Biodiscovery of natural products from microbes associated with Irish coastal sponges

Marine sponges have been an abundant source of new metabolites in recent years. The symbiotic association between the bacteria and the sponge has enabled scientists to access the bacterial diversity present within the bacterial/sponge ecosystem. This study has focussed on accessing the bacterial diversity in two Irish coastal marine sponges, namely Amphilectus fucorum and Eurypon major. A novel species from the genus Aquimarina has been isolated from the sponge Amphilectus fucorum. The study has also resulted in the identification of an α–Proteobacteria, Pseudovibrio sp. as a potential producer of antibiotics. Thus a targeted based approach to specifically cultivate Pseudovibrio sp. may prove useful for the development of new metabolites from this particular genus. Bacterial isolates from the marine sponge Haliclona simulans were screened for anti–fungal activity and one isolate namely Streptomyces sp. SM8 displayed activity against all five fungal strains tested. The strain was also tested for anti–bacterial activity and it showed activity against both against B. subtilis and P. aeruginosa. Hence a combinatorial approach involving both biochemical and genomic approaches were employed in an attempt to identify the bioactive compounds with these activities which were being produced by this strain. Culture broths from Streptomyces sp. SM8 were extracted and purified by various techniques such as reverse–phase HPLC, MPLC and ash chromatography. Anti–bacterial activity was observed in a fraction which contained a hydroxylated saturated fatty acid and also another compound with a m/z 227 but further structural elucidation of these compounds proved unsuccessful. The anti–fungal fractions from SM8 were shown to contain antimycin–like compounds, with some of these compounds having different retention times from that of an antimycin standard. A high–throughput assay was developed to screen for novel calcineurin inhibitors using yeast as a model system and three putative bacterial extracts were found to be positive using this screen. One of these extracts from SM8 was subsequently analysed using NMR and the calcineurin inhibition activity was con rmed to belong to a butenolide type compound. A H. simulans metagenomic library was also screened using the novel calcineurin inhibitor high–throughput assay system and eight clones displaying putative calcineurin inhibitory activity were detected. The clone which displayed the best inhibitory activity was subsequently sequenced and following the use of other genetic based approaches it became clear that the inhibition was being caused by a hypothetical protein with similarity to a hypothetical Na+/Ca2+ exchanger protein. The Streptomyces sp. SM8 genome was sequenced from a fragment library using Roche 454 pyrosequencing technology to identify potential secondary metabolism clusters. The draft genome was annotated by IMG/ER using the Prodigal pipeline. The Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMPN00000000. The genome contains genes which appear to encode for several polyketide synthases (PKS), non–ribosomal peptide synthetases (NRPS), terpene and siderophore biosynthesis and ribosomal peptides. Transcriptional analyses led to the identification of three hybrid clusters of which one is predicted to be involved in the synthesis of antimycin, while the functions of the others are as yet unknown. Two NRPS clusters were also identified, of which one may be involved in gramicidin biosynthesis and the function of the other is unknown. A Streptomyces sp. SM8 NRPS antC gene knockout was constructed and extracts from the strain were shown to possess a mild anti–fungal activity when compared to the SM8 wild–type. Subsequent LCMS analysis of antC mutant extracts confirmed the absence of the antimycin in the extract proving that the observed anti–fungal activity may involve metabolite(s) other than antimycin. Anti–bacterial activity in the antC gene knockout strain against P. aeruginosa was reduced when compared to the SM8 wild–type indicating that antimycin may be contributing to the observed anti–bacterial activity in addition to the metabolite(s) already identified during the chemical analyses. This is the first report of antimycins exhibiting anti–bacterial activity against P. aeruginosa. One of the hybrid clusters potentially involved in secondary metabolism in SM8 that displayed high and consistent levels of gene–expression in RNA studies was analysed in an attempt to identify the metabolite being produced by the pathway. A number of unusual features were observed following bioinformatics analysis of the gene sequence of the cluster, including a formylation domain within the NRPS cluster which may add a formyl group to the growing chain. Another unusual feature is the lack of AT domains on two of the PKS modules. Other unusual features observed in this cluster is the lack of a KR domain in module 3 of the cluster and an aminotransferase domain in module 4 for which no clear role has been hypothesised.

Empirical analysis and improved modelling of natural gas demand in Ireland

Countries across the world are being challenged to decarbonise their energy systems in response to diminishing fossil fuel reserves, rising GHG emissions and the dangerous threat of climate change. There has been a renewed interest in energy efficiency, renewable energy and low carbon energy as policy‐makers seek to identify and put in place the most robust sustainable energy system that can address this challenge. This thesis seeks to improve the evidence base underpinning energy policy decisions in Ireland with a particular focus on natural gas, which in 2011 grew to have a 30% share of Ireland’s TPER. Natural gas is used in all sectors of the Irish economy and is seen by many as a transition fuel to a low-carbon energy system; it is also a uniquely excellent source of data for many aspects of energy consumption. A detailed decomposition analysis of natural gas consumption in the residential sector quantifies many of the structural drives of change, with activity (R2 = 0.97) and intensity (R2 = 0.69) being the best explainers of changing gas demand. The 2002 residential building regulations are subject to an ex-post evaluation, which using empirical data finds a 44 ±9.5% shortfall in expected energy savings as well as a 13±1.6% level of non-compliance. A detailed energy demand model of the entire Irish energy system is presented together with scenario analysis of a large number of energy efficiency policies, which show an aggregate reduction in TFC of 8.9% compared to a reference scenario. The role for natural gas as a transition fuel over a long time horizon (2005-2050) is analysed using an energy systems model and a decomposition analysis, which shows the contribution of fuel switching to natural gas to be worth 12 percentage points of an overall 80% reduction in CO2 emissions. Finally, an analysis of the potential for CCS in Ireland finds gas CCS to be more robust than coal CCS for changes in fuel prices, capital costs and emissions reduction and the cost optimal location for a gas CCS plant in Ireland is found to be in Cork with sequestration in the depleted gas field of Kinsale.

The right to humanitarian assistance in natural and human-made disasters: progress and challenges for an emerging international legal framework

Natural and human-made disasters cause on average 120,000 deaths and over US$140 billion in damage to property and infrastructure every year, with national, regional and international actors consistently responding to the humanitarian imperative to alleviate suffering wherever it may be found. Despite various attempts to codify international disaster laws since the 1920s, a right to humanitarian assistance remains contested, reflecting concerns regarding the relative importance of state sovereignty vis-à-vis individual rights under international law. However, the evolving acquis humanitaire of binding and non-binding normative standards for responses to humanitarian crises highlights the increasing focus on rights and responsibilities applicable in disasters; although the International Law Commission has also noted the difficulty of identifying lex lata and lex ferenda regarding the protection of persons in the event of disasters due to the “amorphous state of the law relating to international disaster response.” Therefore, using the conceptual framework of transnational legal process, this thesis analyses the evolving normative frameworks and standards for rights-holders and duty-bearers in disasters. Determining the process whereby rights are created and evolve, and their potential internalisation into domestic law and policy, provides a powerful analytical framework for examining the progress and challenges of developing accountable responses to major disasters.

The synthesis and biological evaluation of lanostane and cholestane-type natural products

The main objective of this thesis is to outline the synthetic chemistry involved in the preparation of a range of novel lanostane and cholestane derivatives, and subsequent investigation into their biological activity in cancer cells. The biological results obtained throughout the project have driven the strategic synthesis of new compounds, in an effort to optimise the anti cancer potential of lanostane and cholestane derivatives. The first chapter begins with an overview of steroidal compounds and details a literature review of the natural sources of these moieties, as well as their biosynthesis and reported synthetic derivatives. The biological activity of interesting natural and synthetic analogues is also discussed. In addition, an insight into some currently prescribed pharmaceutical compounds, with functional groups relevant to this project, is presented. The second chapter discusses the methods employed for the synthesis of these novel lanostane and cholestane derivatives, and comprises three main sections. Firstly, various oxidation products of lanosterol are synthesised, mainly via epoxidations of the C-8,9 and C- 24,25 alkenes, and also allylic oxidations at these positions. Secondly, amine derivatives of lanosterol are formed by cleaving the lanostane side chain, thereby yielding a new cholestane nucleus, and performing several reductive aminations on the resulting key aldehyde intermediates. Various amines such as piperidine, morpholine, diethylamine and aniline are employed in the reductive amination reactions to yield novel cholestane steroids with amine side chains. Finally, starting from stigmasterol and proceeding with the same methodology of cleaving the steroidal side chain and subsequently performing reductive aminations, novel cholestane derivatives of the biologically active amines are synthesised. The cytotoxicity of these compounds against CaCo-2 and U937 cell lines is presented in terms of percentage viability of cells, IC50 value and apoptosis. The MTT assay is used to determine the percentage viability of cells, and the IC50 data is generated from the MTT results. Apoptosis is measured in terms of fold increase relative to a carrier control. In summary, the compounds formed are discussed in terms of chemical synthesis, spectroscopic interpretation and biological activity. The main reaction pathways involved in the chemistry within this project are various oxidations and reductive amination. The final chapter is a detailed account of the full experimental procedures for the compounds synthesised during this work, including characterisation using spectroscopic and analytical data.

The treatment of landfill leachate using natural systems

Leachate may be defined as any liquid percolating through deposited waste and emitted from or contained within a landfill. If leachate migrates from a site it may pose a severe threat to the surrounding environment. Increasingly stringent environmental legislation both at European level and national level (Republic of Ireland) regarding the operation of landfill sites, control of associated emissions, as well as requirements for restoration and aftercare management (up to 30 years) has prompted research for this project into the design and development of a low cost, low maintenance, low technology trial system to treat landfill leachate at Kinsale Road Landfill Site, located on the outskirts of Cork city. A trial leachate treatment plant was constructed consisting of 14 separate treatment units (10 open top cylindrical cells [Ø 1.8 m x 2.0 high] and four reed beds [5.0m x 5.0m x 1.0m]) incorporating various alternative natural treatment processes including reed beds (vertical flow [VF] and horizontal flow [HF]), grass treatment planes, compost units, timber chip units, compost-timber chip units, stratified sand filters and willow treatment plots. High treatment efficiencies were achieved in units operating in sequence containing compost and timber chip media, vertical flow reed beds and grass treatment planes. Pollutant load removal rates of 99% for NH4, 84% for BOD5, 46% for COD, 63% for suspended solids, 94% for iron and 98% for manganese were recorded in the final effluent of successfully operated sequences at irrigation rates of 945 l/m2/day in the cylindrical cells and 96 l/m2/day in the VF reed beds and grass treatment planes. Almost total pathogen removal (E. coli) occurred in the final effluent of the same sequence. Denitrification rates of 37% were achieved for a limited period. A draft, up-scaled leachate treatment plant is presented, based on treatment performance of the trial plant.

Engineering design of a bioprocess for the production of natural red colourants by submerged fermentation of the thermophilic fungus Penicillium purpurogenum GH2

The development of a new bioprocess requires several steps from initial concept to a practical and feasible application. Industrial applications of fungal pigments will depend on: (i) safety of consumption, (ii) stability of the pigments to the food processing conditions required by the products where they will be incorporated and (iii) high production yields so that production costs are reasonable. Of these requirements the first involves the highest research costs and the practical application of this type of processes may face several hurdles until final regulatory approval as a new food ingredient. Therefore, before going through expensive research to have them accepted as new products, the process potential should be assessed early on, and this brings forward pigment stability studies and process optimisation goals. Only ingredients that are usable in economically feasible conditions should progress to regulatory approval. This thesis covers these two aspects, stability and process optimisation, for a potential new ingredient; natural red colour, produced by microbial fermentation. The main goal was to design, optimise and scale-up the production process of red pigments by Penicillium purpurogenum GH2. The approach followed to reach this objective was first to establish that pigments produced by Penicillium purpurogenum GH2 are sufficiently stable under different processing conditions (thermal and non-thermal) that can be found in food and textile industries. Once defined that pigments were stable enough, the work progressed towards process optimisation, aiming for the highest productivity using submerged fermentation as production culture. Optimum production conditions defined at flask scale were used to scale up the pigment production process to a pilot reactor scale. Finally, the potential applications of the pigments were assessed. Based on this sequence of specific targets, the thesis was structured in six parts, containing a total of nine chapters. Engineering design of a bioprocess for the production of natural red colourants by submerged fermentation of the thermophilic fungus Penicillium purpurogenum GH2.

Phosphorus export from a restored wetland ecosystem in response to natural and experimental hydrologic fluctuations

Wetland restoration is a commonly used approach to reduce nutrient loading to freshwater and coastal ecosystems, with many wetland restoration efforts occurring in former agricultural fields. Restored wetlands are expected to be effective at retaining or removing both nitrogen and phosphorus (P), yet restoring wetland hydrology to former agricultural fields can lead to the release of legacy fertilizer P. Here, we examined P cycling and export following rewetting of the Timberlake Restoration Project, a 440 ha restored riverine wetland complex in the coastal plain of North Carolina. We also compared P cycling within the restored wetland to two minimally disturbed nearby wetlands and an adjacent active agricultural field. In the restored wetland we observed increased soluble reactive phosphorus (SRP) concentrations following initial flooding, consistent with our expectations that P bound to iron would be released under reducing conditions. SRP concentrations in spring were 2.5 times higher leaving the restored wetland than a forested wetland and an agricultural field. During two large-scale drawdown and rewetting experiments we decreased the water depth by 1 m in ∼10 ha of inundated wetland for 2 weeks, followed by reflooding. Rewetting following experimental drainage had no effect on SRP concentrations in winter, but SRP concentrations did increase when the experiment was repeated during summer. Our best estimates suggest that this restored wetland could release legacy fertilizer P for up to a decade following hydrologic restoration. The time lag between restoration and biogeochemical recovery should be incorporated into management strategies of restored wetlands. Copyright 2010 by the American Geophysical Union.

Natural streamflow regime alterations: Damming of the Piave river basin (Italy)

A novel approach is proposed to estimate the natural streamflow regime of a river and to assess the extent of the alterations induced by dam operation related to anthropogenic (e.g., agricultural, hydropower) water uses in engineered river basins. The method consists in the comparison between the seasonal probability density function (pdf) of observed streamflows and the purportedly natural streamflow pdf obtained by a recently proposed and validated probabilistic model. The model employs a minimum of landscape and climate parameters and unequivocally separates the effects of anthropogenic regulations from those produced by hydroclimatic fluctuations. The approach is applied to evaluate the extent of the alterations of intra-annual streamflow variability in a highly engineered alpine catchment of north-eastern Italy, the Piave river. Streamflows observed downstream of the regulation devices in the Piave catchment are found to exhibit smaller means/modes, larger coefficients of variation, and more pronounced peaks than the flows that would be observed in the absence of anthropogenic regulation, suggesting that the anthropogenic disturbance leads to remarkable reductions of river flows, with an increase of the streamflow variability and of the frequency of preferential states far from the mean. Some structural limitations of management approaches based on minimum streamflow requirements (widely used to guide water policies) as opposed to criteria based on whole distributions are also discussed. Copyright © 2010 by the American Geophysical Union.

The potential of natural, photosynthetic pigments to improve the efficiency of dye-sensitized solar cells

Gemstone Team Grenergy

Natural hybridization between genera that diverged from each other approximately 60 million years ago.

A fern from the French Pyrenees-×Cystocarpium roskamianum-is a recently formed intergeneric hybrid between parental lineages that diverged from each other approximately 60 million years ago (mya; 95% highest posterior density: 40.2-76.2 mya). This is an extraordinarily deep hybridization event, roughly akin to an elephant hybridizing with a manatee or a human with a lemur. In the context of other reported deep hybrids, this finding suggests that populations of ferns, and other plants with abiotically mediated fertilization, may evolve reproductive incompatibilities more slowly, perhaps because they lack many of the premating isolation mechanisms that characterize most other groups of organisms. This conclusion implies that major features of Earth's biodiversity-such as the relatively small number of species of ferns compared to those of angiosperms-may be, in part, an indirect by-product of this slower "speciation clock" rather than a direct consequence of adaptive innovations by the more diverse lineages.

Temporal Stability of Molecular Diversity Measures in Natural Populations of Drosophila pseudoobscura and Drosophila persimilis

Many molecular ecological and evolutionary studies sample wild populations at a single point in time, failing to consider that data they collect represents genetic variation from a potentially unrepresentative snapshot in time. Variation across time in genetic parameters may occur quickly in species that produce multiple generations of offspring per year. However, many studies of rapid contemporary microevolution examine phenotypic trait divergence as opposed to molecular evolutionary divergence. Here, we compare genetic diversity in wild caught populations of Drosophila persimilis and D. pseudoobscura collected 16 years apart at the same time of year and same site at four X-linked and two mitochondrial loci to assess genetic stability. We found no major changes in nucleotide diversity in either species, but we observed a drastic shift in Tajima’s D between D. pseudoobscura timepoints at one locus associated with the increased abundance of a set of related haplotypes. Our data also suggests that D. persimilis may have recently accelerated its demographic expansion. While the changes we observed were modest, this study reinforces the importance of considering potential temporal variation in genetic parameters within single populations over short evolutionary timescales.

Conversion of natural forests to managed forest plantations decreases tree resistance to prolonged droughts

© 2015 Published by Elsevier B.V.Throughout the southern US, past forest management practices have replaced large areas of native forests with loblolly pine plantations and have resulted in changes in forest response to extreme weather conditions. However, uncertainty remains about the response of planted versus natural species to drought across the geographical range of these forests. Taking advantage of a cluster of unmanaged stands (85-130year-old hardwoods) and managed plantations (17-20year-old loblolly pine) in coastal and Piedmont areas of North Carolina, tree water use, cavitation resistance, whole-tree hydraulic (Ktree) and stomatal (Gs) conductances were measured in four sites covering representative forests growing in the region. We also used a hydraulic model to predict the resilience of those sites to extreme soil drying. Our objectives were to determine: (1) if Ktree and stomatal regulation in response to atmospheric and soil droughts differ between species and sites; (2) how ecosystem type, through tree water use, resistance to cavitation and rooting profiles, affects the water uptake limit that can be reached under drought; and (3) the influence of stand species composition on critical transpiration that sets a functional water uptake limit under drought conditions. The results show that across sites, water stress affected the coordination between Ktree and Gs. As soil water content dropped below 20% relative extractable water, Ktree declined faster and thus explained the decrease in Gs and in its sensitivity to vapor pressure deficit. Compared to branches, the capability of roots to resist high xylem tension has a great impact on tree-level water use and ultimately had important implications for pine plantations resistance to future summer droughts. Model simulations revealed that the decline in Ktree due to xylem cavitation aggravated the effects of soil drying on tree transpiration. The critical transpiration rate (Ecrit), which corresponds to the maximum rate at which transpiration begins to level off to prevent irreversible hydraulic failure, was higher in managed forest plantations than in their unmanaged counterparts. However, even with this higher Ecrit, the pine plantations operated very close to their critical leaf water potentials (i.e. to their permissible water potentials without total hydraulic failure), suggesting that intensively managed plantations are more drought-sensitive and can withstand less severe drought than natural forests.

Respuestas fenológicas de dos gramíneas C3 y dos C4, del pastizal natural de San Luis (Argentina), a variaciones de agua y temperatura

La temperatura y las precipitaciones son factores claves en la fenología de especies vegetales y su productividad. Los pastizales de la provincia de San Luis son especialmente interesantes porque coexisten gramíneas con vías metabólicas C3 (invernales) y C4 (estivales). Para evaluar cómo se relacionan la producción y la fenología reproductiva de las especies frente a cambios de temperatura y disponibilidad de agua, evalué cuatro gramíneas representativas de la región (dos C3 y dos C4) en dos estudios: 1) observacional a campo, y 2) manipulativo en invernáculo con dos niveles de temperatura y dos niveles de agua. En el estudio observacional comparé diez años de relevamientos fenológicos históricos realizados entre 1976 y 1986, con relevamientos fenológicos propios entre 2008 y 2010, todos llevados a cabo sobre el mismo sitio de estudio. En esta comparación encontré que una de las cuatro especies (Poa ligularis), atrasó significativamente su ciclo reproductivo (38 días la floración y 16 días la diseminación de semillas). La temperatura tuvo menor efecto sobre la producción, pero reflejó un claro control sobre la fenología en las cuatro especies estudiadas. En general, altas temperaturas estivales atrasaron el fin y aumentaron el largo del ciclo reproductivo de todas las especies. En cambio, primaveras más cálidas adelantaron el inicio reproductivo de las C4, pero retrasaron el de las C3. Por otra parte, la mayor disponibilidad de agua incrementó la producción, adelantó el comienzo floral y extendió entre 15 y 30 días el ciclo reproductivo de las cuatro especies. Inviernos lluviosos adelantaron el inicio floral de las especies C3, mientras que años con elevadas precipitaciones estivales retrasaron y extendieron el ciclo reproductivo de las C4. Estos resultados aportan valiosa información sobre las respuestas de la vegetación al clima, y pueden servir de insumo en el diseño de estrategias de manejo sustentable de estos pastizales.

Plan de marketing para la inserción de una bebida natural a base de yacón con piña, en el mercado de Lima, Perú

El consumo de productos naturales en el mundo es cada vez más creciente, y dentro de ello las bebidas funcionales que desde sus orígenes han tenido una performance relativamente satisfactoria. El yacón (Smallanthus sochifolius)es una planta perene, herbácea, que crece en toda la serranía peruana. Su demanda en el mercado exterior ha hecho que cada vez tenga mayor importancia. En el 2014 se exportó USD 2 899.398,00 en forma de polvo, jarabe, deshidratado; sin embargo su consumo en el mercado nacional es bajo, a pesar del gran potencial nutritivo que posee. El mercado de Lima moderna se presenta como un importante atractivo de mercado que podría consumir la bebida. Por ello el objetivo del presente trabajo es Realizar un Plan de Marketing para el mercado de Lima moderna a fin de evaluar la factibilidad de insertar una bebida nueva: yacón con piña, para fomentar el consumo interno de este producto. La metodología utilizada fue de carácter exploratorio, descriptivo cuantitativa y cualitativa mediante información primaria y secundaria que ayudó a su desarrollo. Dentro de la información primaria se utilizó como herramienta la encuesta y la información secundaria a través de informes y estudios que se hicieron para el sector de bebidas no alcohólicas. El sector de bebidas no alcohólicas en el Perú, muestra un importante crecimiento en lo referente a bebidas saludables. En el último año (2014) el crecimiento de este sector fue de 6 por ciento, motivados por la percepción del consumidor en los beneficios saludables de estas bebidas. La zona de Lima moderna acoge al 72,2 por ciento del nivel socio económico A y B, este segmento concentra el más alto promedio de ingresos. En el análisis de Porter, se describe a la empresa como una industria, el cual para este rubro las barreras de ingreso son altas por la inversión en activos físicos y la inversión en publicidad y promoción por ser un producto nuevo. A través del estudio realizado se encontró que existe demanda para este producto, el 31,1 por ciento de los encuestados compraría definitivamente, 25,5 por ciento una vez por semana y el 18,3 por ciento todos los días. La estrategia utilizada en el presente trabajo fue la diferenciación, por las características saludables de la bebida y única en el mercado. La bebida es un producto de introducción en el mercado, el precio fijado es alto porque se orienta a un segmento de mercado selectivo y además fijado en la imagen y calidad del producto. La distribución se realizará a través de los supermercados, minimarkets, hoteles categorizados. Y por último se concluye que la introducción de un producto novedoso, podría marcar estándares en el mercado de bebidas no alcohólicas y permitir la creación de una nueva categoría de productos Premium naturales y funcionales.