Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Molecular assessment of muscle health and function : The effect of age, nutrition and physical activity on the human muscle transcriptome and metabolom
    Hangelbroek, Roland W.J. - \ 2017
    Wageningen University. Promotor(en): A.H. Kersten; C.P.G.M. de Groot, co-promotor(en): M.V. Boekschoten. - Wageningen : Wageningen University - ISBN 9789463437103 - 205
    muscles - age - nutrition - physical activity - transcriptomes - metabolomes - elderly - creatine - phosphocreatine - vitamin d - atrophy - spieren - leeftijd - voeding - lichamelijke activiteit - transcriptomen - metabolomen - ouderen - creatine - fosfocreatine - vitamine d - atrofie

    Prolonged lifespan and decreased fertility will lead to an increased proportion of older adults in the world population (population aging). An important strategy to deal with population aging has been to promote healthy aging; not only to prevent mounting health care costs, but also to maintain independence and quality of life of older populations for as long as possible. Close to the opposite of the healthy aging is frailty. A major component of (physical) frailty is sarcopenia: age-related loss of muscle mass. Decreased muscle size and strength has been associated with a wide variety of negative health outcomes, including increased risk of hospitalization, physical disability and even death. Therefore, maintaining muscle size and strength is very important for healthy aging. Nutrition and physical activity are possible strategies to maintain or even improve muscle function with age.

    The effect of nutrition, age, frailty and physical activity on the function of skeletal muscle is complex. A better understanding of the molecular mechanisms involved can provide new insights in potential strategies to maintain muscle function over the life course. This thesis aims to investigate these mechanisms and processes that underlie the effects of age, frailty and physical activity by leveraging the sensitivity and comprehensiveness of transcriptomics and metabolomics.

    Chapter 2 and 3 describe the effects of age, frailty and resistance-type exercise training on the skeletal muscle transcriptome and metabolome. Both the transcriptome and metabolome show significant differences between frail and healthy older adults. These differences are similar to the differneces between healthy young men and healthy older adults, suggesting that frailty presents itself as a more pronounced form of aging, somewhat independent of chronological age. These age and frailty related differences in the transcriptome are partially reversed by resistance-type exercise training, in accordance with the observed improvement in muscle strength. Regression analysis revealed that the protocadherin gamma gene cluster may be important to skeletal muscle function. Protocadherin gamma is involved in axon guidance and may be upregulated due to the denervation-reinnervation cycles observed in skeletal muscle of older individuals. The metabolome suggested that resistance-type exercise training led to a decrease in branched-chain amino acid oxidation, as shown by a decrease in amino acid derived carnitines. Lastly, the blood metabolome showed little agreement with the metabolome in skeletal muscle, indicating that blood is a poor read-out of muscle metabolism.

    We assessed the effect of knee immobilization with creatine supplementation or placebo on the skeletal muscle transcriptome and metabolome in chapter 4. Knee immobilization caused muscle mass loss and strength loss in all participants, with no differences between creatine and placebo groups. Knee immobilization appeared to induce the HDAC4-myogenin axis, which is primarily associated with denervation and motor neuron diseases. The metabolome showed changes consistent with the decreased expression of energy metabolism genes. While acyl-carnitine levels tended to decrease with knee immobilization, one branched-chain amino acid-derived acyl carnitine was increased after knee immobilization, suggesting increased amino acid oxidation.

    Vitamin D deficiency is common among older adults and has been linked to muscle weakness. Vitamin D supplementation has been proposed as a strategy to improve muscle function among older populations. In chapter 5, supplementation with vitamin D (calcifediol, 25(OH)D) is investigated as nutritional strategy to improve muscle function among frail older adults. However, we observed no effect of vitamin D on the muscle transcriptome. These findings indicate the effects of vitamin D supplementation on skeletal muscle may be either absent, weak, or limited to a small subset of muscle cells.

    Transcriptomic changes due to different forms of muscle disuse are compared in chapter 6 (primarily knee immobilization and bed rest). The goal was to determine the similarities and differences among various causes of muscle atrophy in humans (primarily muscle disuse). Both knee immobilization and bed rest led to significant changes in the muscle transcriptome. However, the overlap in significantly changed genes was relatively small. Knee immobilization was characterized by ubiquitin-mediated proteolysis and induction of the HDAC4/Myogenin axis, whereas bed rest revealed increased expression of genes of the immune system and increased expression of lysosomal genes. Knee immobilization showed the highest similarity with age and frailty-related transcriptomic changes. This finding suggests that knee immobilization may be the most suitable form of disuse atrophy to assess the effectiveness of strategies to prevent age-related muscle loss in humans.

    The transcriptome and metabolome are incredibly useful tools in describing the wide array of biological systems within skeletal muscle. These systems can be modulated using physical activity (or lack thereof) as well as nutrition. This thesis describes some of these processes and highlights several unexplored genes and metabolites that may be important for maintaining or even optimizing muscle function. In the future, it may be possible to optimize both exercise and nutrition for each individual using these techniques; or even better, cheaper and less invasive alternatives.

    Identification of metabolites involved in heat stress response in different tomato genotypes
    Paupière, Marine J. - \ 2017
    Wageningen University. Promotor(en): R.G.F. Visser, co-promotor(en): A.G. Bovy; Y.M. Tikunov. - Wageningen : Wageningen University - ISBN 9789463431842 - 168
    solanum lycopersicum - tomatoes - genotypes - heat stress - heat tolerance - pollen - metabolomes - metabolites - metabolomics - solanum lycopersicum - tomaten - genotypen - warmtestress - hittetolerantie - stuifmeel - metabolomen - metabolieten - metabolomica

    Tomato production is threatened by climate change. High temperatures lead to a decrease of fruit set which correlates with a decrease of pollen fertility. The low viability of tomato pollen under heat stress was previously shown to be associated with alterations in specific metabolites. In this thesis, we used untargeted metabolomics approaches to broaden the identification of metabolites affected by heat stress. We assessed the suitability of pollen isolation methods for metabolomics analysis and considered the pitfalls for our further analysis. We explored the developmental metabolomes of pollen and anthers of different tomato genotypes under control and high temperature conditions and identified that microsporogenesis is a critical developmental stage for the production of mature and fertile pollen grain under heat stress. Several metabolites were putatively associated with tolerance to high temperature such as specific flavonoids, polyamines and alkaloids. These metabolites can be further used as markers in breeding programs to develop new genotypes tolerant to high temperatures.

    The secondary metabolome of the fungal tomato pathogen Cladosporium fulvum
    Griffiths, S.A. - \ 2015
    Wageningen University. Promotor(en): Pierre de Wit; Pedro Crous, co-promotor(en): Jerome Collemare. - Wageningen : Wageningen University - ISBN 9789462575813 - 167
    passalora fulva - secundaire metabolieten - metabolomen - genen - genomica - biologische activiteit - biosynthese - natuurlijke producten - passalora fulva - secondary metabolites - metabolomes - genes - genomics - biological activity - biosynthesis - natural products

    Secondary metabolites (SMs) are biologically active organic compounds that are biosynthesised
    by many plants and microbes. Many SMs that affect the growth, behaviour or survival of other
    organsisms have been re-purposed for use as medicinal drugs, agricultural biocides and animal
    growth promoters. The majority of our anti-infective and anti-cancer drugs are currently derived
    from Streptomyces, bacteria that are free living, filamentous, and ubiquitous in terrestrial habitats.
    Genome sequencing and mature in silico approaches to genome mining has revealed that filamentous
    fungi contain very large numbers of genes related to SM production. Yet these genes are typically
    silent under laboratory conditions. There are now many tools and strategies available to activate
    or clone silent SM genes. This thesis details our efforts to apply various methods to define and
    then manipulate SM genes in Cladosporium fulvum, a biotrophic pathogenic fungus of tomato
    containing many silent SM genes and gene clusters.

    In chapter 1, the relevance of SMs to medicine and agriculture is considered. Filamentous fungi
    are presented as untapped sources of potential useful SMs, as their genomes are often rich in SM
    biosynthetic genes that are silent under most conditions. Methods to activate these silent genes and
    increase the chemical diversity of fungi are detailed. These include the deletion or over-expression
    of genes encoding regulatory proteins, the use of chemical inhibitors, and the manipulation
    of growth conditions. Heterologous expression of silent SM genes in a production host is also
    discussed as a tool for bypassing host regulatory mechanisms altogether. C. fulvum is introduced
    as an organism that has been intensively studied as a biotrophic plant pathogen. Genomic analysis
    showed that this fungus has twenty-three core SM genes, a large catalogue composed of 10
    polyketide synthases (PKSs), 10 non-ribosomal peptide synthases (NPS), one PKS-NPS hybrid
    and one dimethylallyl tryptophan synthase (DMATS). Transcriptional profiling showed that the
    majority was silent during growth on tomato and in vitro. Cladofulvin is introduced as the sole
    detectable SM produced by C. fulvum during growth in vitro. This presented an opportunity to
    apply the aforementioned strategies to induce these silent genes and obtain new compounds. The
    importance of cladofulvin and structurally related anthraquinones are briefly discussed as potential
    medicines. The value of the cladofulvin biosynthetic gene cluster is also emphasised as a potential
    source of novel biosynthetic enzymes.

    In chapter 2 the SM gene catalogue identified during the analysis of the C. fulvum genome was
    analysed in further detail. Each locus containing a core SM gene was inspected for other biosynthetic
    genes linked to SM production, such as those encoding decorating enzymes and regulators. Products
    of these SM genes or gene clusters were speculated, based on their similarity to those characterized
    in other fungi. Six gene clusters were located in the genome of C. fulvum that are conserved in other
    fungal species. Remarkably, two predicted functional gene clusters were linked to the production
    of elsinochrome (PKS1) and cercosporin (PKS7), toxic perylenequinones that generate reactive
    oxygen species (ROS). We profiled the expression of core SM genes during the growth of C. fulvum
    under several in vitro conditions. Expression of each core SM gene was measured by RT-qrtPCR
    and the resulting SM profile was determined by LC-MS and NMR analyses. Confirming previous
    findings, the majority of SM genes remained silent and only cladofulvin was detected. During
    growth on tomato only two core genes, PKS6 and NPS9, were clearly expressed, but both were
    significantly down-regulated during colonization of the mesophyll tissue of tomato leaves. We
    confirmed that cladofulvin does not cause necrosis on solanaceous plants when infiltrated into
    their leaves. In contrast to other biotrophic fungi that have a reduced SM production capacity, our
    studies of C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent
    another mechanism associated with a biotrophic lifestyle.

    In chapter 3 our efforts to activate cryptic pathways in C. fulvum are described, with the aim
    of discovering new compounds. Many Ascomycete-specific global regulators of SM production
    and morphological development in other fungi were identified in C. fulvum. We investigated
    three intensively studied regulators, VeA, LaeA and HdaA. Deleting or over-expressing the genes
    encoding these regulators in C. fulvum yielded no new detectable SMs. Cladofulvin biosynthesis
    was strongly affected by each regulator; HdaA is an activator while VeA and LaeA are repressors of
    cladofulvin production. Attempts were made to stimulate SM production in the mutants and wild
    type strains by growing them on different carbon sources, but only cladofulvin biosynthesis was
    affected. Interestingly, cladofulvin production was stimulated by carbon limitation and strongly
    repressed in the presence of saccharose. Similar to observations made in other fungi, the deletion of
    VeA or LaeA did not affect viability, but maturation and conidiation were affected. Sporulation was
    not overtly affected by the loss of HdaA, but Δhdaa deletion mutants did not produce cladofulvin.
    This suggests that cladofulvin production is not required for asexual reproduction. The main
    finding of this chapter is that global regulator manipulation cannot considered to be a universal
    tool to discover new fungal natural products.

    In chapter 4, anthraquinones and closely related compounds such as anthrones, anthracyclines
    and xanthones are considered. Emodin is perhaps the most well characterised anthraquinone that
    is produced by many fungi and plants. Once synonymous only with constipation, this former
    laxative has since been investigated for its useful anti-cancer, anti-diabetic, anti-infective and antiinflammatory properties. Cladofulvin is a homodimeric anthraquinone composed of nataloe-emodin joined in a remarkably asymmetrical configuration. Dimeric anthraquinones and xanthones are also bioactive, most commonly tested for anti-infective and anti-cancer activities. Despite the ubiquity and medicinal qualities of anthraquinones and related compounds, very few of their biosynthetic pathways are known. No enzymes capable of dimerizing anthraquinones had yet been identified. In this chapter we demonstrated that cladofulvin was very cytotoxic towards human cancer cell-lines, crucially, up-to 300-fold more than its monomeric precursor nataloe-emodin against certain celllines. This became an added incentive to elucidate the cladofulvin pathway and identify the enzyme responsible for dimerizing nataloe-emodin. We confirmed earlier predictions that PKS6/claG is the core gene which starts cladofulvin biosythesis. Deletion of claG abolished cladofulvin production
    and no related metabolites were observed. A route to cladofulvin biosynthesis was proposed, guided
    by the work performed on the monodictyphenone biosynthetic pathway in Aspergillus nidulans.

    We predicted early acting cladofulvin genes and cloned them for heterologous expression in A.
    oryzae strain M-2-3. Using this approach we were able to confirm the first five genes in cladofulvin
    biosynthesis, claBCFGH, which yielded a reduced and dehydrated form of emodin. This is the
    point at which the pathways to cladofulvin and monodictyphenone production diverge. It was
    speculated that this emodin-related intermediate might be converted into nataloe-emodin by claK
    and/or claN. Finally, it was confirmed that the final step in the cladofulvin pathway is encoded by
    claM. Targeted deletion of claM yielded a mutant that accumulated nataloe-emodin and emodin
    but no cladofulvin. We discuss how the sequence of claM and ClaM will accelerate the discovery
    of functionally similar genes and enzymes, providing a template to engineer enzymes capable of
    forming novel dimers from existing monomers.

    In chapter 5 the natural role of cladofulvin was considered. This SM is consistently produced by
    C. fulvum and global regulator mutants in vitro. The respective biosynthetic genes appear most
    active during early and late stages of infection of tomato, but are down-regulated during biotrophic
    growth phase (chapter 2). The Δclag mutants (chapter 3) were not overtly different from the wild
    type during growth in vitro. We inoculated tomato plants with this mutant in order to test whether
    or not cladofulvin was required for normal infection. Simultaneously, we inoculated a C. fulvum
    transformant carrying an extra copy of the cladofulvin pathway-specific relulator, OE.claE, fused
    to the promoter region of the Avr9 effector gene. The strain was expected to produce cladofulvin
    once the fungal hyphae penetrate host stomata and begin to colonise the apoplastic space. In this
    way, we aimed to test the effect of cladofulvin over-production on disease symptom development.
    The growth of each strain on tomato plants was monitored by RT-qrtPCR at 4, 8 and 12 days post
    inoculation (dpi). At each time point the infections were inspected microscopically to detect any
    phenotypic abnormalities. We report that the loss of claG did not result an abnormal infection.
    Both wild type and ΔclaG mutants sporulated without causing necrosis or dessication of host leaves.
    In distinct contrast, brown spots appeared on leaves infected by the OE.claE transformant between
    8 – 12 dpi. This was accompanied by much stronger fungal growth and significant accumulation
    of cladofulvin. The leaves became desiccated and brittle before the fungus conidiated. Possible
    reasons for this phenotype are discussed. A small suite of in vitro experiments was performed on the
    Δclag and wild type strains in order to test the role of cladofulvin in survival. Consistent with the
    absence of a photoprotective pigment, Δclag spores were considerably more sensitive to ultraviolet
    (UV) radiation. Suggesting a role in protection against low temperatures, Δclag spores were less
    resistant to repeated cycles of freezing and thawing. Cladofulvin biosynthesis was stimulated and
    repressed by cold and heat shocking mature C. fulvum colonies, respectively. Altogether, these
    results suggested that cladofulvin confers resistance to abiotic stress.

    In chapter 6 the results obtained in this thesis are discussed in a broader context. Particularly,
    the discovery of the cytochrome P450 that is involved in dimerization of anthraquinones might
    enable discovery of homologous genes encoding enzymes with different specificities. Combining
    bioinformatic and functional analyses should prove to be a powerful strategy for discovering
    compounds with new biological activities, or enzymes relevant to metabolic engineering.

    Potato genetical genomics: investigating the genetic basis of primary metabolism and its relationship to the phenotype
    Carreño Quintero, N. - \ 2013
    Wageningen University. Promotor(en): Harro Bouwmeester; Richard Visser, co-promotor(en): Joost Keurentjes; Christian Bachem. - Wageningen : Wageningen University - ISBN 9789461738110 - 180
    solanum tuberosum - aardappelen - genomica - metabolisme - genetische analyse - metabolomica - metabolomen - fenotypen - loci voor kwantitatief kenmerk - solanum tuberosum - potatoes - genomics - metabolism - genetic analysis - metabolomics - metabolomes - phenotypes - quantitative trait loci

    Primary metabolism is essential for plant growth and survival and it is therefore involved in all physiological processes of the plant. In the past years the advancements in large-scale and high-throughput technologies have enhanced our ability to characterize the plant metabolome. The development of methods for the simultaneous analysis of many different plant metabolites and the necessary software for subsequent data analysis have further expanded the possibilities to investigate plant responses from a system-oriented perspective. This allows the comparison of genetic and phenotypic variation at different molecular levels, enabling us to find associations between genotype and phenotype and their intermediate levels of information transduction. Metabolomics has become increasingly important for the characterization of the metabolic status of plants under different environmental and genetic perturbations. The economic importance of potato and the increasing availability of genetic and molecular resources have stimulated research on many different aspects of the physiology of this crop and the regulation of complex traits. We used the available tools to explore the genetic basis of the composition and content of primary metabolites in a potato population. In this research, the possibilities to combine metabolite profiling with genetic information are explored to identify the genetic factors determining primary metabolism and to infer links between metabolites and agronomic phenotypes.

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