Basal-like breast cancer represents a subgroup of mammary cancers associated with a particularly poor prognosis, as they are refractory to current targeted therapies employed for the treatment of breast cancer. In this work I aimed to explore the therapeutic potential of selective estrogen receptor modulators (SERMs), a targeted breast cancer treatment, in combination with epigallocatechin gallate (EGCG), for the treatment of basal-like breast cancer, using MDA-MB-231 cell as an in vitro model of the disease. A significant reduction in MDA-MB-231 cell number and a significant increase in cytotoxicity was observed following treatment with 25 [mu]M of EGCG in combination with 1 [mu]M of 4-hydroxytamoxifen (4-OHT) (EGCG+4-OHT) or 4 [mu]M of raloxifene (EGCG+Ral) over a 36 h time course. However, these effects were not resolved in time, with an increase in G₁-phase cell cycle arrest. Changes in the metabolism of EGCG were dismissed as a possible mechanism through which the combination treatments may be eliciting the cytotoxicity. Changes in the expression and phosphorylation of various signaling proteins, important for the proliferation and survival of basal-like breast cancer, were investigated through Western blotting. Interestingly, the two combination treatments produced very similar results; reductions in the phosphorylation of EGFR and AKT occurred after 6, 12, and 18 h with EGCG+4-OHT and 6, 12, 18 and 24 h with EGCG+Ral, while a reduction in S6K phosphorylation was observed following 6, 12, 18 and 24 h of both combination treatments. Interestingly, both SERMs contributed significantly to the net reduction in S6K phosphorylation, induced by the combination treatments. Both combination treatments were also associated with a significant increase in the phosphorylation and total expression of stress activated protein kinases, p38 and JNK1/2 following 12, 18 and 24 h of treatment. As changes were observed at an intracellular signaling level, the effect of the combination treatments were investigated at the transcriptomic level after 18 h of treatment, using human oligonucleotide microarrays. This transcriptomic analysis revealed that both combination treatments reduced the transcript expression of five enzymes involved with cholesterol synthesis, which was confirmed through qRT-PCR. Cholesterol is an important component of the plasma membrane and is critical for the transduction of extracellular signals. Furthermore, both combination treatments induced the transcriptomic expression of the zinc coordinating metallothionein (MT) proteins. This was associated with an increased nuclear localization of MTF-1, the transcription factor responsible for MT expression, after 6, 12 and 18 h of both combination treatments. Finally, nuclear Western blotting of the NF-[kappa]B subunit, p65, revealed that both combination treatments reduced the nuclear localization of NF-[kappa]B following 6, 12 and 18 h. In collating this data, it appears that the combination treatments of EGCG+4-OHT and EGCG+Ral are inducing cytotoxicity through various mechanisms, including reduced cellular signaling through EGFR, AKT and S6K, increased stress signaling through JNK1/2 and p38 and altered gene expression of MTs and enzymes involved with cholesterol synthesis. Therefore, the combination treatment of EGCG+SERMs exhibits therapeutic potential in MDA-MB-231 cells, a model of basal-like breast cancer.

A phytochemical investigation on methanolic extract of roots of yellow rose led to the isolation of a catechin gallate. The crude extract first underwent fractionation using petroleum ether, chloroform, ethylacetate, butanol with water as solvents in their respective order. The emerging solvent fractions were subjected to further separation using lipophilic sephadex (LH-20), and silica gel column chromatography to isolate pure compounds. Analytical thin layer chromatography (TLC) was used to confirm the presence of a catechin in butanol fraction. Purified catechin compound was subjected to 2,2-diphenyl-1-picrylhydrazyl (DPPH) experiment to determine its Radical Scavenging Capacity, which was found quite promising. Chemical structure of purified compound was established by using Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) experiments.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 102-115). Also available on the World Wide Web.

Thesis (M. Sc.)--University of Canterbury, 2008. / Typescript (photocopy). "June 2008." Includes bibliographical references (leaves 65-72). Also available via the World Wide Web.

Zugl.: Stuttgart, Univ., Diss., 2004.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20236号 / 医博第4195号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 松原 和夫, 教授 川村 孝 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Plusieurs aliments fonctionnels sont et seront développés à partir de molécules aux propriétés nutraceutiques. Parmi ces molécules, l’épigallocatéchine-3-gallate (EGCG), principal antioxydant du thé vert, présente un grand intérêt en raison de ses propriétés antioxydantes qui pourraient jouer un rôle préventif vis-à-vis de certaines pathologies. Cependant, sa biodisponibilité est faible à cause de son instabilité en milieu intestinal. De son côté, la β-Lactoglobuline (β-Lg) est connue pour sa capacité à lier des molécules bioactives les protégeant de l’oxydation. L’objectif général de ce projet de recherche vise à étudier les interactions entre l’EGCG et la β-Lg et l'impact de ces interactions sur les propriétés fonctionnelles du système formé. La caractérisation moléculaire des interactions entre l’EGCG et la β-Lg, à l’état natif et dénaturé thermiquement, a été réalisée par des méthodes spectroscopiques. Les résultats montrent que l’EGCG interagit avec la β-Lg au niveau moléculaire en formant un complexe 1:1 induisant un changement de la structure moléculaire de la β-Lg. L'interaction provoque une diminution de l’activité antioxydante de l’EGCG dans toutes les conditions étudiées. Cependant, on observe que la β-Lg assure une protection limitée à l'EGCG contre la dégradation dans le temps : la forme dénaturée de la protéine à pH 7 étant légèrement plus efficace. L’impact de l’EGCG sur le comportement thermique de la β-Lg et sur la microstructure des gels obtenus a été étudié par spectroscopie infrarouge à transformée de Fourier (FTIR) et par microscopie électronique. La présence d’EGCG change le profil d’agrégation de la protéine. Cependant, ces interactions n'entraînent pas de changements majeurs dans la microstructure des gels formés, qu'il s'agisse des gels filamenteux ou particulaires. Cette étude démontre que la β-Lg peut lier l’EGCG et maintenir une activité antioxydante non négligeable dans les deux types des gels formés par la protéine. / Molecules with nutraceutical properties can be used to develop functional foods. The major antioxidant in green tea, epigallocatechin-3-gallate (EGCG), could be valuable for preventing certain pathologies. However, its bioavailability in the intestine needs to be improved. The milk protein β-lactoglobulin is known for its ability to protect bioactive molecules from oxidation. The general aim of this research was to study EGCG–β - lactoglobulin interactions and their effects on nutraceutical functionality using spectroscopic methods. The results show that EGCG forms complexes with β-lactoglobulin in a 1:1 ratio and induces changes in the molecular structure of the protein. This interaction causes a decrease in the antioxidant activity of EGCG. However, EGCG degradation is slowed, more in the case of thermally denatured β-lactoglobulin. Using Fourier transform infrared spectroscopy (FTIR) and electron microscopy to study β-lactoglobulin thermal behaviour and gel microstructure, it was determined that binding EGCG changes the protein aggregation pattern but has little effect on the microstructure of filamentous gels or particulate gels. This study shows that both types of β-lactoglobulin gel can bind EGCG and maintain its antioxidant activity.

Les porphyrines sont des composés macrocycliques naturels étudiés de manière approfondie en tant que mimétiques enzymatiques ou catalyseurs d'oxydation en milieu homogène. L'incorporation de porphyrines dans des réseaux de polymères de coordination métal-organique (MOFs), qui constituent une famille de composés cristallins poreux connus pour leur diversité structurelle, pourrait conduire à des matériaux constitués de réseaux étendus présentant les mêmes propriétés que les systèmes homogènes. Toutefois de nombreux travaux ont montré que la limitation majeure pour des applications à grande échelle des MOFs est leur stabilité.La stabilité de ces matériaux peut être accrue en employant des cations métalliques de valence élevée dans l'unité de construction inorganique (tels que Fe3+, Al3+, Zr4+) et / ou en modifiant la fonction coordinante du ligand organique.Ce travail est relatif à l'étude de la réactivité de ligands porphyriniques portant diverses fonctions coordinantes, avec des ions métalliques de valence élevée. L'influence des paramètres de synthèse et la caractérisation approfondie de ces MOFs ont été réalisées par une combinaison de techniques expérimentales (diffraction des rayons X, analyse thermogravimétrique, spectroscopie UV-vis, spectroscopie infrarouge, études de sorption, etc.). Dans un premier temps, l'étude s'est focalisée sur des MOFs porphyriniques à base de groupements carboxylates stables connus, ce qui a conduit à de nouvelles variantes de ces matériaux. Dans un second temps il est démontré que les topologies des réseaux observées sur un ligand porphyrinique à base carboxylate peuvent être étendues à d'autres fonctionnalités avec la synthèse d'un nouveau MOF à base de tétrazolate. L'amélioration de la stabilité a également été explorée avec des ligands portant des fonctions phénol qui ont abouti à l'élaboration de nouveaux réseaux de coordination. La synthèse et l'étude de stabilité d'un MOF porphyrinique à base de gallate est rapportée. L'évaluation préliminaire de l'activité catalytique de certains des MOFs élaborés est également décrite / Porphyrins are important macrocyclic compounds which are prevalent in nature and have been extensively studied by chemists in homogeneous catalysis as enzyme mimics. Incorporating porphyrins in metal-organic frameworks (MOFs) offer an ideal opportunity to obtain material with extended frameworks possessing the same properties as the homogenous systems. Much work has been done on porphyrinic MOFs but their stability remains a problem and a major limitation for possible wide scale applications. In literature, more stable MOFs have been realised using high valent metal ions in the inorganic building unit (such as Fe3+, Al3+, Zr4+) and/or by using more basic functionalities in the organic linker. However, regarding porphyrinic MOFs, little work is reported with ligands based on functionalities other than carboxylic acid groups. Therefore, our work focused on investigating the reactivity of porphyrinic ligands carrying various functionalities with high valent metal ions. More precisely, we focused on the design, synthesis optimisation and characterisation of such materials. This included studying existing stable carboxylate porphyrinic frameworks with a goal of incorporating new functionalities, which led to new variations of these materials. Our work also demonstrated that the framework topologies observed with carboxylate based porphyrinic ligand can be expanded to other functionalities with the synthesis of a new tetrazolate based porphyrinic MOF. MOF synthesis was also investigated with phenolate functionalised ligands and resulted in the first gallate based porphyrinic MOF reported. The stability of this new material was assessed. This manuscript discusses the synthesis and the characterisation of these MOFs via a combination of experimental techniques (X-ray diffraction, TGA analysis, UV-vis spectroscopy, IR-spectroscopy, sorption studies etc.). The preliminary evaluation of the catalytic activity of some of the MOFs is also described

Propyl gallate (PG) is a significant synthetic antioxidant and preservative. Its determination has been studied at carbon paste electrode (CPE) using differential pulse voltammetry (DPV) and high performance liquid chromatography with electrochemical detection (HPLC-ED) and with UV spectrometric detection (HPLC-UV). Concentration dependences were measured in the media of Britton-Robinson buffer (pH 5) and methanol (20 %, v/v) by DPV and the limit of detection 0,6110-7 moldm-3 was obtained. Using HPLC with a mobile phase consisting of 0,01 moldm-3 phosphate buffer (pH 4) and methanol (50 %, v/v) with potential of working electrode E = +0,8 V and detection wavelength λ = 280 nm, concentration dependences were measured. Limit of detection was determined to 0,39 moldm-3 for HPLC-ED and 4,95 moldm-3 for HPLC-UV. After verification of the extraction procedure PG was determined in vegetable oil. The resulting value of 3,2 mgkg-1 corresponds with permited limits.

O chá verde vem sendo utilizado na prevenção e tratamento de variadas doenças infecciosas e imunoinflamatórias, por apresentar efeitos benéficos decorrentes da presença de Epigalocatequina-3-galato (EGCG). Esta catequina apresenta papel antiinflamatório, anti-oxidante, anti-microbiano e mineralizador e poderia ser utilizado no tratamento da doença periapical. O objetivo do presente trabalho foi avaliar o efeito uma pasta à base de EGCG utilizada como curativo de demora em lesões periapicais experimentalmente induzidas em cães. Um total de 80 raízes de pré-molares de cão com rizogênese completa e lesões periapicais experimentalmente induzidas, foram aleatoriamente divididos em 4 grupos submetidos à diferentes protocolos de tratamento endodôntico: 1) curativo de demora com pasta à base de EGCG; 2) curativo de demora com EGCG em veículo aquoso; 3) curativo de demora com pasta à base hidróxido de cálcio (Pasta Calen); 4) tratamento endodôntico em sessão única. Para avaliação da resposta tecidual, os dentes foram avaliados radiograficamente e histopatologicamente. As imagens radiográficas obtidas antes e 120 dias após o tratamento, foram fotografadas e digitalizadas para o programa Image J 1.28 para mensuração das áreas (mm&sup2;) radiolúcidas periapicais. Aos 120 dias após a obturação doa canais radiculares os animais foram eutanasiados e os espécimes foram processados histotecnicamente, corados com HE e avaliados com microscopia de luz convencional e de fluorescência (infiltrado inflamatório, espessamento do ligamento periodontal e reabsorção de tecidos mineralizados). Os resultados obtidos foram analisados estatisticamente utilizando os testes quiquadrado, Fisher, Anova e pós teste de Tukey, com nível de significância de 5% em todos os testes. De acordo com os resultados radiográficos, o percentual médio de redução da área radiolúcida (± erro padrão da média) foi semelhante (p>0,05) nos grupos EGCG em veículo aquoso (64,57%; ±7,514); pasta à base de EGCG (59,95%; ±8,023) e pasta à base de hidróxido de cálcio (58,65%; ±6,192). Todos foram superiores ao grupo tratado em sessão única (19,49%; ±2,881) (p<0,01). A análise histopatológica mostrou que os grupos que receberam aplicação de curativo de demora com EGCG ou hidróxido de cálcio, apresentaram reaparação da lesão periapical com semelhança em todos os parâmetros avaliados. Por outro lado, o tratamento em sessão única resultou em manutenção da lesão periapical, com maior espessura do ligamento periodontal (p<0,001), persistência de infiltrado inflamatório moderado ou severo (p<0,01) e presença de reabsorção óssea e cementária (p<0,0001). Foi possível concluir que a pasta à base de EGCG proporcionou o reparo de lesões periapicais, constituindo possível medicação intracanal alternativa / Green tea has been used in the prevention and treatment of various infectious and immunoinflammatory diseases, since it has beneficial effects due to the presence of Epigallocatechin-3-gallate (EGCG). This catequin present anti-inflammatory, antimicrobial, antioxidant and mineralizing role that could be usefull in the treatment of periapical disease. The aim of the present study was to evaluate the effect of the intracanal dressing with an EGCG paste-based im periapical lesions experimentaly induced in dogs. A total of 80 dog premolar roots with complete rhizogenesis and experimentally induced periapical lesions were randomly divided into 4 groups submitted to different endodontic treatment protocols:1) intracanal dressing with EGCG based paste; 2) intracanal dressing with EGCG in aqueous vehicle; 3) intracanal dressing with based paste on calcium hydroxide (Calen paste); 4) single session endodontic treatment. To evaluate the tissue response, the teeth were evaluated radiographically and histopathologically. Radiographic images are selected before 120 days after treatment, were photographed and scanned for the program Image J 1.28 for the measurement of periapical radiolucent areas (mm2). To 120 days after the root canal filling animals were euthanasied and the specimens processed, stained in the rotin HE and evaluated under conventional and fluorescence microscopy (inflammatory infiltrate, periodontal ligament space and mineralized tissue resorption). The results were compared statistically using chisquare, Fisher, Anova and Tukey post-test, with a significance level of 5% in all tests. According to the radiographic results, the mean percentage reduction of the radiolucent area (standard pattern of the mean) was similar (p> 0.05) in the EGCG groups in aqueous vehicle (64.57%; ± 7.514); EGCG based paste (59.95%, ± 8.023) and calcium hydroxide based paste (58.65%, ± 6.192). All groups were higher for the group treated in single session (19.49%, ± 2.881) (p <0.01). The histopathological analysis showed that the groups that received the prescription of intracanal dressings with EGCG or calcium hydroxide, resulted in periapical lesion repair and were similar in all the evaluated parameters. However, treatment in single session did not repair the periapical lesion resulting in greatest measure of the periodontal ligament (p <0.001), persistence of moderate or severe inflammatory infiltrate (p <0.01) and presence of bone and cementum resorption (p <0.0001). It is possible to conclude that EGCG paste-based allows the periapical lesions repair, constituting possible alternative intracanal medication