Paul D Fraser

Royal Holloway, University of London, Эгхем, England, United Kingdom

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Publications (121)512.35 Total impact

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    ABSTRACT: Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.
    New Phytologist 07/2015; 208(1). DOI:10.1111/nph.13532 · 7.67 Impact Factor
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    ABSTRACT: To produce commercially valuable ketocarotenoids in Solanum tuberosum, the 4, 4' β-oxygenase (crtW) and 3, 3' β-hydroxylase (crtZ) genes from Brevundimonas spp. have been expressed in the plant host under constitutive transcriptional control. The CRTW and CRTZ enzymes are capable of modifying endogenous plant carotenoids to form a range of hydroxylated and ketolated derivatives. The host (cv. Désirée) produced significant levels of nonendogenous carotenoid products in all tissues, but at the apparent expense of the economically critical metabolite, starch. Carotenoid levels increased in both wild-type and transgenic tubers following cold storage; however, stability during heat processing varied between compounds. Subcellular fractionation of leaf tissues revealed the presence of ketocarotenoids in thylakoid membranes, but not predominantly in the photosynthetic complexes. A dramatic increase in the carotenoid content of plastoglobuli was determined. These findings were corroborated by microscopic analysis of chloroplasts. In tuber tissues, esterified carotenoids, representing 13% of the total pigment found in wild-type extracts, were sequestered in plastoglobuli. In the transgenic tubers, this proportion increased to 45%, with esterified nonendogenous carotenoids in place of endogenous compounds. Conversely, nonesterified carotenoids in both wild-type and transgenic tuber tissues were associated with amyloplast membranes and starch granules. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
    Plant Biotechnology Journal 04/2015; DOI:10.1111/pbi.12365 · 5.75 Impact Factor
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    ABSTRACT: The aim of this study was to assess whether endosperm-specific carotenoid biosynthesis influenced core metabolic processes in maize embryo and endosperm and how global seed metabolism adapted to this expanded biosynthetic capacity. Although enhancement of carotenoid biosynthesis was targeted to the endosperm of maize kernels, a concurrent up-regulation of sterol and fatty acid biosynthesis in the embryo was measured. Targeted terpenoid analysis, and non-targeted metabolomic, proteomic, and transcriptomic profiling revealed changes especially in carbohydrate metabolism in the transgenic line. In-depth analysis of the data, including changes of metabolite pools and increased enzyme and transcript concentrations, gave a first insight into the metabolic variation precipitated by the higher up-stream metabolite demand by the extended biosynthesis capacities for terpenoids and fatty acids. An integrative model is put forward to explain the metabolic regulation for the increased provision of terpenoid and fatty acid precursors, particularly glyceraldehyde 3-phosphate and pyruvate or acetyl-CoA from imported fructose and glucose. The model was supported by higher activities of fructokinase, glucose 6-phosphate isomerase, and fructose 1,6-bisphosphate aldolase indicating a higher flux through the glycolytic pathway. Although pyruvate and acetyl-CoA utilization was higher in the engineered line, pyruvate kinase activity was lower. A sufficient provision of both metabolites may be supported by a by-pass in a reaction sequence involving phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.
    Journal of Experimental Botany 03/2015; 66(11). DOI:10.1093/jxb/erv120 · 5.53 Impact Factor
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    ABSTRACT: Proteolysis is a complex and dynamic process which takes place throughout the whole dry-cured processing due to the action of endogenous muscle peptidases, and results in the generation of a high number of small peptides and free amino acids responsible for the final quality of dry-cured ham. In this study, a total of sixty-eight peptides derived from the ubiquitin-60S ribosomal protein have been identified in dry-cured ham at 2, 3.5, 5, 6.5, and 9 months of processing using various chromatographic separations and a quadrupole/time-of-flight mass spectrometer in tandem. Some of the identified peptides have been detected during the whole process, whereas a total of fourteen of them were exclusively identified at 9 months of curing. The presence of any of these peptides could be a good indicative that dry-cured ham pieces have reached a minimum curing process of 9 months. The study of the generated peptides has contributed both to a better knowledge of proteolysis evolution and the endogenous enzymes participating, and to determine their potential to be used as quality markers to monitor the processing time.
    Food Control 02/2015; 48:102-107. DOI:10.1016/j.foodcont.2013.12.029 · 2.81 Impact Factor
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    ABSTRACT: Astaxanthin is a high value carotenoid produced by some bacteria, a few green algae, several fungi but only a limited number of plants from the genus Adonis. Astaxanthin has been industrially exploited as a feed supplement in poultry farming and aquaculture. Consumption of ketocarotenoids, most notably astaxanthin, is also increasingly associated with a wide range of health benefits, as demonstrated in numerous clinical studies. Currently astaxanthin is produced commercially by chemical synthesis or from algal production systems. Several studies have used a metabolic engineering approach to produce astaxanthin in transgenic plants. Previous attempts to produce transgenic potato tubers biofortified with astaxanthin have met with limited success. In this study we have investigated approaches to optimising tuber astaxanthin content. It is demonstrated that the selection of appropriate parental genotype for transgenic approaches and stacking carotenoid biosynthetic pathway genes with the cauliflower Or gene result in enhanced astaxanthin content, to give six-fold higher tuber astaxanthin content than has been achieved previously. Additionally we demonstrate the effects of growth environment on tuber carotenoid content in both wild type and astaxanthin-producing transgenic lines and describe the associated transcriptome and metabolome restructuring. Crown Copyright © 2015. Published by Elsevier Ireland Ltd. All rights reserved.
    Plant Science 02/2015; 234. DOI:10.1016/j.plantsci.2015.01.014 · 3.61 Impact Factor
  • Margit Drapal · Laura Perez-Fons · Paul R. Wheeler · Paul D. Fraser ·
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    ABSTRACT: In order to decipher the complex biological networks underlying biochemical and physiological processes, cellular regulation at all levels must be studied. The metabolites determined by metabolomics represent the end-point of cellular regulation and thus vital components of any integrative network. In the case of pathogenic agents such as M. tuberculosis metabolomics offers an ideal opportunity to gain a better understanding of how this species adapts to environmental conditions and antimicrobial treatments. In the present study a metabolite profiling protocol for Mycobacterium including optimised quenching, extraction and analysis has been devised. These methods have been applied to three different Mycobacterium spp demonstrating potential translation across the genus. Steady-state levels of metabolites during growth have been determined for M. smegmatis, M. phlei and M. bovis BCG (Bacillus Calmette-Guérin). The changes of designated biomarkers emphasised phenotypical differences (e.g. nitrogen metabolism) and similarities (e.g. cysteine biosynthesis) between the bacteria. Each time point showed distinguishable metabolic characteristics from early lag to late stationary phase/beginning of non-replicating phase. The combination of the metabolic results with published "omics" data indicated that transcription appeared to be the most predominant mode of cellular regulation utilised by these bacteria studied.
    Journal of Microbiological Methods 11/2014; 106. DOI:10.1016/j.mimet.2014.07.037 · 2.03 Impact Factor
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    ABSTRACT: Bacillus indicus and Bacillus firmus synthesize C30 carotenoids via farnesyl pyrophosphate (FPP) forming apophytoene the first committed step in the pathway. The end products of the pathways are derivedvia diapolycopen-monoic acid or diapolycopen-dioic acid, respectively, as intermediates. The genomes of both bacteria have been sequenced and the genes for their early terpenoid and specific carotenoid pathways annotated. All genes for a functional 1-deoxy-D-xylulose 5-phosphate synthase pathway were identified in both species whereas the genes of the mevalonate pathway were absent. The gene for direct carotenoid synthesis and conversion were found on a gene cluster which are differently organised. The genes involved in the formation of the carotenoid cores have been assigned by functional complementation in Escherichia coli. This bacterium was co-transformed with a plasmid mediating the formation of the putative substrate and a second plasmid with the gene of interest. Carotenoid products in the transformants were determined by HPLC. By this approach, the genes for a diapophytoene synthase, crtM, for a diapophytoene desaturase, crtNa, for a diapolycopene ketolase, crtNb, and for a diapolycopene aldehyde oxidase, crtNc, were identified. The three crtN genes are closely related and belong to the crtI gene family with a similar reaction mechanism of their enzyme products. Additional genes for the modification of the carotenoid skeleton, diapolycopenoic acids encoding glycosyl transferases and acyl transferases were identified in comparison to the corresponding genes from other bacteria.
    Microbiology 10/2014; 161(Pt_1). DOI:10.1099/mic.0.083519-0 · 2.56 Impact Factor
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    The Journal of Antibiotics 10/2014; 67(10):731-732. DOI:10.1038/ja.2014.52 · 1.73 Impact Factor
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    ABSTRACT: Application of mass spectrometry enables the detection of metabolic differences between groups of related organisms. Differences in the metabolic fingerprints of wild-type Solanum lycopersicum and three monogenic mutants, ripening inhibitor (rin), non-ripening (nor) and Colourless non-ripening (Cnr), of tomato are captured with regard to ripening behaviour. A high-resolution tandem mass spectrometry system coupled to liquid chromatography produced a time series of the ripening behaviour at discrete intervals with a focus on changes post-anthesis. Internal standards and quality controls were used to ensure system stability. The raw data of the samples and reference compounds including study protocols have been deposited in the open metabolomics database MetaboLights via the metadata annotation tool Isatab to enable efficient re-use of the datasets, such as in metabolomics cross-study comparisons or data fusion exercises.
    09/2014; Scientific Data 1, Article number: 140029(1):140029. DOI:10.1038/sdata.2014.29
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    ABSTRACT: The global demand for food, feed, energy and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.
    Journal of Visualized Experiments 05/2014; DOI:10.3791/51393 · 1.33 Impact Factor
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    ABSTRACT: Extensive proteolysis takes place during the processing of dry-cured ham due to the action of muscle peptidases. The aim of this work was to study the degradation of LIM domain binding protein 3 (LDB3), which is located at the Z-lines of the sarcomere, at different times during the Spanish dry-cured ham processing (2, 3.5, 5, 6.5, and 9months). A total of 107 peptides have been identified by mass spectrometry, most of them generated from the first region of the protein sequence (position 1-90) providing evidence for the complexity and variability of proteolytic reactions throughout the whole process of dry-curing. Methionine oxidation has been observed in several peptides by the end of the process. The potential of some of the identified peptides to be used as biomarkers of dry-cured ham processing has also been considered.
    Food Chemistry 04/2014; 149:121-8. DOI:10.1016/j.foodchem.2013.10.076 · 3.39 Impact Factor
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    N. Dağüstü · P. Fraser · E. Enfıssi · P. Bramley ·
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    ABSTRACT: The work described in this paper has used bacterial transgenes (crtI and Hmgr-CoA) which have the potential to increase oil quality in sunflower (Helianthus annuus L.) if an efficient transformation procedure was in place. Optimized procedures for the callus induction from hypocotyl and cotyledon explants, regeneration capacity of sunflower genotypes and transformation to intact embryogenic axis have been established in order to facilitate studies of transformation and production of genetic variability. Callus formation was induced easily from hypocotyl and cotyledon explants. Although cotyledon explants produced low amount of callus per explant, the somatic embryo and direct shoot regeneration capacity of cotyledons were generally much higher than that experienced with hypocotyl explants. Only root regeneration was obtained from hypocotyl explants. Regeneration of embryo and shoot varied from 0 - 29% depended on the genotype and explant. For transformation of sunflower, intact embryogenic axis were dissected from seeds and cocultivated with Agrobacterium tumefaciens. Transgenic sunflower lines expressing either the Erwinia uredovora phytoene desaturase (crtI) gene or hydroxymethylglutaryl-CoA (Hmgr-CoA) reductase genes have been obtained. Possible transformants were selected by their ability to grow on kanamycin. Transformation was confirmed by PCR and nptII. Green shoots were transferred to rooting medium and showed early flowering in in vitro culture conditions. The transformation system is more efficient (90-100%) than previous reports and has shown the incorporation of effector transgenes.
    Biotechnology & Biotechnological Equipment 04/2014; 22(4):933-937. DOI:10.1080/13102818.2008.10817582 · 0.30 Impact Factor
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    ABSTRACT: Plant natural products (PNPs) are unique in that they represent a vast array of different structural features, ranging from relatively simple molecules to very complex ones. Given the fact that many plant secondary metabolites exhibit profound biological activity, they are frequently used as fragrances and flavors, medicines, as well as industrial chemicals. As the intricate structures of PNPs often cannot be mimicked by chemical synthesis, the original plant providers constitute the sole source for their industrial, large-scale production. However, sufficient supply is not guaranteed for all molecules of interest, making the development of alternative production systems a priority. Modern techniques, such as genome mining and thorough biochemical analysis, have helped us gain preliminary understanding of the enzymatic formation of the valuable ingredients in planta. Herein, we review recent advances in the application of biocatalytical processes, facilitating generation of complex PNPs through utilization of plant-derived specific enzymes and combinatorial biochemistry. We further evaluate the options of employing heterologous organisms harboring PNP biosynthetic pathways for the production of secondary metabolites of interest.
    Biotechnology Journal 03/2014; 9(3). DOI:10.1002/biot.201300059 · 3.49 Impact Factor
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    Laura Perez-Fons · Peter M. Bramley · Paul D. Fraser ·
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    ABSTRACT: The rapid advances in sequencing technologies over the last decade have enabled routine sequencing of microbial genomes. Despite notable achievements, metabolomics/metabolite profiling has not progressed with the same rapidity, which in part is due to the intrinsic complex chemical nature of the metabolome. However, well characterised metabolomes are essential if a comprehensive understanding of biological function and biotechnological applications are to be revealed and implemented. In the present study a hyphenated MS metabolite profiling procedure has been developed, predominantly for Bacillus species. The approach has been systematic in its development, delivering optimised procedures for the quenching of bacterial metabolism, extraction of metabolites, the separation and detection of components as well as data analysis, integration and visualisation workflows. Collectively, the procedure has enabled the detection of 27 % of the predicted Bacillus subtilis metabolome in the industrial HU36 strain. The analytical platform developed has been used to assess the chemotype of commercially used probiotic Bacillus strains, including a novel pigmented Bacillus strain HU36 that has potential either as a probiotic or source of antioxidants. The results are discussed in a biochemical context, revealing: (i), specific metabolic networks associated with pigment biosynthesis in HU36 and (ii), biotechnological applications through the demonstration of substantial equivalence.
    Metabolomics 02/2014; 10(1). DOI:10.1007/s11306-013-0553-6 · 3.86 Impact Factor
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    ABSTRACT: The processing of dry-cured ham results in an intense proteolysis that leads to the generation of peptides of different sizes and composition, which have been shown to be biologically active. In this study, a total of ninety-three peptides mainly derived from actin, β-enolase, myosin heavy chain, and creatine kinase proteins have been identified from a size-exclusion chromatography (SEC) fraction that resulted as antioxidant by using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF) and nESI-ion trap mass spectrometry. Several of the identified peptides have been synthesised and their antioxidant activity tested in vitro by using DPPH radical-scavenging assay and reducing power analysis. The peptide with sequence SNAAC showed the best results with an IC50 of 75.2 μM in DPPH radical-scavenging assay and 205 μM in ferric-reducing antioxidant power analysis, very good when comparing with the positive control 2,6-di-tert-butyl-4-methyl phenol (BHT) that showed an IC50 of 358.5 μM and 90.3 μM, respectively, in the different assays. These results suggest that Spanish dry-cured ham represents an important source of powerful antioxidant peptides which due to their natural characteristics may represent a highly valuable alternative in human health.
    Food Research International 02/2014; 56:68–76. DOI:10.1016/j.foodres.2013.12.001 · 2.82 Impact Factor
  • Ron Wehrens · Elisabete Carvalho · Paul D. Fraser ·
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    ABSTRACT: For those chemical compounds absorbing in the UV–Vis region and not readily applicable to routine mass spectrometry ionisation methods, liquid chromatography coupled to diode array detection is a convenient platform to perform metabolite profiling. Data processing by hand is labour-intensive and error prone. In the present study a strategy based on multivariate curve resolution, and its implementation in an R package called alsace are described. The final result of an analysis is a table containing peak heights or peak areas for all features of the individual injections. The capabilities of the software, providing elements such as splitting the data into separate, possibly overlapping time windows, merging the results of the individual time windows, and parametric time warping to align features, are illustrated using a cassava-derived data set.
    Metabolomics 02/2014; 11(1). DOI:10.1007/s11306-014-0683-5 · 3.86 Impact Factor
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    ABSTRACT: Tomato and its processed products are one of the most widely consumed fruits. Its domestication, however, has resulted in the loss of some 95% of the genetic and chemical diversity of wild relatives. In order to elucidate this diversity, exploit its potential for plant breeding, as well as understand its biological significance, analytical approaches have been developed, alongside the production of genetic crosses of wild relatives with commercial varieties. In this article, we describe a multi-platform metabolomic analysis, using NMR, mass spectrometry and HPLC, of introgression lines of Solanum pennellii with a domesticated line in order to analyse and quantify alleles (QTL) responsible for metabolic traits. We have identified QTL for health-related antioxidant carotenoids and tocopherols, as well as molecular signatures for some 2000 compounds. Correlation analyses have revealed intricate interactions in isoprenoid formation in the plastid that can be extrapolated to other crop plants.
    Scientific Reports 01/2014; 4:3859. DOI:10.1038/srep03859 · 5.58 Impact Factor
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    ABSTRACT: Metabolic engineering of the carotenoid pathway in recent years has successfully enhanced the carotenoid contents of crop plants. It is now clear that only increasing biosynthesis is restrictive, as mechanisms to sequestrate these increased levels in the cell or organelle should be exploited. In this study, biosynthetic pathway genes were overexpressed in tomato (Solanum lycopersicum) lines and the effects on carotenoid formation and sequestration revealed. The bacterial Crt carotenogenic genes, independently or in combination, and their zygosity affect the production of carotenoids. Transcription of the pathway genes was perturbed, whereby the tissue specificity of transcripts was altered. Changes in the steady state levels of metabolites in unrelated sectors of metabolism were found. Of particular interest was a concurrent increase of the plastid-localized lipid monogalactodiacylglycerol with carotenoids along with membranous subcellular structures. The carotenoids, proteins, and lipids in the subchromoplast fractions of the transgenic tomato fruit with increased carotenoid content suggest that cellular structures can adapt to facilitate the sequestration of the newly formed products. Moreover, phytoene, the precursor of the pathway, was identified in the plastoglobule, whereas the biosynthetic enzymes were in the membranes. The implications of these findings with respect to novel pathway regulation mechanisms are discussed.
    The Plant Cell 11/2013; 25(11). DOI:10.1105/tpc.113.116210 · 9.34 Impact Factor
  • Leticia Mora · Paul D. Fraser · Fidel Toldrá ·
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    ABSTRACT: Dry-cured meat products experience an intense proteolysis phenomenon during their processing. Products like dry-cured ham, that experience processing times longer than 10 months, show an extensive breakdown of major proteins and the generation of a high number of small peptides and finally, large amounts of free amino acids. Proteomic techniques have been successfully applied to the identification of the generated peptides and their sequencing. These data are essential for a better understanding of proteolytic enzymes and their reaction during processing. This approach should reveal key biomarker peptides controlling the process and establish strategies to drive and optimise enzyme reactions for the production of optimal quality products.
    Food Research International 11/2013; 54(1):1292-1297. DOI:10.1016/j.foodres.2012.09.042 · 2.82 Impact Factor
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    ABSTRACT: The diversity of plant natural product (PNP) molecular structures is reflected in the variety of biochemical and genetic pathways that lead to their formation and accumulation. Plant secondary metabolites are important commodities, and include fragrances, colorants, and medicines. Increasing the extractable amount of PNP through plant breeding, or more recently by means of metabolic engineering, is a priority. The prerequisite for any attempt at metabolic engineering is a detailed knowledge of the underlying biosynthetic and regulatory pathways in plants. Over the past few decades, an enormous body of information about the biochemistry and genetics of biosynthetic pathways involved in PNPs production has been generated. In this review, we focus on the three large classes of plant secondary metabolites: terpenoids (or isoprenoids), phenylpropanoids, and alkaloids. All three provide excellent examples of the tremendous efforts undertaken to boost our understanding of biosynthetic pathways, resulting in the first successes in plant metabolic engineering. We further consider what essential information is still missing, and how future research directions could help achieve the rational design of plants as chemical factories for high-value products.
    Biotechnology Journal 10/2013; 8(10):1159-1171. DOI:10.1002/biot.201300224 · 3.49 Impact Factor

Publication Stats

6k Citations
512.35 Total Impact Points


  • 1998-2015
    • Royal Holloway, University of London
      • • Department of Biological Sciences
      • • Division of Biochemistry
      Эгхем, England, United Kingdom
  • 1995-2015
    • University of London
      • School of Biological Sciences
      Londinium, England, United Kingdom
  • 2009-2012
    • University of Surrey
      Guilford, England, United Kingdom
    • Japan Women's University
      Edo, Tōkyō, Japan
  • 2010
    • University of Oulu
      • Department of Biology
      Uleoborg, Northern Ostrobothnia, Finland
  • 1992
    • Universität Konstanz
      • Plant Physiology and Plant Biochemistry
      Constance, Baden-Württemberg, Germany
  • 1991
    • Universidad del País Vasco / Euskal Herriko Unibertsitatea
      Leioa, Basque Country, Spain