Virgin Olive Oil: Losses of Antioxidant Polar Phenolic Compounds due to Storage, Packaging, and Culinary Uses

Virgin olive oil is a high quality natural product obtained only by physical means. In addition to triacylglycerols it contains nutritionally important polar and non-polar antioxidant phenols and other bioactive ingredients. The polar fraction is a complex mixture of phenolic acids, simple phenols, derivatives of the glycosides oleuropein and ligstroside, lignans, and flavonoids. These compounds contribute significantly to the stability, flavor, and biological value of virgin olive. In the various stages of production, during storage and in the culinary uses, polar phenols and other valuable bioactive ingredients may be damaged. Oxidation, photo-oxidation, enzymic hydrolysis and heating at frying temperatures have a serious adverse effect. Due to the biological importance of the oil and its unique character, analytical methods have been developed to evaluate antioxidant activity or analyse complex phenol mixtures. These are based on radical scavenging assays and chromatographic techniques. Hyphenated methods are also used including liquid chromatography-mass spectrometry and liquid chromatography-nuclear magnetic resonance spectroscopy.

Toluene Dioxygenase Catalysed synthesis and reactions of cis-diol metabolites derived from 2 and 3-methoxy phenols

Using toluene dioxygenase as biocatalyst, enantiopure cisdihydrodiol and cis-tetrahydrodiol metabolites, isolated as their ketone tautomers, were obtained from meta and ortho methoxyphenols. Although these isomeric phenol substrates are structurally similar, the major bioproducts from each of these biotransformations were found at different oxidation levels. The relatively stable cyclohexenone cis-diol metabolite from meta methoxyphenol was isolated, while the corresponding metabolite from ortho methoxyphenol was rapidly bioreduced to a cyclohexanone cis-diol. The chemistry of the 3-methoxycyclohexenone cis-diol product was investigated and elimination, aromatization, hydrogenation, regioselective O-exchange, Stork−Danheiser transposition and O-methylation reactions were observed. An offshoot of this technology provided a two-step chemoenzymatic synthesis, from meta methoxyphenol, of a recently reported chiral fungal metabolite; this synthesis also established the previously unassigned absolute configuration.

Concomitant osmotic and chaotropicity-induced stresses in Aspergillus wentii: compatible solutes determine the biotic window

Whereas osmotic stress response induced by solutes has been well-characterized in fungi, less is known about the other activities of environmentally ubiquitous substances. The latest methodologies to define, identify and quantify chaotropicity, i.e. substance-induced destabilization of macromolecular systems, now enable new insights into microbial stress biology (Cray et al. in Curr Opin Biotechnol 33:228–259, 2015a, doi:10.​1016/​j.​copbio.​2015.​02.​010; Ball and Hallsworth in Phys Chem Chem Phys 17:8297–8305, 2015, doi:10.​1039/​C4CP04564E; Cray et al. in Environ Microbiol 15:287–296, 2013a, doi:10.​1111/​1462-2920.​12018). We used Aspergillus wentii, a paradigm for extreme solute-tolerant fungal xerophiles, alongside yeast cell and enzyme models (Saccharomyces cerevisiae and glucose-6-phosphate dehydrogenase) and an agar-gelation assay, to determine growth-rate inhibition, intracellular compatible solutes, cell turgor, inhibition of enzyme activity, substrate water activity, and stressor chaotropicity for 12 chemically diverse solutes. These stressors were found to be: (i) osmotically active (and typically macromolecule-stabilizing kosmotropes), including NaCl and sorbitol; (ii) weakly to moderately chaotropic and non-osmotic, these were ethanol, urea, ethylene glycol; (iii) highly chaotropic and osmotically active, i.e. NH4NO3, MgCl2, guanidine hydrochloride, and CaCl2; or (iv) inhibitory due primarily to low water activity, i.e. glycerol. At ≤0.974 water activity, Aspergillus cultured on osmotically active stressors accumulated low-M r polyols to ≥100 mg g dry weight−1. Lower-M r polyols (i.e. glycerol, erythritol and arabitol) were shown to be more effective for osmotic adjustment; for higher-M r polyols such as mannitol, and the disaccharide trehalose, water-activity values for saturated solutions are too high to be effective; i.e. 0.978 and 0.970 (25 ºC). The highly chaotropic, osmotically active substances exhibited a stressful level of chaotropicity at physiologically relevant concentrations (20.0–85.7 kJ kg−1). We hypothesized that the kosmotropicity of compatible solutes can neutralize chaotropicity, and tested this via in-vitro agar-gelation assays for the model chaotropes urea, NH4NO3, phenol and MgCl2. Of the kosmotropic compatible solutes, the most-effective protectants were trimethylamine oxide and betaine; but proline, dimethyl sulfoxide, sorbitol, and trehalose were also effective, depending on the chaotrope. Glycerol, by contrast (a chaotropic compatible solute used as a negative control) was relatively ineffective. The kosmotropic activity of compatible solutes is discussed as one mechanism by which these substances can mitigate the activities of chaotropic stressors in vivo. Collectively, these data demonstrate that some substances concomitantly induce chaotropicity-mediated and osmotic stresses, and that compatible solutes ultimately define the biotic window for fungal growth and metabolism. The findings have implications for the validity of ecophysiological classifications such as ‘halophile’ and ‘polyextremophile’; potential contamination of life-support systems used for space exploration; and control of mycotoxigenic fungi in the food-supply chain.

Powder semiconductor photocatalysis in aqueous solution: An overview of kinetics-based reaction mechanisms

A brief, historical overview of 10 apparently different, although in some cases, upon inspection, closely related, popular proposed reaction mechanisms and their associated rate equations, is given and in which the rate expression for each mechanism is derived from basic principles, Appendix A. In Appendix B, each of the 5 main mechanisms are tested using datasets, comprising initial reaction rate vs. organic pollutant concentration, [P] and incident irradiance, ρ, data, reported previously for TiO2, where P is phenol, 4-chlorophenol and formic acid. The best of those tested, in terms of overall fit, simplicity, usefulness and versatility is the disrupted adsorption kinetic model proposed by Ollis. The usual basic assumptions made in constructing these mechanisms are reported and the main underlying concerns explored.

A Revised Disrupted Langmuir-Adsorption Model of Photocatalysis

The increasingly popular disrupted Langmuir–adsorption (DLA) kinetic model of photocatalysis does not contain an explicit function for the dependence of rate on the irradiance, ρ, but instead has a term αρθ, where, α is a constant of the system, and θ is also a constant equal to 1 or 0.5 at low or high ρ values, respectively. Several groups have recently replaced the latter term with an explicit function of the form χ1(−1 + (1 + χ2ρ)1/2), where χ1 and χ2, are constants that can be related to a proposed reaction scheme. Here the latter schemes are investigated, and revised to create a more credible form by assuming an additional hole trapping step. The latter may be the oxidation of water or a surface saturated with O2–. Importantly, this revision suggests that it is only applicable for low quantum yield/efficiency processes. The revised disrupted Langmuir–adsorption model is used to provide good fits to the kinetic data reported for a number of different systems including the photocatalytic oxidation of nitric oxide (NO), phenol (PhOH), and formic acid (FA).

Re-inoculation of autoclaved soil as a non-sterile treatment for xenobiotic sorption and biodegradation studies

Autoclaved soil is commonly used for the study of xenobiotic sorption and as an abiotic control in biodegradation experiments. Autoclaving has been reported to alter soil physico-chemical and xenobiotic sorption characteristics such that comparison of autoclaved with non-autoclaved treatments in soil aging and bioavailability studies may yield misleading results. Experiments could be improved by using autoclaved soil re-inoculated with indigenous microorganisms as an additional or alternative non-sterile treatment for comparison with the sterile, autoclaved control. We examined the effect of autoclaving (3 x 1 h, 121°C, 103.5 KPa) on the physico-chemical properties of a silt loam soil (pH 7.2, 2.3% organic carbon) and the establishment of indigenous microorganisms reintroduced after autoclaving. Sterilisation by autoclaving significantly (p ≤ 0.05) decreased pH (0.6 of a unit) and increased concentrations of water-soluble organic carbon (WSOC; nontreated = 75 mg kg^-1; autoclaved = 1526 mg kg^-1). The initial first-order rate of ¹⁴C-2,4-dichloro-UL-phenol (2,4-DCP) adsorption to non-treated, autoclaved and re-inoculated soil was rapid (K₁ = 16.8-24.4 h^-1) followed by a slower linear phase (K₂). In comparison with autoclaved soil (0.038% day^-1), K₂ values were higher for re-inoculated (0.095% day^-1) and nontreated (0.181% day^-1) soil. This was attributed to a biological process. The Freundlich adsorption coefficient (K(f)) for autoclaved soil was significantly (p ≤ 0.05) higher than for re-inoculated or non-treated soil. Increased adsorption was attributed to autoclaving-induced changes to soil pH and solution composition. Glucose-induced respiration of autoclaved soil after re-inoculation was initially twice that in the non-treated control, but it decreased to control levels by day 4. This reduction corresponded to a depletion of WSOC. 2,4-DCP mineralisation experiments revealed that the inoculum of nonsterile soil (0.5 g) contained 2,4-DCP-degrading microorganisms capable of survival in autoclaved soil. The lag phase before detection of significant 2,4-DCP mineralisation was reduced (from 7 days to ≤3 days) by pre-incubation of re-inoculated soils for 7 and 14 days before 2,4-DCP addition. This was attributed to the preferential utilisation of WSOC prior to the onset of 2,4-DCP mineralisation. Cumulative ¹⁴CO₂ evolved after 21 days was significantly lower (p ≤ 0.05) from non-treated soil (25.3%) than re-inoculated soils (ca 45%). Experiments investigating sorption-biodegradation interactions of xenobiotics in soil require the physico-chemical properties of sterile and non-sterile treatments to be as comparable as possible. For fundamental studies, we suggest using re-inoculated autoclaved soil as an additional or alternative non-sterile treatment.

Shear Strength and Durability Testing of Adhesive Bonds in Cross-Laminated Timber

The paper addresses the quality of the interface and edge bonded joints in layers of cross-laminated timber (CLT) panels. The shear performance was studied to assess the suitability of two different adhesives, Polyurethane (PUR) and Phenol-Resorcinol-Formaldehyde (PRF), and to determine the optimum clamping pressure. Since there is no established testing procedure to determine the shear strength of the surface bonds between layers in a CLT panel, block shear tests of specimens in two different configurations were carried out, and further shear tests of edge bonded specimen in two configurations were performed. Delamination tests were performed on samples which were subjected to accelerated aging to assess the durability of bonds in severe environmental conditions. Both tested adhesives produced boards with shear strength values within the edge bonding requirements of prEN 16351 for all manufacturing pressures. While the PUR specimens had higher shear strength values, the PRF specimens demonstrated superior durability characteristics in the delamination tests. It seems that the test protocol introduced in this study for crosslam bonded specimens, cut from a CLT panel, and placed in the shearing tool horizontally, accurately reflects the shearing strength of glue lines in CLT.

EFFECTS OF ENVIRONMENTAL ANTIOXIDANTS ON PRO-ANGIOGENIC ROS SIGNALLING IN ENDOTHELIAL COLONY FORMING CELLS IN VITRO

Introduction. Endothelial colony-forming cells (ECFCs) hold great cytotherapeutic potential for ischaemic disease. Whilst increasing evidence supports a key role for reactive oxygen species (ROS), specifically those derived from Nox NADPH oxidases, in the underlying angiogenic processes of these and other endothelial cells, such studies investigating the role of redox signalling may be hampered by the standard inclusion of antioxidant agents in endothelial cell media, such as phenol red. Aims. To study the effects of antioxidants present in culture media on pro-angiogenic function of ECFCs in vitro. Methods. Human ECFCs isolated from umbilical cord blood were maintained in media with and without antioxidant components (EGM2 and phenol red-free DMEM, respectively) prior to treatment with pro-oxidant PMA and assessment of their in vitro migratory capacity using a scratch-wound assay to measure pro-angiogenic activity. Results. Our previous work in our group indicated that PMA (500nM) increased ECFC migration in a both a superoxide and NADPH oxidase-dependent manner (control 18.6±2.8, PMA 32.7±6.6% wound closure; n=6, P<0.05), as indicated by attenuation with PEG-SOD and VAS2870. However, inconsistencies in the data generated under varying experimental conditions led us to hypothesise that antioxidant agents in the standard ECFC media may be influencing these effects. Indeed, a direct comparison of cell migration between ECFCs incubated in EGM2 DMEM demonstrated a clear trend towards higher migration in the latter (EGM2 9.0±4.5, DMEM 22.7±6.4%; n=3, P=NS). Similar to our previous EGM2 studies, cell migration was potentiated by PMA (control 11.6±1.6, PMA 25.1±2.8%; n=3, P<0.05), but at a lower dose (100nM), which is consistent with a reduction in media antioxidants. Notably, this response was attenuated by VAS2870 (PMA 37.6±7.3, PMA+VAS2870 10.3±2.9%; n=6, P<0.05), underlining a likely role for Nox NADPH oxidases. Conclusion. Taken together, these data indicate that ECFC migration is sensitive to different endothelial cell growth media, which appears to be dependent upon their antioxidant content. Although further experiments, such as quantification of cellular superoxide generation by dihydroethidium fluorescence may be required to confirm a specific role for antioxidants, such blunting of ROS signalling in vitro is clearly an important consideration which may significantly impact upon data interpretation.

Synthesis and biological activities of polyphenolic hybrids - evaluation of anti-cancer, anti-inflammatory and antioxidant activities

Os compostos polifenólicos constituem uma classe de metabolitos secundários de plantas, mas existe também uma enorme quantidade de derivados sintéticos ou semi-sintéticos contendo múltiplas unidades fenólicas. Estes compostos apresentam importantes características biológicas, que dependem das suas estruturas básicas. Certos derivados desta família de compostos, tais como flavonoides, cromonas e cumarinas contribuem para os benefícios da dieta humana, e partilham o núcleo de benzopiran-(2 e 4)-ona ou benzofuran-3-ona. A presente dissertação inclui uma introdução geral e três capítulos que descrevem as novas rotas sintéticas estabelecidas para a preparação de novos híbridos de diversos compostos polifenólicos, assim como a sua elucidação estrutural e termina com a presentação dos resultados da avaliação biológica desses mesmos compostos. No segundo capítulo discute-se a preparação de híbridos de pirimidina- e imidazolidina-polifenóis, especialmente a síntese diastereoseletiva de novos híbridos benzofuran-3-ona-hidantoína e derivados de uracilo. A rota sintética envolve a ação de carbodiimidas sobre os ácidos cromona-(2- e 3)-carboxílicos num só passo ou em dois passos sequenciais, catalisada por uma base orgânica ou inorgânica. O terceiro capítulo descreve reações do tipo adições conjugadas 1,4 - hetero-ciclisações em cascata de compostos 1,3-dicarbonílicos em ácido cromona-3-carboxílico catalisadas por uma base orgânica, que originaram novas cromonas, cromanonas e flavonas polissubstituídas. As bispiranonas [bispiran-2 e 4)-onas] foram elaboradas numa reacção de acoplamento da 4-hidroxicumarina ou da lactona do ácido triacético com o ácido cromona-3-carboxílico ou precursores formil-funcionalizados (ω-formil-2’-hydroxy acetofenonas e cromona-3-carbaldeídos) utilizando organocatálise básica. Finalmente, alargou-se o estudo das adições conjugadas 1,4 para uma variedade de 4-hidroxipiran-2-onas e cetonas α,β-insaturadas para originar novos análogos de warfarina. Obteve-se uma variedade de estruturas complexas por hibridação das unidades de 4-hidroxicumarina ou da lactona do ácido triacético com os novos derivados de cromonas polissubstituídas. Todos as reações foram executadas em condições suaves e ambientalmente favoráveis, utilizando a 4-pirrolidinopiridina como organocatalisador básico. As estruturas dos novos híbridos polifenólicos foram caracterizados por técnicas espectroscópicas de alta resolução, incluindo espectroscopia de ressonância magnética nuclear (1D e 2D) e por difractometria de raios-X, que nos permitiram resolver o complexidade estrutural dos compostos sintetizados. O quarto capítulo apresenta os resultados da avaliação biológica obtidos com os híbridos polifenólicos sintetizados neste trabalho, mostrando a possibilidade de seu envolvimento na terapia do cancro. A maioria dos compostos foram avaliados quanto ao seu efeito sobre a citotoxicidade e proliferação de células leucémicas e ao seu envolvimento na regulação de via pró-inflamatória NF-kB, na qual, os híbridos de biscumarinas exibiram actividades elevadas (IC50 = 6-19 μM para inibição de NF-kB depois de 8 horas de incubação e IC50 = 15-39 μM para efeitos citotóxicos em células cancerosas, após 24 horas de incubação). Uma inibição moderada das enzimas HDAC e Cdc25 foi induzida pelos derivados de benzofuran-3-ona-hidantoína. Catorze dos novos derivados polifenólicos polissubstituídos, tendo como estrutura básica a benzopiran-4-ona, foram avaliados pela sua actividade quimiopreventiva do cancro mediada pela indução de sinalização citoprotectora Nrf2 (fator 2 relacionado com o fator nuclear da proteína E2) e capacidade para inibir a proliferação das células de cancro da mama. Os derivados da classe das cromanonas foram identificados como os indutores mais potentes da actividade Nrf2. As concentrações necessárias para aumentar a actividade de luciferase em 10 vezes (C10) foram de 2,8-21,3 μM. Todos os novos híbridos polifenólicos que apresentam atividade citotóxica e anti-proliferativa não afectam o crescimento de células saudáveis periféricas do sangue (PBMC) (IC50 > 50 μM), indicando a sua seletividade para as células cancerosas e sugerindo que alguns deles são estruturalmente interessantes para posteriores análises. A avaliação da atividade antioxidante utilizando os testes do radical livre DPPH e o poder redutor do ião férrico FRAP foram realizados em algumas estruturas híbridas polifenólicas.

Structural analysis of glycans and development of microarrays from Helcobacter pylori cell surface glycome

Helicobacter pylori is a bacterial pathogen that affects more than half of the world’s population with gastro-intestinal diseases and is associated with gastric cancer. The cell surface of H. pylori is decorated with lipopolysaccharides (LPSs) composed of three distinct regions: a variable polysaccharide moiety (O-chain), a structurally conserved core oligosaccharide, and a lipid A region that anchors the LPS to the cell membrane. The O-chain of H. pylori LPS, exhibits unique oligosaccharide structures, such as Lewis (Le) antigens, similar to those present in the gastric mucosa and are involved in interactions with the host. Glucan, heptoglycan, and riban domains are present in the outer core region of some H. pylori LPSs. Amylose-like glycans and mannans are also constituents of some H. pylori strains, possibly co-expressed with LPSs. The complexity of H. pylori LPSs has hampered the establishment of accurate structure-function relationships in interactions with the host, and the design of carbohydrate-based therapeutics, such as vaccines. Carbohydrate microarrays are recent powerful and sensitive tools for studying carbohydrate antigens and, since their emergence, are providing insights into the function of carbohydrates and their involvement in pathogen-host interactions. The major goals of this thesis were the structural analysis of LPSs from H. pylori strains isolated from gastric biopsies of symptomatic Portuguese patients and the construction of a novel pathogen carbohydrate microarray of these LPSs (H. pylori LPS microarray) for interaction studies with proteins. LPSs were extracted from the cell surface of five H. pylori clinical isolates and one NCTC strain (26695) by phenol/water method, fractionated by size exclusion chromatography and analysed by gas chromatography coupled to mass spectrometry. The oligosaccharides released after mild acid treatment of the LPS were analysed by electrospray mass spectrometry. In addition to the conserved core oligosaccharide moieties, structural analyses revealed the presence of type-2 Lex and Ley antigens and N-acetyllactosamine (LacNAc) sequences, typically found in H. pylori strains. Also, the presence of O-6 linked glucose residues, particularly in LPSs from strains 2191 and NCTC 26695, pointed out to the expression of a 6-glucan. Other structural domains, namely ribans, composed of O-2 linked ribofuranose residues were observed in the LPS of most of H. pylori clinical isolates. For the LPS from strain 14382, large amounts of O-3 linked galactose units, pointing to the occurrence of a galactan, a domain recently identified in the LPS of another H. pylori strain. A particular feature to the LPSs from strains 2191 and CI-117 was the detection of large amounts of O-4 linked N-acetylglucosamine (GlcNAc) residues, suggesting the presence of chitin-like glycans, which to our knowledge have not been described for H. pylori strains. For the construction of the H. pylori LPS microarray, the structurally analysed LPSs, as well as LPS-derived oligosaccharide fractions, prepared as neoglycolipid (NGL) probes were noncovalently immobilized onto nitrocellulosecoated glass slides. These were printed together with NGLs of selected sequence defined oligosaccharides, bacterial LPSs and polysaccharides. The H. pylori LPS microarray was probed for recognition with carbohydratebinding proteins (CBPs) of known specificity. These included Le and blood group-related monoclonal antibodies (mAbs), plant lectins, a carbohydratebinding module (CBM) and the mammalian immune receptors DC-SIGN and Dectin-1. The analysis of these CBPs provided new information that complemented the structural analyses and was valuable in the quality control of the constructed microarray. Microarray analysis revealed the occurrence of type-2 Lex and Ley, but not type-1 Lea or Leb antigens, supporting the results obtained in the structural analysis. Furthermore, the H. pylori LPSs were recognised by DC-SIGN, a mammalian lectin known to interact with this bacterium through fucosylated Le epitopes expressed in its LPSs. The -fucose-specific lectin UEA-I, showed restricted binding to probes containing type-2 blood group H sequence and to the LPSs from strains CI-117 and 14382. The presence of H-type-2, as well Htype- 1 in the LPSs from these strains, was confirmed using specific mAbs. Although H-type-1 determinant has been reported for H. pylori LPSs, this is the first report of the presence of H-type-2 determinant. Microarray analysis also revealed that plant lectins known to bind 4-linked GlcNAc chitin oligosaccharide sequences bound H. pylori LPSs. STL, which exhibited restricted and strong binding to 4GlcNAc tri- and pentasaccharides, differentially recognised the LPS from the strain CI-117. The chitin sequences recognised in the LPS could be internal, as no binding was detected to this LPS with WGA, known to be specific for nonreducing terminal of 4GlcNAc sequence. Analyses of the H. pylori LPSs by SDS-PAGE and Western blot with STL provided further evidence for the presence of these novel domains in the O-chain region of this LPS. H. pylori LPS microarray was also applied to analysis of two human sera. The first was from a case infected with H. pylori (H. pylori+ CI-5) and the second was from a non-infected control.The analysis revealed a higher IgG-reactivity towards H. pylori LPSs in the H. pylori+ serum, than the control serum. A specific IgG response was observed to the LPS isolated from the CI-5 strain, which caused the infection. The present thesis has contributed to extension of current knowledge on chemical structures of LPS from H. pylori clinical isolates. Furthermore, the H. pylori LPS microarray constructed enabled the study of interactions with host proteins and showed promise as a tool in serological studies of H. pyloriinfected individuals. Thus, it is anticipated that the use of these complementary approaches may contribute to a better understanding of the molecular complexity of the LPSs and their role in pathogenesis.

Infection mechanism of Diplodia corticola

Diplodia corticola is regarded as the most virulent fungus involved in cork oak decline, being able to infect not only Quercus species (mainly Q. suber and Q. ilex), but also grapevines (Vitis vinifera) and eucalypts (Eucalyptus sp.). This endophytic fungus is also a pathogen whose virulence usually manifests with the onset of plant stress. Considering that the infection normally culminates in host death, there is a growing ecologic and socio-economic concern about D. corticola propagation. The molecular mechanisms of infection are hitherto largely unknown. Accordingly, the aim of this study was to unveil potential virulence effectors implicated in D. corticola infection. This knowledge is fundamental to outline the molecular framework that permits the fungal invasion and proliferation in plant hosts, causing disease. Since the effectors deployed are mostly proteins, we adopted a proteomic approach. We performed in planta pathogenicity tests to select two D. corticola strains with distinct virulence degrees for our studies. Like other filamentous fungi D. corticola secretes protein at low concentrations in vitro in the presence of high levels of polysaccharides, two characteristics that hamper the fungal secretome analysis. Therefore, we first compared several methods of extracellular protein extraction to assess their performance and compatibility with 1D and 2D electrophoretic separation. TCA-Acetone and TCA-phenol protein precipitation were the most efficient methods and the former was adopted for further studies. The proteins were extracted and separated by 2D-PAGE, proteins were digested with trypsin and the resulting peptides were further analysed by MS/MS. Their identification was performed by de novo sequencing and/or MASCOT search. We were able to identify 80 extracellular and 162 intracellular proteins, a milestone for the Botryosphaeriaceae family that contains only one member with the proteome characterized. We also performed an extensive comparative 2D gel analysis to highlight the differentially expressed proteins during the host mimicry. Moreover, we compared the protein profiles of the two strains with different degrees of virulence. In short, we characterized for the first time the secretome and proteome of D. corticola. The obtained results contribute to the elucidation of some aspects of the biology of the fungus. The avirulent strain contains an assortment of proteins that facilitate the adaptation to diverse substrates and the identified proteins suggest that the fungus degrades the host tissues through Fenton reactions. On the other hand, the virulent strain seems to have adapted its secretome to the host characteristics. Furthermore, the results indicate that this strain metabolizes aminobutyric acid, a molecule that might be the triggering factor of the transition from a latent to a pathogenic state. Lastly, the secretome includes potential pathogenicity effectors, such as deuterolysin (peptidase M35) and cerato-platanin, proteins that might play an active role in the phytopathogenic lifestyle of the fungus. Overall, our results suggest that D. corticola has a hemibiotrophic lifestyle, switching from a biotrophic to a necrotrophic interaction after plant physiologic disturbances.This understanding is essential for further development of effective plant protection measures.

Antioxidant activities of eight Algerian plant extracts and two essential oils

Total phenol, hydroxycinnamic acid derivatives, flavone/flavonol and flavanones/dihydroflavonol contents of hydro-alcoholic extracts, obtained by sonication, from the aerial parts of Artemisia campestris L., Anthemis arvensis L., Haloxylon scoparium Pomel, Juniperus phoenicea L., Arbutus unedo L., Cytisus monspessulanus L., Thymus algeriensis Boiss et Reut, Zizyphus lotus L (Desf.) collected in Djebel Amour (Sahara Atlas, Algeria) were quantified by spectrophotometric methods. The chemical composition of the essential oils obtained by hydrodistillation from Artemisia campestris L. and Juniperus phoenicea I aerial parts were also evaluated by gas chromatography (GC) and gas chromatography coupled to mass spectrometry (GC-MS). The antioxidant activity of the extracts and essential oils was assessed measuring the capacity for preventing lipid peroxidation using two lipidic substrates (egg yolk and liposomes), the capacity for scavenging DPPH, ABTS, superoxide anion radicals, hydroxyl radicals and peroxyl radicals. Anti-inflammatory activity was assessed by measuring the capacity for inhibiting lipoxygenase. Reducing power and chelating capacity were also assayed. The results showed different amounts of total phenols depending on the method used: A. campestris extract had the highest levels of total phenols when the measurement was made at lambda = 280 nm, whereas H. scoparium and A. unedo extracts showed the highest levels of total phenols with Folin-Ciocalteau. C. monspessulanus had the highest levels of flavones/flavonols and flavanones/dihydroflavonols. The essential oils of A. campestris and J. phoenicea were mainly constituted by alpha-pinene, beta-pinene and sabinene; and a-pinene, respectively. The methods used for assaying the capacity for preventing lipid peroxidation revealed to be inadequate for extracts due to the great interferences detected. The essential oils were more active than the generality of extracts for scavenging peroxyl radicals and for inhibiting lipoxygenase, whereas A. unedo extract was the most active for scavenging ABTS, DPPH, superoxide anion radicals and it also had the best reducing capacity. In a general way, the great majority of the antioxidant activities correlated well with the phenol content although such correlation was not so clear with the flavonoid content. (c) 2013 Elsevier B.V. All rights reserved.

Gallium compounds forthe design of (nano)radiophamarceuticals

Tese de doutoramento, Química (Química Inorgânica), Universidade de Lisboa, Faculdade de Ciências, 2014

Determination of tartaric acid in wines by FIA with tubular tartrate-selective electrodes

A flow injection analysis (FIA) system comprising a tartrate- (TAT) selective electrode has been developed for determination of tartaric acid in wines. Several electrodes constructed for this purpose had a PVC membrane with a complex of quaternary ammonium and TAT as anion exchanger, a phenol derivative as additive, and a more or less polar mediator solvent. Characterization of the electrodes showed behavior was best for membranes with o-nitrophenyl octyl ether as solvent. On injection of 500 μL into a phosphate buffer carrier (pH = 3.1; ionic strength 10–2 mol/L) flowing at 3 mL/min, the slope was 58.06 ± 0.6 with a lower limit of linear range of 5.0 × 10–4 mol/L TAT and R2 = 0.9989. The interference of several species, e.g. chloride, bromide, iodide, nitrate, gallic acid, tannin, sucrose, glucose, fructose, acetate, and citrate, was evaluated in terms of potentiometric selectivity coefficients. The Hofmeister series was followed for inorganic species and the most interfering organic ion was citrate. When red and white wines were analyzed and the results compared with those from an independent method they were found to be accurate, with relative standard deviations below 5.0%.

Extração e caracterização de extratos de Jatropha gossypiifolia L.: avaliação da sua atividade antimicrobiana e antioxidante

Trabalho Final de Mestrado para obtenção do grau de mestre em Engenharia Química e Biológica