Fungos endofíticos constituem uma fonte promissora e ainda pouco explorada de novas substâncias potencialmente bioativas, que podem ter aplicações na medicina, agricultura e biologia química. Neste trabalho foram isolados cinco fungos endofíticos das folhas de Tithonia diversifolia, os quais foram cultivados em pequena escala e avaliados em diversos bioensaios. O fungo endofítico Phoma sorghina foi cultivado em escala ampliada, produzindo nove substâncias, sendo três derivados antraquinônicos inéditos na literatura. Três fungos endofíticos associados à Viguiera robusta foram estudados. De Colletotrichum gloeosporioides foram isoladas oito substâncias, sendo um derivado dicetopiperazínico e um derivado aminoaçúcar inéditos na literatura. Diaporthe phaesolorum produziu três derivados dicetopiperazinicos, um inédito na literatura. Chaetomium globosum produziu treze substâncias, sendo oito derivados azaphilônicos inéditos. Algumas linhagens endofíticas foram avaliadas em experimentos de biotransformação de três naftoquinonas e da 1-tetralona, catalisando reações de oxidação e redução. O fungo Diaphorte phaseolorum catalisou uma reação de metilação inédita na literatura de biotransformação. / Endophytic fungi are a promising and untapped source of potential biologically active compounds that might be useful as therapeutic and agrochemical agents, or chemical biology tools. In this thesis five endophytic fungi were isolated from the leaves Tithonia diversifolia. All the strains have been cultivated in small scale and the extracts submitted to bioassays. The culture of the endophyte Phoma sorghina was scaled up, affording six known compounds and three novel anthraquinone derivatives. Three endophytes from Viguiera robusta have also been studied as sources of natural products. Colletotrichum gloeosporioides produced six known compounds, one novel aminosugar and one new diketopiperazine derivative. Diaporthe phaesolorum biosynthesized three diketopiperazine derivatives, one novel in the literature. Five known compounds and eight novel azaphilone derivatives were isolated from Chaetomium globosum. Some endophytic strains have been evaluated in biotransformation experiments of three naphthoquinones and 1-tetralone, catalyzing oxidation and reduction reactions. The endophyte Diaphorte phaseolorum catalyzed a novel methylation reaction in the biotransformation literature.

This dissertation reports the preparation of several types of anthraquinones structurally related to adriamycin. It describes the synthesis of two types of 2-aminoquinizarin compounds. It also presents two new syntheses of a heterocyclic tetracyclic ring system, similar to the aglicone ring system of adriamycin. A series of 2-aminoquinizarins was prepared by adding several primary amines to quinizarin. Quinizarin was shown to be essentially inert toward secondary amines. Several secondary amine adducts with quinizarin have been prepared, however, by treating the bis-boroacetate ester of quinizarin with the amines. Both types of 2-aminoquinizarin compounds exhibit outstanding potential for possessing antineoplastic activity, and several have been submitted to the National Cancer Institute for testing in their screening program for antineoplastic agents.

Baicalein is known to be a potent antifungal agent and induces programmed cell death in Candida albicans. In the present study, we found that baicalein also inhibited the growth of C. krusei isolates. The minimal inhibitory concentrations of baicalein against eight C. krusei isolates were 1.35--2.70 microg/ml. One-hour exposure to baicalein elicited a consistent and moderate post-antifungal effect on the C. krusei isolates. Further flow cytometric study demonstrated a depolarization of mitochondrial membrane potential. However, both the levels of reactive oxygen species and DNA fragmentation were not significantly changed after baicalein treatment in C. krusei. It can be concluded that the antifungal activity of baicalein was mitochondria-dependent in both C. krusei and C. albicans, but the antifungal mechanism was different. Reactive oxygen species may not play a direct role and baicalein does not initiate programmed cell death or apoptosis in C. krusei. The structure-activity relationship study showed that the three hydroxyl groups in baicalein were essential for its antifungal potency. / Candidiasis has become a serious infection with very high mortality and morbidity in the world if not providing effective treatments. However, due to clinical limitation and resistance of the current antifungal agents, there is an urgent need to search for novel antifungals. In this study, after screening a compound library (n=400) for antifungal activity, three members (metergoline, purpurin and baicalein) were chosen for further study. Their antifungal characteristics and the antifungal mechanisms were investigated. / Metergoline, a serotonin receptor antagonist, was found to have potent antifungal activity against the intrinsically fluconazole-resistant human fungal pathogen Candida krusei. The minimal inhibitory concentration and minimal fungicidal concentration of metergoline against C. krusei were 4 microg/ml and 8 microg/ml respectively. Metergoline induced post-antifungal effect. Significant synergism was found in combination of metergoline with amphotericin B by a checkerboard assay, which may be due to the perturbation of cell permeability and increase in the intracellular accumulation of antifungal agents. Metergoline also inhibited extracellular phospholipase production in C. krusei. To gain insights into the mechanisms, intracellular changes that accompany apoptosis were examined by flow cytometry and spectrophotometry. The results showed an increase in the level of reactive oxygen species, depolarization of mitochondrial membrane potential, phosphatidylserine externalization, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling in the metergoline-treated C. krusei . Taken together, we conclude that metergoline may promote apoptosis in C. krusei through reactive oxygen species production and perturbation in mitochondrial homeostasis, implying its antifungal potential to treat candidiasis. / The antifungal activity of purpurin, a natural red anthraquinone pigment in madder root (Rubia tinctorum L.), was evaluated against Candida isolates by a broth microdilution assay. The minimal inhibitory concentrations of purpurin against Candida species isolates were 1.28--5.12 microg/ml. Mechanistic studies indicated that purpurin inhibited energy-dependent efflux pumps of Candida isolates. Furthermore, purpurin demonstrated a depolarization of mitochondrial membrane potential, suggesting a possible linkage of the antifungal mechanism of purpurin to Candida apoptosis. / Kang, Kai. / Adviser: Fong Wing Ping. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 98-123). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Das Bodenbakterium Rhodococcus opacus 1CP ist für sein Potenzial bekannt, organische Schadstoffe abzubauen. In dieser Studie lag der Fokus auf zwei Genprodukten, den farbstoffabbauenden Peroxidasen DypA und DypB, welche unter Anwendung von Klonierungs- und Expressionsstrategien mit hohen spezifischen Aktivitäten in reiner Form gewonnen werden konnten. Eine umfassende biochemische Analyse zeigte, dass beide Enzyme in der Lage sind, Farbstoffe unterschiedlicher Klassen, insbesondere jedoch, die als schwer abbaubar geltenden Anthraquinone, strukturell anzugreifen. Damit stellen sie lohnenswerte enzymatische Systeme zum Abbau toxischer Farbstoffe beispielsweise in industriellen Abwässern dar.

A series of anthraquinone-linked DNA oligonucleotides was prepared and the efficiency of long-distance radical cation migration was measured. In one set of oligonucleotides, two GG steps are separated by either a TATA or an ATAT bridge. In these two compounds, the efficiency of radical cation migration from GG to GG differs by more than an order of magnitude. Replacement of the thymines in the TATA or ATAT bridges with 3-methyl-2-pyridone (t, a thymine analog) results in the much more efficient radical cation migration across the bridge in both cases. This is attributed to a decrease in the oxidation potential of t to a value below that of A. In contrast, replacement of the thymines in the TATA or ATAT bridges with difluorotoluene (f, a thymine analog with high oxidation potential) does not measurably affect radical cation migration. These findings are readily accommodated by the phonon-assisted polaron-hopping mechanism for long-distance charge transfer in duplex DNA and indicate that DNA in solution behaves as a polaronic semiconductor. Oligomers containing thiophene-pyrrole-thiphene (SNS) monomers were covalently linked to the nucleobases of DNA. Treatment of these oligomers with horseradish peroxidase and hydrogen peroxide lead to the formation of conducting oligomers conjoined to the DNA. The DNA template aligns the oligomers along one strand of the duplex and limits the intermolecular reaction of monomers. This method enables utilization of the unique self-recognizing properties and programmability of DNA to create tailored oligomers.

Das Bodenbakterium Rhodococcus opacus 1CP ist für sein Potenzial bekannt, organische Schadstoffe abzubauen. In dieser Studie lag der Fokus auf zwei Genprodukten, den farbstoffabbauenden Peroxidasen DypA und DypB, welche unter Anwendung von Klonierungs- und Expressionsstrategien mit hohen spezifischen Aktivitäten in reiner Form gewonnen werden konnten. Eine umfassende biochemische Analyse zeigte, dass beide Enzyme in der Lage sind, Farbstoffe unterschiedlicher Klassen, insbesondere jedoch, die als schwer abbaubar geltenden Anthraquinone, strukturell anzugreifen. Damit stellen sie lohnenswerte enzymatische Systeme zum Abbau toxischer Farbstoffe beispielsweise in industriellen Abwässern dar.

À l’heure où l’intégration des enjeux environnementaux dans le développement de procédés éco-efficients joue un rôle essentiel dans le moteur de l’innovation responsable, la chimie verte est devenue l’un des sujets de préoccupation majeure. Ainsi, le développement de nouveaux procédés éco-respectueux pour la production d’ingrédients naturels issus de matières premières végétales renouvelables est devenu une démarche incontournable dans le modèle de recherche. L’objectif de cette thèse a consisté au développement d’une stratégie d’éco-valorisation innovante employant les fluides sub/supercritiques pour l’extraction, la caractérisation, la production et l’imprégnation sur support cosmétique de produits naturels d’origine végétale. Pour cela, nous avons utilisé comme modèle végétal : les graines oléagineuses de la plante Kniphofia uvaria, sélectionnée pour des applications cosmétiques grâce à ses propriétés bioactives antioxydantes et anti-âge. Dans un premier temps, le développement de méthodes complémentaires en SFC ainsi que le développement du couplage SFC-MS a été réalisé à l’aide de la source APCI afin d’identifier les molécules responsables des activités bioactives des graines de Kniphofia uvaria. Ainsi, le développement d’un système hybride (U)HPLC/SFC-HRMS a été réalisé afin de mettre en place ce couplage. Des optimisations en termes de proportion et nature de solvant make-up ainsi qu’un travail au niveau des paramètres SFC et MS ont été faits afin de d’améliorer la sensibilité et la spécificité des analyses lipidiques. Dans un second temps, nous nous sommes attachés au développement d’une stratégie d’enrichissement en composés bioactifs à l’aide des méthodes : SFE et CPC. Ainsi, en SFE, des optimisations en termes de température, pression, nature/proportions de co-solvant dans le fluide ont été réalisées alors qu’en CPC, des optimisations au niveau de l’injection ont été faites. Des conditions optimales pour le fractionnement sélectif des anthraquinones et des triglycérides ont été déterminées en SFE et CPC. Dans un dernier temps, ce travail a consisté à développer un couplage en-ligne pour extraire et imprégner sélectivement sur silice cosmétique : les anthraquinones. Le développement et l’optimisation de ce procédé en-ligne ont été réalisés à l’échelle du laboratoire et ont démontré la faisabilité de ce couplage ainsi qu’un intérêt certain pour l’obtention de produits naturels sous une première forme galénique, destinée à une future incorporation dans la formulation de cosmétiques. / Nowadays, green chemistry is a great challenge. It seeks innovation in the development of eco-efficient processes. The production of natural products from renewable materials by these new environmentally friendly processes is more and more used. The aim of this Ph.D thesis is to develop an eco-valuation strategy to extract, characterize, produce and impregnate natural products onto a cosmetic support using sub/supercritical fluids. Consequently, we used oleaginous plant seeds from Kniphofia uvaria as a plant model, which was selected for its interesting cosmetic properties such as antioxidant or anti-ageing. Firstly, the SFC-MS hyphenation with the APCI as an ionization source was developed to screen bioactive molecules; responsible of cosmetic properties. This coupling was performed by the hybrid combination of (U)HPLC/SFC-HRMS. Various optimizations in terms of the solvent make-up (nature and proportion), modulation with SFC and MS parameters were carried out in order to improve sensitivity and selectivity of lipid analysis. Secondly, an enrichment strategy to concentrate bioactive compounds in the final extract was developed by SFE and CPC. Thus, in SFE, experimental parameters (temperature, pressure, nature/proportion of the modifier in the CO2 fluid) were optimized while in CPC, the injection optimization was realized. Methods for the selective fractionation of anthraquinones and triglycerides were obtained in CPC and SFE. Finally, an on-line sub/supercritical extraction-impregnation process was developed to extract and for simultaneously impregnating anthraquinones onto a cosmetic silica. Development and optimization of this process was realized on a laboratory scale. Consequently, this study demonstrated the feasibility of this concept and it presents a great interest to provide natural products as a galenic form, which could be used in the cosmetic formulation.

AQ4N (banoxantrone) is a prodrug that, under hypoxic conditions, is enzymatically converted to a cytotoxic DNA-binding agent, AQ4. Incorporation of AQ4N into conventional chemoradiation protocols therefore targets both oxygenated and hypoxic regions of tumors, and potentially will increase the effectiveness of therapy. This current pharmacodynamic and efficacy study was designed to quantify tumor exposure to AQ4 following treatment with AQ4N, and to relate exposure to outcome of treatment. A single dose of 60 mg/kg AQ4N enhanced the response of RT112 (bladder) and Calu-6 (lung) xenografts to treatment with cisplatin and radiation therapy. AQ4N was also given to separate cohorts of tumor-bearing mice 24 hours before tumor excision for subsequent analysis of metabolite levels. AQ4 was detected by high performance liquid chromatography/mass spectrometry in all treated samples of RT112 and Calu-6 tumors at mean concentrations of 0.23 and 1.07 microg/g, respectively. These concentrations are comparable with those shown to be cytotoxic in vitro. AQ4-related nuclear fluorescence was observed in all treated tumors by confocal microscopy, which correlated with the high performance liquid chromatography/mass spectrometry data. The presence of the hypoxic marker Glut-1 was shown by immunohistochemistry in both Calu-6 tumors and RT112 tumors, and colocalization of AQ4 fluorescence and Glut-1 staining strongly suggested that AQ4N was activated in these putatively hypoxic areas. This is the first demonstration that AQ4N will increase the efficacy of chemoradiotherapy in preclinical models; the intratumoral levels of AQ4 found in this study are comparable with tumor AQ4 levels found in a recent phase I clinical study, which suggests that these levels could be potentially therapeutic.

Yes / Topoisomerase inhibitors are in common use as chemotherapeutic agents although they can display reduced efficacy in chemotherapy-resistant tumours, which have inactivated DNA damage response (DDR) genes, such as ATM and TP53. Here, we characterise the cellular response to the dual-acting agent, Alchemix (ALX), which is a modified anthraquinone that functions as a topoisomerase inhibitor as well as an alkylating agent. We show that ALX induces a robust DDR at nano-molar concentrations and this is mediated primarily through ATR- and DNA-PK- but not ATM-dependent pathways, despite DNA double strand breaks being generated after prolonged exposure to the drug. Interestingly, exposure of epithelial tumour cell lines to ALX in vitro resulted in potent activation of the G2/M checkpoint, which after a prolonged arrest, was bypassed allowing cells to progress into mitosis where they ultimately died by mitotic catastrophe. We also observed effective killing of lymphoid tumour cell lines in vitro following exposure to ALX, although, in contrast, this tended to occur via activation of a p53-independent apoptotic pathway. Lastly, we validate the effectiveness of ALX as a chemotherapeutic agent in vivo by demonstrating its ability to cause a significant reduction in tumour cell growth, irrespective of TP53 status, using a mouse leukaemia xenograft model. Taken together, these data demonstrate that ALX, through its dual action as an alkylating agent and topoisomerase inhibitor, represents a novel anti-cancer agent that could be potentially used clinically to treat refractory or relapsed tumours, particularly those harbouring mutations in DDR genes.