Controlling the self-assembly of protein polymers via heterodimer-forming modules
Domeradzka, Natalia Eliza - \ 2016
Wageningen University. Promotor(en): Frans Leermakers, co-promotor(en): Renko de Vries; Frits de Wolf. - Wageningen : Wageningen University - ISBN 9789462578661 - 166
polymers - nanotechnology - pichia pastoris - modules - mass spectrometry - microscopy - sds-page - rheology - fluorescence emission spectroscopy - protein purification - fermentation - chromatography - polymeren - nanotechnologie - pichia pastoris - modules - massaspectrometrie - microscopie - sds-page - reologie - fluorescentie-emissiespectroscopie - eiwitzuivering - fermentatie - chromatografie
Supramolecular assemblies formed by protein polymers are attractive candidates for future biomaterials. Ideally, one would like to be able to define the nanostructure, in which the protein polymers should self-assemble, and then design protein polymer sequences that assemble exactly into such nanostructures. Despite progress towards ‘programmability’ of protein polymer self-assembly, we do not yet have such control. This holds especially for hierarchical structures such as self-assembled fibril bundles, where one would like to have independent control over the structures at the different length-scales. In this thesis we explore the use of heterodimerization as a strategy to control self-assembly of protein polymers at multiple length-scales. We tested a selected set of heterodimer-forming peptide modules. The heterodimer-forming modules are genetically incorporated at the C-terminus of protein polymers with a previously characterized self-assembly behavior. Several newly constructed protein polymers were biosynthesized in the yeast Pichia pastoris and, for these new protein polymers we investigated whether the inclusion of the heterodimer-forming blocks improved the control over the assembly of nanostructures.
The incorporation of heterodimer-forming modules into protein polymers is not the only tool that can be used for improving programmability of assembly. In Chapter 2 we present an overview of several tools that can be use, and we highlighted their advantages and disadvantages.
In Chapter 3 we test de novo designed heterodimerizing coiled coils DA = LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK and DB = LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK. These peptides were fused to hydrophilic random coil protein polymer (CP4) and homotrimer forming protein polymer (T9-CP4). We present data on the production, characterization and functionality for four new protein polymers: CP4-DA, CP4-DB, T9-CP4-DA and T9-CP4-DB. When the new protein polymers were produced using the fermentation process established previously for other protein polymers such as CP4 (i.e. standard fermentation), we found the new protein polymers to be partly degraded. The use of a protease deficient strain, as well as changes in aeration or pH were found ineffective in preventing degradation, but nearly intact products were obtained from a fermentation in which the induction was done at 20 ˚C and in which the medium was supplemented with casamino acids. With respect to the physical properties of the new protein polymers, size exclusion chromatography (SEC) showed that an equimolar mixture of CP4-DA and CP4-DB contained mostly dimers, whereas unmixed CP4-DA and CP4-DB contained only monomers. However, we also found that CP4-DB forms homooligomers at concentrations ≥100 µM. A mixture of T9-CP4-DA and T9-CP4-DB forms a hydrogel, most probably due to both homotypic and heterotypic DA/DB associations. We conclude that when used at low concentration, this pair of coiled coils seems to be suitable to control self-assembly of protein polymers produced in Pichia Pastoris.
Next, in Chapter 4 we test another pair of de novo designed coiled coils. These are much shorter and have lower reported values of the association constant as compared to the DA/DB coiled coils. The systems consist of a peptide DE = (EIAALEK)3 and a peptide DK = (KIAALKE)3. The two peptides were C-terminally fused to protein polymers CP4 and T9-CP4. The standard fermentations resulted in intact CP4-DE and T9-CP4-DE, but protein polymers CP4-DK and T9-CP4-DK were found to be partly degraded. The degradation of variants with DK module could not be readily resolved by fermentation at higher pH or using proteases deficient strain. For CP4-DK, ion exchange chromatography showed that about 40% of protein polymer (by mass) was intact. We find that for this pair of coiled-coils, homotypic interactions are so strong that they can drive gel formation in the case of T9-CP4-DE, and a strong increase in viscosity for T9-CP4-DK. Mixtures of the complimentary triblocks also form hydrogels, but it is not yet clear to what extent this is due to homotypic DE/ DE and DK/ DK associations, and to what extent it is due to DE/ DK heterodimer formation.
A very different type of heterodimer-forming block is the so-called WW domain that is found in many natural proteins, and which forms heterodimers with proline-rich peptides PPxY. In Chapter 5 we test the interaction between a naturally occurring WW domain (DWW) and its proline-rich ligand (DPPxY). Both were C-terminally fused to the hydrophilic random coil protein polymer CP4. The new protein polymers CP4-DWW and CP4-DPPxY were produced intact during standard fermentations, but CP4-DPPxY was shown to be glycosylated. Using genetic engineering, we mutated the CP4-DPPxY protein polymer sequence by the substitution Ser12→Ala. A standard fermentation resulted in an intact and non-glycosylated protein polymer CP4-DPPxY*. Interaction studies (ITC and steady state tryptophan fluorescence quenching), showed that both CP4-DPPxY and CP4-DPPxY* bind to CP4-DWW with an equilibrium dissociation constant on the order of mM.
Finally, to demonstrate that heterodimer-forming blocks can be used to independently control protein polymer self-assembly at multiple length-scales, we selected the heterodimer-forming modules DA and DB to control the lateral interactions of fibrils self-assembled from the previously designed triblock protein polymer C2-SH48-C2. In Chapter 6 we construct the protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. The C2-SH48-C2 protein polymers assemble into long and stiff fibrils at neutral pH. The aim of the C-terminal attachment of the DA/DB blocks was to be able to control subsequent physical cross-linking and bundling of the fibrils. Both protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB were produced intact and with high yield during fermentation at optimal conditions as discussed in Chapter 3. Using Atomic Force Microscopy (AFM) we show that at neutral pH, fibrils consisting of 100% C2-SH48-C2-DA or C2-SH48-C2-DB protein polymers bundle up and cross-link via homotypic DA/DA and DB/DB associations. Control over the degree of cross-linking and bundling can be obtained by using mixed fibrils consisting of C2-SH48-C2 with controlled amounts of the newly developed protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. While the effect of the heterodimers on the structure of the fibril network as judged from AFM is very strong, oscillation rheology shows that the inclusion of the heterodimer forming blocks merely leads to a moderate increase in gel stiffness.
In order to place the research discussed in this thesis into the broader perspective, in Chapter 7 we provide a General Discussion. We discuss several general strategies that can be used to control protein polymer self-assembly and discuss why and when there is a need for using heterodimer forming blocks. After providing an overview over results obtained in this thesis, we highlight the most urgent questions that need to be answered next. This is followed by a discussion on the benefits that heterodimer-driven self-assembly may bring to possible future applications of protein polymers as biomaterials. We also discuss the possible risks for human health end environment that might arise from the use of protein polymers technology. Finally we present some speculations about the future of the field of self-assembling protein polymers.
IAG ring test animal proteins 2016
Raamsdonk, L.W.D. van; Rhee, N.E. van de; Scholtens-Toma, I.M.J. ; Prins, T.W. ; Vliege, J.J.M. ; Pinckaers, V.G.Z. - \ 2016
Wageningen : RIKILT Wageningen UR (RIKILT report 2016.008) - 31
ring test - animal proteins - analytical methods - microscopy - fish feeding - animal health - polymerase chain reaction - ringtest - dierlijke eiwitten - analytische methoden - microscopie - visvoeding - diergezondheid - polymerase-kettingreactie
The annual ring test for the detection of animal proteins in animal feed of the IAG - International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy was organized by RIKILT - Wageningen UR, The Netherlands. The aim of the ring study was to provide the participants information on the performance of the local implementation of the detection method for their local quality systems. A further aim was to gather information about the application of the microscopic method. The current 2016 version of the IAG ring test for animal proteins facilitated the full scenario with the methods for microscopy and PCR as published in Regulation (EC) 51/2013 amending Annex VI of Regulation (EC) 152/2009 together with accompanying SOPs. All four samples were based on an artificial feed mimicking a formulation for ruminant feed. Two samples were labelled as fish feed (B and D), which was effectuated by adding 2% of a general fish meal. Adulteration was achieved by adding 0.1% pig MBM (B), 0.1% ruminant MBM (D) and a combination of 0.1% ruminant MBM and 0.1% fish meal (C). This combination of different spikes allowed the diverse application of the detection methods. Forty eight participants enrolled for the ring test, of which 45 submitted microscopic results. Of these, 20 participants applied the combination of microscopic and PCR analysis. Three participants submitted exclusively PCR results.
Krachtpatsers in de kas
Goud, J.C. - \ 2015
Gewasbescherming 46 (2015)4. - ISSN 0166-6495 - p. 122 - 123.
tuinbouw - glastuinbouw - biologische bestrijding - chemische bestrijding - sierteelt - chrysanthemum - microscopie - tentoonstellingen - insectenbestrijding - thrips - frankliniella occidentalis - nematoda - steinernema feltiae - horticulture - greenhouse horticulture - biological control - chemical control - ornamental horticulture - chrysanthemum - microscopy - exhibitions - insect control - thrips - frankliniella occidentalis - nematoda - steinernema feltiae
Op 3 september 2015 opende het museum Micropia, samen met BASF, een nieuwe opstelling over biologische gewasbescherming. De tentoonstelling ‘Krachtpatsers in de kas’ laat het grote publiek zien hoe microscopisch kleine insectenjagers worden ingezet bij de bestrijding van trips in de sierteelt. Californische trips (Frankliniella occidentalis) is een moeilijk te bestrijden probleem in veel kasgewassen. De insecten zijn zeer klein en kruipen weg in de groeipunten van het gewas. Hierdoor ontsnappen ze vaak aan chemische middelen.
IAG ring test feed composition 2015
Raamsdonk, L.W.D. van; Rhee, N.E. van de; Pinckaers, V.G.Z. ; Vliege, J.J.M. - \ 2015
RIKILT Wageningen UR (RIKILT report 2015.017) - 25
feed formulation - pig feeding - ring test - microscopy - voersamenstelling - varkensvoeding - ringtest - microscopie
A ring test was organized for the microscopic determination of botanic composition in animal feed in the framework of the annual ring tests of the IAG - International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy. The organizer of the ring test was RIKILT Wageningen UR, The Netherlands. The aim of the ring study was to provide the participants information on the performance of the local implementation of the method for composition analysis of feed.
IAG ring test animal proteins 2015
Raamsdonk, L.W.D. van; Rhee, N.E. van de; Scholtens-Toma, I.M.J. ; Prins, T.W. ; Vliege, J.J.M. ; Pinckaers, V.G.Z. - \ 2015
Wageningen : RIKILT Wageningen UR (RIKILT report 2015.016) - 31
ring test - microscopy - animal proteins - cattle feeding - pig feeding - animal health - ringtest - microscopie - dierlijke eiwitten - rundveevoeding - varkensvoeding - diergezondheid
A ring test was organized for the detection of animal proteins in animal feed by microscopy in the framework of the annual ring tests of the IAG - International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy. The organizer of the ring test was RIKILT - Wageningen UR, The Netherlands. The aim of the ring study was to provide the participants information on the performance of the local implementation of the detection method for their local quality systems. A further aim was to gather information about the application of the microscopic method. The current 2015 version of the IAG ring test for animal proteins is the first one in the IAG series of ring tests applying the full new method for microscopy as published in Regulation (EC) 51/2013 amending Annex VI of Regulation (EC) 152/2009 together with accompanying SOPs.
IAG ring test feed composition 2014
Raamsdonk, L.W.D. van; Pinckaers, V.G.Z. ; Vliege, J.J.M. - \ 2014
Wageningen : RIKILT Wageningen UR (RIKILT report 2014.010) - 21
voersamenstelling - pluimveevoeding - ringtest - microanalyse - microscopie - kippen - feed formulation - poultry feeding - ring test - microanalysis - microscopy - fowls
A ring test was organized for the microscopic determination of composition in animal feed in the framework of the annual ring tests of the IAG – International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy. The aim of the ring study was to provide the participants information on the performance of the local implementation of the method for composition analysis of feed.
IAG ring test animal proteins 2014
Raamsdonk, L.W.D. van; Pinckaers, V.G.Z. ; Scholtens-Toma, I.M.J. ; Prins, T.W. ; Voet, H. van der; Vliege, J.J.M. - \ 2014
Wageningen : RIKILT Wageningen UR (RIKILT report 2014.011) - 35
ringtest - microscopie - dierlijke eiwitten - rundveevoeding - pluimveevoeding - diergezondheid - ring test - microscopy - animal proteins - cattle feeding - poultry feeding - animal health
A ring test was organized for the detection of animal proteins in animal feed by microscopy in the framework of the annual ring tests of the IAG – International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy. The aim of the ring study was to provide the participants information on the performance of the local implementation of the detection method for their local quality systems. A further aim was to gather information about the application of the microscopic method.
IAG ring test animal proteins 2013
Raamsdonk, L.W.D. van; Pinckaers, V.G.Z. ; Scholtens-Toma, I.M.J. ; Prins, T.W. ; Vliege, J.J.M. - \ 2013
Wageningen : Rikilt - Institute of Food Safety (RIKILT-report 2013.016) - 35
dierlijk eiwit - dierlijke eiwitten - voer - diervoeding - ringtest - microscopie - animal protein - animal proteins - feeds - animal nutrition - ring test - microscopy
A ring test was organized for the detection of animal proteins in animal feed by microscopy in the framework of the annual ring tests of the IAG - International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy. The organizer of the the ring study was to provide the participants information on the performance of the local implementation of the detection method for their local quality systems. A further aim was to gather information about the application of the microscopic method.
Animal proteins in feed : IAG ring rest 2012
Raamsdonk, L.W.D. van; Pinckaers, V.G.Z. ; Vliege, J.J.M. - \ 2012
Wageningen : RIKILT (Report / RIKILT 2012.009) - 39
dierlijke eiwitten - diervoedering - voer - microscopie - analytische methoden - animal proteins - animal feeding - feeds - microscopy - analytical methods
A ring test was organized for the detection of animal proteins in animal feed by microscopy in the framework of the annual ring tests of the Inernational Association for Feeding stuff Analysis, Section Feeding stuff Microscopy.
Animal proteins in feed : IAG ring rest 2010
Raamsdonk, L.W.D. van; Pinckaers, V.G.Z. ; Hekman, W.E. ; Vliege, J.J.M. ; Ruth, S.M. van - \ 2010
Wageningen : RIKILT (Report / RIKILT 2010.009) - 38
analytische methoden - microscopie - dierlijke eiwitten - kwaliteitscontroles - analytical methods - microscopy - animal proteins - quality controls
A ring test was organized for the detection of animal proteins in animal feed by microscopy in the framework of the annual ring tests of the IAG - International Association for Feeding stuff Analysis, Section Feeding stuff Microscopy. The organizer of the ring test was RIKILT - Institute of food safety, Wageningen University and Research Centre, The Netherlands. The aim of the ring study was to provide the participants information on the performance of the method in their laboratory. This is essential information for their individual quality systems. A further objective was to gather information about a set of analytical parameters of the microscopic method.
The 2008 Dutch NRL/IAG proficiency test for detection of animal proteins in feed
Raamsdonk, L.W.D. van; Hekman, W.E. ; Vliege, J.J.M. ; Pinckaers, V.G.Z. ; Ruth, S.M. van; Voet, H. van der - \ 2008
Wageningen : RIKILT (Rapport / RIKILT, Instituut voor Voedselveiligheid 2008.0007) - 31
dierlijk eiwit - dierlijke eiwitten - voer - analytische methoden - ringtest - microscopie - animal protein - animal proteins - feeds - analytical methods - quality controls - microscopy
The results of a proficiency test for the detection of animal proteins in animal feed by microscopy, PCR (DNA detection) and immunoassay methods are presented in this report.
A microscopic analysis of Arabidopsis chromatin
Willemse, J.J. - \ 2007
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Hans de Jong; Joan Wellink. - [S.l.] : S.n. - ISBN 9789085045960 - 119
chromatine - arabidopsis - microscopie - biologische technieken - dna - histonen - fluorescentie - worteltopjes - chromatin - arabidopsis - microscopy - biological techniques - dna - histones - fluorescence - root tips
Genetic information of eukaryotic organisms is stored as DNA in the nuclei of their cells. Nuclear DNA is associated with several proteins, which together form chromatin. The most abundant chromatin proteins arehistones,they arrange the initial packaging step of the DNA. DNA staining reveals two cytogenetically different versions of chromatin; lightly stained euchromatin and intensely stained heterochromatin. Heterochromatinization is used to keep DNA elements selectively repressed. Several modifications on histones and/or DNA are used to distinguish eu- from heterochromatin. The histone modifications are collectively called the histone code. HP1 is one of the proteins that can bind histone modifications; it has a bi-partite organization to allow simultaneous binding to histones as well as to other proteins. Arabidopsis thaliana , the model plant to investigate chromatin organization, has a small genome and a simple distribution of eu- and heterochromatin allowing an easy distinction between the two forms of chromatin by light microscopy. The fixed division pattern of the Arabidopsis root allows monitoring of the chromatin organization through developmental progression. The dynamic chromatin organization in Arabidopsis is investigated in this thesis.Chapter 2 describes a semi-automated method to quantify fluorescence intensity in intact organs and tissues, composed of several cell layers. The method has been developed andtestedon whole mount preparations of Arabidopsis root tips containing Propidium Iodide stained nuclei. With a diameter of less than 150 mm the root tip is thin enough for standard confocal 3D microscopy, which makes the organ very suitable for whole mount imaging. Advantages of the root as model system for such a study are the lack of chlorophyll and the presence of transparent cell walls with only little background fluorescence. In addition the technique enables structural and quantitative analyses of stereotype tissue patterns and cell position, thus providing important information about the developmental history of every cell. With our method we now can measure DNA amounts in spatially reconstructed nuclei of a complete root tip. In our novel averaging 3D method we calculate the mean of the summed fluorescence intensities of all nuclear sections of one nucleus and interpolate the missingsections, thereby avoiding small detection problems with accuracy comparable with the existing 3D methods. The quantification showed that vascular tissue cells endoreduplicate after the first cell division from the stem cell. Furthermore, cortical and endodermal cells progress through the cell cycle at comparable velocity as mother and daughter cells, as visualized by groups of cells containing increased amounts of DNA. The organizational changes in chromatin during development led us to investigate mobility of principal parts of chromatin like histones and histone binding proteins.Histones are the proteins organizing the first step of folding DNA into chromatin fibers. These organizing proteins need to be flexibly positioned on the DNA to allow accession to the DNA for several processes. By making use of H2B-YFP expressing plants in a wt and in a DNA methylation mutant (ddm1) background, the mobility of the core histone H2B was analysed using FRAP (Chapter 3). In both transgenic plants the heterochromatic sequences appeared, in the majority of the nuclei, as distinct spots which allowed us to determine the mobility of H2B in euchromatic, heterochromatic, and centromeric regions. The mobility of H2B was measured on a time scale of about half an hour in living cells of intact roots and three distinguishable fractions of H2B were found; a euchromatic mobile fraction, a less mobile heterochromatic fraction and an immobile fraction. The half time of recovery in euchromatin was aboud 80 seconds, therefore the binding time of a H2B protein inside a nucleosome is around 2 minutes in these regions. Heterochromatic mobility of H2B was slower with a half time of recovery of aboud 7 minutes. The centromeric H2B was shown not to be mobile at all (immobile fraction 95%). Since histones seem to be reasonably fixed in position especially in heterochromatin we wondered about the dynamics of histone binding proteins.AtLHP1 the HP1 homologue in Arabidopsis was shown to be located in foci in the euchromatic area of interphase root cell nuclei in which relatively high levels of H3K9m3 and/or H3K27m3 occur (Chapter 4), and by using FRET-FLIM, to closely interact with DNA. The interaction with DNA was quite loose as FRAP data shows an average binding time of 1.2 seconds. The mobility of AtLHP1 did not differ between foci and interfoci, and also no distinction in DNA binding efficiency was observed. The AtLHP1 containing foci most likely represent chromatin complexes controlling the expression of genes present in these foci.Using mutated versions of AtLHP1 from which the Chromodomain (CD), Chromoshadow domain (CSD), the Acidic domain (AD), or the conserved part of the hinge (H) were deleted we assessed the functions of these separate domains (Chapter 5). Localization studies showed that the CD and H are essential for foci formation, whereas no influence of deletions of the CSD or AD were observed. FRET-FLIM data showed that the CSD and AD are essential for DNA binding anywhere in the nucleus, while deletion of the CD did not have any effect on the DNA binding capacity of AtLHP1. FRAP data indicated that the AD is essential to keep AtLHP1 mobile, whereas the CD, and CSD are essential for binding. Based on these results a hypothetical model of AtLHP1 functioning is presented (Chapter 5).In Chapter 6 the results obtained in the preceding chapters are discussed. Limits of the microscopical techniques used in this thesis are evaluated. Furthermore the histone displacement caused by transcription is discussed.
The Wageningen 2005 meeting of the IAG working group Microscopy
Raamsdonk, L.W.D. van - \ 2006
Wageningen : RIKILT (Report / RIKILT 2006.007) - 20
microscopie - voer - analytische methoden - kwaliteitscontroles - microscopy - feeds - analytical methods - quality controls
Het zijn de kleine dingen die het doen
Norde, W. - \ 2006
Wageningen : Wageningen Universiteit - 36
fysische chemie - microscopie - micellen - biotechnologie - nanotechnologie - physical chemistry - microscopy - micelles - biotechnology - nanotechnology
The organization of training courses for microscopic detection of animal proteins in feeds
Raamsdonk, L.W.D. van; Beaten, V. ; Boix, A. ; Jorgenson, J.S. ; Holst, Chr. van - \ 2005
Wageningen : RIKILT (Report / RIKILT 2005.009)
microscopie - dierlijke eiwitten - scholingscursussen - microscopy - animal proteins - training courses
Ontwikkeling en routinematige implementatie van een moleculaire detectietechniek voor stengelaaltjes (Ditylenchus dipsaci) en witrot (Sclerotium cepivorum) in grondmonsters
Landeweert, R. ; Helder, J. ; Elsen, S.J.J. van den; Staps, R.V. ; Zwaardemaker, N. ; Keidel, H. - \ 2005
In: Programma Najaarsvergadering KNPV Samenvattingen Hoogtepunten 2004-2005, Wageningen, 30-11-2005 Wageningen : - p. 270 - 270.
gewasbescherming - ditylenchus dipsaci - nematoda - stromatinia cepivora - schimmels - analyse - kwalitatieve analyse - grondanalyse - detectie - microscopie - genen - plant protection - ditylenchus dipsaci - nematoda - stromatinia cepivora - fungi - analysis - qualitative analysis - soil analysis - detection - microscopy - genes
Samenvatting van de voordracht te houden op 30 november 2005 tijdens de Najaarsvergadering van de KNPV (Koninklijke Nederlandse Plantenziektekundige Vereniging).
Magnetic resonance imaging of plants: plant water status and drought stress response
Weerd-Meulenkamp, L. van der - \ 2002
Wageningen University. Promotor(en): T.J. Schaafsma; H. van As. - S.l. : S.n. - ISBN 9789058087072 - 119
planten - waterbalans - droogte - droogteresistentie - waterstress - plant-water relaties - membraanpermeabiliteit - celgroei - kernmagnetische resonantie - microscopie - computersimulatie - plantenfysiologie - plants - water balance - drought - drought resistance - water stress - plant water relations - membrane permeability - cell growth - nuclear magnetic resonance - microscopy - computer simulation - plant physiology
This Thesis presents an approach for the study of plant water balance during drought stress, using a combination of in vivo NMR experiments and computer simulations. The ultimate aim is the interpretation of the NMR parameters in terms of physiologically relevant characteristics, such as cell dimensions and membrane permeability. Especially the latter has raised a growing interest in plant science, and up to now the measurement of this parameter in vivo was limited to single cells and short experiment time spans.
NMR microscopy of plants yields information on various levels of organisation. The NMR images provide clear anatomical details, which have been used to monitor the response of stem growth rates to osmotic stress. On the tissue and cell levels, the NMR parameters T 2 (transverse spin relaxation time) and D app (apparent diffusion coefficient) provide information on the physical and chemical properties. Correct quantitative values for T 2 and D app are crucial for a useful interpretation. Therefore, Chapter 2 evaluates the accuracy of different fitting procedures.
The physical and chemical properties can vary considerably between and within different tissues, cells, and intracellular compartments, resulting in distinctly different relaxation and diffusion characteristics for these compartments. The interpretation of these parameters is not straightforward. A numerical model of restricted diffusion and relaxation behaviour was therefore developed, based on Fick's second law of diffusion (Chapter 3). This model expands previous one-dimensional models to a two-dimensional space, consisting of multiple concentric cylindrical compartments, separated by membranes. Numerical simulation experiments using this model demonstrate the importance of modelling two-dimensional diffusion in relation to the effects of spatial restrictions, and spin exchange between the different compartments.
This model has been applied to investigate the effects of diffusive exchange on the transverse spin relaxation times, the apparent diffusion coefficients, and the NMR signal amplitudes of water in plant cells (Chapter 4). For different multi-compartment model systems a Pulsed Field Gradient Multiple Spin Echo (PFG-MSE) experiment was simulated, and intrinsic physiological parameters, i.e. the bulk diffusion constant, the cell radius and the membrane permeability were afterwards extracted using common theoretical models. The results justify the use of these models to interpret the in vivo experiments, since meaningful diffusion constants, cell radii and membrane permeabilities can be extracted for a large range of conditions. This is still true if not all conditions of the theory are known or met, e.g . for intact plants.
Chapters 5 and 6 study the effect of mild osmotic stress on maize and pearl millet by in vivo1H NMR microscopy, and water uptake measurements. Single NMR parameter images of (i) the water content, (ii) the transverse relaxation time ( T 2 ) and (iii) the apparent diffusion coefficient ( D app ) were used to follow the water status of the stem apical region during osmotic stress. The results are interpreted using the multi-compartment model (Chapter 4), tailored to suit plant cells. For this particular case, an equation was derived to describe the relation between the observed T 2 , the cell dimensions, the bulk T 2 , and the membrane permeability, based on the Brownstein & Tarr theory. Experimentally determined T 2 values of non-stressed stem tissue are indeed correlated to the cell dimensions, which is in agreement with the derived equation. The T 2 of maize cells is higher than the T 2 of equally sized millet cells, implying that the membrane permeability of the latter is higher.
The growth rate was strongly inhibited by mild stress in both species, even though the water uptake was only mildly affected. During stress, there are hardly any changes in water content or T 2 of the stem region of maize. In contrast, the apical tissue of pearl millet showed a ~30% decrease of T 2 within 48 hours of stress, whereas the water content and D app hardly changed. This decrease in T 2 can be caused by a decreasing cell radius, a decreasing bulk T 2 , and/or an increasing membrane permeability for water. To distinguish between these contributions, additional scanning electron microscopy was used, showing no apparent changes in cell size. Transverse spin relaxation measurements of a wide range of sugar solutions showed only very small effects of osmotic adjustment on the bulk T 2 . Together, these results point to an increase in membrane permeability during stress. This conclusion is confirmed by numerical simulations of the plant cell model, which showed that only an increasing membrane permeability yields a similar combination of water content, T 2 , and D app values during stress.
Under severe osmotic stress, the effects on the plant water balance are naturally larger (Chapter 7). During stress, no significant changes occurred in the maize stem, though the leaves wilted, and the plant died after two days of stress. Pearl millet showed again changes in T 2 , especially in the secondary shoots, which were more pronounced than during mild stress. Furthermore, the stem tissue shrunk, implying that the cell dimensions changed; the secondary shoots showed far less decrease in water content, however. Despite these changes, the plants recovered once stress was relieved. In the framework of the plant cell model, the decreasing T 2 is interpreted as the result of a combination of decreasing cell size and increasing membrane permeability. The latter can result in a higher tissue conductance, thereby facilitating water re-allocation to young, expanding tissues to prevent irreparable damage.
The combination of experimental data and simulations as presented in this Thesis has proven to be an effective tool to link NMR information to physiology (Chapter 8). This approach promises to be of great use to plant science, and to NMR microscopy in general.
Decision Support Systems ter ondersteuning van microscopisch onderzoek
Raamsdonk, L.W.D. van - \ 2001
Wageningen : RIKILT (Rapport / RIKILT 2001.008) - 26
kwaliteitscontroles - microscopie - expertsystemen - microscoopglaasjes - melkpoeder - zetmeel - beslissingsondersteunende systemen - quality controls - microscopy - expert systems - microscope slides - dried milk - starch - decision support systems
Microscopische schatten: de RIKILT beeldbank in het programma TREASURY
Raamsdonk, L.W.D. van - \ 2000
Wageningen : Rijkskwaliteitsinstituut voor Land- en Tuinbouwproducten (RIKILT) (Rapport RIKILT-DLO 2000.007) - 20
microscopie - microscoopglaasjes - melkpoeder - besmetters - zetmeelproducten - zetmeel - graan - zaden - microscopy - microscope slides - dried milk - contaminants - starch products - starch - grain - seeds
Betrouwbaarheid en herhaalbaarheid microscopische analysemethode diermeel
Voet, H. van der; Jong, J. de; Pinckaers, V.G.Z. - \ 1999
Wageningen : DLO-Rijks-Kwaliteitsinstituut voor land- en tuinbouwprodukten (RIKILT-DLO) (Rapport / RIKILT-DLO 99.015) - 13
analyse - microscopie - statistische analyse - detectie - voer van dierlijke oorsprong - beendermeel - herhaalbaarheid - nauwkeurigheid - analysis - microscopy - statistical analysis - detection - feed of animal origin - bone meal - repeatability - accuracy