Saccharomyces Cerevisiae as a biotechnological tool for ageing research:studies on translation and metabolism

The yeast Saccharomyces cerevisiae is an important model organism for the study of cell biology. The similarity between yeast and human genes and the conservation of fundamental pathways means it can be used to investigate characteristics of healthy and diseased cells throughout the lifespan. Yeast is an equally important biotechnological tool that has long been the organism of choice for the production of alcoholic beverages, bread and a large variety of industrial products. For example, yeast is used to manufacture biofuels, lubricants, detergents, industrial enzymes, food additives and pharmaceuticals such as anti-parasitics, anti-cancer compounds, hormones (including insulin), vaccines and nutraceuticals. Its function as a cell factory is possible because of the speed with which it can be grown to high cell yields, the knowledge that it is generally recognized as safe (GRAS) and the ease with which metabolism and cellular pathways, such as translation can be manipulated. In this thesis, these two pathways are explored in the context of their biotechnological application to ageing research: (i) understanding translational processes during the high-yielding production of membrane protein drug targets and (ii) the manipulation of yeast metabolism to study the molecule, L-carnosine, which has been proposed to have anti-ageing properties. In the first of these themes, the yeast strains, spt3?, srb5?, gcn5? and yTHCBMS1, were examined since they have been previously demonstrated to dramatically increase the yields of a target membrane protein (the aquaporin, Fps1) compared to wild-type cells. The mechanisms underlying this discovery were therefore investigated. All high yielding strains were shown to have an altered translational state (mostly characterised by an initiation block) and constitutive phosphorylation of the translational initiation factor, eIF2a. The relevance of the initiation block was further supported by the finding that other strains, with known initiation blocks, are also high yielding for Fps1. A correlation in all strains between increased Fps1 yields and increased production of the transcriptional activator protein, Gcn4, suggested that yields are subject to translational control. Analysis of the 5´ untranslated region (UTR) of FPS1 revealed two upstream open reading frames (uORFs). Mutagenesis data suggest that high yielding strains may circumvent these control elements through either a leaky scanning or a re-initiation mechanism. In the second theme, the dipeptide L-carnosine (ß-alanyl-L-histidine) was investigated: it has previously been shown to inhibit the growth of cancer cells but delay senescence in cultured human fibroblasts and extend the lifespan of male fruit flies. To understand these apparently contradictory properties, the effects of L-carnosine on yeast were studied. S. cerevisiae can respire aerobically when grown on a non-fermentable carbon source as a substrate but has a respiro-fermentative metabolism when grown on a fermentable carbon source; these metabolisms mimic normal cell and cancerous cell metabolisms, respectively. When yeast were grown on fermentable carbon sources, in the presence of L-carnosine, a reduction in cell growth and viability was observed, which was not apparent for cells grown on a non-fermentable carbon source. The metabolism-dependent mechanism was confirmed in the respiratory yeast species Pichia pastoris. Further analysis of S. cerevisiae yeast strains with deletions in their nutrient-sensing pathway, which result in an increase in respiratory metabolism, confirmed the metabolism-dependent effects of L-carnosine.

Dynamic restructuring of Pd nanoparticles and single crystals during catalytic selective alcohol oxidation

The aerobic selective oxidation (selox) of alcohols represents an environmentally benign and atom efficient chemical valorisation route to commercially important allylic aldehydes, such as crotonaldehyde and cinnamaldehyde, which find application in pesticides, fragrances and food additives. Palladium nanoparticles are highly active and selective heterogeneous catalysts for such oxidative dehydrogenations, permitting the use of air (or dioxygen) as a green oxidant in place of stoichiometric chromate permanganate saltsor H2O2. Here we discuss how time-resolved, in-situ X-ray spectroscopies (XAS and XPS) reveal dynamic restructuring of dispersed Pd nanoparticles and Pd single-crystals in response to changing reaction environments, and thereby identify surface PdO as the active species responsible for palladium catalysed crotyl alcohol selox (Figure 1); on-stream reduction to palladium metal under oxygen-poor regimes thus appears the primary cause of catalyst deactivation. This insight has guided the subsequent application of surfactant-templating and inorganic nanocrystal methodologies to optimize the density of desired active PdO sites for the selective oxidation of natural products such as sesquiterpenoids.

Propriedades antioxidantes de constituintes fenólicos de macroalgas marinhas

Desde tempos remotos que as macroalgas marinhas são utilizadas por comunidades humanas, nomeadamente as orientais, como parte importante da sua dieta alimentar. A composição química das diferentes macroalgas marinhas (algas castanhas – Phaeophyta, algas vermelhas – Rhodophyta e algas verdes – Chlorophyta) confirma que além de terem um valor nutricional satisfatório podem ser uma fonte muito interessante de compostos bioativos como, por exemplo, os compostos fenólicos. Quimicamente os compostos fenólicos caracterizam-se por apresentarem um ou mais grupos hidroxilo ligados a um anel aromático. Estes compostos englobam desde moléculas simples até moléculas poliméricas de grandes dimensões. A maioria dos compostos fenólicos apresenta atividade antioxidante. O interesse pelo estudo de metabolitos secundários das macroalgas com propriedades antioxidantes surgiu, numa primeira fase, como uma tentativa de encontrar substitutos para os antioxidantes sintéticos usados como aditivos alimentares (nomeadamente o BHA e o BHT) que demonstravam possuir efeitos carcinogénicos. No entanto, rapidamente a comunidade científica reconheceu que a aplicação de novos compostos fenólicos naturais é muito mais vasta. Sabe-se hoje que é crucial para a promoção da saúde de um indivíduo que se verifique a manutenção do equilíbrio entre a produção de radicais livres e as respetivas defesas antioxidantes. Quando esse equilíbrio se altera e ocorre uma acumulação de radicais livres no organismo, este entra em stress oxidativo, situação que pode conduzir a danos dos lípidos celulares, proteínas e ácidos nucleicos, o que favorece o aparecimento de diversas doenças e acelera o envelhecimento celular. Assim, atualmente existe um crescente interesse por parte da indústria farmacêutica e da indústria da cosmética no estudo dos compostos fenólicos isolados de macroalgas. De entre estes, destacam-se os florotaninos, que para além das propriedades antioxidantes têm demonstrado possuir outras atividades farmacológicas importantes, tais como atividade antibacteriana, anti-viral, antineoplásica, anti-hipertensora e anti-diabética. Este trabalho consiste numa revisão bibliográfica sobre os diversos compostos fenólicos com atividade antioxidante isolados de macroalgas marinhas e que demonstraram vantagens na sua incorporação, quer em formulações cosméticas, quer em medicamentos, debatendo as suas ações farmacológicas, mecanismos de ação e possíveis aplicações futuras.

New biomass derived carbon catalysts for biomass valorization

Due to diminishing petroleum reserves, unsteady market situation and the environmental concerns associated with utilization of fossil resources, the utilization of renewables for production of energy and chemicals (biorefining) has gained considerable attention. Biomass is the only sustainable source of organic compounds that has been proposed as petroleum equivalent for the production of fuels, chemicals and materials. In fact, it would not be wrong to say that the only viable answer to sustainably convene our future energy and material requirements remain with a bio-based economy with biomass based industries and products. This has prompted biomass valorization (biorefining) to become an important area of industrial research. While many disciplines of science are involved in the realization of this effort, catalysis and knowledge of chemical technology are considered to be particularly important to eventually render this dream to come true. Traditionally, the catalyst research for biomass conversion has been focused primarily on commercially available catalysts like zeolites, silica and various metals (Pt, Pd, Au, Ni) supported on zeolites, silica etc. Nevertheless, the main drawbacks of these catalysts are coupled with high material cost, low activity, limited reusability etc. – all facts that render them less attractive in industrial scale applications (poor activity for the price). Thus, there is a particular need to develop active, robust and cost efficient catalytic systems capable of converting complex biomass molecules. Saccharification, esterification, transesterification and acetylation are important chemical processes in the valorization chain of biomasses (and several biomass components) for production of platform chemicals, transportation fuels, food additives and materials. In the current work, various novel acidic carbons were synthesized from wastes generated from biodiesel and allied industries, and employed as catalysts in the aforementioned reactions. The structure and surface properties of the novel materials were investigated by XRD, XPS, elemental analysis, SEM, TEM, TPD and N2-physisorption techniques. The agro-industrial waste derived sulfonic acid functionalized novel carbons exhibit excellent catalytic activity in the aforementioned reactions and easily outperformed liquid H2SO4 and conventional solid acids (zeolites, ion-exchange resins etc). The experimental results indicated strong influence of catalyst pore-structure (pore size, pore-volume), concentration of –SO3H groups and surface properties in terms of the activity and selectivity of these catalysts. Here, a large pore catalyst with high –SO3H density exhibited the highest esterification and transesterification activity, and was successfully employed in biodiesel production from fatty acids and low grade acidic oils. Also, a catalyst decay model was proposed upon biodiesel production and could explain that the catalyst loses its activity mainly due to active site blocking by adsorption of impurities and by-products. The large pore sulfonated catalyst also exhibited good catalytic performance in the selective synthesis of triacetin via acetylation of glycerol with acetic anhydride and out-performed the best zeolite H-Y with respect to reusability. It also demonstrated equally good activity in acetylation of cellulose to soluble cellulose acetates, with the possibility to control cellulose acetate yield and quality (degree of substitution, DS) by a simple adjustment of reaction time and acetic anhydride concentration. In contrast, the small pore and highly functionalized catalysts obtained by hydrothermal method and from protein rich waste (Jatropha de-oiled waste cake, DOWC), were active and selective in the esterification of glycerol with fatty acids to monoglycerides and saccharification of cellulosic materials, respectively. The operational stability and reusability of the catalyst was found to depend on the stability of –SO3H function (leaching) as well as active site blocking due to adsorption of impurities during the reaction. Thus, our results corroborate the potential of DOWC derived sulfated mesoporous active carbons as efficient integrated solid acid catalysts for valorization of biomass to platform chemicals, biofuel, bio-additive, surfactants and celluloseesters.

In vitro experimental environments lacking or containing soil disparately affect competition experiments of Aspergillus flavus and co-occurring fungi in maize grains

In vitro experimental environments are used to study interactions between microorganisms, and predict dynamics in natural ecosystems. This study highlights that experimental in vitro environments should be selected to closely match the natural environment of interest during in vitro studies to strengthen extrapolations about aflatoxin production by Aspergillus and competing organisms. Fungal competition and aflatoxin accumulation was studied in soil, cotton wool or tube (water-only) environments, for Aspergillus flavus competition with Penicillium purpurogenum, Fusarium oxysporum or Sarocladium zeae within maize grains. Inoculated grains were incubated in each environment at two temperature regimes (25oC and 30oC). Competition experiments showed interaction between main effects of aflatoxin accumulation and environment at 25oC, but not so at 30oC. However, competition experiments showed fungal populations were always interacting with their environments. Fungal survival differed after the 72-hour incubation in different experimental environments. Whereas, all fungi incubated within the soil environment survived; in the cotton-wool environment, none of the competitors of A. flavus survived at 30 oC. With aflatoxin accumulation, F. oxysporum was the only fungus able to interdict aflatoxin production at both temperatures. This occurred only in the soil environment and fumonisins accumulated instead. Smallholder farmers in developing countries face serious mycotoxin contamination of their grains, and soil is a natural reservoir for the associated fungal propagules, and a drying and storage surface for grains on these farms. Studying fungal dynamics in the soil environment and other environments in vitro can provide insights into aflatoxin accumulation post harvest.

Banana peel: an effective biosorbent for aflatoxins

This work reports the application of banana peel as a novel bioadsorbent for in vitro removal of five mycotoxins (aflatoxins (AFB1, AFB2, AFG1, AFG2) and ochratoxin A). The effect of operational parameters including initial pH, adsorbent dose, contact time and temperature were studied in batch adsorption experiments. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and point of zero charge (pHpzc) analysis were used to characterise the adsorbent material. Aflatoxins’ adsorption equilibrium was achieved in 15 min, with highest adsorption at alkaline pH (6–8), while ochratoxin has not shown any significant adsorption due to surface charge repulsion. The experimental equilibrium data were tested by Langmuir, Freundlich and Hill isotherms. The Langmuir isotherm was found to be the best fitted model for aflatoxins, and the maximum monolayer coverage (Q0) was determined to be 8.4, 9.5, 0.4 and 1.1 ng mg−1 for AFB1, AFB2, AFG1 and AFG2 respectively. Thermodynamic parameters including changes in free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) were determined for the four aflatoxins. Free energy change and enthalpy change demonstrated that the adsorption process was exothermic and spontaneous. Adsorption and desorption study at different pH further demonstrated that the sorption of toxins was strong enough to sustain pH changes that would be experienced in the gastrointestinal tract. This study suggests that biosorption of aflatoxins by dried banana peel may be an effective low-cost decontamination method for incorporation in animal feed diets. © 2016 Informa UK Limited, trading as Taylor & Francis Group.

Extraction of betacyanins from Gomphrena globosa L. flowers: choosing na acid as adjuvant

Betalains are plant derived natural pigments that are presently gaining popularity for use as natural colorants in food industry. Although being betalains from red beetroot already used as food colorant (E- 162), these compounds are not as well studied as compared to other natural pigments such as anthocyanins, carotenoids or chlorophylls (I]. Since food additives are on the focus of public interest, it is becoming increasingly important to meet consumers' expectations for natural and healthy products. Hence, the search for new plant-derived colorants for the food industry is still necessary [2]. Betalains were originally called 'nitrogenous anthocyanins', which incorrectly implied structural similarities between the two pigment classes. There are two structurally different types of betalains: the yellow/orange betaxanthins which are the condensation products of betalamic acid and assorted amino compounds, and the red betacyanins which are formed by glycosylation and acylation of cyclo-DOPA [3]. Looking at the chemical structure of the pigment, the addition of an acid to the extraction solvent will increase the affinity of the pigment with the solvent. The aim of this study was to use Gomphrena globosa L. flowers, as an alternative plant source to obtain these pigments and to evaluate the best acid to be used within the extraction procedure. For that purpose three different acids (acetic, hydrochloric and phosphoric acids, all ofthem allowed by the food industry), adjusted at the same pH, were tested during a maceration extraction procedure. After the extraction a purification through C18 column was performed in order to obtain a more concentrate extract in betacyanins. The results were analysed by HPLC-PDA-MSIESI. The betacyanin profile allowed the identification of gomphrenin IIJIII and isogomphrenin IIIIII and the best results were achieved by performing the extraction procedure using hydrochloric acid (6.6 mg/g extract), while phosphoric acid only presented trace amounts of these compounds. When acetic acid was used, the pigment extracted was 6.8 times less (0.97 mg/g extract) when compared to HCI. In conclusion hydrochloric acid can be considered the most suitable acid to be applied in the extraction procedure of these pigments.

Effects of aluminum on cells and tissues

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X-ray fluorescence spectroscopic determination of heavy metals and trace elements in aerial parts of Origanum sipyleum L from Turkey

Purpose: To determine the heavy metal and trace element composition of the powdered aerial parts of Origanum sipyleum L. and its water extract. Methods: The heavy metal and trace elements content of the powdered plant material and 2 % aqueous extract were evaluated by x-ray fluorescence spectroscopy with silicon drift detector SDD at a resolution of 145 eV and 10,000 pulses. The process conditions were 0.1 g sample weight, process time of 300 s at a voltage of 25 kV and 50 kV, and at a current of 0.5 and 1.0 mA under helium atmosphere. Results: The major elements, K, Ca and Na, known as macronutrients, constituted 11990, 10490 and 970 ppm of the powdered drug and 8910, 2991 and 810 ppm of the water extract, respectively. Among other constituents, arsenic, lead and uranium levels were < 1, 2.1 and < 3 ppm, respectively, in the powdered material while in the aqueous extract, the levels were < 1, < 2 and 200 ppm, respectively. Conclusion: O. sipyleum is a potential source of macro- and micronutrients from which useful food additives and health supplements can be derived.

Natural additives and agricultural wastes in biopolymer formulations for food packaging

The main directions in food packaging research are targeted toward improvements in food quality and food safety. For this purpose, food packaging providing longer product shelf-life, as well as the monitoring of safety and quality based upon international standards, is desirable. New active packaging strategies represent a key area of development in new multifunctional materials where the use of natural additives and/or agricultural wastes is getting increasing interest. The development of new materials, and particularly innovative biopolymer formulations, can help to address these requirements and also with other packaging functions such as: food protection and preservation, marketing and smart communication to consumers. The use of biocomposites for active food packaging is one of the most studied approaches in the last years on materials in contact with food. Applications of these innovative biocomposites could help to provide new food packaging materials with improved mechanical, barrier, antioxidant, and antimicrobial properties. From the food industry standpoint, concerns such as the safety and risk associated with these new additives, migration properties and possible human ingestion and regulations need to be considered. The latest innovations in the use of these innovative formulations to obtain biocomposites are reported in this review. Legislative issues related to the use of natural additives and agricultural wastes in food packaging systems are also discussed.