH-induced unfolding were investigated using fluorescence spectroscopy, highlighting the high stability of TFL in a wide range of conditions. Exploiting isothermal titration calorimetry, more information about sugar-binding were acquired, identifying N-acetylgalactosamine as the best candidate, which has micromolar affinity for TFL. It has also been observed that Tremella fuciformis lectin shows no cytotoxicity on malignant and healthy cells and interestingly, it seems to reduce skin malignant cells migration and to have a positive effect in the upregulation of certain genes involved in cancer arresting (SBPP1). In addition, immunomodulatory activity of TFL was analyzed by means of ELISA essay, resulting in the up-regulation of the most important inflammatory markers (IL-6, TNFα), that could be usefully exploited in order to kill intracellular microorganisms and to help the human body to develop a stronger tumoral resistance. In conclusion, this work provides a new and interesting insight into cancer treatment, being an essential prerequisite for future in vitro and in vivo experiments. The encouraging results obtained so far, alongside with the fact that TFL has been produced also heterologously in E.coli, pave the way for further studies, including the feasibility to perform mutagenesis and to explore the possibility to further encapsulate the protein inside tailor-made nanoparticles, with the aim of reducing the amount of protein used, increasing its therapeutical efficiency, and preventing side effects.

博士
中山醫學大學
牙醫學系碩士班
99
Euphorbia Fisheriana Steudal (EF) and Saussurea Involucrata (SI) are rare plants which were used in ancient Eastern medicine. In this study, the antitumor properties of EF and SI were assessed by MTT assay, Western immunobloting, DNA fragmentation and nuclei staining methods. We have found that both of the plant extracts promote the apoptotic death of tumor cells by activation of proapoptotic regulators of Bcl-2 family and MAPK pathway, especially SAPK/JNK2. In case when EF was employed, the initiation of apoptosis was linked to the activation of intrinsic pathway and p38. No visible changes were detected in relation to ERK and JNK1. P38 inhibition and gene silencing by siJNK2 caused an inhibition of Bax and p53, at the same time procaspase-9 was distinctly up-regulated in contrast to caspase-9. Different outcome was observed after induction with SI. Apoptosis was triggered via activation of caspase-8 and caspase-9; no changes were detected in relation to p38 and ERK. The specific aspect of SI treatment was in inhibition of JNK1. Gene silencing by siJNK2 down-regulated Bax and p53; and up-regulated procaspase-9. These results suggest that EF and SI are potent inhibitors in the growth of cancer cells, and the apoptotic mechanism might be related to the activation of JNK2, rather than p38. Additionally, SI is an effective inhibitor of JNK1. Euphorbia Fisheriana Steudal and Saussurea Involucrata would be novel natural drugs nowadays, less toxic and with a higher cost benefit in comparison to chemical drugs in cancer treatment.

博士
國立中興大學
食品科學系
91
This study evaluated antioxidant and Anti-tumor properties of extracts from Antrodia camphorata in submerged culture (ACSC). Chapter 2 focuses on the studies of biologically active compounds and the antioxidant activity as well as free radical scavenging effects of dry matter of cultural medium (DMCM), filtrate (DMF) and different solvent extracts of mycelia from Antrodia camphorata in submerged culture (ACSC). DMF showed the strongest inhibition of lipid peroxidation as a function of its concentration, and was comparable to the antioxidant activity of BHA at the same concentration of 0.2 mg/ml. The hexane extract of mycelia had the weakest antioxidant ability, whereas other mycelial extracts exhibited a modest inhibition of lipid peroxidation. DMF and water extract of mycelia (WEM) showed marked activity in free radical scavenging. The antioxidant activities of filtrate and mycelial extracts were correlated to the presence of total polyphenols, the crude triterpenoid and the protein/polysaccharide ratio of the crude polysaccharide. It was found that DMCM had lower antioxidant ability than DMF in different model systems; indicating that the major antioxidant components in DMF must be derived from the secondary metabolites of mycelia. The results presented herein indicated that DMF could possibly act as a chemopreventing agent with respect to free radical-related diseases. Chapter 3 focuses on studies of the protective effects of DMF from Antrodia camphorata in ACSC on H2O2-induced cytotoxicity in HepG2 and carbon tetrachloride (CCl4)-induced hepatotoxicity in SD rats, and the possible mechanisms involved in this protection were also investigated. The preliminary study showed that the inhibitory effect of DMF and its crude triterpenoids on lipid peroxidation was in a dose-response manner in AAPH/linoleic acid system. HepG2 cells were pretreated with DMF at the concentration of 0.10 mg/ml for 4 h, significantly (p<0.05) decreased the lipid peroxidation which was measured by the formation of malondialdehyde induced by 1 h treatment of H2O2 (100 M). The oral pretreatment with DMF (0.25 and 0.50 mg/kg b.w.) for 5 consecutive days prior to the administration of a single dose of 40 % CCl4 (0.10 ml/100g b.w., ip) significantly prevented the increase in serum levels of hepatic enzyme markers (alanine and aspartate aminotransferase) and liver lipid peroxidation (p<0.05). The histopathological evaluation of the rat liver revealed that DMF reduced the incidence of liver lesions including neutrophil infiltration, hydropic swelling and necrosis induced by CCl4 in rats. Moreover, GSH-dependent enzymes (Glutathione peroxidase, Glutathione reductaser and Glutathione S-transferase) and the GSH/GSSG ratio were significantly improved in the oral pretreatment DMF of rats (p<0.01). Based on the above results, we speculate that DMF may play a role in preventing oxidative damage in living systems by up-regulating hepatic GSH-dependent enzymes to preserve the normal GSH/GSSH ratio and directly scavenging free radicals formed during CCl4 metabolism. Chapter 4 focuses on the studies of the effect of methanolic extracts of mycelial (MEM) inhibited cell viability and the mechanism of MEM-induced cytotoxic in hepatoma cells. The IC50 of MEM on the proliferation of HepG2 (wild type p53) and Hep3B (delete p53) was 49.5 and 62.7 g/ml, respectively, on day 2. The effect of MEM on inhibiting cell viability was about 0.2-fold of that methanolic extracts of Antrodia camphorata fruiting bodies (MEAF). There is no observable cytotoxicity of MEM in Chang liver cells and rat primary hepatocytes at the concentration of 100 g/ml. MEM-mediated HepG2 and Hep3B cells death by apoptosis were determined by cell morphological changes, DNA fragmentation and cell cycle analysis. Cell cycle analysis revealed that MEM induced apoptosis on HepG2 via G0/G1 cell cycle arrest. HepG2 and Hep3B treated with MEM (100 g/ml) for 72 h, the apoptotic cells were 98.3 and 39.5%, respectively. It was speculated that MEM-induced cells apoptosis in HepG2 was mediated by p53-dependent that was why HepG2 was more susceptible than Hep3B. The results suggest that MEM induced HepG2 apoptosis through inhibition cell growth and up-regulation of Fas/FasL to activate the pathway of caspase-3 and -8 cascade. Chapter 5 focuses on the studies of purification and identification of antitumor components of methanol extracts of mycelia (MEM) from ACSC. The fractions separated by HPLC from MEM were examined to evaluate the antitumor effect of inhibition cell viability on Hep3B. Then, the antitumor fraction in MEM, which was high in amount and possessed potent antitumor activity, was selected to precede purification and identification. There were six fractions (A-F) in MEM separated by analytical column of HPLC. Based on the integration of peak area, fraction C was the most abundant fraction (40%), fractions B (16%) and C (13%) was the next, and the other fraction were about 4-8% in quantity. Hep3B was treated with fraction B, C, D, and E for 24 h, and the IC50 on Hep3B was about 26-45 g/ml. However, fractions A and F showed no inhibition effect on cell viability in Hep3B. Fraction C was further separated and purified by the preparative HPLC and TLC, respectively. The molecular formula and weight of compound C were C16H22O3 and 262.2, respectively and was identified by UV, MS, IR and 1H-, and 13C-NMR. Keywords: Antrodia camphorata in submerged culture; antioxidant; anti-tumor; apoptosis; hepatoma cells

Yes
An efficient four-step synthesis of tetracyclic lactones from 1,4-benzodioxine-2-carboxylic acid was developed. Ellipticine derivatives exhibit antitumor activity however only a few derivatives without carbazole subunit have been studied to date. Herein, several tetracyclic lactones were synthesized and biologically evaluated. Several compounds (2a, 3a, 4a and 5a) were found to be inhibitors of the Kras-Wnt pathway. The lactone 2a also exerted a potent inhibition of Tau protein translation and was shown to have capacity for CYP1A1-bioactivation. The results obtained are further evidence of the therapeutic potential of tetracyclic lactones related to ellipticine. Molecular modeling studies showed that compound 2a is inserted between helix α3 and α4 of the KRas protein making interactions with the hydrophobic residues Phe90, Glu91, Ile9364, Hie94, Leu133 and Tyr137and a hydrogen bond with residue Arg97.
The Spanish Minister (CTQ2011-29285-C02-02) the SGR(2014)-1017 Generalitat de Catalunya and the Laboratories Servier (France)

Marine macroalgae have a long tradition of usage and applications among the far Eastern populations, either for direct consumption and nutrition or for medicinal purposes. During the recent years, these marine organisms have received increasing popularity among the Western populations which are becoming aware of their potential to be exploited as a source of valuable bioactive compounds. Among these compounds are the phlorotannins, which are a group of phloroglucinol-derived phenolic compounds occurring exclusively on brown seaweeds and claimed for their promising health-promoting effects. Although several bioactive properties have been demonstrated for these compounds, there is still a long way to go before understanding the mechanisms behind them, and this is particularly true for the anti-inflammatory and antitumor properties of phlorotannins from Fucus vesiculosus. Moreover, little is known about the fate of these compounds when crossing the gastrointestinal tract. In this context, the aim of this work was to maximize the extraction of phlorotannins from this species for further characterization through UHPLC DAD-ESI-MSn analysis and evaluation of the bioactive properties that could be relevant from a performance point of view throughout the gastrointestinal tract, namely antioxidant, anti-inflammatory, antitumor and prebiotic effects. Through the response-surface methodology, the conditions determined for maximum recovery of F. vesiculosus phlorotannins were acetone 67% (v/v) in a proportion of 70 mL/g of seaweed powder at 25 °C. After an intermediate purification step, the phlorotannin-rich fraction (EtOAc) revealed a complex chromatographic profile composed of several fucols, fucophlorethols, fuhalols and several other phlorotannin derivatives. Additionally, three potential new phlorotannin derivatives, namely fucofurodiphlorethol, fucofurotriphlorethol and fucofuropentaphlorethol have been tentatively identified in this extract. Both the crude extract and EtOAc revealed good antioxidant activity, particularly against the nitric oxide radical, which is as very important player in the inflammatory signalling cascade. Indeed, strong anti-inflammatory activity was confirmed in a cellular system of inflammation using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. From the EtOAc further subfractions with phlorotannins of increasing molecular weights were obtained and tested in the same cell line, revealing some differences between their anti-inflammatory activity which could be related to their complexity and molecular weight. A remarkably strong anti-inflammatory activity was observed for a particular subfraction, F2, characterized by the presence of a major phlorotannin derivative with molecular weight of 508 g/mol. At 200 µg/mL this subfraction was capable of inhibiting IκBα phosphorylation and degradation, and consequently blocking the pro-inflammatory signalling cascade at the transcriptional level. The EtOAc and some of its subfractions, namely F1 and F5, also demonstrated promising antitumor properties exerting a selective cytotoxic effect against gastric (MKN-28) and colon cancer cells (Caco 2 and HT-29) but not on normal cells. From these samples, F5, characterized for the presence of fucols, fucophlorethols, a fucofurodiphlorethol and eckstolonol, revealed the strongest effect in all the three tumor cell lines, exhibiting IC50 values of 56.3 ± 14.7, 97.4 ± 11.6 and 118.8 ± 19.7 µg/mL for MKN-28, Caco-2 and HT 29, respectively. Interestingly, while the EtOAc displayed this activity via induction of cell cycle arrest and activation of cell death mechanisms, the subfraction F1 only caused cell cycle arrest and the subfraction F5 only triggered cell death via activation of the apoptotic/necrotic mechanisms. Finally, these compounds were found to exert promising inhibitory capacity against some digestive enzymes, particularly α-glucosidase, in which the IC50 values were 45 a 250 times lower compared to acarbose, thus revealing a therapeutical potential for the treatment of type-II diabetes. However, similar to plant polyphenols, phlorotannins were found to be susceptible to degradation and loss of antioxidant activity during their passage through the gastrointestinal tract, with less than 15% of the initial total phlorotannins becoming bioaccessible and available for absorption. In turn, although the non-bioaccessible fraction of F. vesiculosus samples only contributed with a modest positive effect for the modulation of human microbiota, they could stimulate the production of propionate and butyrate,’pç which are two short-chain fatty acids with well established health-promoting properties for the host. In conclusion, this study not only allowed better understanding of the phlorotannin composition of F. vesiculosus, but also demonstrates that its extracts and/or purified fractions may have a relevant impact in the maintenance of a positive gastrointestinal health acting at different levels. Ultimately, this work contributes to the valorization of this species as a possible supply of natural compounds with great bioactive potential and applicability.
Desde a antiguidade que as macroalgas marinhas têm sido tradicionalmente usadas pelos povos de extremo oriente, quer para consumo direto, como fonte de nutrientes, como para fins medicinais. Nos últimos anos estes organismos marinhos têm vindo a ganhar uma crescente popularidade entre as populações ocidentais que começam a estar cada vez mais conscientes do potencial que estas possuem como uma possível fonte de compostos bioativos de grande interesse. De entre os seus compostos destacam-se os florotaninos, um grupo de compostos fenólicos derivados do floroglucinol, reivindicados pelos seus benefícios para a saúde, e cuja ocorrência é exclusiva das macroalgas castanhas. Apesar de várias propriedades bioativas terem já sido demonstradas para estes compostos, existe ainda um longo percurso a percorrer até à compreensão dos mecanismos por detrás destas, sendo isto particularmente verdade no que diz respeito às propriedades anti-inflamatórias e antitumorais dos florotaninos provenientes da macroalga Fucus vesiculosus. Além disso, pouco se sabe acerca do destino destes compostos durante a sua passagem pelo trato gastrointestinal. Neste contexto, neste trabalho pretendeu-se otimizar a extração de florotaninos a partir desta macroalga e elucidar a composição destes extratos através da técnica de UHPLC-DAD-ESI-MSn , bem como clarificar as potencialidades bioativas de extrato bruto ou frações do ponto de vista de atuação ao longo to trato gastrointestinal, nomeadamente os efeitos antioxidante, anti-inflamatório, antitumoral e prébiotico. Recorrendo à técnica de superfície-resposta, as condições determinadas para o máximo de recuperação de florotaninos da alga F. vesiculosus foram: acetona a 67% (v/v) numa proporção de 70 mL/g de farinha de alga a 25 ºC. Após um passo de purificação intermédio, a fração rica em florotaninos (EtOAc) revelou um complexo perfil cromatográfico composto por vários fucóis, fucofloretóis, fualóis e outros derivados de florotaninos. Foram ainda detetados três potenciais novos derivados de florotaninos, nomeadamente o fucofurodifloretol, fucofurotrifloretol e o fucofuropentafloretol. Tanto o extrato bruto como a EtOAc revelaram boa atividade antioxidante, em particular contra o radical de óxido nítrico, um importante interveniente na cascata sinalizadora da inflamação. De facto, uma atividade anti-inflamatória pronunciada foi confirmada em células RAW 264.7 estimuladas com lipopolissacarídeo (LPS). A partir da EtOAc foram ainda obtidas 9 sub-frações de florotaninos com diferentes pesos moleculares que, após testadas na mesma linha celular, revelaram algumas diferenças ao nível da atividade anti inflamatória que possivelmente estarão relacionados com a sua complexidade e peso molecular. Um efeito anti-inflamatório bastante marcado foi ainda observado para uma sub-fração em particular, a F2, caracterizada pela presença de um composto derivado de florotaninos com peso molecular de 508 g/mol. Esta fração, a 200 µg/mL, foi capaz de inibir a fosforilação e degradação da proteína IκBα, consequentemente bloqueando a cascata sinalizadora da inflamação ao nível transcricional. A fração EtOAc bem como algumas das posteriores sub-frações, nomeadamente F1 e F5, demostraram ainda propriedades antitumorias promissoras, provocando um efeito citotóxico seletivo apenas contra células tumorais de cancro gástrico (MKN-28) e cancro do colon (Caco-2 e HT-29), não afetando células normais. Das três amostras, a F5, caracterizada pela presença de fucóis, fucofloretóis, um fucofurodifloretol e eckstolonol, foi a que revelou o efeito mais acentuado em todas as linhas celulares tumorais, demonstrando valores de IC50 de 56.3 ± 14.7, 97.4 ± 11.6 e 118.8 ± 19.7 µg/mL para MKN-28, Caco-2 e HT-29, respetivamente. Curiosamente, enquanto a EtOAc exerceu este efeito através do bloqueio do ciclo celular e ativação de mecanismos de morte celular, a sub-fração F1 apenas promoveu o bloqueio do ciclo celular enquanto a sub-fração F5 apenas ativou a morte celular via ativação de mecanismos de apoptose/necrose. Por fim, verificou-se uma boa capacidade inibidora das enzimas digestivas por parte destes compostos, em particular da enzima α-glucosidase cujo IC50 foi 45 a 250 vezes inferior ao da acarbose, revelando potencial terapêutico contra a diabetes do tipo-II. No entanto, à semelhança dos polifenóis das plantas, os florotaninos revelaram-se suscetíveis a degradação e perda de atividade antioxidante durante a sua passagem ao pelo trato gastrointestinal, com menos de 15% dos florotaninos totais da amostra inicial ficando bioacessíveis e disponíveis para absorção. Em contrapartida, apesar de a fração não bioacessível das amostras de F. vesiculosus terem apenas contribuído com um efeito modestamente positivo na modulação da microbiota humana, estas demonstraram uma capacidade interessante de estimular a produção de propionato e butirato, dois ácidos gordos de cadeia curta com propriedades benéficas para a saúde do hospedeiro bem estabelecidas. Em conclusão, este estudo não só permitiu compreender melhor a composição dos florotaninos presentes na alga F. vesiculosus, como também demonstrou que os seus extratos e/ou frações purificadas podem ter um impacto relevante na manutenção de uma boa saúde gastrointestinal atuando em diferentes níveis. Em última instância, este trabalho contribui para a valorização da espécie F. vesiculosus como uma possível fonte de compostos naturais com grande potencial biológico e de aplicabilidade.
Programa Doutoral em Química Sustentável

Tese de doutoramento em Ciências (ramo de conhecimento em Química)
In this study, seven wild mushroom species (Ganoderma lucidum, Coprinopsis atramentaria, Rhodotus palmatus, Lactarius bertillonii, Lactarius vellereus, Xerocomus chrysenteron and Morchella esculenta), were characterized for their nutritional value (macronutrients and energy) and chemical composition (fatty acids, tocopherols, sugars and organic acids). A special focus was given to the analysis of phenolic acids and related compounds in mushroom fruiting bodies, spores and in vitro produced mycelia, by using chromatographic and mass spectrometry techniques. After mushrooms ingestion, the mentioned compounds suffer conjugation reactions, such as methylation and glucuronidation, and their structure can vary, modifying or not, their bioactive properties. With that aim, circulating metabolites of phenolic acids found in humans, such as methyl and glucuronide derivatives, were prepared by chemical synthesis and further characterized by the usual methods (melting point, 1H and 13C-nuclear magnetic resonance, and high resolution mass spectrometry). The bioactive properties of the synthesized compounds were evaluated and compared with the parent phenolic acid, and also with the mushroom phenolic extract. The bioactivities assessed were: i) antioxidant activity, measured by free radicals scavenging activity, reducing power and lipid peroxidation inhibition in vitro assays; ii) antimicrobial activity, including antibacterial and antifungal properties with evaluation of demelanizing capacity, determined by disc diffusion and microdilution methods; and iii) inhibitory growth activity against different human tumor cell lines, and also against non-tumor porcine liver primary cells culture to evaluate toxicity, using sulforhodamine B assay. The studied edible mushroom species are a good choice to include in our daily diet due to their richness in nutrients such as carbohydrates and proteins, and bioactive molecules, being also low caloric foods due to the low fat and energy contents. Furthermore, mushroom phenolic extracts showed antioxidant and antimicrobial properties, and also the capacity to inhibit the growth of human tumor cell lines. The synthesized methylated and acetyl glucuronated methyl esters derivatives revealed, in general, higher inhibitory growth activity against human tumor cell lines and antimicrobial activity than their parent compounds being, in the latter case, even higher than the one showed by standard antibiotics. Moreover, all the tested compounds revealed no toxicity against non-tumor cells. This means that the inclusion of methyl and acetyl glucuronic methyl ester groups in the molecule can, in some cases, increase the phenolic acids bioactivity. Nevertheless, future studies are needed in order to understand and clarify the specific mechanistic pathways of these important metabolites or metabolite derivatives.
Neste trabalho, caracterizaram-se sete espécies de cogumelos silvestres (Ganoderma lucidum, Coprinopsis atramentaria, Rhodotus palmatos, Lactarius bertillonii, Lactarius vellereus, Xerocomus chrysenteron, e Morchella esculenta) quanto ao seu valor nutricional (macronutrientes e energia) e composição química (ácidos gordos, tocoferóis, açúcares e ácidos orgânicos). Deu-se uma maior relevância aos ácidos fenólicos e compostos relacionados que foram analisados utilizando técnicas de cromatografia e de espetrometria de massa no corpo frutífero, esporos e ainda no micélio produzido in vitro. Após a ingestão de cogumelos, os compostos mencionados sofrem reações de conjugação, tais como metilação e glucuronidação, o que pode alterar a sua estrutura e, consequentemente, as suas propriedades bioativas. Assim, sintetizaram-se possíveis metabolitos ou derivados de metabolitos de ácidos fenólicos que circulam no organismo humano, nomeadamente compostos metilados e ésteres metílicos do ácido glucurónico acetilado. Estes compostos foram completamente caracterizados utilizando técnicas de ponto de fusão, ressonância magnética nuclear 1H e 13C, e ainda espetrometria de massa de alta resolução. As propriedades bioativas dos compostos sintetizados foram avaliadas e comparadas com as dos ácidos fenólicos parentais, assim como com as dos extratos fenólicos de cogumelos. As bioatividades analisadas foram: i) atividade antioxidante, em ensaios in vitro de avaliação da atividade captadora de radicais livres, poder redutor e inibição da peroxidação lipídica; ii) atividade antimicrobiana incluindo propriedades antibacterianas e antifúngicas com determinação da capacidade desmelanizante, avaliadas por métodos de difusão em disco e de microdiluição; e iii) actividade inibitória do crescimento em diferentes linhas celulares tumorais humanas, e em células não tumorais de fígado de porco, usando uma cultura primária para avaliar a sua toxicidade, usando o método da sulforrodamina B. As espécies de cogumelos comestíveis estudadas mostraram ser uma boa opção para inclusão na nossa dieta diária dada a sua riqueza em nutrientes como glúcidos e proteínas, baixos teores calóricos e energéticos, e também por serem ricos em moléculas bioativas. Para além disso, os extratos fenólicos de cogumelos revelaram atividade antioxidante, antimicrobiana e também mostraram inibir o crescimento em linhas celulares tumorais humanas. Em geral, os derivados metilados e ésteres metílicos do ácido glucurónico acetilado mostraram maior actividade inibitória do crescimento (em linhas celulares tumorais humanas) e atividade antimicrobiana do que os respetivos ácidos fenólicos. De salientar que a atividade antimicrobiana destes possíveis metabolitos foi superior à dos antibióticos utilizados como padrões. Os resultados obtidos permitiram concluir que a inserção de grupos metilo e ésteres metílicos do ácido glucurónico acetilado nos ácidos fenólicos aumentam a sua bioatividade. No entanto, são ainda necessários mais estudos que permitam elucidar completamente os mecanismos de ação envolvidos na bioatividade destes importantes metabolitos e derivados.
Fundação para a Ciência e Tecnologia (FCT)-Portugal for financial support to the Portuguese NMR network and to FCT and FEDER-COMPETE/QREN/EU for the financial support through the research project (PTDC/AGR-ALI/110062/2009) and for my PhD grant (SFRH/BD/70304/2010) that gave me the opportunity to perform this work.

Tese de Doutoramento em Ciências (Especialidade em Química)
According their empirical relevance in traditional medicine and diets, six wild species from the Portuguese medicinal flora were selected as important sources of phenolic compounds, namely flavonoids. The present study aimed to characterize the phenolic composition and evaluate bioactive properties (antioxidant, antitumor and antiangiogenic) of methanolic extracts, infusions and decoctions prepared from Chamaemelum nobile and Matricaria recutita, and Prunus spinosa, Arbutus unedo, Rosa micrantha and Rosa canina fruits; and to synthesize derivatives/metabolites of some flavonoids present in the studied samples. The main phenolic compounds in C. nobile extract, decoction and infusion were flavonols, flavones, phenolic acids and derivatives. The extract gave the highest antioxidant activity in the β-carotene bleaching and TBARS formation inhibition assays. The highest radical scavenging activity and reducing power were observed in the infusion. The extract showed the highest antitumor (in different human tumor cell lines) and antiangiogenic (phosphorylation inhibition of VEGFR-2) activities. M. recutita infusion and decoction showed better results than the extract in the radical scavenging activity and β-carotene bleaching inhibition assays. The antitumor activity of the extract and infusion showed to be selective for HCT-15 and HeLa cell lines. Luteolin-O-acylhexoside was the most abundant flavonoid in the three preparations. 3- O-Caffeoylquinic acid was the most abundant phenolic compound in P. spinosa, quercetin 3-O-glucoside in A. unedo, and taxifolin in R. micrantha and R. canina. P. spinosa revealed to be the most rich in anthocyanins. Two different enriched phenolic extracts were prepared, in order to compare their bioactivity: non-anthocyanin phenolic compounds enriched extract (PE) and anthocyanins enriched extract (AE). A. unedo (PE) and R. canina (AE) presented the highest antioxidant activity in all the assays, and the highest antitumor activity was observed in A. unedo (PE and AE). The synthesis of derivatives/metabolites of flavonoids was performed using quercetin or quercetin 3-Orutinoside (rutin) (identified in the studied samples) affording O-tri and different O-tetra protected (benzyl or methyl) quercetins that were submitted to glucuronidation attempts using acetobromo-α-ᴅ-glucuronic acid methyl ester in order to obtain possible precursors of human metabolites. As complex mixtures were obtained, further studies are needed to allow the formation of higher amounts of the required glucuronide derivatives to enable their isolation in a pure form.
De acordo com a sua relevância empírica na medicina tradicional e utilização em dietas, foram selecionadas seis espécies silvestres da flora medicinal Portuguesa, como fontes importantes de compostos fenólicos, nomeadamente flavonoides. O presente estudo teve como objetivo caracterizar a composição fenólica e avaliar as propriedades bioativas (antioxidante, antitumoral e antiangiogénica) de extratos, infusões e decocções preparadas a partir de Chamaemelum nobile e Matricaria recutita, e frutos de Prunus spinosa, Arbutus unedo, Rosa micrantha e Rosa canina; e sintetizar derivados/ metabolitos de alguns flavonoides presentes nas espécies estudadas. Os compostos fenólicos mais abundantes no extrato, decocção e infusão de C. nobile foram flavonóis, flavonas, ácidos fenólicos e derivados. O extrato mostrou a maior atividade antioxidante nos ensaios de inibição da descoloração do β-caroteno e da formação de TBARS. A maior atividade captadora de radicais livres e o maior poder redutor foram observados na infusão. O extrato mostrou maior atividade antitumoral (nas diferentes linhas celulares tumorais) e antiangiogénica (inibição da fosforilação do VEGFR-2). A infusão e a decocção de M. recutita mostraram maior atividade captadora de radicais livres e inibição da descoloração do β-caroteno do que o extrato. Os efeitos antitumorais do extrato e da infusão mostraram ser seletivos para as linhas celulares HeLa e HCT-15. A luteolina-Oacil- hexósido foi o flavonoide mais abundante nas três preparações. O ácido 3-Ocafeoilquínico foi o composto fenólico mais abundante em P. spinosa, enquanto que a quercetina 3-O-glucósido foi o maioritário em A. unedo, e a taxifolina foi o mais abundante em R. canina e R. micrantha. P. spinosa revelou o maior teor em antocianinas. Prepararam-se dois extratos fenólicos diferentes de forma a comparar a sua bioatividade: um extrato enriquecido em compostos fenólicos sem antocianinas (PE) e um extrato enriquecido em antocianinas (AE). A. unedo (PE) e R. canina (AE) apresentaram a maior atividade antioxidante em todos os ensaios, e a maior atividade antitumoral foi observada em A. unedo (PE e AE). A síntese de derivados/metabolitos dos flavonoides foram realizados usando a quercetina e rutina (quercetina 3-O-rutinósido) (identificados nas amostras estudadas) obtendo-se O-tri e diferentes O-tetra (benzil ou metil) quercetinas protegidas, que foram submetidas a tentativas de glucuronidação usando o éster metílico do ácido acetobromo-α-ᴅ-glucurónico a fim de obter possíveis precursores de metabolitos humanos. Tendo-se obtido misturas complexas, são necessários mais estudos para permitir a formação de quantidades mais elevadas dos glucuronidos requeridos, que possibilitem o isolamento dos compostos numa forma pura.
Fundação para a Ciência e Tecnologia (FCT) - Portugal for the financial support through to the Portuguese NMR network and through PEst-C/QUI/UI0686/2011- 2012 and 2013-2014, PEst-OE/AGR/UI0690/2011-2012, 2013 and 2014, supporting the research centres (CQUM and CIMO, respectively) and through my PhD grant (SFRH/BD/ 8307/2011) also supported by POPH-QREN and FSE.

碩士
國立嘉義大學
應用化學系研究所
101
Abstract Here, we synthesize two new Roussin’s Red Esters (RREs) : RRE(ALA-TEG) (1) and RRE(ALA-PEG400) (2). These complexes have used IR, EA and TEM to prove their comformation and micelle form. We have also used NO analyer to demonstrate complexes (1) and (2) that can released NO by UV. In addition, we demonstrated that NO can cleavage DNA in direct proportion to its concentration.

Mestrado de dupla diplomação com a Université Moulay Taher
Honey is considered a natural sweet substance produced by honeybees, from the nectars of plant flowers and honeydews. Honey has always been regarded as a food that is beneficial for human health with several therapeutic qualities described. The quality of honey is still a top concern for experts as no good method has been defined so far for the simultaneous detection of different types of honey. Consequently, the development of easy, quick, precise analytical tools that may give data for assessing honey authenticity, is important. Because of that, it is essential to inform consumers of the mislabeling of honey with lower quality. This study aimed to evaluate the physicochemical characteristics and to assess the quality of Algerian honey from different botanical and geographical origins. For that, ten samples of honey with different marked botanical origins were analyzed, including three samples from rosemary honey, three from tamarisk honey, three from milk thistle honey and one multiflora honey. The quality of the samples was determined through different parameters. Melissopalynological and physicochemical analyses (color, moisture, pH, acidity, electrical conductivity, diastase index, proline, 5-hydroxymethylfurfural, mineral content, proteins, carbohydrates, energy, and ash) were performed, as well as the profile evaluation of sugar and phenolic compounds. Antioxidant activity (reducing power and DPPH free radical scavenging activity) antitumor and anti-inflammation activity were also evaluated. Finally, the presence of antibiotics, recurrent residues in honey, such as tetracyclines and sulphonamides were screened using the multi-analyte receptor assay system Charm II. The melissopalinological analysis showed the presence of 10 major types of pollen grains, with Rosmarinus officinalis, Cytisus stratitus and Centaurea sp. pollens as the most abundant. Furthermore, since no honeydew elements were detected, all the samples were classified as nectar honeys. Samples R1, R2, and R3 were classified as rosemary monofloral honey; samples T1, T2 and MF were classified as Cytisus striatus honey; CH1-CH3 were classified as Centaurea sp. and T3 as multifloral, which not always agreed with the labeled botanical origin. Generally, honey samples presented values of moisture, free acidity, 5-HMF, proline content, and diastase index within the limits of the legal requirements, suggesting that the honey was extracted at a correct ripeness stage. The results showed that almost all honey samples have light amber color, except the rosemary honeys which presented an extra white amber color. Although exhibiting a normal diastase index, the R2 and R3 samples presented a 5-HMF value higher than the admitted in the legislation, suggesting that less adequate heat treatments and/or conservation methods might have been employed. The most common minerals were potassium, sodium, calcium, manganese, while copper and Manganese were present in some samples in minor quantities and the heavy metals (cadmium and lead) are absent from all samples. The sugar profile, analyzed by high pressure liquid chromatography with refractive index detection (HPLC-RI), showed that fructose and glucose were the most abundant compounds, representing more than 60% of total sugars. Other sugars, such as turanose, maltulose and maltose were also detected in a lower proportion. Regarding the phenolic profile, nineteen compounds (eight phenolic acids and seven flavonoids), two isoprenoid compounds (trans, trans- and cis, trans- abscisic acid), one spermidine and one phenolic diterpene were identified. T2 sample showed a higher amount of phenolic acids than flavonoids. However, the most abundant compounds were the benzoic acid derivative which was detected in all samples. Concerning the evaluation of the antitumor activity and anti-inflammatory activity the samples showed a significant potential. Finally, concerning the antibiotics screening, not all the samples showed negative results.
O mel é considerado uma substância doce natural produzida pelas abelhas, a partir dos néctares das flores das plantas e de meladas. O mel sempre foi considerado um alimento benéfico para a saúde, com várias qualidades terapêuticas descritas. A sua qualidade ainda é uma das principais preocupações para os especialistas, pois não há um método ideal para a classificação simultânea de diferentes tipos de mel. Consequentemente, é importante o desenvolvimento de ferramentas analíticas simples, rápidas e precisas que possam fornecer dados que permitam avaliar a autenticidade do mel. Por esse motivo, é essencial informar os consumidores da incorreta rotulagem de méis com baixa qualidade. O objetivo deste estudo foi avaliar as características físico-químicas e desse modo aferir a qualidade de méis argelinos com diferentes origens botânicas e geográficas. Para isso, foram recolhidas dez amostras de méis rotulados com diferentes origens botânicas, nomeadamente: três de mel de alecrim, três de mel de tamarino, três de mel de cardo e um de mel multifloral. A qualidade dos méis foi aferida através de diferentes parâmetros. Foram realizadas análises melissopalinológicas e físico-químicas (cor, humidade, pH, acidez, condutividade elétrica, índice diastático, prolina, 5-hidroximetilfurfural, conteúdo em minerais, proteínas, hidratos de carbono, energia e cinzas), bem como a avaliação do perfil em açúcares e compostos fenólicos. Também foi avaliada a atividade antioxidante (poder redutor e poder bloqueador de radicais livres) e atividade antitumoral e antiinflamatório. Finalmente, a presença de antibióticos, resíduos recorrentes no mel, como tetraciclinas e sulfonamidas, foram investigados através do sistema de despistagem Charm II. A análise melissopalinológica mostrou a presença de 10 tipos de grãos de pólen maioritários, sendo os pólenes de Rosmarinus officinalis, Cytisus stratitus e Centaurea sp. os mais abundantes. Além disso, e como não foram detetados elementos de melada, as amostras analisadas foram classificadas como méis de néctar: as amostras R1, R2 e R3 foram classificadas como mel monofloral de alecrim; as amostras T1, T2 e MF foram classificadas como mel de Cytisus striatus; CH1-CH3 foram classificados como de Centaurea sp. e T3 como multifloral, nem sempre coincidindo com a classificação utilizada no rótulo. De uma forma geral, as amostras de mel apresentaram valores de humidade, acidez livre, 5-HMF, teor de prolina e índice de diástase dentro dos limites requeridos legalmente, sugerindo que os méis foram extraídos no nível de maturação correto. Os resultados mostram que quase todas as amostras de mel apresentaram uma color âmbar clara, exceto o mel de alecrim que apresentou uma color âmbar extra clara. Apesar de apresentarem um índice de diástase normal, as amostras R2 e R3 apresentaram um valor de 5-HMF superior ao admitido na legislação, sugerindo a utilização de tratamentos térmicos e/ou métodos de conservação menos adequados. Os minerais mais comuns identificados foram o potássio, sódio, cálcio, enquanto cobre e manganês estiveram presentes em algumas amostras em quantidades menores e os metais pesados (cádmio e chumbo) estão ausentes em todas as amostras. O perfil dos açúcares, analisado por cromatografia líquida de alta pressão com deteção de índice de refração (HPLC-RI), mostrou que a frutose e a glucose foram os compostos mais abundantes, representando mais de 60% dos açúcares totais. Outros açúcares, como a turanose, a maltulose e a maltose, também foram detetados em menor proporção. Em relação ao perfil fenólico, foram identificados dezanove compostos (oito ácidos fenólicos e sete flavonóides), dois compostos isoprenóides (ácido trans, trans- e cis, trans-abscísico), uma espermidina e um diterpeno fenólico. No que diz respeito à avaliação da atividade antitumoral e atividade anti-inflamatória, as amostras apresentam potencial significativo. Finalmente, após o estudo de deteção de antibióticos verificou-se que nem todas as amostras estão isentas de resíduos.