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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    Real-Time Assembly of Viruslike Nucleocapsids Elucidated at the Single-Particle Level
    Marchetti, Margherita ; Kamsma, Douwe ; Cazares Vargas, Ernesto ; Hernandez García, Armando ; Schoot, Paul van der; Vries, Renko de; Wuite, Gijs J.L. ; Roos, Wouter H. - \ 2019
    Nano Letters 19 (2019)8. - ISSN 1530-6984 - p. 5746 - 5753.
    acoustic force spectroscopy - artificial virus - biophysics - optical tweezers - physical virology - Self-assembly

    While the structure of a multitude of viral particles has been resolved to atomistic detail, their assembly pathways remain largely elusive. Key unresolved issues are particle nucleation, particle growth, and the mode of genome compaction. These issues are difficult to address in bulk approaches and are effectively only accessible by the real-time tracking of assembly dynamics of individual particles. This we do here by studying the assembly into rod-shaped viruslike particles (VLPs) of artificial capsid polypeptides. Using fluorescence optical tweezers, we establish that small oligomers perform one-dimensional diffusion along the DNA. Larger oligomers are immobile and nucleate VLP growth. A multiplexed acoustic force spectroscopy approach reveals that DNA is compacted in regular steps, suggesting packaging via helical wrapping into a nucleocapsid. By reporting how real-time assembly tracking elucidates viral nucleation and growth principles, our work opens the door to a fundamental understanding of the complex assembly pathways of both VLPs and naturally evolved viruses.

    Light on phloem transport (an MRI approach)
    Prusova, Alena - \ 2016
    Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Henk van As. - Wageningen : Wageningen University - ISBN 9789462579156 - 130
    solanum lycopersicum - phloem - light - flow - photoperiod - nuclear magnetic resonance - biophysics - magnetic resonance imaging - solanum lycopersicum - floëem - licht - stroming - fotoperiode - kernmagnetische resonantie - biofysica - kernspintomografie

    This thesis (Light on phloem transport – an MRI approach) aims to answer the question whether phloem transport can be a limiting factor for photosynthesis efficiency (and ultimately causing a bottleneck towards achieving higher yields). To answer this key question, we manipulated the source: sink ratio within tomato (Solanum lycopersicum L.) while measuring phloem transport with magnetic resonance imaging (MRI) flowmetry. Additionally we compared phloem flow characteristics of two potato plants (Solanum tuberosum L.) which differed in source : sink ratio. In Chapter 2, the source strength was manipulated by varying the light intensity. An increase in phloem sap volume flow under higher light intensities was observed. However, under all light intensities applied, the phloem flow velocity was found to be constant (as has previously been suggested in other studies) although a clear diurnal pattern was observed. This finding does not fit in current models to describe the mechanism of phloem transport and a different mechanism must be at play. The results of this chapter demonstrate that increased levels of photo-assimilates are transported in sieve tubes, which are activated when needed by the plant. This is the first study which shows that plants activate individual sieve tubes when more photo-assimilates are available, yet maintain constant velocity. Those observations were in a tomato plant with pruned fruit trusses (i.e., in a simplified system). In Chapter 3, we investigated whether tomato plants still exhibit constant phloem flow velocity (with a diurnal pattern) under normal conditions, i.e., with strong sinks (tomato fruits) still attached. This was tested for both a long and short photoperiod by measuring flow characteristics with MRI flowmetry. We simultaneously monitored other plant processes like xylem flow rates with a heat balance sensor, net photosynthesis with gas exchange and stem diameter changes with a linear motion potentiometer. With this integrated approach, we revealed a correlation between night phloem volume flow, dark respiration and stem growth. We also conclusively showed that phloem volume flow performs a diurnal pattern under a variety of source-sink ratios which appears to be a normal behaviour for tomato plants growing under moderately-high light conditions. In chapters 2 and 3 we learned that under higher source strength a greater amount of phloem sap is transported, but the changes in flow were not accompanied by changes in velocity. To further our understanding of the mechanisms driving phloem transport, it is of interest to know how the sucrose concentration in phloem sap relates to phloem flow. In Chapter 4 we used an average T2 relaxation time in the phloem vascular tissue region to reveal the plant’s phloem carbon status under source manipulation. In this chapter we demonstrated that T2 relaxation time, when measured in parallel with phloem flow, can provide additional information about phloem region carbon status, i.e., changes in the T2 relaxation time are correlated with changes in sucrose concentration in the whole phloem region.

    When studying phloem transport in plants with magnetic resonance imaging (MRI) flowmetry, plants which are relatively easy to manipulate (e.g. fruit pruning) like tomato have so far been used. However, tomato plants (used in all three previous chapters) have relatively low sink strength beneath the MRI measurement site. A potentially preferable approach is to work with plants with strong sinks beneath the measurement site. In Chapter 5 we studied potato as a potentially better test subject for MRI flowmetry as it possesses strong sink below the MRI measurement site (i.e., developing tubers). For that purpose we used two potato plants (cv. Desiree) both with several developing tubers. One of the plants overexpressed the StSWEET gene (35S:StSWEET) which appears to have altered its source : sink ratio. As a result, the 35S:StSWEET plant transported 60% more phloem sap than Desiree WT. Strikingly, the average phloem flow velocity in both plants was the same and the greater amount of transported phloem sap in the 35S:StSWEET plant was accommodated by more sieve tubes than in Desiree WT. This finding agrees with the hypothesis about the conserved nature of phloem flow velocity, where volume flow is regulated by the number of active sieve tubes (Chapter 2 and 3). In this chapter we also demonstrate that a potato plant with developing tubers represents a good subject to study phloem transport with MRI flowmetry. We concluded that under optimal conditions (which are commonly met in greenhouses) phloem transport is likely to reach its maximum capacity and therefore photosynthesis could be limited by the export and transport of photo-assimilates because of the finite number of sieve tubes and constant flow velocity.

    Climate Change: Global Risks, Challenges and Decisions
    Richardson, K. ; Steffen, W. ; Liverman, D. ; Barker, T. ; Jotzo, F. ; Kammen, D.M. ; Leemans, R. ; Lenton, T.M. ; Munasinghe, M. ; Osman-Elasha, B. ; Schellnhuber, H.J. ; Stern, N. ; Vogel, C. ; Waever, O. - \ 2011
    Cambridge : Cambridge University Press - ISBN 9780521198363 - 524
    klimaatverandering - koolstofcyclus - beleid - mitigatie - risico - klimaat - biofysica - economie - ethiek - climatic change - carbon cycle - policy - mitigation - risk - climate - biophysics - economics - ethics
    Providing an up-to-date synthesis of knowledge relevant to the climate change issue, this book ranges from the basic science documenting the need for policy action to the technologies, economic instruments and political strategies that can be employed in response to climate change. Ethical and cultural issues constraining the societal response to climate change are also discussed. This book covers a very wide range of disciplines – core biophysical sciences involved with climate change (geosciences, atmospheric sciences, ocean sciences, ecology/biology) as well as economics, political science, health sciences, institutions and governance, sociology, ethics and philosophy, and engineering.
    New approach to analyse spin probe and spin trap ESR
    Makarova, K. - \ 2011
    Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Henk van As. - [S.l. : S.n. - ISBN 9789085858409 - 145
    paramagnetische elektronenresonantiespectroscopie - chemische reacties - vrije radicalen - biofysica - electron paramagnetic resonance spectroscopy - chemical reactions - free radicals - biophysics
    The goal of this thesis is to develop new comprehensive methods for the analysis of ESR spectra and interpretation of magnetic parameters. A new approach for the analysis of fast isotropic spectra is proposed. It is based on a combination of an experimental approach (multifrequency ESR) and accurate spectra simulation using an improved model, that will be further introduced below. The determined magnetic parameters of the spin probe are directly interpreted in terms of structural information about the spin probe surroundings (lipid bilayer). The obtained magnetic parameters of various spin traps are interpreted by artificial neural networks (ANN) in order to obtain information about the identities of trapped radicals. Then, Density Functional Theory (DFT) calculations are applied to study the mechanism of reactions involving free radicals detected by spin trapping ESR and to calculate magnetic parameters of the radical adducts.

    Ketens en associaties
    Leermakers, F.A.M. - \ 2007
    Wageningen : Wageningen Universiteit
    polymeren - neurochemie - neuronen - biofysica - polymers - neurochemistry - neurons - biophysics
    Structure and dynamics of an essential transmembrane segment of the proton translocation channel of V-ATPase
    Duarte, A.M. - \ 2007
    Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Marcus Hemminga; Carlo van Mierlo. - [S.l.] : S.n. - ISBN 9789085047377 - 117
    transmembraaneiwitten - moleculaire structuur - micellen - biofysica - transmembrane proteins - molecular conformation - micelles - biophysics
    In the last decades osteoporosis has become a major subject on the field of drug discovery and design. One of the enzymes recently considered important to use as a target for theses drugs is the enzyme H+-VO-ATPase. This proton pump is located in the osteoclast cells, which are positioned at the bone surface. These enzymes control the proton flux to the bone surface and consequently bone resorption. One major task on drug design is the knowledge of the secondary and tertiary structure of the enzyme under study. The topology of the V-ATPase protein complex has been largely established, however, only the three-dimensional structure of some individual subunits is known up to the time this thesis was printed. The work presented in this thesis focuses on the proton translocation channel located in subunit a of the V-ATPase complex. For this purpose, we designed two peptides, consisting of 25 and 37 amino acid residues, representing the seventh transmembrane segment of subunit a that encompass the proton translocation channel as well as the region of interest for possible inhibitors. Using a combination of NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy we analysed the conformation of these V-ATPase peptides in different membrane-mimicking environments: aqueous solutions of SDS (sodium dodecyl sulphate) and amphipols, and the organic solvent DMSO (dimethylsulphoxide). The conformation of the V-ATPase peptides in SDS micelles was studied by CD spectroscopy, however, due to their low solubility NMR spectroscopy turned out to be impossible. The CD results showed that the size of the peptide can drastically alter the solubilization in SDS. For the 37-residue V-ATPase peptide the overall conformation was a-helical and not dependent on the SDS concentration. On the other hand, the conformation of the 25-residue V-ATPase peptide depended on the peptide to SDS ratio changing between an a-helix and β-sheet conformation. As an alternative solubilising agent for the peptides in aqueous solutions we tested amphipols, a new class of macromolecules that were designed to solubilise transmembrane proteins for NMR and X-ray studies. The CD and tryptophan fluorescence spectroscopy results showed that both peptides aggregated in a β-sheet conformation. The formation of these b‑sheets aggregates might result from the interaction of the arginine residue present in the V-ATPase peptides with the anionic polymer. Such an interaction could prevent the peptide from cross­ing the hydrophobic core of the particle, preventing the formation of an a‑he­lix. High-quality high-resolution NMR spectra of the V-ATPase peptides were obtained in DMSO enabling to analyse the atomic structure of the peptides. The use of DMSO on structural studies of transmembrane polypeptides always raised some debate in the literature. This fact motivated us to perform a molecular dynamics study to investigate the solvation of the 25-residue V-ATPase peptide by DMSO. From this work, we show that DMSO can provide both polar and apolar environments to the peptide, making it a good membrane-mimicking organic solvent. The NMR study of the 37-residues peptide enabled us to confirm the a-helical conformation and to predict that the transmembrane spanning region of the seventh transmembrane segment is larger than expected. Instead of 25 transmembrane residues, we propose a transmembrane region of 32 residues. Furthermore, the NMR study of the 25-residue peptide lead us to postulate the existence of a hinge region located near the cytoplasmic end of the channel. It is proposed that the presence of this hinge allows the opening and closing of the proton translocation channel and provides flexibility for the channel to act as a binding pocket for inhibitors. The resulting NMR data sets for the two V-ATPase peptides are deposited in the BioMagResbank (BMRB) under the access numbers 15025 and 6878, and the calculated structure ensemble for the 25-residue peptide is deposited in the PDB databank with entry 2NVJ.
    ESR Spectroscopy in membrane biophysics
    Hemminga, M.A. ; Berliner, L.J. - \ 2007
    Duitsland : Springer (Biological magnetic resonance vol. 27) - ISBN 9780387250663 - 380
    paramagnetische elektronenresonantiespectroscopie - oppervlakte-eiwitten - biofysica - spectraalanalyse - electron paramagnetic resonance spectroscopy - surface proteins - biophysics - spectral analysis
    Spectroscopic methods are not only important as an analytical tool, they also provide information about fundamental physical and chemical properties of molecules, the molecular and electronic structure, and the dynamic behaviour of molecules. Starting from a comprehensive quantum mechanical description, this book introduces the optical (IR, Raman, UV/Vis, CD, fluorescence and laser spectroscopy) and magnetic resonance (1D and 2D-NMR, ESR) techniques. It is a timely review of the increasing interest in using spin-label ESR as an alternative structural technique for NMR or X-ray diffraction. It is aimed at training an audience to learn ESR spectroscopy to determine membrane protein structures, conformational dynamics and protein-lipid interaction.
    Hyperspectral remote sensing of biochemical and biophysical parameters: the derivate red-edge "double-peak feature", a nuisance or an opportunity?
    Cho, M.A. - \ 2007
    Wageningen University. Promotor(en): Andrew Skidmore, co-promotor(en): Sip van Wieren. - [S.l.] : S.n. - ISBN 9789085046226 - 206
    remote sensing - vegetatie - biochemie - biofysica - remote sensing - vegetation - biochemistry - biophysics

    Improved quantification and monitoring of biophysical and biochemical attributes is required to predict the response of ecosystems to climate change and acquire deeper understanding of the carbon cycle. Remote sensing is widely viewed as a time- and cost-efficient way to proceed with large-scale monitoring of vegetation parameters. For over thirty years, use has been made of broadband sensors such as Landsat TM/ETM+. The advent of hyperspectral remote sensing or imaging spectrometry enlarged the number of available bands within the visible, near-infrared (NIR) and shortwave infrared (SWIR). Hyperspectrally detectable variables associated with 1eaf chlorophyll content, phenological state and vegetation stress such as the spectral shift of the red-edge (670-780 nm) slope and its inflection point termed the red-edge position (REP), are not accessible with broadband sensors. State of the art indices and analytical techniques applied for broad-band remote sensing are not always suitable for information extraction from high dimensional hyperspectral data. This study aimed to develop new hyperspectral indices and propose innovative ways for empirically estimating biochemical and biophysical parameters from hyperspectral data.

    The red edge position is estimated using the first derivative of the spectral curve. Existing curve fitting approaches localise the REP while assuming a derivative curve with a single peak. The proposed linear extrapolation method localises the red edge position while explicitly considering two peaks in the derivative curve. The major contribution of this study is that the linear extrapolation method allows optimised estimates of 1eaf chlorophyll or nitrogen content while minimising the confounding effects of background and the structure of leaves and canopy. By minimising these canopy effects, the linear extrapolation may be useful for detecting early physiological stresses associated with changes in 1eaf chlorophyll/nitrogen levels. The linear extrapolation method also shows high potential for discriminating tree and shrub species at both the leafand canopy scales. Lastly, it could be used as a more stable predictor for monitoring green grass biomass in the Majella National park, Italy compared with two-band vegetation indices. The method is simple to implement, but sensitive to spectral noise. Spectral smoothing is recommended when noise is a problem.

    The study also highlights the utility of partial least squares (PLS) regression based on airborne hyperspectral imagery (HyMap) for estimating grass biomass and beech (Fagus sylvatica L.) forest mean diameter-at-breast height (DBH) in the Majella National Park, Italy. PLS regression produced lower prediction errors for grass biomass and beech forest mean DBH compared with univariate regression involving vegetation indices such as NDVI. NDVI may be simple to implement but could be lacking in terms of exploiting the information content inherent in several narrow bands.

    In a nutshell, this study makes a contribution in the domain of information extraction from hyperspectral data for estimating vegetation parameters such as 1eaf chlorophyll/nitrogen concentration, grass biomass and forest structural parameter using empirical models. Other studies are focused on developing physically based methods given the lack of robustness and portability of empirical models for varying environmental conditions. However, empirical models that are less sensitive to environmental conditions such as models based on the linear extrapolation REP could be used to support the development of physically based models, particularly to estimate the values of the model parameters, or to refine the underlying concepts on which the model is constructed. The future of hyperspectral remote sensing could hinge or enhancing the link between empirical and physically based approaches.

    Biofysica, wetenschap van leven en dood
    Amerongen, H. van - \ 2006
    Wageningen : Wageningen Universiteit - 34
    biofysica - biophysics
    Exploring socio-ecological niches for legumes in western Kenya smallholder farming systems
    Ojiem, J.O. - \ 2006
    Wageningen University. Promotor(en): Ken Giller, co-promotor(en): Nico de Ridder; B. Vanlauwe. - [S.l.] : S.n. - ISBN 9789085045137 - 169
    peulgewassen - aanpassingsvermogen - biofysica - sociale economie - kenya - heterogeniteit - economische analyse - stikstoffixatie - productiviteit - agro-ecosystemen - legumes - adaptability - biophysics - socioeconomics - kenya - heterogeneity - economic analysis - nitrogen fixation - productivity - agroecosystems
    Keywords: adaptability, agro-ecosystems, biophysical and socio-economic heterogeneity, economic benefits, N2-fixation, productivity.

    This thesis explores the potential of using herbaceous and grain legume species to improve soil fertility and farm productivity in the heterogeneous smallholder farming systems of westernKenya. Poor soil fertility is responsible for the limited productivity of the westernKenyasmallholder farming systems. Although legumes have the potential for improving productivity, their sustainable use is impeded by the high degree of biophysical and socio-economic heterogeneity that characterizes the farming systems. The socio-ecological niche concept was proposed as a framework for facilitating the identification and integrated assessment of biophysical and socio-economic factors with potential influence on the choice of sustainable legume technologies for smallholder farmers.

    The utility of the socio-ecological niche concept was tested through on-farm experiments and socio-economic surveys in westernKenya. The on-farm experiments were conducted across three major agro-ecological zones (AEZ), and under different soil fertility conditions, to assess legume emergence, survival, nodulation, diseases tolerance, grain yield, biomass production, atmospheric N 2 -fixation and net N contribution to soil N fertility. In addition, the economic benefits of growing grain and green manure legumes in rotation with maize were assessed to determine how they are influenced by agro-ecological conditions and within farm soil fertility heterogeneity. Socio-economic surveys characterized farmer legume production objectives, as well as socio-cultural, economic and institutional factors with potential impact on the use of legume technologies by westernKenyasmallholder farmers. The biophysical and socio-economic factors were integrated and analysed to identify legume species for different farmer resource endowment groups, agro-ecological conditions and field typologies. Analysis of the alternative legume production scenario was undertaken, to test the utility of the socio-ecological niche concept. 

    Legume grain yield, total dry matter production (TDM) and atmospheric N 2 -fixation increased with rainfall and soil fertility status. TDM ranged from 0.1 Mg ha -1 to 13.9 Mg ha -1 , and was generally less for the grain legumes, compared with the green manure and forage legumes. However, soyabean and groundnut showed greater potential among the grain legumes, producing up to 4.6 Mg ha -1 TDM. While the legume species and varieties showed capacity to form viable nodules with naturally occurring rhizobia, application of P was essential for good nodulation. 

    Generally, the species fixed 23-90% of their N requirements in AEZ 1 (Museno) and AEZ 2 (Majengo), compared to 7-77% of their N requirements in AEZ 3 (Ndori). However, N 2 -fixation by the green manure species (29-232 kg N ha -1 ) was greater than that by grain legume species (3-172 kg N ha -1 ). Net N input by the grain legumes was negatively correlated with grain yield, and legume grain yields of above 1 Mg ha -1 resulted in negative net N inputs. Economic benefits of fitting legumes into the smallholder cropping systems in rotation with maize varied with rainfall, soil fertility and legume species. Yearly maize productivity of these rotations (short and long rains crops) decreased by 47%, from AEZ 1 to AEZ 3, and by 33%, from fertile fields to least fertile fields. Although continuous maize fertilized with both N and P had the largest total maize productivity, returns to land and labour were greatest with grain legume-maize cropping systems. In AEZ 2, where moisture was not limiting during the experimentation period, mean returns to land for grain legume-maize cropping systems were US$ 879 ha -1 , compared with US$ 533 for green manure-maize, and US$ 459 for continuous maize with N and P.

    Rainfall, soil fertility, land, labour, and livestock ownership were identified as the most important factors influencing the choice of appropriate legumes for the smallholder socio-ecological niches. The analysis of the current legume production situation showed that the medium and the low resource endowed farmers were food insecure due to a combination of land and labour scarcity. However, when alternative legumes species selected according to the socio-ecological niche concept were used, maize self-sufficiency increased by 21-48%. This study demonstrated the utility of the socio-ecological niche concept as a useful tool for facilitating the integration of legumes into the westernKenyasmallholder farming systems to improve soil fertility and farm productivity.
    Epigenetics of the locomotory system in zebrafish
    Meulen, T. van der - \ 2005
    Wageningen University. Promotor(en): Johan van Leeuwen, co-promotor(en): Sander Kranenbarg; Henk Franssen. - Wageningen : - ISBN 9789085043225 - 191
    danio rerio - skeletspierstelsel - epigenetica - biologische ontwikkeling - ontogenie - merkergenen - lichaamsbeweging - biofysica - diermodellen - dierproeven - mechanische invloeden - danio rerio - musculoskeletal system - epigenetics - biological development - ontogeny - marker genes - exercise - biophysics - animal models - animal experiments - mechanical influences
    Movements are important. When animals move, they exert mechanical load on their bodies and their muscles and bones respond by the generation of more tissue. Vice versa, a lack of movement will result in a decrease in bone and muscle tissue. During my PhD studies I have looked into the normal development of muscle and bone in the trunk of zebrafish. In addition, I have looked at different levels whether this development can be influenced by mechanical load. I investigated how cells respond to load by subjecting zebrafish cells to strain and observing the effects on cell morphology. The cells appear to orient themselves perpendicularly away from the strain, which results in a lower absolute strain value. During this orientation process, the expression of more than 400 genes is adjusted. In an entire organism I have investigated the normal development of bone and muscle. During development the muscle fibres turn away from the body axis and together they build up a spiral like architecture. I have tried to explain this architecture by means of a computer model in which the equal work theory was implemented. According to this theory, the muscle fibres can only generate equal amounts of work when they can all shorten equally and by doing so function optimally. The observed architecture appears to be adapted to the movement of the embryos in the egg and it is only generated after the embryos start moving. From these observations I hypothesised that mechanical load, represented by the movements of the embryo, could be important for the normal development of the muscle architecture. I tested this hypothesis by making use of mutant zebrafish embryos that are immobile. I found that the morphology of the embryos is indeed affected by this lack of movement, although the spiral like architecture does develop in the mutant embryos. Finally, I made zebrafish larvae perform endurance training by making them swim against a water current. They developed more slow muscle in the trunk muscles, similar to endurance training in humans, but the heart muscle developed more fast muscle. The general conclusion I can draw from the results in this thesis is that mechanical load already during early development is important for fine-tuning genetically predetermined principles.
    Lang leve de natuurkunde
    Mulder, B. - \ 2002
    Wageningen : Wageningen Universiteit - 30
    fysica - biofysica - physics - biophysics
    Oxygen diffusion in fish embryos
    Kranenbarg, S. - \ 2002
    Wageningen University. Promotor(en): J.L. van Leeuwen; J.W.M Osse; M. Muller. - S.l. : s.n. - ISBN 9789058086808 - 183
    vissen - embryo's - embryonale ontwikkeling - embryologie - zuurstof - diffusie - zuurstoftransport - zuurstofconsumptie - voedingsstoffen - beperkingen - grootte - vorm - cardiovasculair systeem - genexpressie - modellen - biofysica - fishes - embryos - embryonic development - embryology - oxygen - diffusion - oxygen transport - oxygen consumption - nutrients - constraints - size - shape - cardiovascular system - gene expression - models - biophysics - cum laude

    All vertebrate embryos pass through a developmental period of remarkably low morphological variability. This period has been called phylotypic period. During the phylotypic period, organogenesis takes place, including blood vessel development. Before the phylotypic period, the embryos rely on diffusion for the internal oxygen transport. Diffusion, however, is an efficient way of transport only over small distances. Analytical models were constructed to investigate whether physical constraints ( i.e. diffusional limitations) demand the development of an internal oxygen transport system as the embryos grow bigger. These models showed that teleost embryos are smaller than their theoretically maximum size during the phylotypic period, based on oxygen diffusion. Lack of oxygen does therefore not demand blood vessel development. Subsequently, numerical models of oxygen diffusion in a zebrafish embryo ( Danio rerio ) were developed, thereby including the realistic shape of the embryo. These models were tested and refined with oxygen micro-electrode measurements of the oxygen partial pressure profile in and around the zebrafish embryo. This numerical-experimental procedure revealed a high oxygen permeability in the yolk of the zebrafish embryo. Furthermore, lowest oxygen partial pressures were found in the head region with a gradient of posteriorly increasing oxygen partial pressures along the midline of the embryo. The three-dimensional oxygen partial pressure profile was compared with the expression pattern of the angiogenic factor ( vegf ), which is known to be expressed under hypoxic conditions. The apparent colocalization of low oxygen partial pressure and the expression of vegf suggests oxygen to play an important role in regulating blood vessel development rather than posing a direct request for its development.

    Biochemistry and biophysics of tolerance systems
    Buitink, J. ; Hoekstra, F.A. ; Leprince, O. - \ 2002
    In: Desiccation and survival in plants: drying without dying / Black, M., Pritchard, H.W., Wallingford : CABI - p. 293 - 318.
    plantenweefsels - verdroging - biochemie - biofysica - tolerantie - plant tissues - desiccation - biochemistry - biophysics - tolerance
    The role of macromolecular stability in desiccation tolerance
    Wolkers, W.F. - \ 1998
    Agricultural University. Promotor(en): L.H.W. van der Plas; F.A. Hoekstra. - S.l. : Wolkers - ISBN 9789054858805 - 244
    vloeistoffen (liquids) - absorptie - emissie - omloop - veroudering - abcissie - degeneratie - necrose - verouderen - verwelking - biochemie - metabolisme - polymeren - moleculaire biologie - biofysica - eiwitten - enzymen - nucleïnezuren - celfysiologie - liquids - absorption - emission - circulation - senescence - abscission - degeneration - necrosis - aging - wilting - biochemistry - metabolism - polymers - molecular biology - biophysics - proteins - enzymes - nucleic acids - cell physiology

    The work presented in this thesis concerns a study on the molecular interactions that play a role in the macromolecular stability of desiccation-tolerant higher plant organs. Fourier transform infrared microspectroscopy was used as the main experimental technique to assess macromolecular structures within their native environment.

    Protein secondary structure and membrane phase behavior of Typha latifolia pollen were studied in the course of accelerated aging. The overall protein secondary structure of fresh pollen highly resembled that of aged pollen, which indicates that endogenous proteins in these pollen are very stable, at least with respect to their conformation. In contrast, large changes in membrane phase behavior were detected between fresh and aged pollen. Membranes isolated from fresh pollen occurred mainly in the liquid crystalline phase at room temperature, whereas the membranes of aged pollen were at least partly in the gel phase (Chapter 2).

    The in situ heat stability of the proteins in this pollen was studied as a function of the water content of the pollen. Temperature-induced denaturation of proteins was accompanied by the formation of intermolecular extendedbeta-sheet structures. Below 0.16 g H 2 O g -1dry weight (DW), the temperature at which the proteins began to denature increased rapidly and the extent of protein structural rearrangements due to heating decreased (Chapter 3).

    Inspection of the overall protein secondary structure of thin slices of embryo axes of onion, white cabbage and radish seeds did not show signs of protein aggregation and denaturation after long-term dry storage. It was concluded that, despite the loss of viability and the long postmortem storage period, secondary structure of proteins in desiccation-tolerant dry seed is very stable and conserved during at least several decades of open storage (Chapter 4).

    Adaptations in overall protein secondary structure in association with the acquisition of desiccation tolerance were studied using isolated immature maize embryos. Isolated immature maize ( Zea mays ) embryos acquire tolerance to rapid drying between 22 and 25 days after pollination (DAP) and to slow drying from 18-DAP onwards. In fresh, viable 20- and 25-DAP embryo axes, the overall protein secondary structure was identical, and this was maintained after flash drying. On rapid drying, 20-DAP axes showed signs of protein breakdown and lost viability. Rapidly dried 25-DAP embryos germinated and had a protein profile similar to the fresh control. On slow drying, thealpha-helical contribution in both the 20- and 25-DAP embryo axes increased when compared with that in the fresh controls, and survival of desiccation was high. The protein profile in dry mature axes resembled that after slow drying of the immature axes. Rapid drying resulted in an almost complete loss of membrane integrity in 20-DAP embryo axes and much less so in 25-DAP axes. After slow drying, membrane integrity was retained in both the 20- and 25-DAP axes. It was concluded that slow drying of excised immature embryos leads to an increased proportion ofalpha-helical protein structures in their axes, which coincides with additional tolerance of desiccation stress (Chapter 5).

    A novel FTIR method was used to study glasses of pure carbohydrates and glasses in the cytoplasm of desiccation-tolerant plant organs. The method is based on a temperature study of the position of the OH-stretching vibration band (vOH). The glass transition temperatures ( Tg s) of several dry carbohydrate glasses determined by this FTIR method resembled those of produced by other methods. FTIR analysis gives additional information on the molecular properties of glassy structures. The shift ofvOH with temperature - the wavenumber-temperature coefficient (WTC) - is indicative of the average strength of hydrogen bonding in glasses. The WTC was found to be higher in sugar glasses having higher Tg . This suggests that carbohydrate glasses are more loosely packed when they have higher Tg . For Typha latifolia pollen and dried Craterostigma plantagineum leaves similarvOH vs temperature plots were obtained as for pure carbohydrate glasses, indicating that a glass transition was observed. The data suggested that the carbohydrates that are present in the cytoplasm of these plant organs are the primary components contributing to the glassy state (Chapter 6).

    In order to find a relation between desiccation tolerance and physical stability, the heat stability of proteins and the properties of the glassy matrix in several dry maturation-defective mutant seeds of Arabidopsis thaliana were studied. Proteins in dried wild-type seeds did not denature up to 150°C. In dried desiccation-sensitive lec1-1 , lec1-3 and abi3-5 seeds, protein denaturation occurs at temperatures below 100°C. In desiccation-tolerant abi3-7 and abi3-1 seeds, protein denaturation commenced above 120 and 135°C, respectively. The maximal rate of change ofvH with temperature was much higher in abi3-5 , lec1-1 and lec1-3 mutant seeds than in wild-type, abi3-1 , and abi3-7 seeds. This was interpreted as a higher molecular packing density in dried desiccation-tolerant than in dried desiccation-sensitive seeds, which is associated with a higher, respectively lower protein denaturation temperature. The generally lower physical stability of the desiccation-sensitive mutant seeds coincides with a lack of biochemical adaptations that normally occur in the later stages of seed development (Chapter 7).

    The relation between physical stability and desiccation tolerance was also studied in slowly dried (desiccation-tolerant) and rapidly dried (desiccation-sensitive) carrot somatic embryos. Although protein denaturation temperatures were similar in the embryos after slow or rapid drying, the extent of protein denaturation was higher in the rapidly dried embryos. Slowly dried embryos are in a glassy state at room temperature, whereas no clearly defined glass transition temperature was observed in the rapidly dried embryos. Moreover, the molecular packing density of the cytoplasmic glassy matrix was higher in the slowly dried embryos. While sucrose is the major soluble carbohydrate after rapid drying, on slow drying, the trisaccharide umbelliferose accumulates at the expense of sucrose. Dry umbelliferose and sucrose glasses have almost similar Tg s. Both umbelliferose and sucrose depressed the transition temperature of dry liposomal membranes equally well; prevented leakage from dry liposomes after rehydration, and preserved the secondary structure of dried proteins. The similar protecting properties in model systems and the apparent interchangeability of both sugars in viable dry somatic embryos suggest no special role for umbelliferose in the improved physical stability of the slowly dried somatic embryos. It was suggested that LEA proteins, which are synthesized during slow drying together with the sugars, are responsible for the increased stability of the slowly dried embryos (Chapter 8).

    The dehydration-sensitive polypeptide, poly-L-lysine was used as a model to study dehydration-induced conformational transitions of this polypeptide as influenced by drying rate and carbohydrates. In solution poly-L-lysine adopts a random coil conformation. Upon slow drying of small droplets of the polypeptide solution over a period of several hours, the polypeptide adopts an extendedbeta-sheet conformation. Upon fast air-drying within 2-3 minutes, the aqueous polypeptide structure is preserved. Slow air-drying in the presence of sugars also preserves the aqueous conformation and results in the formation of a glassy state having a higher Tg than that of sugar alone. The importance of direct sugar - polypeptide interaction in stabilization during slow air-drying was studied by drying the polypeptide in the presence of glucose, sucrose or dextran. Compared to dextran (and sucrose to a lesser extent), glucose gives superior protection, while having the lowest Tg and the best interacting properties. It was suggested that during slow drying, a protectant with sufficient interaction is required for preservation of the aqueous protein structure (Chapter 9).

    The structure of a D-7 LEA (late embryogenesis abundant)-like protein protein isolated from Typha latifolia pollen was studied using FTIR. In solution, the protein adopts a random coil conformation. Fast air-drying (5 minutes) leads to the formation ofalpha-helical structure, whereas slow drying (few hours) leads to bothalpha-helical and intermolecular extendedbeta-sheet structures. When dried in the presence of sucrose, the protein adopts predominantlyalpha-helical conformation, irrespective of drying rate. Drying of a mixture of LEA protein and sucrose results in the formation of a glassy state having higher Tg and a higher average strength of hydrogen bonding than a pure sucrose glass. It was suggested that LEA proteins might be involved in the formation of a tight molecular network in the dehydrating cytoplasm of anhydrobiotic organisms, which may contribute to desiccation tolerance (Chapter 10).

    Taken together, in situ FTIR studies can give additional information on the molecular organization in desiccation-tolerant cells. The added value of this approach is that molecular structures and inter-molecular interactions can be studied in intact biological systems (Chapter 11).

    Transannular cyclisation reactions and the germacrane system mediated by enzymes from Cichorium intybus
    Piet, D.P. - \ 1996
    Agricultural University. Promotor(en): Æ. de Groot; M.C.R. Franssen. - S.l. : Piet - ISBN 9789054855880 - 155
    enzymen - biofysica - cichorium intybus - chemische reacties - cyclische verbindingen - enzymes - biophysics - cichorium intybus - chemical reactions - cyclic compounds

    Chicory ( Cichorium intybus L.), one of the many species of the Compositae family, has been cultivated for the production of the leaves since 300 BC as a food supplement and since the 16th century as a substitute for coffee. The sprouts of the chicory are appreciated for their bitter taste. This bitter taste is associated with the presence of sesquiterpene lactones. The majority of these sesquiterpene lactones possess a guaiane framework, a small number possesses a eudesmane- or a germacrane framework. The abundance of these sesquiterpene lactones is not limited to the leaves of the plant. Considerable amounts of are also present in the root, currently an agricultural waste product. Not only is the root a rich source of sesquiterpene lactones, it also contains a large amount of inulin ( 1 ), a storage carbohydrate which is based on fructose instead of glucose. Fructose, an interesting sweetener, is a versatile building block in the synthesis of several polymers and natural products. The bitter principles in the chicory may find their application as a bitter tasting additive in consumer goods.

    The biosynthesis of the sesquiterpene lactones in the chicory is believed to start from a head to tail cyclisation of farnesyl pyrophosphate ( 23 ) into a germacrane, followed by cyclisation into eudesmanes and guaianes. This thesis deals with the cyclisation of germacrane synthons and natural germacranes, induced by a root homogenate of fresh chicory. The goal is to determine the substrate specificity of the germacrane cyclase of chicory and to obtain more insight in the biosynthesis of the sesquiterpene bitter principles in C.intybus.

    A general introduction on the history, use and contents of the chicory is given in chapter 1. In chapter 2, an overview of the literature on the (bio)synthesis of germacrane sesquiterpenes and their possible biotransformation into a variety of cyclised products, is presented.

    In chapter 3, the synthesis of two (E,E)-cyclodeca-1,5-dienols possessing the germacrane framework ( 100 and 101 ), is described. The cyclisation behaviour of these compounds and the natural germacrane (+)-hedycaryol ( 39 ) towards a chicory root homogenate is discussed. The cyclising enzymes in this homogenate transform the 10-membered ring compounds into products with a eudesmane skeleton by protonation of the C l -C 10 double bond followed by transannular cyclisation and subsequent stereoselective incorporation of a water molecule at C 4 . The flexibility of the 10-membered ring system was demonstrated by the formation of the epimeric diols 117-120 from 100 and 101 . The relatively small hydroxyl function at C 7 permitted inversion of the germacrane framework, enabling cyclisation through two different syn- conformations. The large isopropanol group of 39 prohibits this inversion to such an extent that cyclisation takes place only through one conformation to give cryptomeridiol ( 125 ).

    In chapter 4, the synthesis of the three (E,E)-cyclodeca-1,6-dienols 131-133 and their cyclisation by a chicory root homogenate is described. Two kinds of hydroazulene alcohols were obtained in these reactions arising from 1,5- and 1,7-cyclisation. The 1,5-cyclisation products ( 134-136 ) are formed through an internal nucleophilic displacement of the allylic alcohol moiety by the Cj-CjO double bond, while in the formation of the 1,7-cyclisation products ( 137-139 ), an allylic isomerisation reaction of the (E,E)-cyclodeca-1,6-dienol skeleton into an allylic (E,E)-cyclodeca-1,5- dienol skeleton preceded the internal nucleophilic displacement reaction. Hydroazulenes possessing a C 6 -C 7 double bond like 134 resemble natural products like alismol ( 43) .

    Recently, the structure of a trinor-guaiane, dictamnol ( 140 ), similar to alismol, was published. The ring fusion of dictamnol ( 140 ) was postulated as cis. Since 140 was already synthesised at our laboratory and major discrepancies were found between our NMR spectral data of 140 and those reported in the literature, serious doubt about the stereochemistry and the ring junction arose. Therefore, natural dictamnol was isolated, its stereochemistry was reinvestigated and a structural revision into a transfused hydroazulene ( 152 ) is proposed.

    In chapter 5, the biotransformation of derivatives of germacrone, a readily available sesquiterpene germacrane, is described. In a number of cases, enzyme mediated cyclisation of the chemically epoxidised germacrone derivatives, had to compete with spontaneous cyclisation reactions. However, some selectivity was observed, especially in the biotransformation of germacrone-4,5-epoxide ( 48 ) into neoprocurcumenol ( 161 ). Compound 161 is the only product obtained through an enzyme-mediated cyclisation of 48 . The C l -C 10 double bond in 161 is characteristic for guaiane bitter principles in the chicory. In boiled root samples, the only conversion that was observed was a homofragmentation reaction of 48 into curcumenone ( 160 ).

    The synthesis of isogermacrone ( 166 ) paved the way for studying the influence of the position and the stereochemistry of the double bond on the ring fusion of the cyclisation products. The 4,5-epoxides of isogermacrone ( 167 ) and isogermacrene B ( 176 ) were transformed by a chicory root homogenate into a cis-fused eudesmane and two tricyclo[]sesquiterpenes.

    Chapter 6 deals with the synthesis of (E,Z)-cyclodeca-1,5-dienone 184 and the biotransformation of 184 and structurally related compounds. Transannular cyclisation reactions of (E,Z)-cyclodeca- 1,5-dienes appear to proceed in a different way as compared to the (E,E)-cyclodeca-1,5-dienes in chapter 3-5. Instead of a carbon-carbon bond formation between both double bonds of the germacrane skeleton, ring substituents are involved in the cyclisation process to relieve ring strain. If no additional ring substituents are present, e.g. in E-epoxide 201 , a cis-fused hydroazulene diol ( 202 ) is obtained. The Z-epoxides 211 and 214 were not or not unambiguously transformed by a chicory root homogenate.

    In chapter 7, the biotransformation of farnesyl pyrophosphate ( 23 ) by a partially purified chicory root homogenate is described. Radio-GC and GC-MS analysis of the incubation products obtained from [1- 3H]-farnesyl pyrophosphate revealed that 23 was initially transformed into germacrene A ( 36 ). However, the Cope rearrangement product, (β-elemene, 222) and two cyclisation products of 36 , α- and β-selinene ( 223 and 224 ) were the only products that were detected in the assay, since 36 is sensitive towards acid and elevated temperatures.

    In conclusion, the substrate specificity of the germacrane cyclase is discussed and an active site model for the germacrane cyclase in proposed together with two tentative biosyntheses of the sesquiterpene lactones in chicory. The most likely biosynthesis starts with cyclisation of farnesyl pyrophosphate ( 23 ) in germacrene A ( 36 ) followed by several oxidation steps to give intermediate 227 . Enzyme mediated cyclisation of 227 would start with the protonation and subsequent dehydration of the C 3 -hydroxyl group giving allylic cation 228 . This cation then would give 229 after a 1,5-cyclisation, followed by a selective deprotonation towards the bridgehead carbon atom. Further oxidation of 229 would give the guaianolides 12-17 .

    Glucosidation of the C 3 -hydroxyl function of 227 gives sonchuside A ( 20 ) and cichorioside C ( 21 ) which may be cyclised by germacrane cyclasing enzymes into the corresponding eudesmanolides, e.g. 18 . Presumably, glucosidation of the C 3 -hydroxyl group prevents the 1,5-cyclisation process towards the guaianolides.

    Mitochondrial DNA sequence evolution in shorebird populations
    Wenink, P.W. - \ 1994
    Agricultural University. Promotor(en): A.J. Baker; W.B. van Muiswinkel. - S.l. : Wenink - ISBN 9789090068664 - 136
    mitochondria - moleculaire biologie - genetische code - biofysica - eiwitten - enzymen - nucleïnezuren - genotypen - genetische variatie - evolutie - fylogenie - oorsprong - fylogenetica - wereld - waadvogels - mitochondria - molecular biology - genetic code - biophysics - proteins - enzymes - nucleic acids - genotypes - genetic variation - evolution - phylogeny - origin - phylogenetics - world - waders

    This thesis describes the global molecular population structure of two shorebird species, in particular of the dunlin, Calidris alpina, by means of comparative sequence analysis of the most variable part of the mitochondrial DNA (mtDNA) genome. There are several reasons why mtDNA is the molecule of choice to probe the recent evolutionary history of a species. Most importantly, mtDNA accumulates substitutions at a high average rate that permits the tracing of genealogies within the time frame of speciation. The population structure of shorebirds, like that of arctic- breeding waterfowl (Ploeger, 1968), must have been influenced dramatically by the Pleistocene glaciations (mainly during the last one million years). The fastest evolving part of the mtDNA genome, the non-coding control region, offers sufficient genetic resolution to reveal differentiation of such recent origin. The typical mode of maternal inheritance, the absence of recombination, and the presumed neutrality of substitutions, are characteristics that add to the suitability of mtDNA for the construction of robust phylogenies ( Chapter 1 ).

    Cloning and sequencing of the control region of a turnstone ( Arenaria interpres ) facilitated subsequent amplification and direct sequencing of the homologous region in other turnstones, and dunlins as well. Comparison of this approximately 1200 basepairs (bp) region for several turnstones, dunlins and a chicken ( Gallusdomesticus ) revealed the presence of differentially evolving sequence blocks within the control region. Both shorebird species contain an AC repetitive sequence at the 3' end of the light strand, varying in size (around 100 bp) and composition between individuals. Sequence identity is highest in the central part of the control region, similar to the conservation of this part in other vertebrate species. Most single nucleotide substitutions, as well as insertions and deletions, are restricted to two segments, notably at the beginning and near the end of the control region. Overall, the organization of the avian control region resembles its human counterpart. Sequence comparison of the larger variable segment at the beginning of the control region (CR I) for worldwide samples of 25 tumstones and 25 dunlins demonstrated the utility of this region for the detection of intraspecific differentiation. The turnstone reveals few differences worldwide and identity of clones from distant regions, whereas the dunlin reveals divergent clusters of genotypes that are geographically restricted. It is concluded that the turnstone has been confined historically to one Pleistocene refugium, from which it has dispersed around the world to establish its current biogeography. The dunlin, on the other hand, became divided into several isolated populations during the Pleistocene and has retained a significant amount of intraspecific genetic diversity until the present ( Chapter 2 ).

    This remarkable difference in population genetic structure between the two shorebird species may be explained by their differing ecologies. The turnstone is a high arctic breeder, and depends mainly on cold tundra habitat, whereas the dunlin breeds mostly in the lower arctic and even in temperate zones. Cold tundra habitat may have disappeared almost entirely during the last interglacial (Eemian: around 125,000 years ago) that was characterized by high temperature peaks (Anklin et al., 1993). A very similar lack of global mtDNA differentiation has been observed in the knot ( Calidris canutus ) , another shorebird that is a typical breeder of the high arctic tundra (A.J. Baker and T. Piersma, personal communication).

    Representative samples of dunlin populations from four major regions in the world were analysed for 910 bases of mtDNA sequence from the control region and the cytochrome b gene. The regions comprised the Pacific coast of North America, the Atlantic coast of North America, the Atlantic coast of Europe and arctic central Siberia. Sequence comparison of the three amplified DNA fragments showed that most substitutions are located in the CR I fragment, and substantially less in another control region segment (CR II) and part of the coding sequence of the cytochrome b gene. The 50 substitutions that were found together defined 35 different genotypes. A genealogical tree relating these genotypes revealed five major clusters. Each cluster has high geographic specificity. The cluster containing the most divergent sequences is present along the Atlantic coast of North America and represents the dunlin population breeding in arctic central Canada. Two clusters of genotypes are located principally in western Europe and central Siberia. Evidence for a low level of gene flow between these latter two populations was provided by three individuals whose genotypes suggested they were immigrants. Two other clusters are found along the Pacific coast of North America. Whereas dunlins from southern Alaska assorted to one cluster, dunlins from the southerly wintering population revealed genotypes of both clusters.

    The genetic divergence of these major mtDNA lineages can be dated to the late Pleistocene based on a molecular clock for the control region of birds. Genotypic diversity within the population samples is extensive and the calculated long term effective (female) population sizes argue against strong historical bottlenecks. Overall, there is a negative correlation of mtDNA variation and previously defined morphometric variation in dunlins. This discordance is induced largely by the morphometrical similarity of the genetically most divergent populations from both North American coasts.

    A plausible scenario for the genetic divergence of the major dunlin lineages is the ancestral fragmentation of populations over tundra refugia, that were created by the extensive glaciations of the northern hemisphere during the Pleistocene. Prolonged isolation of populations of reduced size increased the effect of genetic drift and this may have led to the observed mtDNA monophyly. The different lineages continuously diverged by the process of mutation. This ancient subdivision has been retained after retreat of the icesheets, most likely as a result of the strong site-fidelity of dunlins to their breeding ground. Dunlin populations could thus not become homogenized genetically because gene flow is not extensive enough between them ( Chapter 3 ).

    The generally observed lack of genetic population differentiation in birds, in contrast to other vertebrate groups, has been interpreted as a sign of panmixia, caused by the high dispersive capabilities of birds (Cooke and Buckley, 1987). This conclusion is mainly based on the analysis of allozyme data, but more recently also on the analysis of mtDNA restriction polymorphism (Ball et al. 1988). However, allozymes are relatively conserved genetic markers, and thus do not provide resolution at shorter time scales of evolution. The dunlin is not exceptional in its degree of natal philopatry. Rather, the findings in dunlin indicate that population structure in this species is of recent evolutionary origin, that could be detected by virtue of the high rate of nucleotide substitution in the selected mtDNA sequences. In addition, the global coverage of this study is beyond the geographical scope of most avian studies, and thus had a better perspective for detecting major phylogenetic splits within a species.

    To elucidate the geographical distribution of mtDNA lineages over the circumpolar breeding range of the dunlin (intraspecific phylogeography: Avise et al., 1987), many additional samples from interspersed populations were analysed for both control region segments. No additional major lineages turned up among 155 breeding dunlins, but one lineage previously found among wintering dunlins in western North America could be located to the eastern Siberian breeding ground. Samples from breeding birds in Greenland, Iceland, the Baltic, southern Norway, northern Norway, and western Siberia revealed genotypes that cluster together in the major European lineage. The central Siberian lineage was found in northern Russia from the Lena river delta in the east, across the Taymyr peninsula in the middle, to the Yamal Peninsula in the west. A few of these 'central Siberian' genotypes were retrieved from dunlins breeding in Norway and eastern Siberia, indicating a restricted amount of gene flow between these populations. A zone of geographical overlap between the European and the central Siberian phylogeographic groups is present at the Yamal peninsula, where equal numbers of dunlins assorted to these respective major lineages. Dunlins captured in northern, western and southern Alaska all belonged to the same mtDNA lineage and thus constitute one genetic population.

    A large fraction of the total mtDNA variance in dunlins is distributed between the five major phylogeographic regions (76%). Extensive diversity also exists, however, among the individuals of a local population. This is induced by the high rate of substitution in CR I and renders the traditional population genetic correlation measure GST less applicable. Time estimates for the corrected sequence divergence of each phylogeographic group on the basis of a molecular clock indicate a repeated fragmentation of populations, and coincide well with the onset of glacial periods. The ancestral population in central Canada may have been separated from all other dunlin populations for over 200,000 years.

    Phylogeographic groups can be correlated to the global geography of morphometrically defined subspecies in the dunlin. Whereas several disputed subspecies gain support from the genetic data (i.e. C . a. hudsonia in central Canada and C . a. centralis in central Siberia), other subspecies merge into the same phylogeographic group. No major phylogenetic divisions are apparent among the morphometrically dissimilar populations in north-eastern Greenland, Iceland, the Baltic Sea, and Norway (recognized until now as three to four different subspecies). Gauged by the depth of the other phylogenetic splits in dunlins, they can jointly be referred to as C . a. alpina, Similarly, the dunlins from northern and southern Alaska can be merged under C. a. pacifica.

    Detailed comparison of populations in Europe reveals a developing geographic specificity of slightly divergent genotypes of the European genetic cluster. Intermediate genotypic correlation measures between locales are supported by measures of restricted gene flow, particularly for the Icelandic and Baltic populations. The genetic differences between European populations have likely evolved after retreat of the ice sheets, approximately 10,000 years ago. Post-Pleistocene colonization of newly exposed breeding grounds combined with the habit of strong site-fidelity can explain the population differentiation within Europe ( Chapter 4 ).

    It is thus revealed how morphology lacks an evolutionary perspective in the determination of intraspecific taxonomy. For the dunlin, a parallel morphological evolution of genetically divergent populations, as well as the opposing process of morphological divergence of evolutionary closely related populations, is observed. Morphometric characters employed in intraspecific avian taxonomy are suffering from homoplasy, either as a result of character plasticity and environmental induction (James, 1983), or because of very high mutation rates and strong directive selection acting on phenotypes (Turelli et al., 1988). Because morphometrically different dunlin populations are often mixed outside the short breeding season, environmental induction of morphology seems unlikely, although this possibility remains to be investigated. Although the concept of a molecular clock is debatable, general agreement exists as to the neutrality of most nucleotide substitutions in DNA and the cumulative character of the mutation process. On the basis of statistically reliable amounts of substitution, the phylogenetic branching order of intraspecific lineages can therefore be inferred with precision. This applies even more so to the non-coding mitochondrial control region. Although the oldest split in the dunlin mtDNA phylogeny is dated at approximately 200,000 years ago, the species itself is probably much older, in the range of a million years (Baker, 1992). This time discrepancy could imply that many populations have been transient in the intraspecific history of the dunlin. Only populations that radiated during the later part of the Pleistocene have survived until the present. The observed genetic differentiation within Europe thus represents the shallow branch tips in the phylogenetic tree of dunlins. The mtDNA assays suggest that measures to protect declining breeding populations in Europe, like the dunlins breeding around the Baltic Sea, cannot be argued for on the basis of a subspecific status of these populations. Rather, subspecies should be reserved for groups that represent a major source of intraspecific genetic diversity.

    Limited numbers of migratory and wintering dunlins from around the world were sequenced for both control region segments to trace lineages away from the breeding grounds. The mtDNA lineages detected in these birds were identical to those already known from the breeding grounds. Mixtures of major mtDNA lineages are present in different regions of the world. Samples of dunlins from both sides of the Pacific Ocean comprised two lineages that were found separately on the breeding grounds in eastern Siberia and Alaska. The two lineages identified in population samples from the western Palearctic (western Europe and western Asia) correspond to those present on the breeding grounds in Europe and central Siberia. Overall, it appears that dunlin populations breeding in different circumpolar regions occupy overlapping areas on migration and in winter through much of their southern range. Dunlins wintering along the North American west coast can be assigned to the Alaskan as well as to the eastern Siberian breeding grounds. In parallel, it is likely that dunlins migrating along the eastern Pacific coast of Asia originate from northern Siberia as well as from Alaska. Because the Alaskan genotype found in some eastern Asian dunlins occurs in high frequency only in birds breeding in northern Alaska, it appears that the northern and southern Alaskan populations migrate in different directions. The allocation of individual dunlins to their breeding population on the basis of their mtDNA genotype, can only be certain for those lineages that are geographically separated on the breeding grounds. Because of the limited gene flow between the European and central Siberian breeding populations, uncertainty exists in the population assignment of western Palearctic dunlins. Additional characters such as body mass and time of passage during spring migration or the presence of a particular moult pattern during fall migration can be instructive for the discrimination of dunlins of Siberian origin at European staging posts. These characters seem to be correlated with the possession of a central Siberian genotype by individual dunlins. Larger sample sizes remain to be tested, however, to obtain a better estimate of the diagnostic value of each of these methods ( Chapter 5 ).

    It is not clear what underlies the different genetic compositions of dunlin populations at the breeding grounds versus the wintering regions. Dunlins probably have also migrated during the Pleistocene, under the influence of seasonal temperature fluctuations. Their wintering quarters may have been fragmented by extensive glaciations, just as the breeding grounds were. Sharing of wintering grounds would likely have opened a route for exchange of individuals between the different populations. Such gene flow would have hampered the process of stochastic lineage sorting under the influence of genetic drift. The five major flyways that are recognized for dunlins around the world today, may still partially reflect the separate ranges that were occupied by populations throughout the last glacial period. Only the population migrating along the Atlantic coast of North America has remained geographically fully separate. What causes the mixing of the other populations outside their breeding range? The question might simply be reversed. Why does the subdivided population structure still exist over the northern breeding range? This can be explained by the imprinting of natal site-fidelity in the juvenile dunlin. Juvenile dunlins leave the breeding grounds indepently of the adult birds in a rough general direction, that may also be imprinted. Their exact direction of southward migration, however, is likely a learned behaviour and this is more prone to error or change (Rösner, 1990).

    This thesis demonstrates the utility of mtDNA in elucidating the population genetic structure of a bird species. By sequencing the most variable part of the mtDNA genome the major gene pools within a species can be detected together with their phylogenetic relationships. On this basis important insights into the evolutionary history and also the life history of the dunlin Calidris alpina were gained. This method should prove highly valuable not only in the detection and preservation of genetic diversity in dunlins but also in other (endangered) animal species.

    Prediction of production : nutrition induced tissue partitioning in growing pigs
    Greef, K.H. de - \ 1992
    Agricultural University. Promotor(en): M.W.A. Verstegen; B. Kemp. - S.l. : De Greef - 117
    voer - varkens - groei - ontwikkeling - biologie - biochemie - biofysica - moleculaire biologie - vetweefsel - modellen - onderzoek - feeds - pigs - growth - development - biology - biochemistry - biophysics - molecular biology - adipose tissue - models - research

    In several swine growth models, a constant ratio is assumed between lipid deposition rate and protein deposition rate when pigs are fed below their protein deposition capacity. In the present thesis, it was studied whether there is an effect of energy intake, live weight and nutritional history on composition of growth in restrictedly fed pigs. Results showed that all three mentioned factors affected the composition of growth. Fatness of deposited tissue increased both with an increase in energy intake and with live weight. It was concluded that the composition of total tissue is not constant. It was derived that the composition of extra tissue, deposited due to extra energy intake is independent of energy intake. This extra tissue is consistently fatter than total deposited tissue. This explains the increase in fatness of pigs with each increase in energy intake. It was studied which factor (energy intake or body weight) is of major importance for the partitioning of tissue. Composition of growth was affected by energy intake to a considerably larger extent than by live weight. Furthermore, data on the partitioning of deposited protein and lipid within the body showed that, even at low energy intakes, the major part of extra tissue (due to extra energy intake) was deposited in non-lean carcass parts.

    Celbiologie : een tweeluik
    Jongkind, J.F. - \ 1978
    Wageningen : Landbouwhogeschool - 26
    biofysica - celfysiologie - biophysics - cell physiology
    Over eten en over leven
    Wit, C.T. de; Nooij, T.J. - \ 1973
    Wageningen : Landbouwhogeschool - 24
    landbouw - economische sociologie - sociale economie - sociale structuur - biofysica - thermodynamica - calorimetrie - energiebeleid - energie - economie - probleemanalyse - economische sectoren - voedselvoorziening - nederland - landbouw als bedrijfstak - agriculture - economic sociology - socioeconomics - social structure - biophysics - thermodynamics - calorimetry - energy policy - energy - economics - problem analysis - economic sectors - food supply - netherlands - agriculture as branch of economy
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