Browsing by Author "Monteiro, Sara"
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- Assessment of Potential Effects of Common Fining Agents Used for White Wine Protein StabilizationPublication . Chagas, Ricardo; Monteiro, Sara; Ferreira, Ricardo BoavidaA variety of fining agents are commercially available to the wine industry, including proteins and inorganic ion exchangers. These fining agents are essentially used to control the levels of phenolics in wine, but they also have the potential to interact with other wine components, most often as a side effect. They are therefore expected to influence, at least in part, the potential for wine protein haze formation. Six common fining agents—casein, egg albumin, isinglass, chitosan, chitin, and polyvinylpolypyrrolidone (PVPP)—were analyzed to assess their effects on wine protein haze-forming potential and on the levels of proteins and phenolic compounds in a Muscat of Alexandria wine. Bentonite was selected as the positive control, whereas nonfined wine was used as the negative control. Differential results were detected among the selected fining agents when compared to the controls. Egg albumin and chitosan, although incapable of stabilizing the wine, originated a small but significant decrease in the protein haze formed, whereas chitosan and PVPP were second to bentonite in removing the most polyphenols from the wine. Thus, while chitosan fining removes a fraction of polyphenols from the wine and seems to induce a small decrease in its haze-forming potential, PVPP eliminates more polyphenols while leaving its haze-forming potential unaltered. The fining agents analyzed did not significantly affect wine protein content but did remove considerable levels of polyphenols and presented no apparent effect on protein stabilization of the fined wines. Results show that these fining agents do not contribute significantly to protein stabilization in white wines, confirming that bentonite was the most effective agent in wine protein stabilization
- Fusion proteins towards fungi and bacteria in plant protectionPublication . Pinheiro, Ana Margarida; Carreira, Alexandra; Ferreira, Ricardo B.; Monteiro, SaraIn agriculture, although fungi are considered the foremost problem, infections by bacteria also cause significant economical losses. The presence of different diseases in crops often leads to a misuse of the proper therapeutic, or the combination of different diseases forces the use of more than one pesticide. This work concerns the development of a ‘super-Blad’: a chimeric protein consisting of Blad polypeptide, the active ingredient of a biological fungicide already on the market, and two selected peptides, SP10-5 and Sub5, proven to possess biological potential as antibacterial agents. The resulting chimeric protein obtained from the fusion of Blad with SP10-5 not only maintained strong antibacterial activity, especially against Xanthomonas spp. and Pseudomonas syringae, but was also able to retain the ability to inhibit the growth of both yeast and filamentous fungi. However, the antibacterial activity of Sub5 was considerably diminished when fused with Blad, which seems to indicate that not all fusion proteins behave equally. These newly designed drugs can be considered promising compounds for use in plant protection. A deeper and focused development of an appropriate formulation may result in a potent biopesticide that can replace, per se, two conventional chemistries with less impact on the environment
- Insuficiência ovárica prematuraPublication . Monteiro, Sara; Soares, Ana PaulaIntroduction: Premature ovarian insufficiency (POI) has important repercussions on women’s life. As the cure rates of cancers in childhood and young women continue to improve, it is likely that its incidence will rise (1,2). Objective: Review the physiology, etiology, diagnosis, evaluation, treatment options and prevision and prevention methods of POI. Design: Bibliographic search in PubMED using the MeSH terms: Primary ovarian insufficiency, Premature ovarian failure, Premature ovarian dysfunction, Premature menopause, Hypergonadotropic hypogonadism, based in articles published from 2009 until November 2013 and some addicional reference articles quoted in the articles above. Results: POI is a subclass of ovarian dysfunction in which follicular maturation and hormonal production ceases. The main symptom is amenorrhea. It is diagnosed by detecting high follicle-stimulating hormone (FSH) and low estradiol serum concentrations in women younger than 40 years-old. In 90% of the cases the underlying cause is not identified. Half of the women with POI have intermittent ovarian function. The association with autoimmune diseases is frequent. Hormone replacement therapy (HRT) is recommended. Oocyte donation associated with assisted reproduction provide the best rates in pregnancy success. Discussion: Apparently the longer the POI, the bigger the impact on women’s health and the smaller the chances of spontaneous remissions and pregnancy. Oocyte cryopreservation and in vitro maturation could be an option in the future (3–5).
- Maximizing Blad-containing oligomerfungicidal activity in sweet cultivars of Lupinus albus seedsPublication . Cruz, Filipe; Batista-Santos, Paula; Monteiro, Sara; Neves-Martins, J.; Ferreira, Ricardo BoavidaDuring seed germination and plantlet growth, an important aspect of Lupinus β-conglutin proteolysis is the accumulation of blad in the cotyledons. Blad, a 20.4 kDa, 173 residue polypeptide which inhibits fungal growth, is the main subunit of BCO (Blad-containing oligomer), a Lupinus bioactive polypeptide oligomer, which underwent a successful translational research during the last thirty years. The development of this recent broad-spectrum biological fungicide for plant disease control made evident the advantage of being non-toxic to the environment, plants, humans and other animals, an interesting characteristic given the increasing consumer’s concern about food safety. The industrial-scale production of the edible fungicide BCO for agricultural purposes involves germinating and growing for ca. 8 days massive amounts of Lupinus plantlets. Therefore, it becomes economically relevant to maximize/increment the amount of extractable BCO. Although BCO represents an alternative pathway for lupin production in Europe, there is no previous evidence on lupin cultivars concerning BCO activity or its cotyledonary concentration, and therefore on the most promising cultivars for BCO extraction 16 and fungicide production. In this work, the amount of BCO and its level of fungicide activity was evaluated in seven sweet cultivars of L. albus (cvs. Amiga, Energy, Estoril, Ludic, Misak, Multitalia and Rumbo), along 20 days after the onset of germination. In addition, four distinct lots of cv. Energy and six lots of cv Misak, with different harvest years (1997, 2010, 2013 and 2014) and/or different sites of production in Portugal (Alto Alentejo, Baixo Alentejo and Beira Litoral) were also analysed. Quantitative assays demonstrated maximal accumulation of BCO in the cotyledons of 4-days-old L. albus plantlets, apart from significant differences between seeds’ harvest year or production site. The assays also showed that cv. Energy had significant higher accumulation of BCO (31.67 μg mg-1 wet weight) than Rumbo (25.67 μg mg-1) and Misak (22.12 μg mg-1), respectively second and third cultivars with highest accumulation of the oligomer. Additionally, very significant differences were also observed among seeds’ harvest year or production site. These observations reduced dramatically the original purpose of this work on the study of BCO variations at the level of sweet L. albus cultivars, since the ‘noise’ detected in BCO levels caused by the year and location of their production was far greater than its variation among cultivars. The observation that storing for several years the seeds at -20ºC did not seem to decrease BCO activity and suggests that the variation in cotyledonary BCO accumulation is essentially controlled by the prevailing edaphoclimatic conditions during seed formation. Furthermore, the activity tests exhibited significant antifungal activity between 3 and 5 days-old plantlets, with absence of antifungal activity on 8 days-old plantlets or older. Overall this study is of considerable importance to maximize BCO extraction from the cotyledons of sweet L. albus cultivars
- A nontoxic polypeptide oligomer with a fungicide potency under agricultural conditions which is equal or greater than that of their chemical counterpartsPublication . Monteiro, Sara; Carreira, Alexandra; Freitas, Regina; Pinheiro, Ana Margarida; Ferreira, Ricardo BoavidaThere are literally hundreds of polypeptides described in the literature which exhibit fungicide activity. Tens of them have had attempted protection by patent applications but none, as far as we are aware, have found application under real agricultural conditions. The reasons behind may be multiple where the sensitivity to the Sun UV radiation can come in first place. Here we describe a multifunctional glyco-oligomer with 210 kDa which is mainly composed by a 20 kDa polypeptide termed Blad that has been previously shown to be a stable intermediary product of β-conglutin catabolism. This oligomer accumulates exclusively in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination. Blad-oligomer reveals a plethora of biochemical properties, like lectin and catalytic activities, which are not unusual per si, but are remarkable when found to coexist in the same protein molecule. With this vast range of chemical characteristics, antifungal activity arises almost as a natural consequence. The biological significance and potential technological applications of Blad-oligomer as a plant fungicide to agriculture, its uniqueness stems from being of polypeptidic in nature, and with efficacies which are either equal or greater than the top fungicides currently in the market are addressed
- Phaemoniella chlamydospora infection induces changes in phenolic compounds content in Vitis viniferaPublication . Oliveira, Helena; Martin, Nico; Vesentini, Damiano; Rego, Cecilia; Monteiro, Sara; Ferreira, Ricardo BoavidaThe effect of Phaeomoniella chlamydospora infection was evaluated in three grapevine cultivars, in relation to the variation in phenolic compounds within the host. Young plants of Vitis vinifera (cv. Chardonnay, Touriga Nacional and two clones of cv. Aragonez) were infected using two strains of Pa. chlamydospora and were harvested after fi ve months from inoculation. Overall, a localised increment in the amount of total phenolics within a methanolic extract of grapevine wood tissue was observed following infection. Such pattern was also manifested by a localised increase in non-tannin phenolics. HPLC analysis was used to identify the host phenolic compounds, which were mostly affected by the host-pathogen interaction. trans-Resveratrol was found to augment very signifi cantly in both cv. Chardonnay and cv. Touriga Nacional following infection. e-Viniferin also increased considerably in infected plants of cv. Touriga Nacional. Signifi cant differences were encountered among infected plants depending not only on the cultivar, but also on the site of inoculation. Overall, cv. Chardonnay and cv. Touriga Nacional contained a higher amount of infection-induced phenolic compounds than both cv. Aragonez clones. Infection of young shoots also led to a greater accumulation of phenolic compounds than that of the trunk. Results are discussed in view of understanding the role of phenolic compounds and especially trans-resveratrol in grapevine defences against Pa. chlamydospora.
- Reference gene validation for quantitative RT-PCR during biotic and abiotic stresses in Vitis viniferaPublication . Borges, Alexandre Filipe; Fonseca, Catarina; Ferreira, Ricardo Boavida; Lourenço, Ana Maria; Monteiro, SaraGrapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies
- The unique biosynthetic route from Lupinus Beta-conglutin gene to bladPublication . Monteiro, Sara; Freitas, Regina; Rajasekhar, Baru T.; Teixeira, Artur R.; Ferreira, Ricardo BoavidaBackground: During seed germination, b-conglutin undergoes a major cycle of limited proteolysis in which many of its constituent subunits are processed into a 20 kDa polypeptide termed blad. Blad is the main component of a glycooligomer, accumulating exclusively in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination. Principal Findings: The sequence of the gene encoding b-conglutin precursor (1791 nucleotides) is reported. This gene, which shares 44 to 57% similarity and 20 to 37% identity with other vicilin-like protein genes, includes several features in common with these globulins, but also specific hallmarks. Most notable is the presence of an ubiquitin interacting motif (UIM), which possibly links the unique catabolic route of b-conglutin to the ubiquitin/proteasome proteolytic pathway. Significance: Blad forms through a unique route from and is a stable intermediary product of its precursor, b-conglutin, the major Lupinus seed storage protein. It is composed of 173 amino acid residues, is encoded by an intron-containing, internal fragment of the gene that codes for b-conglutin precursor (nucleotides 394 to 913) and exhibits an isoelectric point of 9.6 and a molecular mass of 20,404.85 Da. Consistent with its role as a storage protein, blad contains an extremely high proportion of the nitrogen-rich amino acids
- Transcriptomic changes following the compatible interaction Vitis vinifera-Erysiphe necator. Paving the way towards an enantioselective role in plant defence modulationPublication . Borges, Alexandre Filipe; Ferreira, Ricardo Boavida; Monteiro, SaraThe compatible interaction between Erysiphe necator and Vitis vinifera induces significant alterations in the host transcriptome, affecting essentially those genes involved in signalling and secondary metabolite biosynthetic pathways. The precise transcriptomic changes vary from the early events to later stages of infection. In the present work, suppressive subtraction hybridization (SSH) was used to identify several differentially expressed transcripts in symptomatic and asymptomatic leaves from powdery mildew infected grapevines following a long term interaction. The detected transcripts show little or no correlation with similar expression studies concerning the early stages of infection which suggests distinct host responses occur before and after the infection is established. The transcription level of thirteen genes was assessed through qRT-PCR using appropriately selected and validated normalization genes. With one exception, all these genes underwent moderate levels of differential transcription, with log2- fold change values ranging from 2.65 to 4.36. The exception, a dirigent-like (DIR) protein, was upregulated over 180 fold in symptomatic leaves, suggesting an important role for stereochemical selectivity in the compatible interaction E. necatoreV. vinifera. DIR copy number was determined in the genome of three grapevine cultivars exhibiting high (Carignan), moderate (Fernão Pires) and low (Touriga Nacional) sensitivity to E. necator. It was found to be a two-copy gene in all cultivars analyzed. Further analysis involving DIR metabolic neighbourhood transcripts was performed. The possible physiological significance of the detected DIR upregulation is discussed