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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Tissue Metabolic Changes Drive Cytokine Responses to Mycobacterium tuberculosis
Lachmandas, Ekta ; Rios-Miguel, Ana B. ; Koeken, Valerie A.C.M. ; Pasch, Eva van der; Kumar, Vinod ; Matzaraki, Vasiliki ; Li, Yang ; Oosting, Marije ; Joosten, Leo A.B. ; Notebaart, Richard A. ; Noursadeghi, Mahdad ; Netea, Mihai G. ; Crevel, Reinout van; Pollara, Gabriele - \ 2018
The Journal of Infectious Diseases 218 (2018)1. - ISSN 0022-1899 - p. 165 - 170.
cytokines - functional genomics - human challenge model - immune response - immunometabolism - metabolism - microarrays - transcriptomics - tuberculosis

Cellular metabolism can influence host immune responses to Mycobacterium tuberculosis. Using a systems biology approach, differential expression of 292 metabolic genes involved in glycolysis, glutathione, pyrimidine, and inositol phosphate pathways was evident at the site of a human tuberculin skin test challenge in patients with active tuberculosis infection. For 28 metabolic genes, we identified single nucleotide polymorphisms that were trans-acting for in vitro cytokine responses to M. tuberculosis stimulation, including glutathione and pyrimidine metabolism genes that alter production of Th1 and Th17 cytokines. Our findings identify novel therapeutic targets in host metabolism that may shape protective immunity to tuberculosis.

Differences in transcriptional responses to acute and chronic dietary interventions with fatty acids
Matualatupauw, Juri C. - \ 2017
University. Promotor(en): Sander Kersten, co-promotor(en): Lydia Afman; J. Bouwman. - Wageningen : Wageningen University - ISBN 9789463432078 - 172
fatty acids - gene expression - genotyping - phenotypes - nutritional intervention - transcriptomics - fish oils - apolipoprotein e - adipose tissue - microarrays - polymerase chain reaction - vetzuren - genexpressie - fenotypen - maatregel op voedingsgebied - transcriptomica - visoliën - apolipoproteïne e - vetweefsel - polymerase-kettingreactie

Various types of dietary fatty acids have different effects on human health. The aim of this thesis was to increase our understanding of the molecular mechanisms underlying the effects of dietary fatty acids. To do this, we examined changes in whole genome gene expression profiles upon both acute as well as longer term dietary fatty acid interventions. Furthermore, from previous research, it is clear that large inter-individual differences in the response to dietary fatty acids exist. We used whole genome gene expression analyses to increase our understanding of the mechanisms underlying some of these inter-individual differences.

Many modifiable and non-modifiable factors can be the cause of these inter-individual differences. In chapter 2, we reviewed all studies that examined differences in the transcriptional response to dietary interventions based on the presence of one of these factors. These include gender, age, BMI, body composition, blood lipid levels and gut microbial composition. We conclude that transcriptome analyses are well-suited for studying the underlying mechanisms behind these differences in the response to diet. Nevertheless, the number of studies that use this approach remains limited.

Another factor that may modify the response to a dietary intervention is genetics, e.g. the apolipoprotein E4 (APOE4) variant. People who carry the APOE4 allele have an increased risk of cardiovascular disease. Fish-oil supplementation may help in the prevention of cardiovascular disease, though inter-individual differences in the response to n-3 polyunsaturated fatty acids on gene expression profiles have been observed. In chapter 3, we aimed to assess the impact of APOE4 on peripheral blood mononuclear cell (PBMC) whole genome gene expression at baseline and following a 6-month fish-oil intervention. We observed increased gene expression of IFN signaling and cholesterol biosynthesis pathways in APOE4 carriers, which might explain part of the association between APOE4 and CVD. Furthermore, fish-oil supplementation may be beneficial by decreasing interferon signalling-related gene expression in APOE4 carriers.

Another long-term dietary intervention with fatty acids was studied in chapter 4. We examined the effect of a 12-week high medium-chain saturated fatty acid diet on subcutaneous adipose tissue gene expression profiles. We observed increased expression of genes involved in oxidative energy metabolism and decreased inflammation-related gene expression due to the high medium-chain saturated fatty acid intake. Considering the role of the adipose tissue in sustaining the low-grade inflammation that is associated with obesity, these findings may be indicative of a more anti-inflammatory phenotype of the adipose tissue. We concluded that medium-chain saturated fatty acids may potentially have beneficial effects on adipose tissue functioning.

Besides studying the effects of long-term interventions with fatty acids on whole genome gene expression, we also examined the effects of acute high-fat challenges. In chapter 5, we determined the additional value of determining whole genome gene expression changes in response to a high-fat challenge compared to assessment at fasting only. In addition, we aimed to identify whether a 4 week high-fat high-calorie diet can induce a shift in gene expression profiles in healthy subjects towards a metabolic syndrome-like gene expression profile. We found that fasting whole blood whole genome gene expression profiles are highly responsive to a 4-week high-fat high-calorie diet, with changes in in the direction of a metabolic syndrome-like gene expression profile. High-fat challenge responses in healthy subjects show only minimal changes in gene expression upon the dietary intervention and a marginal shift in the direction of the metabolic syndrome. We concluded that fasting gene expression profiles are more responsive compared to high-fat challenge responses to a 4-week high-fat high-calorie diet.

Besides chapter 5, several other studies have also examined changes in whole genome gene expression in blood cells induced by high-fat challenges. In chapter 6, we combined microarray data from four high-fat challenge studies varying in study population, challenge composition and research laboratory. By performing this meta-analysis, we showed a general PBMC whole genome gene expression response to a high-fat challenge. We concluded that a meta-analysis provides added value for the discovery of consistently differentially expressed genes and pathways compared to selecting only those genes and pathways that are identified in all separate studies.

In conclusion, in this thesis we showed differences in the whole genome gene expression response to fish-oil supplementation in PBMCs of APOE4 carriers vs non-carriers. Furthermore, the effects on whole genome gene expression of the two long-term dietary interventions, i.e. the fish-oil supplementation in PBMCs of APOE4 carriers and the high medium-chain saturated fatty acid diet in adipose tissue, may be beneficial by downregulation of gene expression related to inflammation. We also showed that whole genome gene expression responses to high-fat challenges are affected by a 4-week high-fat high-calorie diet, though changes in fasting gene expression profiles are much more pronounced. Finally, we showed the value of meta-analysis of microarray data in high-fat challenge studies for identifying the general response to a high-fat challenge.

How to measure health improvement? : assessment of subtle shifts in metabolic phenotype
Fazelzadeh, Parastoo - \ 2017
University. Promotor(en): Sander Kersten; John van Duynhoven, co-promotor(en): Mark Boekschoten. - Wageningen : Wageningen University - ISBN 9789463430739 - 187
health promotion - improvement - measurement - metabolic profiling - elderly - obesity - microarrays - rna - peripheral blood mononuclear cells - gezondheidsbevordering - verbetering - meting - metabolische profilering - ouderen - obesitas - perifere mononucleaire bloedcellen

Human health is impacted by a complex network of interactions between biological pathways, mechanisms, processes, and organs, which need to be able to adapt to a continuously changing environment to maintain health. This adaptive ability is called ‘phenotypic flexibility’. It is thought that health is compromised and diseases develop when these adaptive processes fail. As the product of interactions between several factors such as genetic makeup, diet, lifestyle, environment and the gut microbiome, the ‘metabolic phenotype’ provides a readout of the metabolic state of an individual. Understanding these relationships will be one of a major challenges in nutrition and health research in the next decades. To address this challenge, the development of high-throughput omics tools combined with the application of elaborate statistical analyses will help characterize the complex relationship of (bio) chemicals in human systems and their interaction with other variables including environment and lifestyle to produce the measured phenotype. An important aim of this thesis was to identify phenotype shifts by looking at effect of prolonged resistance-type exercise training on skeletal muscle tissue in older subjects and the possible shift toward the features of younger subjects as a reference for a healthier phenotype. A second aim was to identify phenotype shifts by looking at the response to a challenge in obese subjects and the possible shift toward lean subjects as a reference for a healthier phenotype.

Chapter 2 and 3 of this thesis show how the significant remaining plasticity of ageing skeletal muscle can adapt to resistance-type exercise training. The data indicate that frail and healthy older subjects have two distinct phenotypes according to the skeletal muscle tissue metabolite profiles and that exercise training shifts aged muscle towards a younger phenotype. We showed that the effect of exercise on amino acid derived acylcarnitines (AAAC’s) in older subjects points towards decreased branched chain amino acid catabolism, likely due to compromised activation of the branched chain α-keto acid hydrogenase (BCKDH) complex. Furthermore, we found that the protocadherin gamma gene cluster might be involved in aged-muscle denervation and re-innervation. Finally, plasma was found to be a poor indicator of muscle metabolism, emphasizing the need for direct assessment of metabolites in muscle tissue.

Chapter 4 of this thesis examines whether a mixed meal challenge response provides a readout for a shift in phenotype upon weight loss in obese male subjects. We concluded that weight loss moderately affects the mixed meal challenge response of both plasma metabolome and transcriptome of peripheral blood mononuclear cells in obese subjects. Measurements at the fasted and postprandial state also provide us with a different type of information.

In Chapter 5 it is demonstrated that the global testing of pathways could provide a concise summary of the multiple univariate testing approach used in Chapter 4. In Chapter 6 it is discussed how the findings of this thesis increase our understanding of how to measure phenotypic flexibility as a proxy of health. In this thesis it is shown that the correlations between tissue and plasma metabolites are rather weak, emphasising the need to perform organ-specific studies. Availability of less invasive/painful sampling techniques and the use of small amounts of tissue would enable larger scale human studies on adipose tissue and skeletal muscle to more accurately define phenotypical shifts due to diet or lifestyle interventions. With respect to the assessment of phenotypical flexibility by omics approaches, significant complications can be expected in trying to relate plasma metabolism to PBMC gene expression. Organ-focussed approaches that integrate multiple omics levels using system biology approaches are considered to be a lot more promising.

The influence of phase II conjugation on the biological activity of flavonoids
Beekmann, K. - \ 2016
University. Promotor(en): Ivonne Rietjens; Peter van Bladeren, co-promotor(en): L. Actis-Goretta. - Wageningen : Wageningen University - ISBN 9789462577640 - 171 p.
flavonoids - biological activity - in vitro - biosynthesis - peroxisomes - microarrays - daidzein - genistein - oestrogen receptors - isoflavones - quercetin - kaempferol - serine proteinases - threonine - flavonoïden - biologische activiteit - biosynthese - peroxisomen - daidzin - genisteïne - oestrogeenreceptoren - isoflavonen - quercetine - serine proteïnasen

Flavonoid consumption is often correlated with a wide range of health effects, such as the prevention of cardiovascular diseases, neurodegenerative diseases, and diabetes. These effects are usually ascribed to the activity of the parent flavonoid aglycones, even though these forms of the flavonoids generally have a low systemic bioavailability. During uptake, flavonoids undergo phase II metabolism and are present in the systemic circulation nearly exclusively as conjugated metabolites. The aim of this thesis was to study the effect of conjugation on the biological activity of selected flavonoids towards different endpoints relevant for human health. To this end, conjugation with glucuronic acid was taken as the model type of conjugation because this modification is generally observed to be the most important metabolic conjugation reaction for flavonoids in man.

A review of scientific literature published until early 2012 reveals that metabolic conjugation can affect the biological activity of flavonoids in different ways. Conjugation can increase, decrease, inverse or not affect the biological activity, depending on the flavonoid, the type and position of conjugation, the endpoint studied, and the assay system used. Based on the literature reviewed it is concluded that the effect of conjugation has to be studied on a case-by-case basis.

As the research on the biological activity of biologically relevant flavonoid conjugates is often hampered by the generally low commercial availability and high prices of these conjugates, a simple and versatile method for the biosynthesis of metabolically relevant flavonoid conjugates is described. Using this method, relevant conjugates can be prepared from different flavonoid substrates in sufficient quantities for in vitro bioassays. Further, an efficient strategy for the identification of these flavonoid conjugates by LC-MS and 1H-NMR using MetIDB (Metabolite Identification Database), a publicly accessible database of predicted and experimental 1H-NMR spectra of flavonoids, is presented.

To study the effect of conjugation on the biological activities of flavonoids, several different assay systems and endpoints were used to study the activity of different flavonoids and their conjugates. The effects of quercetin, kaempferol, and their main plasma conjugates quercetin-3-O-glucuronide and kaempferol-3-O-glucuronide (K-3G) on different endpoints related to peroxisome proliferator-activated receptor (PPAR)-γ were studied. PPAR-γ activation is reported to have positive health effects related to adipogenesis, insulin resistance and inflammation. The presented results show that the flavonoid aglycones increased PPAR-γ mediated gene expression in a stably transfected reporter gene cell line, and that glucuronidation diminished this effect. These observed increases in reporter gene expression were accompanied by increased PPAR-γ receptor-mRNA expression upon exposure to kaempferol, an effect that was also reduced by glucuronidation. Using the cell-free Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction (MARCoNI) it was demonstrated that, unlike the known PPAR-γ agonist rosiglitazone, neither the flavonoid aglycones nor the conjugates are agonistic ligands of the PPAR-γ receptor. Supporting the hypothesis that the tested compounds have a different mode of action from normal LBD agonism, quercetin appeared to synergistically increase the effect of rosiglitazone in the reporter gene assay. The modes of action behind the observed effects remain to be elucidated and might include effects on protein kinase activities affecting expression of the PPAR-γ receptor, or posttranscriptional modifications of PPAR-γ.

Another type of nuclear receptor known to be targeted by certain flavonoids are the estrogen receptor (ER)α- and ERβ. ERs are the main targets of estrogenic compounds, and upon their activation different transcriptional responses with opposite effects on cell proliferation and apoptosis are elicited; ERα activation stimulates cell proliferation, while ERβ activation causes apoptosis and reduces ERα mediated induction of cell proliferation. Using the MARCoNI assay, the intrinsic estrogenic effects of the two main dietary isoflavones daidzein and genistein, and their plasma conjugates daidzein-7-O-glucuronide and genistein-7-O-glucuronide on the ligand induced coregulator binding of ERα- and ERβ-LBD were studied and compared to the effect of the positive control 17β-estradiol (E2). The results show that the tested isoflavone compounds are less potent agonists of ERα- and ERβ-LBD than E2, although they modulate the LBD-coregulator interactions in a manner similar to E2. Genistein is shown to be a more potent agonist than daidzein for both receptor subtypes. While in the MARCoNI assay genistein had a strong preference for ERβ-LBD activation over ERα-LBD activation, daidzein had a slight preference for ERα-LBD activation over ERβ-LBD activation. Glucuronidation reduced the intrinsic agonistic activities of both daidzein and genistein to induce ERα-LBD and ERβ-LBD - coregulator interactions and increased their average half maximal effective concentrations (EC50s) by 8 to 4,400 times. The results presented further show that glucuronidation changed the preferential activation of genistein from ERβ-LBD to ERα-LBD and increased the preferential activation of daidzein for ERα-LBD; this is of special interest given that ERβ activation, which is counteracting the possible adverse effects of ERα activation, is considered one of the supposedly beneficial modes of action of isoflavones.

Many flavonoids are reported to be inhibitors of protein kinases. To study the effect of conjugation on the inhibition of serine/threonine protein kinases by flavonoids, kaempferol and its main plasma conjugate K-3G were selected as model compounds. Protein kinases are involved in a wide range of physiological processes by controlling signaling cascades and regulating protein functions; modulation of their activities can have a wide range of biological effects. The inhibitory effects of kaempferol, K-3G, and the broad-specificity protein kinase inhibitor staurosporine on the phosphorylation activity of recombinant protein kinase A (PKA) and of a lysate prepared from the hepatocellular carcinoma cell line HepG2 were studied using a microarray platform that determines the phosphorylation of 141 putative serine/threonine phosphorylation sites derived from human proteins. The results reveal that glucuronidation reduces the intrinsic potency of kaempferol to inhibit the phosphorylation activity of PKA and HepG2 lysate on average about 16 and 3.5 times, respectively. It is shown that the inhibitory activity of K-3G in the experiments conducted was not caused by deconjugation to the aglycone. Furthermore, the results show that kaempferol and K-3G, unlike the broad-specificity protein kinase inhibitor staurosporine, did not appear to inhibit all protein kinases present in the HepG2 lysate to a similar extent, indicating that kaempferol selectively targets protein kinases, a characteristic that appeared not to be affected by glucuronidation. The fact that K-3G appeared to be only a few times less potent than kaempferol implies that K-3G does not necessarily need to be deconjugated to the aglycone to exert potential inhibitory effects on protein kinases.

The results obtained in the present thesis support the conclusion that glucuronidation of flavonoids does not necessarily abolish their activity and that flavonoid glucuronides may be biologically active themselves, albeit at higher concentrations than the parent aglycones. In line with the conclusions from the earlier literature review, an updated literature review on the effect of conjugation on the biological activity of flavonoids concludes that that the effect of conjugation on the biological activity of flavonoids depends on the type and position of conjugation, the endpoint studied and the assay system used. Based on the results described and the literature reviewed in this thesis, several recommendations and perspectives for future research are formulated. Several methodological considerations are formulated that need to be taken into account when studying the biological activity of flavonoids and their conjugates to avoid confounding results. Further, the relevance of the gut microbiome for flavonoid bioactivity is highlighted, and considerations regarding the pharmacokinetics and pharmacodynamics of flavonoids in vivo are formulated. Altogether, it can be concluded that circulating flavonoid conjugates may exert biological activities themselves, and that understanding these is a prerequisite to successfully elucidate the mechanisms of action behind the biological activities linked to flavonoid consumption.

Sex-linked transcriptional divergence in the hermaphrodite fungus Neurospora tetrasperma
Samils, N. ; Gioti, A. ; Karlsson, M. ; Sun, Y.Y. ; Kasuga, T. ; Bastiaans, E. ; Wang, Z. ; Li, N. ; Townsend, J.P. ; Johannesson, H. - \ 2013
Proceedings of the Royal Society. B: Biological Sciences 280 (2013)1764. - ISSN 0962-8452
biased gene-expression - mating-type chromosomes - false discovery rates - x-chromosome - evolution - crassa - recombination - drosophila - microarrays - selection
In the filamentous ascomycete Neurospora tetrasperma, a large (approx. 7 Mbp) region of suppressed recombination surrounds the mating-type (mat) locus. While the remainder of the genome is largely homoallelic, this region of recombinational suppression, extending over 1500 genes, is associated with sequence divergence. Here, we used microarrays to examine how the molecular phenotype of gene expression level is linked to this divergent region, and thus to the mating type. Culturing N. tetrasperma on agar media that induce sexual/female or vegetative/male tissue, we found 196 genes significantly differentially expressed between mat A and mat a mating types. Our data show that the genes exhibiting mat-linked expression are enriched in the region genetically linked to mating type, and sequence and expression divergence are positively correlated. Our results indicate that the phenotype of mat A strains is optimized for traits promoting sexual/female development and the phenotype of mat a strains for vegetative/male development. This discovery of differentially expressed genes associated with mating type provides a link between genotypic and phenotypic divergence in this taxon and illustrates a fungal analogue to sexual dimorphism found among animals and plants.
Biomolecule substrate topography for inkjet printed structures
Mujawar, L.H. - \ 2013
University. Promotor(en): Willem Norde, co-promotor(en): Aart van Amerongen. - S.l. : s.n. - ISBN 9789461735478 - 145
microarrays - ruimtelijke verdeling - eiwitten - assays - porositeit - substraten - spatial distribution - proteins - porosity - substrates
Spot morphology of non-contact printed protein molecules on non-porous substrates with a range of hydrophobicities
Mujawar, L.H. ; Norde, W. ; Amerongen, A. van - \ 2013
The Analyst 138 (2013)2. - ISSN 0003-2654 - p. 518 - 524.
microarrays - oligonucleotides - technology - biochip - surface
Non-contact inkjet printing technology is one of the most promising tools for producing microarrays. The quality of the microarray depends on the type of the substrate used for printing biomolecules. Various porous and non-porous substrates have been used in the past, but due to low production cost and easy availability, non-porous substrates like glass and plastic are preferred over porous substrates. On these non-porous substrates, obtaining spot uniformity and a high signal to noise ratio is a big challenge. In our research work, we have modified pristine glass slides using various silanes to produce a range of hydrophobic glass substrates. The hydrophobicities of the slides expressed in the contact angle (¿) of a sessile drop of water were 49°, 61°, 75°, 88° and 103°. Using a non-contact inkjet printer, microarrays of biotinylated biomolecules (BSA and IgG) were produced on these modified glass substrates, pristine (untreated) glass and also on HTA polystyrene slides. The uniformity of the spots, reflecting the distribution of the biomolecules in the spots, was analyzed and compared using confocal laser scanning microscopy (CLSM). The quality of the spots was superior on the glass slide with a contact angle of [similar]75°. We also investigated the influence of the hydrophobicity of the substrate on a two-step, real diagnostic antibody assay. This nucleic acid microarray immunoassay (NAMIA) for the detection of Staphylococcus aureus showed that on highly hydrophilic (¿ <10°) and hydrophobic substrates (¿ > 100°) the assay signal was low, whereas an excellent signal was obtained on the substrates with intermediate contact angles, ¿ [similar] 61° and ¿ [similar] 75°, respectively. Graphical abstract: Spot morphology of non-contact printed protein molecules on non-porous substrates with a range of hydrophobicities
Functional analysis of inter-individual transcriptome differential expression in pig longissimus muscle
Zhao, S. ; Hulsegge, B. ; Harders, F.L. ; Bossers, R. ; Keuning, E. ; Hoekman, A.J.W. ; Hoving-Bolink, A.H. ; Pas, M.F.W. te - \ 2013
Journal of Animal Breeding and Genetics 130 (2013)1. - ISSN 0931-2668 - p. 72 - 78.
meat quality - microarrays - discovery - traits - fibers - gene - pork
Selection of pigs for increased meat production or improved meat quality changes muscle mass and muscle composition. This will be related to transcriptome expression profile changes in muscle tissue, generating inter-individual differences. This study investigated the differentially expressed genes in the transcriptome profiles of the longissimus muscle of 75 Large White–Duroc cross sows and castrates. The use of a common reference design enabled to investigate the inter-individual transcriptome expression profile differences between the animals as compared with the means of all animals. The aim of the study was to identify the biological processes related to these inter-individual differences. It was expected that these processes underlie the selection effects. In total, 908 transcripts were differentially expressed. Among them, 762 were mainly downregulated and 146 were mainly upregulated. Gene Ontology and Pathways analyses indicated that the differentially expressed genes belong to three groups of processes involved in protein synthesis and amino acid–protein metabolism, energy metabolism and muscle-specific structure and activity processes. Comparing the functional biological analysis results with previously reported data suggested that the protein synthesis, energy metabolism and muscle-specific structure would contribute to meat production and the meat quality
Copper-Free Click Biofunctionalization of Silicon Nitride Surfaces via Strain-Promoted Alkyne-Azide Cycloaddition Reactions
Manova, R.K. ; Pujari, S.P. ; Weijers, C.A.G.M. ; Zuilhof, H. ; Beek, T.A. van - \ 2012
Langmuir 28 (2012)23. - ISSN 0743-7463 - p. 8651 - 8663.
self-assembled monolayers - terminated monolayers - organic monolayers - one-step - functionalization - chemistry - films - immobilization - microarrays - dna
Cu-free "click" chemistry is explored on silicon nitride (Si3N4) surfaces as an effective way for oriented immobilization of biomolecules. An omega-unsaturated ester was grafted onto Si3N4 using UV irradiation. Hydrolysis followed by carbodiimide-mediated activation yielded surface-bound active succinimidyl and pentafluorophenyl ester groups. These reactive surfaces were employed for the attachment of bicyclononyne with an amine spacer, which subsequently enabled room temperature strain-promoted azide alkyne cycloaddition (SPAAC). This stepwise approach was characterized by means of static water contact angle, X-ray photoelectron spectroscopy, and fluorescence microscopy. The surface-bound SPAAC reaction was studied with both a fluorine-tagged azide and an azide-linked lactose, yielding hydrophobic and bioactive surfaces for which the presence of trace amounts of Cu ions would have been problematic. Additionally, patterning of the Si3N4 surface using this metal-free click reaction with a fluorescent azide is shown. These results demonstrate the ability of the SPAAC as a generic tool for anchoring complex molecules onto a surface under extremely mild, namely ambient and metal-free, conditions in a clean and relatively fast manner.
Improved functional immobilization of llama single-domain antibody fragments to polystyrene surfaces using small peptides
Harmsen, M.M. ; Fijten, H.P.D. - \ 2012
Journal of Immunoassay and Immunochemistry 33 (2012)3. - ISSN 1532-1819 - p. 234 - 251.
linked-immunosorbent-assay - escherichia-coli - passive-immunization - mouth-disease - in-vitro - microarrays - strategies - bivalent - pigs - hydrophobins
We studied the effect of different fusion domains on the functional immobilization of three llama single-domain antibody fragments (VHHs) after passive adsorption to polystyrene in enzyme-linked immunosorbent assays (ELISA). Three VHHs produced without any fusion domain were efficiently adsorbed to polystyrene, which, however, resulted in inefficient antigen binding. Functional VHH immobilization was improved by VHH fusion to a consecutive myc-His6-tag and was even more improved by fusion to the llama antibody long hinge region containing an additional His6-tag (LHc-His6). The partial dimerization of VHH-LHc-His6 fusion proteins through LHc-mediated disulfide-bond formation was not essential for their improved functional immobilization. VHH fusions to specific polystyrene binding peptides, hydrophobins, or other, unrelated VHH domains were less suitable for increasing functional VHH immobilization because of reduced microbial expression levels. Thus, VHH-LHc-His6 fusion proteins are most suited for functional passive adsorption in ELISA.
A DNA-based strategy for dynamic positional enzyme immobilization inside fused silica microchannels
Vong, T.H. ; Schoffelen, S. ; Dongen, S.F.M. van; Beek, T.A. van; Zuilhof, H. ; Hest, J.C.M. van - \ 2011
Chemical Science 2 (2011)7. - ISSN 2041-6520 - p. 1278 - 1285.
directed immobilization - microarrays - proteins - reactors - monoliths
A three-enzyme cascade reaction was successfully realized in a continuous flow microreactor. The first enzyme (Candida antarctica lipase B, also known as Pseudozyma antarctica lipase B) and the third enzyme (horseradish peroxidase) of the cascade process were immobilized in a mild non-contact manner via ssDNA-ssDNA interaction in discrete zones on the capillary wall, whereas the second enzyme (glucose oxidase) was kept in the mobile phase. The unique combined feature of patterning, possibility of loading and stripping, and modularity in a fused silica microchannel is demonstrated. By changing the distance between the two enzyme patches, the reaction time available for glucose oxidase could be independently and modularly varied. The reusability of the enzymatic microfluidic system was shown by using the hybridization and dehybridization capabilities of DNA as a tool for subsequent enzyme immobilization and removal
Een op padlock probe gebaseerde universele micro-array-detectie voor meerdere Phytopthora-soorten
Gaszczyk, K. ; Verstappen, E.C.P. ; Mendes, O. ; Schoen, C.D. ; Bonants, P.J.M. - \ 2011
Gewasbescherming 42 (2011)4. - ISSN 0166-6495 - p. 182 - 182.
phytophthora - detectie - moleculaire detectie - microarrays - detection - molecular detection
Binnen dit project is een diagnostische methode ontwikkeld die toe te passen is in planta, en die ook de meest recent beschreven (quarantaine-) soorten. Ook worden meerdere Phytophthora-soorten tegelijkertijd gedetecteerd.
Patterning of Peptide Nucleic Acids Using Reactive Microcontact Printing
Calabretta, A. ; Wasserberg, D. ; Posthuma-Trumpie, G.A. ; Subramaniam, V. ; Amerongen, A. van; Corradini, R. ; Tedeschi, T. ; Sforza, S. ; Reinhoudt, D.N. ; Marchelli, R. ; Huskens, J. ; Jonkheijm, P. - \ 2011
Langmuir 27 (2011)4. - ISSN 0743-7463 - p. 1536 - 1542.
stereogenic centers - dna biosensors - pna - microarrays - nanoparticle - recognition - immobilization - hybridization - handedness - proteins
PNAs (peptide nucleic acids) have been immobilized onto surfaces in a fast, accurate way by employing reactive microcontact printing. Surfaces have been first modified with aldehyde groups to react with the amino end of the synthesized PNAs. When patterning fluorescein-labeled PNAs by reactive microcontact printing using oxygen-oxidized polydimethylsiloxane stamps, homogeneous arrays were fabricated and characterized using optical methods. PNA-patterned surfaces were hybridized with complementary and mismatched dye-labeled oligonucleotides to test their ability to recognize DNA sequences. The stability and selectivity of the PNA-DNA duplexes on surfaces have been verified by fluorescence microscopy, and the melting curves have been recorded. Finally, the technique has been applied to the fabrication of chips by spotting a PNA microarray onto a flat PDMS stamp and reproducing the same features onto many slides. The chips were finally applied to single nucleotide polymorphism detection on oligonucleotides.
Comparison of two GM maize varieties with a near-isogenic non-GM variety using transcriptomics, proteomics and metabolomics
Barros, E. ; Lezar, S. ; Anttonen, M.J. ; Dijk, J.P. van; Rohlig, R.M. ; Kok, E.J. ; Engel, K.H. - \ 2010
Plant Biotechnology Journal 8 (2010)4. - ISSN 1467-7644 - p. 436 - 451.
gene-expression - h-1-nmr spectroscopy - safety assessment - food - tool - nmr - hybridization - microarrays - mutants - plants
P>The aim of this study was to evaluate the use of four nontargeted analytical methodologies in the detection of unintended effects that could be derived during genetic manipulation of crops. Three profiling technologies were used to compare the transcriptome, proteome and metabolome of two transgenic maize lines with the respective control line. By comparing the profiles of the two transgenic lines grown in the same location over three growing seasons, we could determine the extent of environmental variation, while the comparison with the control maize line allowed the investigation of effects caused by a difference in genotype. The effect of growing conditions as an additional environmental effect was also evaluated by comparing the Bt-maize line with the control line from plants grown in three different locations in one growing season. The environment was shown to play an important effect in the protein, gene expression and metabolite levels of the maize samples tested where 5 proteins, 65 genes and 15 metabolites were found to be differentially expressed. A distinct separation between the three growing seasons was also found for all the samples grown in one location. Together, these environmental factors caused more variation in the different transcript/protein/metabolite profiles than the different genotypes.
Food Allergens Profiling with an Imaging Surface Plasmon Resonance-Based Biosensor
Rebe, S. ; Liu, H. ; Norde, W. ; Bremer, M.G.E.G. - \ 2010
Analytical Chemistry 82 (2010)20. - ISSN 0003-2700 - p. 8485 - 8491.
peanut allergens - immunoassay - protein - milk - microarrays - system
Food allergy is a growing health concern, which currently affects approximately 4% of adults and 8% of infants. For consumer protection purposes, food producers are required by law to disclose on the product label whether a major allergen is used during the production process. The commonly employed monitoring methods are highly laborious, time-consuming, and often expensive when screening for multiple allergens. Here, we utilize imaging surface plasmon resonance (iSPR) in combination with antibody array for rapid, quantitative, and multianalyte food allergens detection. We demonstrate how the use of this technology provides a complete allergen profile within short measurement time and with adequate sensitivity. The successful applicability of this approach is demonstrated by analyzing cookies and dark chocolate products from different manufacturers. Hazelnut content of the tested food products is also determined by enzyme linked immunosorbent assay and is found to correlate well with the hazelnut content determined by iSPR. This newly developed method opens the door to automated and high-throughput allergen analysis, ultimately aiming at providing the consumer with safer food.
Genome-wide gene expression surveys and a transcriptome map in chicken
Nie, H. - \ 2010
University. Promotor(en): Martien Groenen; Mari Smits, co-promotor(en): Richard Crooijmans. - [S.l.] : S.n. - ISBN 9789085856221 - 164
kippen - pluimvee - dierveredeling - genomen - genexpressie - transcriptie - genetische kartering - genexpressieanalyse - microarrays - dna microarrays - transcriptomics - marker assisted breeding - moleculaire veredeling - fowls - poultry - animal breeding - genomes - gene expression - transcription - genetic mapping - genomics - molecular breeding
The chicken (Gallus gallus) is an important model organism in genetics, developmental biology, immunology, evolutionary research, and agricultural science. The completeness of the draft chicken genome sequence provided new possibilities to study genomic changes during evolution by comparing the chicken genome to that of other species. The development of long oligonucleotide microarrays based on the genome sequence made it possible to survey genome-wide gene expression in chicken. This thesis describes two gene expression surveys across a range of healthy chicken tissues in both adult and embryonic stages. Specifically, we focus on the mechanisms of regulation of gene transcription and their evolution in the vertebrate genome.

Chapter 1 provides a brief history of the chicken as a model organism in biological and genomics research. In particular a brief overview is presented about expression profiling experiments, followed by an introduction to gene transcription regulation in general. Finally, the aim and outline of this thesis is presented.

An important aim of this thesis is to generate surveys of genome-wide gene expression data in chicken using microarrays. In chapter 2, we introduce microarray data normalization including background correction, within-array normalization and between-array normalization. Based on these results an analysis approach is recommended for the analysis of two-color microarray data as performed in the experiments described in this thesis. We also briefly explain the relevant methodology for the identification of differentially expressed genes and how to translate resulting gene lists into biological knowledge. Finally, specific issues related to updating microarray probe annotation in farm animals, is discussed. For the analysis of the microarray data in this thesis re-annotation of the probes on the chicken 20K oligoarray was done using the oligoRAP, analysis pipeline.

The vast amount of data generated from a single transcriptomics study makes it impossible to extract meaningful biological knowledge by manually going through individual genes from a list with hundreds and thousands of differentially expressed genes. In chapter 3, we present a practical approach using a collection of R/Bioconductor packages to extract biological knowledge from a microarray experiment in farm animals. Furthermore, a locally adaptive statistical procedure (LAP) analysis approach is used to identify differentially expressed chromosomal regions in a microarray experiment.

Chapter 4 presents a genome-wide gene expression survey across eight different tissues (brain, bursa of Fabricius, kidney, liver, lung, small intestine, spleen, and thymus from 10-week old chickens) in adult birds using a chicken 20K microarray. To a certain extent, most genes show some tissue-specific pattern of expression. Housekeeping and tissue-specific genes are identified based on gene expression patterns across the eight different tissues. The results show that housekeeping genes are more compact, i.e. are smaller, with shorter, coding sequence length, intron length, and smaller length of the intergenic regions. This observed compactness of housekeeping genes may be a result of selection on economy of transcription during evolution. Furthermore, a comparative analysis of gene expression among mouse, chicken, and frog showed that the expression patterns of orthologous genes are conserved during evolution between mammals, birds, and amphibians.

The chicken embryo has been a very popular model for developmental biology. To study the overall gene expression pattern in whole chicken embryos at different developmental stages and/or embryonic tissues, a genome-wide gene expression survey across different developmental and embryonic stages was performed (chapter 5). The study included four different developmental stages (HH stage 3, 10, 15, 22) and eight different embryonic tissues (brain, bursa of Fabricius, heart, kidney, liver, lung, small intestine, and spleen from HH stage 36). We were able to identify several embryonic stage- and tissue-specific genes in our analysis. Genomic features of genes widely expressed under these 12 conditions suggest that widely expressed genes are more compact than tissue-specific genes, confirming the findings described in chapter 4. The analysis of the differentially expressed genes during the different developmental stages of whole embryo indicates a gradual change in gene expression during embryo development. A comparison of the gene expression profiles between the same organs, of adults and embryos reveals both striking similarities as well as differences.

The overall goal of this thesis was to improve our understanding of the mechanisms of transcriptional regulation in the chicken. In chapter 6, a transcriptome map for all chicken chromosomes is presented based on the expression data described in chapter 4. The results reveal the presence of two distinct types of chromosomal regions characterized by clusters of highly or lowly expressed genes respectively. Furthermore, these regions show a high correlation with a number of genome characteristics, like gene density, gene length, intron length, and GC content. A comparative analysis between the chicken and human transcriptome maps suggests that the regions with clusters of highly expressed genes are relatively conserved between the two genomes. Our results revealed the presence of a higher order organization of the chicken genome that affects gene expression, confirming similar observations in other species.

Finally, in chapter 7 I summarize the main findings and discuss some of the limitations of the analyses described in this thesis. I also discuss the different merits and shortcomings of studying gene expression using either microarrays or next-generation sequencing technology and propose directions for future research. The rapid developments in new-generation sequencing technology will facilitate better coverage and depth of the chicken genome. This will provide a better genome assembly and an improved genome annotation. The sequence-based approaches for studying gene expression will reduce noise levels compared to hybridization-based approaches. Overall, next-generation sequencing is already providing greatly enhance tools to further improve our understanding of the chicken transcriptome and its regulation.


Site-Specific Immobilization of DNA in Glass Microchannels via Photolithography
Vong, T. ; Maat, J. ter; Beek, T.A. van; Lagen, B. van; Giesbers, M. ; Hest, J.C.M. van; Zuilhof, H. - \ 2009
Langmuir 25 (2009)24. - ISSN 0743-7463 - p. 13952 - 13958.
organic monolayers - covalent attachment - silicon surfaces - micrometer-scale - alkyl monolayers - electrochemistry - lithography - microarrays - strategies - h-si(111)
For the first time, it microchannel was photochemically patterned with it functional linker. This simple method was developed for the site-specific attachment of DNA via this linker onto silicon oxide surfaces (e.g., fused silica and borosilicate glass), both onto a flat surface and onto the inside of a fused silica microchannel. Sharp boundaries in the micrometer range between modified and unmodified zones were demonstrated by the attachment Of fluorescently labeled DNA oligomers. Studies of repeated hybridization-deliybridization cycles revealed selective and reversible binding of cDNA strands at the explicit locations. On average, similar to 7 x 10(11) fluorescently labeled DNA molecules were hybridized per square centimeter. The modified surfaces were characterized with X-ray photoelectron spectroscopy, infrared microscopy, static contact angle measurements, confocal laser scanning microscopy, and fluorescence detection (to quantify the attachment of the fluorescently labeled DNA).
Web services for transcriptomics
Neerincx, P. - \ 2009
University. Promotor(en): Jack Leunissen. - [S.l. : S.n. - ISBN 9789085854647 - 184
bio-informatica - internet - moleculaire biologie - computers - datacommunicatie - gegevensverwerking - transcriptomics - computernetwerken - microarrays - genexpressieanalyse - datamining - bioinformatics - molecular biology - data communication - data processing - computer networks - genomics - data mining
Transcriptomics is part of a family of disciplines focussing on high throughput molecular biology experiments. In the case of transcriptomics, scientists study the expression of genes resulting in transcripts. These transcripts can either perform a biological function themselves or function as messenger molecules containing a copy of the genetic code, which can be used by the ribosomes as templates to synthesise proteins. Over the past decade microarray technology has become the dominant technology for performing high throughput gene expression experiments.
A microarray contains short sequences (oligos or probes), which are the reverse complement of fragments of the targets (transcripts or sequences derived thereof). When genes are expressed, their transcripts (or sequences derived thereof) can hybridise to these probes. Many thousand copies of a probe are immobilised in a small region on a support. These regions are called spots and a typical microarray contains thousands or sometimes even more than a million spots. When the transcripts (or sequences derived thereof) are fluorescently labelled and it is known which spots are located where on the support, a fluorescent signal in a certain region represents expression of a certain gene. For interpretation of microarray data it is essential to make sure the oligos are specific for their targets. Hence for proper probe design one needs to know all transcripts that may be expressed and how well they can hybridise with candidate oligos. Therefore oligo design requires:
1. A complete reference genome assembly.
2. Complete annotation of the genome to know which parts may be transcribed.
3. Insight in the amount of natural variation in the genomes of different individuals.
4. Knowledge on how experimental conditions influence the ability of probes to hybridise with certain transcripts.
Unfortunately such complete information does not exist, but many microarrays were designed based on incomplete data nevertheless. This can lead to a variety of problems including cross-hybridisation (non-specific binding), erroneously annotated and therefore misleading probes, missing probes and orphan probes.
Fortunately the amount of information on genes and their transcripts increases rapidly. Therefore, it is possible to improve the reliability of microarray data analysis by regular updates of the probe annotation using updated databases for genomes and their annotation. Several tools have been developed for this purpose, but these either used simplistic annotation strategies or did not support our species and/ or microarray platforms of interest. Therefore, we developed OligoRAP (Oligo Re- Annotation Pipeline), which is described in chapter 2. OligoRAP was designed to take advantage of amongst others annotation provided by Ensembl, which is the largest genome annotation effort in the world. Thereby OligoRAP supports most of the major animal model organisms including farm animals like chicken and cow. In addition to support for our species and array platforms of interest OligoRAP employs a new annotation strategy combining information from genome and transcript databases in a non-redundant way to get the most complete annotation possible.
In chapter 3 we compared annotation generated with 3 oligo annotation pipelines including OligoRAP and investigated the effect on functional analysis of a microarray experiment involving chickens infected with Eimeria bacteria. As an example of functional analysis we investigated if up- or downregulated genes were enriched for Terms from the Gene Ontology (GO). We discovered that small differences in annotation strategy could lead to alarmingly large differences in enriched GO terms.
Therefore it is important to know, which annotation strategy works best, but it was not possible to assess this due to the lack of a good reference or benchmark dataset. There are a few limited studies investigating the hybridisation potential of imperfect alignments of oligos with potential targets, but in general such data is scarce. In addition it is difficult to compare these studies due to differences in experimental setup including different hybridisation temperatures and different probe lengths. As result we cannot determine exact thresholds for the alignments of oligos with non-targets to prevent cross-hybridisation, but from these different studies we can get an idea of the range for the thresholds that would be required for optimal target specificity. Note that in these studies experimental conditions were first optimised for an optimal signal to noise ratio for hybridisation of oligos with targets. Then these conditions were used to determine the thresholds for alignments of oligos with non-targets to prevent cross-hybridisation.
Chapter 4 describes a parameter sweep using OligoRAP to explore hybridisation potential thresholds from a different perspective. Given the mouse genome thresholds were determined for the largest amount of gene specific probes. Using those thresholds we then determined thresholds for optimal signal to noise ratios. Unfortunately the annotation-based thresholds we found did not fall within the range of experimentally determined thresholds; in fact they were not even close. Hence what was experimentally determined to be optimal for the technology was not in sync with what was determined to be optimal for the mouse genome. Further research will be required to determine whether microarray technology can be modified in such a way that it is better suited for gene expression experiments. The requirement of a priori information on possible targets and the lack of sufficient knowledge on how experimental conditions influence hybridisation potential can be considered the Achiles’ heels of microarray technology.
Chapter 5 is a collection of 3 application notes describing other tools that can aid in analysis of transcriptomics data. Firstly, RShell, which is a plugin for the Taverna workbench allowing users to execute statistical computations remotely on R-servers. Secondly, MADMAX services, which provide quality control and normalisation of microarray data for AffyMetrix arrays. Finally, GeneIlluminator, which is a tool to disambiguate gene symbols allowing researchers to specifically retrieve literature for their genes of interest even if the gene symbols for those genes had many synonyms and homonyms.

Web services

High throughput experiments like those performed in transcriptomics usually require subsequent analysis with many different tools to make biological sense of the data. Installing all these tools on a single, local computer and making them compatible so users can build analysis pipelines can be very cumbersome. Therefore distributed analysis strategies have been explored extensively over the past decades. In a distributed system providers offer remote access to tools and data via the Internet allowing users to create pipelines from modules from all over the globe.
Chapter 1 provides an overview of the evolution of web services, which represent the latest breed in technology for creating distributed systems. The major advantage of web services over older technology is that web services are programming language independent, Internet communication protocol independent and operating system independent. Therefore web services are very flexible and most of them are firewall-proof. Web services play a major role in the remaining chapters of this thesis: OligoRAP is a workflow entirely made from web services and the tools described in chapter 5 all provide remote programmatic access via web service interfaces. Although web services can be used to build relatively complex workflows like OligoRAP, a lack of mainly de facto standards and of user-friendly clients has limited the use of web services to bioinformaticians. A semantic web where biologists can easily link web services into complex workflows does n
Effective use of enzyme microreactors : thermal, kinetic and ethical guidelines
Swarts, J.W. - \ 2009
University. Promotor(en): Remko Boom; Michiel Korthals, co-promotor(en): Anja Janssen. - [S.l. : S.n. - ISBN 9789085853732 - 146
bioreactoren - enzymen - enzymactiviteit - microfiltratie - temperatuur - vloeistofmechanica - industriële enzymen - microtechnieken - microarrays - bioreactors - enzymes - enzyme activity - microfiltration - temperature - fluid mechanics - industrial enzymes - microtechniques
Microreactor technology is reported to have many benefits over regular chemical methods. Due to the small dimensions over which temperature and concentration gradients can exist, mass and heat transfer can be very quick. This could minimize the time needed for heating and mixing, due to a reduction in diffusion limitation. Furthermore, a very low fluid to chip volume ratio could facilitate a very stable fluid temperature.

The goal of this thesis research was to investigate the effect of the use of microreactors on enzyme kinetics and the thermal behaviour of fluids inside the chip. First, the effect of the design and use of a microsystem on the fluid temperature inside the microfluidic chip was investigated experimentally and with computer models. A stable and predictable temperature is of great importance for running (enzymatic) processes in a microchip. Next, we used model enzyme reactions to investigate whether the enzyme kinetics were different on micro and bench scale, and when diffusion would play a role. Furthermore, some social and ethical aspects of microreactor technology applications were studied.

To ensure a stable and predictable temperature of the fluids inside the microreactor, the microsystem should be properly designed and used. To test these two aspects, we investigated the effect of practical use (chapter 2) and design parameters (chapter 3) on this fluid temperature. The micro system used in this research consisted of a PEEK chipholder, a relatively small heater, a glass microchip, and surrounding air. We conducted experiments and used computational fluid dynamics models to understand the effect of all varied parameters. In the design of the system, the chipholder shape and material (with its density, specific heat, and thermal conductivity) dominated the temperature of the fluid inside the chip. A temperature gradient as large as 40°C was observed over the length of the chip. This temperature profile at fluid level can be changed by adapting the geometry and material of the chipholder. The results show that a uniform temperature is highly dependent on the correct design of the integrated system of chip, chipholder, and heater. The practical use of the chip with moderate air flow over the chip and moderate fluid flow rates through the channel had no effect on the fluid temperature. A well designed micro system can therefore be considered thermally robust under moderate processing conditions.

The microsystem from chapters 2 and 3 was used for enzyme reactions on micro scale. The kinetic parameters of a lipase catalyzed esterification reaction (chapter 4) and a β-galactosidase catalyzed hydrolysis reaction (chapter 5) on this micro scale were the same as those found on bench scale. Kinetic and thermal (in-)activation results obtained on micro scale can be used for large scale processing. This can bring down optimization costs by reducing the required amount of enzyme and chemicals.

Next, we found that at residence times below a few seconds, diffusion effects limited the reaction rate and therefore reduced the conversion per volume of enzyme microreactor. This effect of diffusion on the conversion increased quadratically with channel width, increased with enzyme concentration, and decreased with substrate concentration. When an enzyme microreactor system should be run efficiently, these factors should be explored to avoid diffusion limitation and subsequent reduced volumetric productivity.

With microreactor technology reaching maturity, a wider application of the technology could be imagined. With increasing application the impact it will have on society will also increase. In chapter 6, three examples of microreactor technology applications in nutrition, in medicine, and in energy carrier supply were investigated. The benefits and costs, and their distribution were discussed for these examples. Furthermore, possible strategies of communication surrounding a public introduction of such a novel technology were considered. The applications proposed in this chapter were only three out of an infinite number of possibilities. However, the discussion of these examples can be used as a framework for discussing future applications as they might be developed in the future. A societal backlash as with the GMO-scare in 1990s, can be avoided when the relevant issues are communicated appropriately and timely. This could improve the chances of success of this technology in the market.

In this thesis we have shown that microreactors can be a useful tool for reaction engineering. Their use could reduce the required amount of enzyme and chemicals for optimization. Furthermore, they can be used to study processes with a very short residence time. To use microreactor technology effectively, one does have to consider whether the scale is appropriate, and whether that the system, including chipholder, interfaces to the outer world and thermal actuators, is properly designed and used.

Bayesian networks for omics data analysis
Gavai, A.K. - \ 2009
University. Promotor(en): Jack Leunissen; Michael Muller, co-promotor(en): Guido Hooiveld; P.J.F. Lucas. - [S.l.] : S.n. - ISBN 9789085853909 - 98
bio-informatica - waarschijnlijkheidsmodellen - bayesiaanse theorie - netwerkanalyse - genexpressie - roken - vluchtige verbindingen - biochemische omzettingen - voedingsonderzoek bij de mens - genexpressieanalyse - microarrays - netwerken - nutrigenomica - bioinformatics - probabilistic models - bayesian theory - network analysis - gene expression - smoking - volatile compounds - biochemical pathways - human nutrition research - genomics - networks - nutrigenomics
This thesis focuses on two aspects of high throughput technologies, i.e. data storage and data analysis, in particular in transcriptomics and metabolomics. Both technologies are part of a research field that is generally called ‘omics’ (or ‘-omics’, with a leading hyphen), which refers to genomics, transcriptomics, proteomics, or metabolomics. Although these techniques study different entities (genes, gene expression, proteins, or metabolites), they all have in common that they use high-throughput technologies such as microarrays and mass spectrometry, and thus generate huge amounts of data. Experiments conducted using these technologies allow one to compare different states of a living cell, for example a healthy cell versus a cancer cell or the effect of food on cell condition, and at different levels.
The tools needed to apply omics technologies, in particular microarrays, are often manufactured by different vendors and require separate storage and analysis software for the data generated by them. Moreover experiments conducted using different technologies cannot be analyzed simultaneously to answer a biological question. Chapter 3 presents MADMAX, our software system which supports storage and analysis of data from multiple microarray platforms. It consists of a vendor-independent database which is tightly coupled with vendor-specific analysis tools. Upcoming technologies like metabolomics, proteomics and high-throughput sequencing can easily be incorporated in this system.
Once the data are stored in this system, one obviously wants to deduce a biological relevant meaning from these data and here statistical and machine learning techniques play a key role. The aim of such analysis is to search for relationships between entities of interest, such as genes, metabolites or proteins. One of the major goals of these techniques is to search for causal relationships rather than mere correlations. It is often emphasized in the literature that "correlation is not causation" because people tend to jump to conclusions by making inferences about causal relationships when they actually only see correlations. Statistics are often good in finding these correlations; techniques called linear regression and analysis of variance form the core of applied multivariate statistics. However, these techniques cannot find causal relationships, neither are they able to incorporate prior knowledge of the biological domain. Graphical models, a machine learning technique, on the other hand do not suffer from these limitations.
Graphical models, a combination of graph theory, statistics and information science, are one of the most exciting things happening today in the field of machine learning applied to biological problems (see chapter 2 for a general introduction). This thesis deals with a special type of graphical models known as probabilistic graphical models, belief networks or Bayesian networks. The advantage of Bayesian networks over classical statistical techniques is that they allow the incorporation of background knowledge from a biological domain, and that analysis of data is intuitive as it is represented in the form of graphs (nodes and edges). Standard statistical techniques are good in describing the data but are not able to find non-linear relations whereas Bayesian networks allow future prediction and discovering nonlinear relations. Moreover, Bayesian networks allow hierarchical representation of data, which makes them particularly useful for representing biological data, since most biological processes are hierarchical by nature. Once we have such a causal graph made either by a computer program or constructed manually we can predict the effects of a certain entity by manipulating the state of other entities, or make backward inferences from effects to causes. Of course, if the graph is big, doing the necessary calculations can be very difficult and CPU-expensive, and in such cases approximate methods are used.
Chapter 4 demonstrates the use of Bayesian networks to determine the metabolic state of feeding and fasting mice to determine the effect of a high fat diet on gene expression. This chapter also shows how selection of genes based on key biological processes generates more informative results than standard statistical tests. In chapter 5 the use of Bayesian networks is shown on the combination of gene expression data and clinical parameters, to determine the effect of smoking on gene expression and which genes are responsible for the DNA damage and the raise in plasma cotinine levels of blood of a smoking population. This study was conducted at Maastricht University where 22 twin smokers were profiled. Chapter 6 presents the reconstruction of a key metabolic pathway which plays an important role in ripening of tomatoes, thus showing the versatility of the use of Bayesian networks in metabolomics data analysis.
The general trend in research shows a flood of data emerging from sequencing and metabolomics experiments. This means that to perform data mining on these data one requires intelligent techniques that are computationally feasible and able to take the knowledge of experts into account to generate relevant results. Graphical models fit this paradigm well and we expect them to play a key role in mining the data generated from omics experiments.
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