Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 59 pages. Includes Vita. Includes bibliographical references.

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Orientador: Lourdes Campaner dos Santos / Banca: Maysa Furlan / Banca: Marcelo Aparecido da Silva / Resumo: As espécies da família Eriocaulaceae apresentam estudos químicos na literatura que descrevem a presença de substâncias com potencial biológico, com destaque para a planifolina em Paepalanthus planifolius. Apesar disso, existem poucos dados sobre a química dos microrganismos associados a essas espécies, como fungos endofíticos. Para esse estudo, selecionamos o fungo endofítico Microsphaeropsis arundinis, isolado dos capítulos de P. planifolius. O endófito foi cultivado em meios de cultivo líquido (Caldo Batata Dextrose, Caldo YM, Extrato de Malte, Caldo Nutrient e Caldo Czapek-Dox) e sólido (arroz parboilizado e milho canjica) e os extratos acetato de etila obtidos foram analisados quimicamente quanto a seu perfil cromatográfico por HPLC-PDA e biologicamente a partir da atividade antitumoral contra linhagens de câncer de mama e de câncer de pulmão. O extrato acetato de etila cultivado em arroz parboilizado demonstrou-se o mais ativo frente as linhagens de adenocarcinoma de mama murino (IC50 = 39,91 μg mL-1) e adenocarcinoma de mama humano (IC50 = 51,93 μg mL-1), sendo cultivado em escala ampliada e estudado fitoquimicamente buscando-se o isolamento das substâncias produzidas. A utilização de técnicas espectroscópicas e espectrométricas permitiu a identificação de oito substâncias, sendo as substâncias 1, 2, 3 e 7 inéditas na literatura e a substância 5 descrita pela primeira vez como produto natural. Uma análise biossintética mostra que as substâncias são provenientes da via d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Species of Eriocaulaceae family showed some chemical studies that describe the presence of compounds with biological potential, such as planifolin in Paepalanthus planifolius . Despite these compounds, there are few data about the chemistry of the microorganisms associated with these species, such as endophytic fungi. For this study, we selected the endophytic fungus Microsphaeropsis arundinis, isolated from P. planifolius capitula. The endophyte was cu ltivate in liquid culture media (Potato Dextrose Broth, YM Broth, Malt Extract, Nutrient Broth and Czapek - Dox Broth) and solid culture media ( parboiled rice and corn) and the ethyl acetate extracts were chemically analyzed by HPLC - PDA and biologically eval uated against adenocarcinoma cell lines using MTT assay. The ethyl acetate extrac t cultivated in parboiled rice was the most active one against murine breast adenocarcinoma (IC 50 = 39,91 μg mL - 1 ) and human breast adenocarcinoma (IC 50 = 51,93 μg mL - 1 ), being select to cultivated on an enlarged scale and fractionated result in the isolation of major compounds . The use of spectroscopic and spectrometric techniques result in the ide ntification of eight compounds, with 1 ( 2Z, 5E ) - 4,7,9 - trihydroxy - 2,5 - decadienoic acid) 2 ( ( 2Z, 5E ) - 4 - acetoxy - 7,9 - dihydroxydeca - 2,5 - dienoic acid ), 3 ( ( 2Z,5E ) - 7 - acetoxy - 4,9 - dihydroxydeca - 2,5 - dienoic acid) and 7 ( 1 - (2,5 - dihydroxypheny l) - 3' - oxobutan - 2' ' - yl acetate) unpublished in ... (Complete abstract click electronic access below) / Mestre

This thesis is regarded with the biomimetic total synthesis of phenolic natural products and describes two different projects. Chapter 1 introduces phenolic natural products, the most important biosynthetic pathways for their formation and some examples of relevant biomimetic syntheses. A short introduction to dimeric and pseudo-dimeric natural products can also be found in this chapter. Specific introductions can be found at the start of Chapters 2 and 3. Chapter 2 describes the total synthesis of a dimeric thymol derivative isolated from Arnica sachalinensis (which we have named “thymarnicol”). Inspired by the biosynthesis proposed by Passreiter and co-workers, we tested and confirmed the feasibility of a key hetero-Diels–Alder dimerisation step. During our investigations, we gained significant new insights into the origin and reactivity of thymarnicol. The final oxidative cyclisation has been found to occur spontaneously upon exposure to visible light in air. Chapter 3 discusses our efforts to develop a divergent biomimetic synthetic strategy towards a family of prenylated phenylpropanoid natural products isolated from Illicium genus plants. Our first biomimetic approach revealed the chemical instability of our proposed key intermediates. Therefore, a revised approach was trialled, allowing the total synthesis of a small set of natural products and related structures. We envisage that this strategy could be exploited by accessing a large number of members of this family of compounds.

Synthèse d’inhibiteurs potentiels non phosphorylés de la désoxyxylulose phosphate réductoisomérase et étude de la voie de biosynthèse des unités isopréniques chez Acanthamoeba polyphaga. De nombreux microorganismes pathogènes utilisent la voie du méthylérythritol phosphate (MEP) pour la synthèse des unités isopréniques (IPP et DMAPP). Absente chez l’homme, cette voie constitue une cible de choix pour lutter contre ces microorganismes. La fosmidomycine est un des meilleurs inhibiteurs connus à ce jour de la désoxyxylulose phosphate réductoisomérase (DXR), deuxième enzyme de cette voie. Afin d’améliorer d’une part la biodisponibilité de ce type d’inhibiteur et d’autre part de lutter contre le phénomène de résistance à cet antibiotique, nous avons synthétisé des analogues de la fosmidomycine où le groupement phosphonate est remplacé par un groupement tétrazole ou par un groupement squaryle. Les molécules synthétisées ont été testées sur la DXR d’ Escherichia coli et n’ont pas révélé d’activité inhibitrice significative.Par ailleurs, nous avons montré, par des expériences d’incorporation de glucose marqué au 13C, que l’amibe Acanthamoeba polyphaga, utilise la voie du mévalonate pour synthétiser les unités isopréniques nécessaires à la synthèse de ses stérols / Synthesis of potential non-phosphonate inhibitors of the deoxyxylulose phosphate reductoisomerase and study of the biosynthetic pathway for isoprenoids units synthesis using by Acanthamoeba polyphagaMany pathogenic microorganisms synthesize their isoprenoid units (IPP and DMAPP) via the methylerythritol phosphate pathway (MEP pathway). Absent in man, all enzymes of this metabolic route are potential targets for the design of new antimicrobials. This pathway is present in pathogenic bacteria, but absent in mammals. Hence, the development of small-molecule inhibitors for the MEP enzymes constitutes a novel approach for the design of new antimicrobials. Fosmidomycin is the most efficient inhibitor of the the deoxyxylulose phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway. In an attempt to improve the pharmacological properties and the bioavailability of this antibiotic, we synthesized analogues of the fosmidomycin by replacing the phosphonate group by tetrazole or squaryl moieties. These synthesized compounds were tested on the DXR isolated from Escherichia coli.Otherwise, we showed by achieving incorporation experiments with 13C labeled glucose that the amoeba Acanthamoeba polyphaga utilize the mevalonate pathway to synthesize its sterols.

Rhodotorula (Rhodosporidium) toruloides is an oleaginous basidiomycete yeast with great biotechnological potential. Capable of accumulating lipid up to 76 % of its dry biomass and well suited to the metabolism of lignocellulosic hydrolysate, it is a good candidate for production of advanced biofuels as well as a host of other potential roles in industry. However, molecular genetic tools for manipulation of this yeast are lacking and its high genomic GC content can make routine cloning difficult. Agrobacterium tumefaciens-mediated transformation of R. toruloides CBS 14 was demonstrated, and plasmid vectors were developed for transformation of R. toruloides, including elements for Saccharomyces cerevisiae in-yeast assembly. In-yeast assembly is robust to the manipulation of GC-rich DNA and of large plasmids. Using these vectors and an EGFP reporter, a screen to identify inducible promoters was performed, and promoters from the genes NAR1, ICL1, CTR3, and MET16 identified. These promoters have independent induction/repression conditions and different levels and rates of induction. Minimal inducible promoters were determined, which are as small as 200 bp. As well as showing tight regulation of the EGFP marker, the NAR1 promoter was able to drive conditional rescue of a leu2 mutant strain. In parallel, as a proof of principle for production of advanced biofuels, hydrocarbon biosynthesis pathways were expressed in R. toruloides and analysed by GC-MS. After co-expression of Synechococcus elongatus fatty acyl-ACP reductase and fatty aldehyde decarbonylase, and E. coli ferredoxin and ferredoxin reductase, production of the alkane heptadecane was observed. To increase the availability of free fatty acids (FFA) for production of hydrocarbons by other pathways, Thermomyces lanuginosus lipase 2 was expressed, resulting in a 1.3-fold increase in the concentration of FFAs.

Orientador: Maysa Furlan / Coorientador: Silvia Noelí López / Banca: Isabele Rodrigues Nascimento / Banca: Carlos Alberto Labate / Banca: Maria Fátima das Graças Fernandes da Silva / Banca: Luciana Gonzaga de Oliveira / Resumo: Três espécies de Piper foram escolhidas como modelos para estudos biossintéticos, transcriptômicos e proteômicos: P. tuberculatum, P. nigrum e P. crassinervium. As duas primeiras espécies acumulam principalmente metabólitos secundários da classe das amidas e essas substâncias foram alvo de experimentos biossintéticos. Foram monitoradas as atividades de diversas enzimas envolvidas na via fenilpropanoídica, dentre elas a fenilalanina amônia liase (PAL) e a cinamato 4-hidroxilase (C4H). Observou-se também atividade de uma enzima que catalisa a conversão do ácido ferúlico ao ácido 3,4-metilenodioxicinâmico, denominada 3,4- metilenodioxicinâmico sintase. Esta é provavelmente uma enzima monooxigenase-dependente do citocromo P450. A principal questão biossintética está centrada na sequência na qual as reações ocorrem: o grupo metilenodioxílico pode ser formado antes ou depois da porção amídica. Para elucidar esta questão, sintetizou-se dois possíveis precursores contendo o grupo 4-hidróxi-3- metóxifenílico: o ácido ferupérico e a feruperina. No entanto, experimentos de atividade enzimática in vitro utilizando estes dois precursores não foram esclarecedores, devido a existência de piperina endógena no extrato enzimático proveniente de folhas de P. nigrum, o que impediu observar a formação de produto. Foi avaliado também a participação de ácido malônico e L-valina na biossintese destas amidas: incorporou-se precursores isotopicamente marcados em plântulas de P. tuberculatum: ácido 2-[13C]-malônico, ácido malônico-U-2H e 15N-L-valina). Após análise por CLAEEM, conclui-se que o ácido malônico participa da reação de condensação com fenilpropanóides, gerando os derivados C6-C5 e L-valina é precursor do grupo isobutilamina. Considerando todos as evidências... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Three Piper species were chosen as a models for biosynthetic, transcriptomic and proteomics studies: P. tuberculatum, P. nigrum and P. crassinervium. The first two species accumulate mainly amides as secondary metabolites. This class of compounds was targeted by biosynthetic experiments. It was monitored the enzymatic activities of several enzymes involved in the phenylpropanoid pathway, including phenylalanine ammonia-lyase and cinnamate 4-hydroxylase. It was also observed the activity of an enzyme that catalyzes the conversion of ferulic acid to 3,4- methylenedioxycinnamic acid, named 3,4 methylenedioxycinnamic acid synthase. The main question was centered on the biosynthetic sequence: whether the methylenedioxy group can be formed before or after the amide portion. In order to elucidate this question, two possible precursors containing the group 4-hydroxy-3-methoxyphenyl were synthesized: feruperic acid and feruperine. However, experiments of in vitro enzymatic activity using these two precursors were not enlightening, due to the existence of piperine as endogenous in the enzymatic extract from leaves of P. nigrum, which prevented observe the formation of product. It was also evaluate the participation of malonic acid and L-valine in the biosynthesis of these amides, by incorporating isotopically labeled precursors in plants of P. tuberculatum: 2-[13C]-malonic acid, malonic-U-2H acid and 15N-Lvaline. After analysis by HPLC-MS, it was concluded that the malonic acid participates in the condensation reaction with phenylpropanoids, generating the C6-C5 derivatives and L-valine is the putative precursor of the isobutylamine group. Considering all the experimental evidence, the biosynthetic pathway proposed was that in which the methylenedioxy group is formed before the amide. In the second step, it carried out the ... (Complete abstract click electronic access below) / Doutor

Sequenced diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum belong to the chromist algae harboring secondary plastids, which display distinct evolutionary history when compared to photosynthetic organelles from rhodophytes, green algae and plants. Via secondary endosymbiosis, heterotrophic eukaryotic ancestor of diatoms engulfed red alga, and in addition to the new organelle, it obtained fitness increasing peculiarities in the chimerical cell metabolism and lifestyle. We examined phylogeny and in silico localization of the nuclear-encoded but plastid located enzymes of tryptophan biosynthesis. We suggest that the diatom tryptophan pathway represents an extreme in the trend of plastid (cyanobacterial) enzymes to be replaced by eukaryotic isoforms. In addition, the gene napped during the endosymbiotic gene transfer from the diatom plastid genome to the diatom nucleus (psb28) was described.