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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.

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A novel approach for anthropogenic heat flux estimation from space
Chrysoulakis, Nektarios ; Heldens, Wieke ; Gastellu-Etchegorry, Jean Philippe ; Grimmond, Sue ; Feigenwinter, Christian ; Lindberg, Fredrik ; Frate, Fabio Del ; Klostermann, Judith ; Mitraka, Zina ; Esch, Thomas ; Albitar, Ahmad ; Gabey, Andrew ; Parlow, Eberhard ; Olofson, Frans - \ 2016
In: International Geoscience and Remote Sensing Symposium (IGARSS). - Institute of Electrical and Electronics Engineers Inc. - ISBN 9781509033324 - p. 6774 - 6777.
Sentinels - urban climate - urban energy budget

The recently launched H2020 project URBANFLUXES (URBan ANthrpogenic heat FLUX from Earth observation Satellites) investigates the potential of EO to retrieve anthropogenic heat flux, as a key component in the Urban Energy Budget (UEB). URBANFLUXES advances existing Earth Observation (EO) based methods for estimating spatial patterns of turbulent sensible and latent heat fluxes, as well as urban heat storage flux at city scale and local scale. Independent methods and models are engaged to evaluate the derived products and statistical analyses provide uncertainty measures. Optical, thermal and SAR data are exploited to improve the accuracy of the UEB components spatial distribution calculation. Synergistic use of different types and of various resolution EO data allows estimates in local and city scale. Ultimate goal of the URBANFLUXES is to develop a highly automated method for estimating UEB components to use with Copernicus Sentinel data, enabling its integration into applications and operational services.

Efficacy of a pandemic (H1N1) 2009 virus vaccine in pigs against the pandemic influenza virus is superior to commercially available swine influenza vaccines.
Loeffen, W.L.A. ; Stockhofe-Zurwieden, N. ; Weesendorp, E. ; Zoelen-Bos, D.J. van; Heutink, R. ; Quak, J. ; Goovaerts, D. ; Heldens, J. ; Maas, H.A. ; Moormann, R.J.M. ; Koch, G. - \ 2011
Veterinary Microbiology 152 (2011)3-4. - ISSN 0378-1135 - p. 304 - 314.
fever virus - new-jersey - fort-dix - a-virus - transmission - pathogenesis - isolations - infection - quantification - outbreak
In April 2009 a new influenza A/H1N1 strain, currently named “pandemic (H1N1) influenza 2009¿ (H1N1v), started the first official pandemic in humans since 1968. Several incursions of this virus in pig herds have also been reported from all over the world. Vaccination of pigs may be an option to reduce exposure of human contacts with infected pigs, thereby preventing cross-species transfer, but also to protect pigs themselves, should this virus cause damage in the pig population. Three swine influenza vaccines, two of them commercially available and one experimental, were therefore tested and compared for their efficacy against an H1N1v challenge. One of the commercial vaccines is based on an American classical H1N1 influenza strain, the other is based on a European avian H1N1 influenza strain. The experimental vaccine is based on reassortant virus NYMC X179A (containing the hemagglutinin (HA) and neuraminidase (NA) genes of A/California/7/2009 (H1N1v) and the internal genes of A/Puerto Rico/8/34 (H1N1)). Excretion of infectious virus was reduced by 0.5–3 log10 by the commercial vaccines, depending on vaccine and sample type. Both vaccines were able to reduce virus replication especially in the lower respiratory tract, with less pathological lesions in vaccinated and subsequently challenged pigs than in unvaccinated controls. In pigs vaccinated with the experimental vaccine, excretion levels of infectious virus in nasal and oropharyngeal swabs, were at or below 1 log10 TCID50 per swab and lasted for only 1 or 2 days. An inactivated vaccine containing the HA and NA of an H1N1v is ably to protect pigs from an infection with H1N1v, whereas swine influenza vaccines that are currently available are of limited efficaciousness. Whether vaccination of pigs against H1N1v will become opportune remains to be seen and will depend on future evolution of this strain in the pig population. Close monitoring of the pig population, focussing on presence and evolution of influenza strains on a cross-border level would therefore be advisable.
Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells
Metz, S.W.H. ; Geertsema, C. ; Martina, Byron E. ; Andrade, Paulina ; Heldens, J. ; Oers, M.M. van; Goldbach, R.W. ; Vlak, J.M. ; Pijlman, G.P. - \ 2011
Virology journal 8 (2011). - ISSN 1743-422X - 12 p.
semliki-forest-virus - equine encephalitis-virus - envelope fusion protein - swine-fever virus - n-linked glycans - sindbis virus - structural proteins - baculovirus vectors - nucleotide-sequence - signal peptide
Background - Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results - Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions - Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.
Biochemical and molecular analysis of pink tomatoes: deregulated expression of the gene encoding transcription factor SlMYB12 leads to pink tomato fruit colour
Ballester, A.R. ; Molthoff, J.W. ; Vos, C.H. de; Lintel Hekkert, B. ; Orzaez, D. ; Fernandez-Moreno, J.P. ; Tripodi, S. ; Grandillo, S. ; Martin, C. ; Heldens, J. ; Ykema, M. ; Granell, A. ; Bovy, A.G. - \ 2010
Plant Physiology 152 (2010). - ISSN 0032-0889 - p. 71 - 84.
arabidopsis-thaliana - flavonoid biosynthesis - in-vivo - anthocyanin - carotenoids - health - accumulation - inheritance - metabolome - mutations
The color of tomato fruit is mainly determined by carotenoids and flavonoids. Phenotypic analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum 'Moneyberg' and the wild species Solanum chmielewskii revealed three ILs with a pink fruit color. These lines had a homozygous S. chmielewskii introgression on the short arm of chromosome 1, consistent with the position of the y (yellow) mutation known to result in colorless epidermis, and hence pink-colored fruit, when combined with a red flesh. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-colored flavonoid naringenin chalcone in the fruit peel, while carotenoid levels are not affected. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone was down-regulated in pink fruit, suggesting that the candidate gene underlying the pink phenotype encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and basic helix-loop-helix transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the MYB12 gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink- and red-fruited genotypes during ripening. Genetic mapping and segregation analysis showed that MYB12 is located on chromosome 1 and segregates perfectly with the characteristic pink fruit color. Virus-induced gene silencing of SlMYB12 resulted in a decrease in the accumulation of naringenin chalcone, a phenotype consistent with the pink- colored tomato fruit of IL1b. In conclusion, biochemical and molecular data, gene mapping, segregation analysis, and virus-induced gene silencing experiments demonstrate that the MYB12 transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit and suggest strongly that SlMYB12 is a likely candidate for the y mutation.
The ATF6-Met [67] Val substitution is associated with increased plasma cholesterol levels
Meex, S.J. ; Weissglas-Volkov, D. ; Kallen, C.J. ; Thuerauf, D.J. ; Greevenbroek, M.M. ; Schalkwijk, C.G. ; Stehouwer, C.D. ; Feskens, E.J.M. ; Heldens, J. ; Ayoubi, T.A. ; Hofker, M.H. ; Wouters, B.G. ; Vlietinck, R. ; Sinsheimer, J.S. ; Taskinen, M.R. ; Kuusisto, J. ; Laakso, K. ; Bruin, T.W. ; Pajukanta, P. ; Glembotski, C.C. - \ 2009
Arteriosclerosis Thrombosis and Vascular Biology 29 (2009). - ISSN 1079-5642 - p. 1322 - 1327.
transcription factor - in-vivo - stress - domain - activation - atf6 - degradation - binding - er
Objective— Activating transcription factor 6 (ATF6) is a sensor of the endoplasmic reticulum stress response and regulates expression of several key lipogenic genes. We used a 2-stage design to investigate whether ATF6 polymorphisms are associated with lipids in subjects at increased risk for cardiovascular disease (CVD). Methods and Results— In stage 1, 13 tag-SNPs were tested for association in Dutch samples ascertained for familial combined hyperlipidemia (FCHL) or increased risk for CVD (CVR). In stage 2, we further investigated the SNP with the strongest association from stage 1, a Methionine/Valine substitution at amino-acid 67, in Finnish FCHL families and in subjects with CVR from METSIM, a Finnish population-based cohort. The combined analysis of both stages reached region-wide significance (P=9x10–4), but this association was not seen in the entire METSIM cohort. Our functional analysis demonstrated that Valine at position 67 augments ATF6 protein and its targets Grp78 and Grp94 as well as increases luciferase expression through Grp78 promoter. Conclusions— A common nonsynonymous variant in ATF6 increases ATF6 protein levels and is associated with cholesterol levels in subjects at increased risk for CVD, but this association was not seen in a population-based cohort. Further replication is needed to confirm the role of this variant in lipids. We report the association of the ATF6-methionine [67]valine amino-acid substitution with plasma cholesterol levels. Association analyses in 2674 subjects and functional data suggest that the ATF6 gene may influence cholesterol levels in subjects at increased risk to develop cardiovascular disease.
Sequence and organization of the Spodoptera exigua multicapsid nucleopolyhedrovirus genome
IJkel, W.F.J. ; Strien, E.A. van; Heldens, J.G.M. ; Broer, R. ; Zuidema, D. ; Goldbach, R.W. ; Vlak, J.M. - \ 1999
Journal of General Virology 80 (1999). - ISSN 0022-1317 - p. 3289 - 3304.
Recombination of baculovirus DNA following lipofection in insect larvae.
Hajos, J.P. ; Zuidema, D. ; Kulcsar, P. ; Heldens, J.G.M. ; Zavodszky, P. ; Vlak, J.M. - \ 1998
Archives of Virology 143 (1998). - ISSN 0304-8608 - p. 2045 - 2050.
Specificity of multiple homologous regions in Spodoptera exigua nucleopolyhedrovirus DNA replication.
Broer, R. ; Heldens, J.G.M. ; Strien, E.A. van; Zuidema, D. ; Vlak, J.M. - \ 1998
Journal of General Virology 79 (1998). - ISSN 0022-1317 - p. 1563 - 1572.
Functional analysis of DNA replication origins in the genome of Spodoptera exigua baculovirus.
Ijkel, W.F.J. ; Broer, R. ; Heldens, J.G.M. ; Goldbach, R.W. ; Vlak, J.M. ; Zuidema, D. - \ 1998
In: 17th Annual Meeting of the American Society for Virology, Vancouver, Canada - p. 65 - 65.
A highly conserved genomic region in baculoviruses: sequence analysis of an 11.3 kbp DNA fragment (46.5-55.1 m.u.) of the Spodoptera exigua multicapsid nucleopolyhedrovirus.
Heldens, J.G.M. ; Liu, Y. ; Zuidema, D. ; Goldbach, R.W. ; Vlak, J.M. - \ 1998
Virus Research 55 (1998). - ISSN 0168-1702 - p. 187 - 198.
Molecular genetics of the Spodoptera exigua multicapsid nucleopolyhedrovirus genome
Heldens, J.G.M. - \ 1998
Agricultural University. Promotor(en): R.W. Goldbach; J.M. Vlak; D. Zuidema. - S.l. : Heldens - ISBN 9789054858447 - 129
baculovirus - kernpolyedervirussen - moleculaire genetica - insecten - plantenplagen - noctuidae - biologische bestrijding - virussen - organismen ingezet bij biologische bestrijding - baculovirus - nuclear polyhedrosis viruses - molecular genetics - insects - plant pests - noctuidae - biological control - viruses - biological control agents

Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is an attractive biological control agent for the beet army worm S. exigua . This baculovirus has a narrow host range and is relatively, compared to other baculoviruses, virulent for beet army worm larvae. The molecular principles that specify the host range and virulence of SeMNPV are unknown. This thesis describes studies aimed at the unravelling of the molecular genetics of this baculovirus and the key steps in the infection and genome replication process.

As a first step SeMNPV was multiplied in an established cell line of S. exigua to obtain a better understanding of the replication process. Polyhedra derived from cell culture were unable to infect S. exigua larvae, although the hemolymph isolated from these larvae did contain budded virus able to infect insect cell lines (Chapter 2). This suggested the generation of a genetic defect in the SeMNPV genome during replication and/or maintenance in cell culture. A partial plasmid library and complete cosmid library were made to construct a physical map of the SeMNPV genome and locate the genetic defect(s). The position of the polyhedrin gene and the transcriptional direction of the p10 gene allowed the pin-pointing of the zero point and orientation of the circular SeMNPV genome. A large deletion (20-25 kilobase pairs), turned out to be the genetic defect, arising upon limited passaging of the virus in cell culture. This deletion was located between map unit 12.9 and 31.3 in the SeMNPV genome. The occurrence of this deletion implied that the construction of SeMNPV recombinant viruses still virulent in vivo cannot be achieved via conventional techniques, i.e. homologous recombination in cell culture (Chapter 2). Alternative recombinationstrategies involving yeast artificial chromosomes and in vivo cloning were considered and partially tested (Chapter 7).

In Chapters 3 and 4 the identification and characterization of cis -acting elements in SeMNPV DNA replication are described. Transient DNA replication assays, sequence analyses and hybridization experiments identified one non- hr ( hr homologous region) and six hr origins of DNA replication. SeMNPV hr s contained one ( hr 4) up to nine ( hr 1) repeated, near-identical 68-bp long palindromes. The SeMNPV hr s, located in non-coding regions, were found dispersed in the viral genome as observed in the genomes of two other baculoviruses, Autographa californica MNPV and Orgyia pseudotsugata MNPV. Transient DNA replication assays in AcMNPV-infected insect cells revealed no replication of SeMNPV- hr s and, in SeMNPV-infected insect cells no replication of AcMNPV- hr s could be observed, suggesting that these elements display specificity (Chapter 3). In the SeMNPV- Xba I library one additional genomic fragment unrelated to hr s and reminiscent of AcMNPV and OpMNPV non- hr origins of DNA replication that underwent SeMNPV dependent DNA replication was identified. By deletion analysis the core of this non- hr origin was mapped within a 800 bp region of non-coding sequence. This sequence contained also several motifs such as multiple palindromes, direct repeats, putative transcriptional factor binding sites and multiple polyadenylation signals, characteristic for baculovirus non- hr and other eukaryotic origins of DNA replication. In contrast to the hr s, the SeMNPV non- hr origin could be replicated by the replication machinery of the heterologous AcMNPV (Chapter 4).

The putative helicase is the most intriguing trans -acting DNA replication factor of baculoviruses, since it may be involved in both DNA replication and host range determination. An open reading frame (ORF) potentially encoding a polypeptide of 143 kDa (p143) with considerable amino acid identity to the putative helicases of AcMNPV, BmNPV and OpMNPV was identified in SeMNPV. Sequence alignment of the SeMNPV p143 indicated that it is somewhat diverged from its AcMNPV, BmNPV and OpMNPV homologs. Whether the protein is also involved in the host range of SeMNPV determination remained unsolved. The ORF is expressed as a 4 kb transcript between 4 and 24 h p.i., starting from an unusual transcriptional initiation site present eleven nucleotides upstream of the translational start. Transient plasmid dependent DNA replication assays showed that not only helicase plays a crucial role in SeMNPV and AcMNPV replication specificity, but also one or more of the other previously mentioned essential trans -acting DNA replication factors. Apparently the interaction between the origins of DNA replication and/or the assembly of the replisome is a highly virus specific process (Chapter 5).

Complete sequence and transcriptional analysis of the 11.3 kb SeMNPV- Xbal-C fragment containing the p143 gene revealed twelve ORFs that all showed high amino acid identity to AcMNPV and OpMNPV homologs. The genetic organization of the SeMNPV- Xba I-C fragment was identical to the AcMNPV and OpMNPV helicase region, although in an antigenomic orientation. In line with recent observations in herpes- and poxvirus genomes, which contain conserved central parts of their genomes and more diverged termini, it is hypothesized that baculoviruses genomes could also contain a highly conserved gene block centered around the p143 gene and a more diverged region at the polyhedrin - p10 loci. This hypothesis is further supported by partial sequence and hybridization data from other baculovirus genomes. The organization of the less conserved polyhedrin-p10 region could be a marker for the genetic relatedness of baculoviruses (Chapter 6). The state of the art on the sequence analysis of the complete SeMNPV genome is described in Chapter 7.

The availability of a physical map, the insght in the genetic organization of the SeMNPV genome and the occurence of the spontaneous deletion mutants in cell culture prompted the development of alternative recombination strategies to bypass the use of the insect cell lines (Chapter 7). To this end a recombination strategy using yeast genetics was employed. Deletion of the yeast autonomous replicating sequences prior to the application of the recombinant baculoviruses in the field is recommended and can be achieved for SeMNPV using a direct in vivo recombination and selection protocol. The strategy proposed is based on the occurrence of homologous recombination of baculoviruses in the insect (Chapter 7).

The research on SeMNPV described in this thesis, has created a good starting point to study the molecular basis of virulence and host range. The development of the recombination system described in chapter 7 could offer a tool for the insertion and deletion of specific (viral) genes for this purpose. Moreover exploiting the proposed recombination system, the improvement of the insecticidal properties of SeMNPV can be pursued.

Functional analysis of DNA replication origins in the genome of Spodoptera exigua multicapsid nucleopolyhedrovirus.
IJkel, W.F.J. ; Heldens, J.G.M. ; Broer, R. ; Goldbach, R.W. ; Vlak, J.M. ; Zuidema, D. - \ 1997
In: Abstract Book of the Annual Meeting of the 'SON werkgemeenschap Nucleïnezuren', Lunteren, the Netherlands - p. 29 - 29.
Specificity of baculovirus trans-acting DNA replication factors: analysis of the Spodoptera exigua MNPV p143 gene.
Heldens, J.G.M. ; Liu, Y. ; Goldbach, R.W. ; Vlak, J.M. - \ 1997
In: Abstract Book of the 30th Annual Meeting of the Society for Invertebrate Pathology, Banff, Canada - p. 26 - 26.
Characterization of a putative Spodoptera exigua multicapsid nucleopolyhedrovirus helicase gene.
Heldens, J.G.M. ; Liu, Y. ; Zuidema, D. ; Goldbach, R.W. ; Vlak, J.M. - \ 1997
Journal of General Virology 78 (1997). - ISSN 0022-1317 - p. 3101 - 3114.
Identification and functional analysis of a non-hr origin of DNA replication in the genome of Spodoptera exigua multicapsid nucleopolyhedrovirus.
Heldens, J.G.M. ; Broer, R. ; Zuidema, D. ; Goldbach, R.W. ; Vlak, J.M. - \ 1997
Journal of General Virology 78 (1997). - ISSN 0022-1317 - p. 1497 - 1506.
The Spodoptera exigua MNPV helicase gene: structural and functional analysis.
Heldens, J.G.M. ; Yi, L. ; Zuidema, D. ; Goldbach, R.W. ; Vlak, J.M. - \ 1997
In: Abstract Book of the 16th Annual Meeting of the American Society for Virology, Bozeman, Montana, USA - p. 120 - 120.
Generation of a p10-based baculovirus expression vector in yeast with infectivity for insect larvae and insect cell culture.
Heldens, J.G.M. ; Kester, H.A. ; Zuidema, D. ; Vlak, J.M. - \ 1997
Journal of Virological Methods 68 (1997). - ISSN 0166-0934 - p. 57 - 63.
Indentification and characterization of multiple hr-like sequences in the genome of Spodoptera exigua MNPV.
Broer, R. ; Heldens, J.G.M. ; Strien, E.A. van; Goldbach, R.W. ; Vlak, J.M. - \ 1997
In: Abstract Book of the 30th Annual Meeting of the Society for Invertebrate Pathology, Banff, Canada - p. 9 - 9.
Wood quality and wood preferences in relation to food preparation and diet composition in central Malawi.
Brouwer, I.D. ; Hartog, A.P. den; Kamwendo, M.O.K. ; Heldens, M.W.O. - \ 1996
Ecology of Food and Nutrition 35 (1996). - ISSN 0367-0244 - p. 1 - 13.
Specificity of baculovirus DNA replication factors.
Heldens, J.G.M. ; Liu, Y. ; Broer, R. ; Zuidema, D. ; Goldbach, R.W. ; Vlak, J.M. - \ 1996
In: Abstract Annual Meeting SON/NWO, Lunteren - p. 2 - 2.
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