Academic literature on the topic 'Hydroxycinnamate'

Bornyl cis-4-hydroxycinnamate, a bioactive compound isolated from Piper betle stems, has the potential for use as an anti-cancer agent. This study investigated the effects of bornyl cis-4-hydroxycinnamate on cell migration and invasion in melanoma cells. Cell migration and invasion were compared in A2058 and A375 melanoma cell lines treated with/without bornyl cis-4-hydroxycinnamate (1–6 µM). To examine whether bornyl cis-4-hydroxycinnamate has a potential anti-metastatic effect on melanoma cells, cell migration and invasion assays were performed using a Boyden chamber assay and a transwell chamber in A2058 and A375 cells. Gelatin zymography was employed to determine the enzyme activities of MMP-2 and MMP-9. Cell lysates were collected for Western blotting analysis of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitors of metalloproteinase-1/2 (TIMP-1/2), as well as key molecules in the mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK)/ phosphatidylinositide-3 kinases (PI3K)/Akt/ mammalian target of rapamycin (mTOR), growth factor receptor-bound protein 2 (GRB2) signaling pathways. Our results demonstrated that bornyl cis-4-hydroxycinnamate is a potentially useful agent that inhibits melanoma cell migration and invasion, and altered melanoma cell metastasis by reducing MMP-2 and MMP-9 expression through inhibition of the FAK/PI3K/Akt/mTOR, MAPK, and GRB2 signaling pathways. Moreover, bornyl cis-4-hydroxycinnamate inhibited the process of the epithelial-to-mesenchymal transition in A2058 and A375 melanoma cells. These findings suggested that bornyl cis-4-hydroxycinnamate has potential as a chemotherapeutic agent, and warrants further investigation for its use in the management of human melanoma.

A “novel” protocol is presented for easy and reliable estimation of soluble hydroxycinnamate levels inCichorium intybusL. leaf tissue in large-scale experiments. Samples were standardized by punching 6 discs per leaf, and hydroxycinnamates were extracted by submerging the discs in 80% ethanol with 5% acetic acid for at least 48 h in the darkness at 4°C. Residual dry mass of the discs was used fora posterioricorrection of compound levels. Chlorophyll was eliminated by chloroform, and the aqueous phases were transferred to microplates, dried, and dissolved in 50% methanol for HPLC analysis and storage. An HPLC program of 8 min was developed for the analysis of the extracts. Comparisons with extractions of liquid nitrogen powders indicated that the novel extraction method was reliable. No degradation of the major hydroxycinnamates—caftaric, chlorogenic, and chicoric acids—was observed, during maceration at ambient temperatures, or after storage for 1 year.

Corn borers are the most important pest affecting maize. Resistance to corn borer attack may compromise plant fitness being detrimental for some important agronomic traits such as yield. Against the attack of this pest, cell wall-bound hydroxycinnamates have been previously described as a possible defense mechanism. In this study, agronomic characterization and cell wall-bound hydroxycinnamates quantification was performed in a subset of Recombinant Inbred Lines (RILs) from a Multiparent Advanced Generation Intercross (MAGIC) population that showed contrasting behavior against corn borer attack. Resistant lines showed greater concentration of p-coumaric acid, the only hydroxycinnamate that could have a role in the resistance in these particular materials. In addition, results indicated that resistant lines showed precocity, low grain moisture at harvest, and reduced plant height, thus, selecting for resistance may be detrimental for yield. In this way, a breeding strategy directly targeting grain yield in order to tolerate corn borer attack would be the recommended one.

Plant phenolic compounds have shown the ability to cooperate with one another at low doses in producing enhanced anticancer effects. This may overcome the limitations (e.g., poor bioavailability and high-dose toxicity) in developing these agents as cancer medicines. We have previously reported that the hydroxycinnamic acid derivative (HCAD) methyl-4-hydroxycinnamate and the phenolic diterpene carnosic acid (CA) can synergistically induce massive calcium-dependent apoptosis in acute myeloid leukemia (AML) at non-cytotoxic concentrations of each agent. Here, we explored the chemical nature of the synergy between HCADs and either CA, in inducing cytotoxicity, or the active metabolite of vitamin D (calcitriol), in enhancing the differentiation of AML cells. This was done by determining the structure–activity relationship of a series of hydroxycinnamic acids and their derivatives (methyl hydroxycinnamates and hydroxybenzylideneacetones) in combination with CA or calcitriol. The HCAD/CA synergy required the following critical structural elements of an HCAD molecule: (a) the para-hydroxyl on the phenolic ring, (b) the carbon C7–C8 double bond, and (c) the methyl-esterified carboxyl. Thus, the only HCADs capable of synergizing with CA were found to be methyl-4-hydroxycinnamate and methyl ferulate, which also most potently enhanced calcitriol-induced cell differentiation. Notably, the C7–C8 double bond was the major requirement for this HCAD/calcitriol cooperation. Our findings may contribute to the rational design of novel synergistically acting AML drugs based on prototype combinations of HCADs with other agents studied here.

A six-coordinate manganese (II) complex with the tripod ligand tris(2-benzimidazolylmethyl) amine (ntb), with composition [Mn(ntb) (4-hydroxycinnamate)](4-hydroxycinnamate) · (DMF)0.5 · (H2O)3, was synthesized and characterized by elemental and thermal analyses, electrical conductivity, IR, and UV/vis spectral measurements. The crystal structure of the complex has been determined by the single-crystal X-ray diffraction. The Mn (II) cation is bonded to an ntb ligand and a 4-hydroxycinnamate ligand through four N atoms and two O atoms, giving a distorted octahedral coordination geometry. Cyclic voltammograms of the complex indicate a quasi-reversible Mn3+/Mn2+ couple. The X-band EPR spectrum of the complex exhibits a six-line manganese hyperfine splitting pattern with g = 2, A = 95, and confirms that the material is high-spin Mn(II).

ABSTRACT Hydroxycinnamates, aromatic compounds that play diverse roles in plants, are dissimilated by enzymes encoded by the hca genes in the nutritionally versatile, naturally transformable bacterium Acinetobacter sp. strain ADP1. A key step in the hca-encoded pathway is activation of the natural substrates caffeate, p-coumarate, and ferulate by an acyl:coenzyme A (acyl:CoA) ligase encoded by hcaC. As described in this paper, Acinetobacter cells with a knockout of the next enzyme in the pathway, hydroxycinnamoyl-CoA hydratase/lyase (HcaA), are extremely sensitive to the presence of the three natural hydroxycinnamate substrates; Escherichia coli cells carrying a subclone with the hcaC gene are hydroxycinnamate sensitive as well. When the hcaA mutation was combined with a mutation in the repressor HcaR, exposure of the doubly mutated Acinetobacter cells to caffeate, p-coumarate, or ferulate at 10−6 M totally inhibited the growth of cells. The toxicity of p-coumarate and ferulate to a ΔhcaA strain was found to be a bacteriostatic effect. Although not toxic to wild-type cells initially, the diphenolic caffeate was itself converted to a toxin over time in the absence of cells; the converted toxin was bactericidal. In an Acinetobacter strain blocked in hcaA, a secondary mutation in the ligase (HcaC) suppresses the toxic effect. Analysis of suppression due to the mutation of hcaC led to the development of a positive-selection strategy that targets mutations blocking HcaC. An hcaC mutation from one isolate was characterized and was found to result in the substitution of an amino acid that is conserved in a functionally characterized homolog of HcaC.

Cerium (III) chloride, sodium 4-hydroxycinnamate, and cerium 4-hydroxycinnamate have been successfully characterized as effective anodic, cathodic, and mixed inhibitors for steel in ethanol fuel blend by electrochemical and surface analyses. Electrochemical and surface analyses indicate that good inhibition performances are due to the formation of protective layer as a result of adsorption between the metal and inhibitor components. In addition, the inhibition mechanism of each inhibitor is also suggested and discussed in detail.

1. alpha-Cyano-4-hydroxycinnamate was coupled to Sepharose CL-4B activated with 1,2:3,4-bisepoxybutane. 2. The low-Km rat liver mitochondrial aldehyde dehydrogenase was specifically bound to this affinity medium, and could subsequently be eluted with alpha-cyano-4-hydroxycinnamate. 3. The enzyme purified in this manner had a subunit molecular mass of 55 kDa and a pI of approx. 6.5. A minor component of approx. 57 kDa was also present and had a significantly higher pI value; this may be the precursor for aldehyde dehydrogenase. 4. alpha-Cyanocinnamate and some related compounds were found to be uncompetitive inhibitors of the enzyme. 5. No cytosolic aldehyde dehydrogenase was bound to the affinity column, but a protein from a rat liver post-mitochondrial supernatant with a molecular mass of approx. 25 kDa was bound, and could be eluted subsequently with alpha-cyano-4-hydroxycinnamate.

Precise tuning of gene expression by transcriptional regulators determines the response to internal and external chemical signals and adjusts the metabolic machinery for many cellular processes. As a part of ongoing efforts by the Midwest Center for Structural Genomics, a number of transcription factors were selected to study protein-ligand and protein-DNA interactions. HcaR, a new member of the MarR/SlyA family of transcription regulators from soil bacteria Acinetobacter sp. ADP1, is an evolutionarily atypical regulator and represses hydroxycinnamate (hca) catabolic genes. Hydroxycinnamates containing an aromatic ring play diverse, critical roles in plant architecture and defense. HcaR regulates the expression of the hca catabolic operon, allowing this and related bacterial strains to utilize hydroxycinnamates: ferulate, p-coumarate, and caffeate as sole sources of carbon and energy. HcaR appears to be capable of responding to multiple aromatic ligands. These aromatic compounds bind to HcaR and reduce its affinity to the specific DNA sites. As a result, the transcription of genes encoding several catabolic enzymes is up-regulated. The HcaR structures of the apo-form and in a complex with several ligands: ferulic acid, 3,4 dihydroxybenzoic acid, vanillin and p-coumaric acid have been determined to understand how HcaR accommodates various aromatic compounds using the same binding pocket. We also have identified a potential DNA site for HcaR in the regulatory region upstream of the genes of the hca catabolic operon in Acinetobacter sp. ADP1 and have confirmed DNA binding by EMSA. The co-crystal structure of HcaR and palindromic 24-mer DNA has been determined for this DNA site. The crystal structures of HcaR, the apo-form, ligand-bound forms, and the specific DNA-bound form provide critical structural basis of protein-ligand (substrates or product) and protein-DNA interactions to understand the regulation of the expression of hydroxycinnamate (hca) catabolic genes. Our studies allow for better understanding of DNA-binding and regulation by this important group of transcription factors belonging to the MarR/SlyA families. This work was supported by National Institutes of Health grant GM094585 and by the U. S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.

The first study was looking into the phytochemical analysis of herbal medicinal plant from Malaysia. Phenolic compounds in an aqueous infusion from leaves of Ficus deltoidea (Moraceae), a well-known herbal tea in Malaysia, were analysed by HPLC coupled to photodiode-array and fluorescence detectors and an electrospray ionization tandem mass spectrometer. Following chromatography of extracts on a reverse phase C12 column 25 flavonoids were characterized and/or tentatively identified, with the main constituents being flavan-3-ol monomers, proanthocyanidins and C-linked flavone glycosides. The proanthocyanidins were dimers and trimers comprising (epi)catechin and (epi)afzelechin units. No higher molecular weight proanthocyanidin polymers were detected. The antioxidant activity of F. deltoidea extract was analysed using HPLC with on-line antioxidant detection. This revealed that 85% of the total antioxidant activity of the aqueous F. deltoidea infusion was attributable to the flavan-3-ol monomers and the proanthocyanidins In terms of the bioavailability of hydroxycinnamates with the high occurrence of p-coumaroylquinic acid in F. deltoidea extract, further study was carried out using radiolabeled caffeic acid. In the study, male Sprague-Dawley rats ingested 140 x 106 dpm of [3-14C]trans-caffeic acid and over the ensuing 72 h period body tissues, plasma, urine and faeces were collected and the overall levels of radioactivity determined. Where sufficient radioactivity had accumulated samples were analysed by HPLC with on-line radioactivity and tandem mass spectrometric detection. Nine labeled compounds were identified, the substrate and its cis isomer, 3¢-O- and 4¢-O-sulphates and glucuronides of caffeic acid, 4¢-O-sulphates and glucuronides of ferulic acid and isoferulic acid-4¢-O-sulphate. Four unidentified metabolites were also detected. After passing down the gastrointestinal tract the majority of the radiolabeled metabolites were excreted in urine with minimal accumulation in plasma. Only relatively small amounts of unidentified 14C-labeled metabolites were expelled in faeces. There was little or no accumulation of radioactivity in body tissues, including the brain. The overall recovery of radioactivity 72 h after ingestion of [3-14C]caffeic acid was ~80% of intake. The role of colonic microflora in the metabolism of caffeic acid was carried using an in vitro model of the human colonic microflora. Caffeic acid (55 µmoles) was incubated with human faecal materials obtained from five Asian donors, and caffeic acid degradation was monitored from 0-8 h. Faecal samples were analysed by GC-MS, where major phenolic acids identified were dihydrocaffeic acid, 3-(3¢-hydroxyphenyl)propionic acid and phenylacetic acid. Caffeic acid was quickly degraded by the colonic microflora, as it disappeared after 4 h of incubation in two of the faecal samples. The degree of degradation of caffeic acid was significantly influenced by the addition of glucose as well as individual variations in the density and the composition of microflora. These findings support extensive metabolism of caffeic acid in the colon, depending on the substrate concentration and the supplement of glucose which resulting the formation of simple phenolics.

Heritage apple cultivars for cider-making are often distinguished by a high concentration of tannins (phenolic compounds), and/or acid. The phenolic content of some cider apples far exceeds that of white wine, however most cider fermentation practices are directly taken from white winemaking, not accounting for effects of high concentrations of phenolic compounds on yeast fermentation. The objective of this study was to determine the impact of ferulic acid, p-coumaric acid, and chlorogenic acid—at concentrations reported in apples—and their interactions with yeast assimilable nitrogen (YAN) on fermentation kinetics and cider aroma. Our hypothesis was that the phenolic compounds present in high-tannin cider apples would negatively impact fermentation kinetics, but not alter the aroma, and that added YAN would reduce these effects. Ferulic acid negatively affected fermentation performance (p < 0.05), but p-coumaric acid and chlorogenic acid did not. p-Coumaric acid led to the greatest changes in cider aroma. Differences were also detected for different concentrations of ferulic acid. Chlorogenic acid did not affect aroma. Yeast strain influenced fermentation performance and cider aroma. Finally, addition of exogenous YAN improved fermentation performance for the low concentration ferulic acid condition, but not for the high concentration. Adding YAN also changed cider aroma in the presence of p-coumaric acid.
Master of Science in Life Sciences

Chez les plantes, la subérine est un biopolymère constitué de composés aliphatiques etaromatiques déposés au niveau de la paroi des cellules de plusieurs tissus comme l’endodermeet le périderme des racines ou encore le manteau des graines. La subérine forme une barrièrehydrophobe permettant entre autres de contrôler les flux d’eau et de solutés, et de protéger laplante de stress environnementaux comme la sécheresse ou les pathogènes. Grâce à desanalyses en LC-MS/MS et en GC-MS, nous avons pu montrer que la majorité des alcools grasprésents dans la fraction soluble de racines d’Arabidopsis thaliana est sous forme d’alkylcaféates et d’alkyl coumarates. De plus, nous avons montré que ces cires associées aupolymère de subérine sont présentes dès les premiers stades du développement de la racine.Une étude de la distribution des chaînes acyles des racines nous a permis de mettre enévidence la contribution majeure des alcools gras dans la composition de la subérine ainsi quel’importance de la subérine dans le métabolisme lipidique des racines. Afin d’identifier desacteurs impliqués dans l’export des précurseurs de la subérine vers l’espace extracellulaire,nous avons mené une approche de génétique inverse en utilisant des lignées mutées pour desgènes codant notamment pour des ABCG transporteurs co-exprimés avec des gènes connuspour participer à la biosynthèse de la subérine. Les résultats de ces analyses ont confirmé quele processus d’export fait intervenir plusieurs protéines pouvant avoir des fonctionsredondantes et ont suggéré l’implication d’un nouveau transporteur dans l’export desprécurseurs de subérine
In plants, suberin is a complex biopolymer made of aliphatic and aromatic compounds.It is deposited in the cell wall of tissues such as the endoderm and the periderm of roots or thecoat of the seeds. Suberin forms a hydrophobic barrier controlling the flow of water andsolutes, and protecting the plant from environmental stresses such as drought or pathogens.Through LC-MS/MS and GC-MS analyses, we have shown that the majority of the fattyalcohols present in the soluble fraction of Arabidopsis roots is in the form of alkyl caffeatesand alkyl coumarates. Such waxes, most probably associated with the suberin polymer, arealready detected at early stages of root development. A study of the distribution of all acylchains present in roots allowed us to highlight the major contribution of fatty alcohols in thecomposition of suberin and the importance of suberin in the global lipid metabolism of theroots. To identify proteins involved in the export of suberin precursors to the extracellularspace, we conducted a reverse genetic approach using lines mutated in genes coding forseveral ABCG transporters which were co-expressed with genes known to participate in thebiosynthesis of suberin. The results of these analyses confirmed that the export processinvolves several proteins that can have redundant functions, and supported the involvement ofa new transporter in the export of the suberin precursors

The potential for malodour in wine caused by the accumulation of ethylphenols has been widely studied with respect to the breakdown of the hydroxycinnamic acids, p-coumaric and ferulic acid, by D. bruxellensis. The presence of esterified hydroxycinnamate conjugates in grapes and wine is well established and they account for a large proportion of the hydroxycinnamate content. There exists the possibility that these conjugates could also provide the potential for spoilage, though they have never been linked to the direct formation of ethylphenols. The research highlighted within this thesis examines the potential role of a number of esterified conjugates in the production of ethylphenols by D. bruxellensis. Two classes of berry derived esters, the tartaric acid and glucose bound hydroxycinnamates, as well as the vinification formed ethyl esters, were synthesised and used for model fermentation experiments. Chapter 2 describes the preparation of a number of protected hydroxycinnamic acid derivatives that were used in the synthesis of the hydroxycinnamoyl tartrate esters (7 and 8) for the first time. Coupling 1-O-chloroacetyl protected p-coumaric and ferulic acids (21 and 22) with di-tert-butyl-L-tartrate (34) followed by selective hydrolysis of the tert-butyl esters yielded p-coumaroyl tartrate (7) and feruloyl tartrate (8). Hydroxycinnamoyl glucose esters (9 and 10) were prepared using the same hydroxycinnamates (21 and 22), esterifying with a prepared trichloroacetimidate glucosyl donor sequence, though purification of the glucose esters resulted in undesired chemical transformations. It was found that photoisomerisation of the glucose esters could be prevented via synthesis under red light, which gave trans-9 and 10, however migration of the hydroxycinnamoyl moiety around the glucose ring, which yielded mainly the 2-O-α- and 6-O-α-esters, was a product of submitting the esters to non-aqueous solvents and could not be avoided. The acyl migration of the glucose esters that was observed in Chapter 2 has been researched at a DFT B3LYP 6-31G* theoretical level in Chapter 3 with respect to both the thermodynamics and kinetics of the transformations. The desired 1-O-β-esters were thermodynamically favoured only in water, while in any other solvent studied the 2-O-α- and 6-O-α-esters would prevail. Kinetically, migration to the 3-O-position involved lower energy barriers which can be equated to a more rapid process, although the ring-flipped conformation needed to achieve the migration would promote subsequent migration to the 6-O-position. Step-wise migration, from the 1-O- to the 2-O-position, was found to be thermodynamically less favoured than other migrations investigated. This effect can be rationalised by the formation of a 5-membered cyclic intermediate in comparison to the 6- membered intermediate produced during 1-O- to 3-O-migration. However, the energy barriers involved in 1-O-β- to 2-O-β-migration better explain the comparative extent of migration observed between the p-coumaroyl and feruloyl glucose esters. The possibility of multiple glucose esters existing in wine was the focus of a brief study, finding two separate p-coumaroyl glucose esters in red and white wine, while a lesser extent of migration in feruloyl glucose limited observation to concentrated wine alone. However, due to co-elution of feruloyl glucose (10) with suspected p-coumaroyl anthocyanin derivatives in red wine, HPLC-MRM was required to detect it, which is the first report of this compound in red wine. Theoretical studies into observed photoisomerisations and the synthesis of cis-hydroxycinnamates are described in Chapter 4. The cis-ethyl hydroxycinnamates were isolated and hydrolysed to give a mixture of cis/trans-hydroxycinnamic acids (3 and 4), which could be separated by flash chromatography, though the pure cis-isomers isomerised rapidly under ambient conditions and slowly under red light back to the trans-isomers. Stable isomeric mixtures were achieved by irradiation with ultra-violet light giving mixtures of 40-50% of the cis-isomer which could be used further in fermentation studies. Computational evidence suggested that isomerisation of the hydroxycinnamic acids was favoured with greater resonance throughout the molecule. Those with deprotonated phenolic moieties possessed the most intramolecular electron movement, decreasing the HOMO-LUMO gap and promoting photoisomerisation. Smaller solvent and substrate effects were also noted, though the nature of the phenol and carboxyl clearly played the most important role in determining stability of each isomer. Fermentation in the presence of the synthesised trans-hydroxycinnamoyl esters (7-12) and investigation into the stereospecificity of D. bruxellensis enzyme activities was performed as detailed in Chapter 5. In Australia, three genetic groups of D. bruxellensis account for 98% of isolates, with the largest of these groups making up 85%. AWRI 1499 is a representative of the largest genetic group, with AWRI 1608 and AWRI 1613 belonging to the two remaining significant genetic groups. In the presence of AWRI 1499, the transethyl- esters (11 and 12) were metabolised to varying extents with the preference for breakdown of ethyl coumarate (11) over ethyl ferulate (12). This selectivity was investigated further and found to be common for both AWRI 1499 and AWRI 1608, while AWRI 1613 was unable to breakdown either ester. The preference for formation of 4- ethylphenol (1) over 4-ethylguaiacol (2) from the ethyl esters could accentuate the ratio of these compounds as seen in wine, initially thought to be brought about by the relative concentration of the precursor acids. Of the berry derived esters, the tartrate esters (7 and 8) were not metabolised by AWRI 1499, and subsequent fermentations with AWRI 1608 and 1613 yielded the same result. This confirmed that the tartrate esters cannot contribute directly to the formation of ethylphenols during exposure to D. bruxellensis. The glucose esters were metabolised by AWRI 1499 to a moderate extent (35% conversion), providing information that these can contribute to the accumulation of ethylphenols during barrel ageing. Furthermore, the isomerisation of the glucose esters lead to studies into the stereoselectivity of D. bruxellensis enzyme activities, whereby the decarboxylase as well as the ethyl esterase showed selectivity for the trans-isomers and that the cis-hydroxycinnamate content of grapes and wine are not important in the accumulation of ethylphenols. The experimental procedures employed throughout Chapters 2-5 are outlined in Chapter 6.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2012

Diabetes is a worldwide health issue that has been expanding mainly in developed countries. It is characterized by abnormal levels of blood sugar due to several factors. The most common are resistance to insulin and the production of defective insulin which exerts little or no effect. Its most common symptoms include tissue damage to several systems due to elevated levels of blood sugar. One of the key enzymes in hydrocarbon metabolism is α-glucosidase (EC 3.2.1.20). It catalyzes the breakdown of complex carbohydrates into their respective monomers (glucose) which allows them to be absorbed. In this work, caffeoyl quinic acids and their metabolites were analyzed as potential inhibitors for α-glucosidase. The search for the best inhibitor was conducted using molecular docking. The affinity of each compound was compared to the inhibitor present in the crystal structure of the protein. As no inhibitor with a similar affinity was´found, a new approach was used, in situ drug design. It was not possible to achieve an inhibitor capable of competing with the one present in the crystal structure of the enzyme, which is also its current commercial inhibitor. It is possible to draw some conclusions as to which functional groups interact best with certain residues of the active site. This work was divided into three main sections. The first section, Diabetes, serves as an introduction to what is Diabetes, its symptoms and/or side effects and how caffeoyl quinic acids could be used as a treatment. The second section, Caffeoylquinic acids and their metabolites as inhibitors for Alfa-glucosidase, corresponds to the search through molecular docking of caffeoyl quinic acids as inhibitors for α-glucosidase and what was possible to draw from this search. The last section, In situ design of an inhibitor for α-glucosidase (EC 3.2.1.20), corresponds to the in situ drug design study and what it achieved. The representation of each of the molecules used as a ligand can be found in the Annexes.
Universidade da Madeira