Light controlled synthesis of nucleic acids

Dissertação apresentada para obtenção do grau de Doutor em Bioquímica - especialidade Biotecnologia, pela Universidade Nova de Lisboa,Faculdade de Ciências e Tecnologia

The transport of carboxylic acids and important role of the Jen1p transporter during the development of yeast colonies

On solid substrates, yeast colonies pass through distinct developmental phases characterized by the changes in pH of their surroundings from acidic to nearly alkaline and vice versa. At the beginning of the alkali phase colonies start to produce ammonia, which functions as a quorum-sensing molecule inducing the reprogramming of cell metabolism. Such reprogramming includes, among others, the activation of several plasma membrane transporters and is connected with colony differentiation. In the present study, we show that colony cells can use two transport mechanisms to import lactic acid: a ‘saturable’ component of the transport, which requires the presence of a functional Jen1p transporter, and a ‘non-saturable’ component (diffusion) that is independent of Jen1p. During colony development, the efficiency of both transport components changes similarly in central and outer colonial cells. Although the lactate uptake capacity of central cells gradually decreases during colony development, the lactate uptake capacity of outer cells peaks during the alkali phase and is also kept relatively high in the second acidic phase. This lactate uptake profile correlates with the localization of the Jen1p transporter to the plasma membrane of colony cells. Both lactic acid uptake mechanisms are diminished in sok2 colonies where JEN1 expression is decreased. The Sok2p transcription factor may therefore be involved in the regulation of non-saturable lactic acid uptake in yeast colonies.

Role of the DHH1 Gene in the Regulation of Monocarboxylic Acids Transporters Expression in Saccharomyces cerevisiae

Previous experiments revealed that DHH1, a RNA helicase involved in the regulation of mRNA stability and translation, complemented the phenotype of a Saccharomyces cerevisiae mutant affected in the expression of genes coding for monocarboxylic-acids transporters, JEN1 and ADY2 (Paiva S, Althoff S, Casal M, Leao C. FEMS Microbiol Lett, 1999, 170∶301–306). In wild type cells, JEN1 expression had been shown to be undetectable in the presence of glucose or formic acid, and induced in the presence of lactate. In this work, we show that JEN1 mRNA accumulates in a dhh1 mutant, when formic acid was used as sole carbon source. Dhh1 interacts with the decapping activator Dcp1 and with the deadenylase complex. This led to the hypothesis that JEN1 expression is post-transcriptionally regulated by Dhh1 in formic acid. Analyses of JEN1 mRNAs decay in wild-type and dhh1 mutant strains confirmed this hypothesis. In these conditions, the stabilized JEN1 mRNA was associated to polysomes but no Jen1 protein could be detected, either by measurable lactate carrier activity, Jen1-GFP fluorescence detection or western blots. These results revealed the complexity of the expression regulation of JEN1 in S. cerevisiae and evidenced the importance of DHH1 in this process. Additionally, microarray analyses of dhh1 mutant indicated that Dhh1 plays a large role in metabolic adaptation, suggesting that carbon source changes triggers a complex interplay between transcriptional and post-transcriptional effects.

Discovery of Ascomycete characteristics in Sporothrix schenckii

Sporothrix schenckii has been studied by light microscopy, and also by transmission and scanning electron microscopy. Characteristics of Ascomycetes have been oibserved at the level of the cell-wall and in the synaptic system of the hyphae. Also the perfect state has been discovered. The four spored asei are formed directly from the mycelium and there is no fructification. Dolichoascus schenckii is the name suggested for this perfect state which constitutes a new genus of the Endomycetaceae.

Bioactivity of phenolic acids: Metabolites versus parent compounds: A review

Phenolic acids are present in our diet in different foods. In particular, mushrooms are a good source of these molecules. Due to their bioactive properties, phenolic acids are extensively studied and there is evidence of their role in disease prevention. Nevertheless, in vivo, these compounds are metabolized and circulate in the organism as glucuronated, sulfated and methylated metabolites, displaying higher or lower bioactivity. To clarify the importance of the metabolism of phenolic acids, the knowledge about the bioactivity of the metabolites is extremely important. In this review, chemical features, biosynthesis and bioavailability of phenolic acids are discussed as well as the chemical and enzymatic synthesis of their metabolites. Finally, the metabolites bioactive properties are compared with that of the corresponding parental compounds.

The contribution of phenolic acids to the anti-inflammatory activity of mushrooms: Screening in phenolic extracts, individual parent molecules and synthesized glucuronated and methylated derivatives

In the present study, the ethanolic extracts of fourteen edible mushrooms were investigated for their anti-inflammatory potential in LPS (lipopolysaccharide) activated RAW 264.7 macrophages. Furthermore the extracts were chemically characterized in terms of phenolic acids and related compounds. The identified molecules (p-hydroxybenzoic, p-coumaric and cinnamic acids) and their glucuronated and methylated derivatives obtained by chemical synthesis were also evaluated for the same bioactivity, in order to establish structure-activity relationships and to comprehend the effects of in vivo metabolism reactions in the activity of the compounds. The extracts of Pleurotus ostreatus, Macrolepiota procera, Boletus impolitus and Agaricus bisporus revealed the strongest anti-inflammatory potential (EC50 values 96 ± 1 to 190 ± 6 µg/mL, and also the highest concentration of cinnamic acid (656 to 156 µg/g), which was also the individual compound with the highest anti-inflammatory activity. The derivatives of p-coumaric acid revealed the strongest properties, specially the derivative methylated in the carboxylic group (CoA-M1) that exhibited similar activity to the one showed by dexamethaxone used as anti-inflammatory standard; by contrast, the derivatives of p-hydroxybenzoic revealed the lowest inhibition of NO production. All in all, whereas the conjugation reactions change the chemical structure of phenolic acids and may increase or decrease their activity, the glucuronated and methylated derivatives of the studied compounds are still displaying anti-inflammatory activity.

Synthesis, antiangiogenesis evaluation and molecular docking studies of 1-Aryl-3-[(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas: Discovery of a new substitution pattern for type II VEGFR-2 Tyr kinase inhibitors

The synthesis and biological evaluation of novel 1-aryl-3-[2-, 3- or 4-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 3, 4 and 5 as VEGFR-2 tyrosine kinase inhibitors, are reported. The 1-aryl-3-[3-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 4a-4h, with the arylurea in the meta position to the thioether, showed the lowest IC50 values in enzymatic assays (10-206 nM), the most potent compounds 4d-4h (IC50 10-28 nM) bearing hydrophobic groups (Me, F, CF3 and Cl) in the terminal phenyl ring. A convincing rationalization was achieved for the highest potent compounds 4 as type II VEGFR-2 inhibitors, based on the simultaneous presence of: (1) the thioether linker and (2) the arylurea moiety in the meta position. For compounds 4, significant inhibition of Human Umbilical Vein Endothelial Cells (HUVECs) proliferation (BrdU assay), migration (wound-healing assay) and tube formation were observed at low concentrations. These compounds have also shown to increase apoptosis using the TUNEL assay. Immunostaining for total and phosphorylated (active) VEGFR-2 was performed by Western blotting. The phosphorylation of the receptor was significantly inhibited at 1.0 and 2.5 microM for the most promising compounds. Altogether, these findings point to an antiangiogenic effect in HUVECs.

Mass transfer effects on reactive separation processes - discovery of reactive arrheotropes

Membrane reactor, reactive membrane separation, arrheotrope, azeotrope, dusty gas model, esterification, residue curve map, distillation, kinetics, singular point, bifurcation

Effects of deoxycholic acids and urodeoxycholic acids on cancerogenic progression in Barrett`s Esophagus

Magdeburg, Univ., Med. Fak., Diss., 2014

Effects of deoxycholic acids and urodeoxycholic acids on cancerogenic progression in Barrett`s Esophagus

Magdeburg, Univ., Med. Fak., Diss., 2014