Anchorage of Innovations: Assessing Dutch efforts to use the greenhouse effect as an energy source

This case study concerns the Dutch glasshouse horticulture sector which is responsible for 10% of the country’s natural gas consumption. Various developments resulted in internal as well as external pressures to bring this down. This has led to a variety of ‘alternative energy approaches’ for the sector. The interactions between niche and regime, however, are not well understood. Building on Loeber (2003) the concept of ‘anchorage’ to analyse this interaction is used. It is concluded that the concept of anchorage provides a useful tool to study the interaction between niche and regime and the crooked pathways of ‘innovation in the making

Proton NMR studies on Megaphaera elsdenii flavodoxin : structure elucidation by 2D-NMR and implications

1H NMR techniques have been applied for a thorough study of the uncrystallizable Megasphaera elsdenii flavodoxin in its three redox states. The aim of the research project described in this thesis was to obtain answers regarding questions concerning the redox potential regulation of FMN by the protein and the observed activation barrier occurring between the oxidized and one-electron reduced redox states of the protein. Detailed information about the structure of the, from a NMR point of view, challenging large protein in solution was gathered.Applying the sequential resonance assignment procedure using phase-sensitive 2D- correlated spectroscopy (DQF-Cosy, HoHaHa, DQ experiments) and phasesensitive nuclear Overhauser enhancement spectroscopy (NOESY) to two-electron reduced M.elsdenii flavodoxin, essentially complete proton assignments for its 137 amino acid residues have been made. In addition all proton resonances of the two-electron reduced flavin were assigned, using 13C-enriched FMN molecules. An exception has to be made for C 3 'OH which exchanges too fast with solvent molecules to be detected. The presence of a fast electron shuttle between the paramagnetic semiquinone and the diamagnetic hydroquinone state of the protein was extensively exploited during the assignment procedure to create a varying proton sphere around the flavin which depended on the semiquinone percentage in the samples. Protons in this sphere are characterized in the 2D-NMR spectra by either complete disappearance or broadening of the corresponding cross peaks thereby simplifying the complex 2D-NMR spectra of the protein and labelling these protons with regard to their distance to the flavin.The assignments of the two-electron reduced state of M.elsdenii flavodoxin provided the starting point of further studies on the protein as described in this thesis. The secondary structure of the protein has been determined by visual, qualitative inspection of sequential connectivities involving CαH, CβH and NH protons observed in NOESY spectra. The global fold of the protein was then established by using nonsequential interresidual NOE connectivities as primary source of information. M.elsdenii flavodoxin consists of a central parallel β-sheet including five strands surrounded on both sides by a pair of α-helices. The flavin is non-covalently bound at the periphery of the molecule. The protein is very stable and compact as reflected by the unexpected observation of several hydroxyl and sulfhydryl groups at 41 °C and pH 8.3 with water as solvent, and the extremely slow exchange of 32 amide protons against deuterium oxide under the same experimental conditions.Comparing the secondary structure elements and the global fold of two-electron reduced M.elsdenii flavodoxin with the crystal structures of oxidized as well as one-electron reduced ClostridiumMP flavodoxin it became clear that there is a high structural similarity between both flavodoxins. Therefore the crystal structure of the semiquinone state of ClostridiumMP flavodoxin was used to build by amino acid residue replacement a plausible starting structure for the restrained molecular dynamics calculations on two-electron reduced M.elsdenii flavodoxin. These calculations, using 509 experimental interresidual NOE distance restraints (including one non- NOE in the flavin binding region) which are all very well spread over the molecule, resulted in a tertiary structure of two-electron reduced M. elsdenii flavodoxin which satisfies the experimental restraints very well (maximum NOE violation 0.66 Å, the potential energy of the structure is -2278 ± 122 kJ mol -1). For the first time the three-dimensional structure of a flavoprotein has been elucidated by NMR.The tertiary structure of M. elsdenii flavodoxin is highly defined with the exception of the flavin. The latter is expected to result from performing the RMD simulation without water molecules and without proper charges on the flavin (phosphate dianionic and N 1 negatively charged). Water plays an important role in regions of the molecule accessible to solvent molecules. The negatively charged phosphate is strongly hydrogen bonded by peptide dipoles in the region Trp7-Thr13 of the protein. Amide protons involved in phosphate binding are characterized by their very lowfield resonance positions. This study shows that these amides are solvent accessible as they exhibit fast exchange with deuterated solvent on the timescale of a NOESY exchange experiment (at pH 8.3, 41 °C). Under the same experimental conditions also part of the isoalloxazine binding region is accessible to solvent molecules. The possibility of measuring exchange characteristics of individual protons by NMR signifies important additional information on protein tertiary structure.Taking into account this solvent accessibility of both the phosphate and part of the isoalloxazine binding region and the stabilizing effects of it on charges, it is proposed that the role of electrostatic interactions between the negatively charged phosphate and N 1 on the redox potential of flavodoxin will be less dominant than proposed. The destabilization of the negatively charged N 1 in two-electron reduced flavodoxin by its local microenvironment as compared to the uncharged N 1 in the semiquinone state (both relative to their energies in water) is expected to be an important contribution to the redox potential. The amide exchange against deuterons and several typical line shapes in the 2D-NMR spectra are consistent with the structure generated.Greatly benefiting from the assignments made of the two-electron reduced state, essentially complete sequential assignments have been made of oxidized M.elsdenii flavodoxin, including flavin assignments. Based on identity in NOEcontacts and in chemical shift positions of the protons, it is concluded that the tertiary structure outside the immediate flavin binding region remains identical on going from the oxidized to the reduced state of the protein. However, functionally important conformation changes in the flavin binding region do occur on reduction as observed by NMR. The orientation of the peptide link between Gly58 and Ser59 is altered (bringing the carbonyl group in close contact to N 5 H) as well as the position of the amide of Glu61. The semiquinone state is stabilized as compared to the oxidized state. These subtle conformation changes account for the activation barrier occurring in the transition from the oxidized to the one-electron reduced state of the protein. On additional reduction of the protein to the two-electron reduced state no further conformation changes are expected as a fast electron exchange between the semiquinone and the hydroquinone state prevents structural rearrangements. M.elsdenii flavodoxin is thereby predestined for oneelectron transfer reactions by shuttling between the semiquinone and hydroquinone states, as has already been shown by previous studies. No difference in water accessibility and flexibility in the flavin binding region are observed on reduction. On two-electron reduction the flavin becomes anti-aromatic as evidenced by a decrease in ring current effect of the pyrazine part of the isoalloxazine moiety on the apo region of the protein.A three-dimensional non-selective Clean TOCSY-NOESY 1H NMR study of M.elsdenii flavodoxin in the oxidized state has been performed to demonstrate the value of the experiment for making sequential resonance assignments in relatively large proteins. An easy and concise way for the analysis of cross peaks has been given and sequential assignments in various secondary structure elements of flavodoxin are made confirming the assignments made by 2D-NMR results. Additional information has also been obtained. Non-selective TOCSY-NOESY has been compared with selective TOCSY-NOESY and non-selective TOCSY-NOESY.Finally, apoflavodoxin of M.elsdenii has been studied using 2D-NMR techniques. The large majority of proton resonances outside the flavin binding region has been assigned. By analyzing sequential and long-range NOE connectivities, which are virtually identical to the connectivities observed for the holoprotein, and based on identity in chemical shifts, it is concluded that the structure of the protein outside the flavin binding region is nearly identical to that of the holoprotein in its three redox states. This is in contrast to results reported from far-uv-circular dichroism studies. Many proton resonances in the isoalloxazine binding region have not (yet) been assigned, possibly resulting from multiple conformations in this region of the protein, preventing structural analysis

Historische en hedendaagse systeem-innovaties in de glastuinbouw en varkenshouderij: Een innovatie-sociologische analyse

Transities en systeem-innovaties in de landbouw zijn het thema van dit rapport. Klimaatverandering, energiegebruik, methaan-emissies, dierenwelzijn, economische concurrentie, voedselschandalen (BSE, dioxine), verzuring, stankproblemen, landschapsinrichting, en rurale ontwikkeling zijn enkele van de socio-economische ontwikkelingen die druk creëren op het landbouwsysteem. Veel van deze ontwikkelingen kunnen (waarschijnlijk) niet binnen de grenzen van het bestaande systeem het hoofd geboden worden. Daarom is het thema van transities naar nieuwe systemen (systeem-innovatie) gestegen op de maatschappelijke en politieke agenda. Wat betreft empirische focus gaat dit rapport over transities in twee sectoren: glastuinbouw en varkenshouderij. Voor beide sectoren is een studie gedaan van historische transities en een studie van contemporaine transities ‘in the making’. Hedentendaage, staat de glastuinbouw onder druk wegens hoog energiegebruik (en dus CO2 emissies en klimaatverandering), vooral gerelateerd aan ruimteverwarming met gas. De varkenssector staat onder druk wat betreft dierenwelzijn (o.a. ruimtegebruik in stallen, onverdoofd castreren van biggetjes), internationale economische concurrentie, en mestproblematiek (verzuring, stank). In reactie op deze druk, zijn in beide sectoren radicale innovatietrajecten gestart die systeemcomponenten aanzienlijk veranderen

Assisting walking balance using a bio-inspired exoskeleton controller

Background: Balance control is important for mobility, yet exoskeleton research has mainly focused on improving metabolic energy efficiency. Here we present a biomimetic exoskeleton controller that supports walking balance and reduces muscle activity. Methods: Humans restore balance after a perturbation by adjusting activity of the muscles actuating the ankle in proportion to deviations from steady-state center of mass kinematics. We designed a controller that mimics the neural control of steady-state walking and the balance recovery responses to perturbations. This controller uses both feedback from ankle kinematics in accordance with an existing model and feedback from the center of mass velocity. Control parameters were estimated by fitting the experimental relation between kinematics and ankle moments observed in humans that were walking while being perturbed by push and pull perturbations. This identified model was implemented on a bilateral ankle exoskeleton. Results: Across twelve subjects, exoskeleton support reduced calf muscle activity in steady-state walking by 19% with respect to a minimal impedance controller (p < 0.001). Proportional feedback of the center of mass velocity improved balance support after perturbation. Muscle activity is reduced in response to push and pull perturbations by 10% (p = 0.006) and 16% (p < 0.001) and center of mass deviations by 9% (p = 0.026) and 18% (p = 0.002) with respect to the same controller without center of mass feedback. Conclusion: Our control approach implemented on bilateral ankle exoskeletons can thus effectively support steady-state walking and balance control and therefore has the potential to improve mobility in balance-impaired individuals.Support Biomechanical Engineerin