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The land of the checkpoints : Study of the daily geographies of checkpoints in the Occupied Palestinian Territories
Rijke, Alexandra - \ 2019
Wageningen University. Promotor(en): C. Minca, co-promotor(en): M.E. Ormond. - Wageningen : Wageningen University - ISBN 9789463951418 - 357
When the Israeli state occupied the Palestinian Territories in 1967, it gradually imposed restrictions on Palestinian movement. At first easily circumvented by Palestinians, these restrictions have become an intricate, multi-layered ‘architecture of occupation’ over the last 50 years. This architecture of occupation includes the Wall, illegal Jewish settlements and an elaborate checkpoint system. As a consequence, many Palestinians and Jewish settlers have to pass through Israeli checkpoints on a daily basis. In this PhD thesis, I have analysed their experiences. I have studied the workings of the checkpoints – its rules and regulations, managers and machines – and the diverse ways in which Palestinian commuters engage with the checkpoints. I conclude that these checkpoints produce arbitrary, mutable and selective regimes of mobility, and that they should be seen as the outcome of the endless interplay between its managers, commuters, rules, material devices and procedures of control.
Microbial treasure trove : Unravelling the potential of class 2 CRISPR–Cas systems
Mohanraju, Prarthana - \ 2019
Wageningen University. Promotor(en): J. van der Oost, co-promotor(en): D. Swarts. - Wageningen : Wageningen University - ISBN 9789463951258 - 318
Animals are known to possess a large repertoire of immune systems with a high degree of sophistication. On the other hand, the immune systems found in bacteria and archaea appeared to be much more rudimentary. However, the ground-breaking discovery of a novel immune system, CRISPR-Cas, proved otherwise. CRISPR-Cas, is unique in being both adaptive and heritable, and it relies on small RNA molecules that specifically guide the defence system to matching invader DNA sequences. This natural defence system has been successfully repurposed into a valuable molecular technology which is a robust, efficient, easy-to-use method to precisely alter DNA sequences of living organisms. The current CRISPR-based technologies, mostly employ the Cas9 protein, for diverse biotechnological applications. Nevertheless, the natural diversity of CRISPR-Cas systems is remarkably extensive, including systems that target DNA, systems that target RNA, and systems that target both DNA and RNA. The diverse class 2 CRISPR nucleases have unique molecular features that contribute to an expansive toolbox for genome and transcriptome engineering. These nucleases differ greatly in their structure and mechanisms. These differences could be exploited as complementary applications creating numerous CRISPR-based technologies possibly with favourable specificity, efficiency and/or delivery. This thesis explores the diversity of Class 2 CRISPR–Cas systems and provides mechanistic insights into different class 2 nucleases. In addition, it describes potential applications to expand the current repertoire of CRISPR-based technologies.
Due to the everlasting arms race between prokaryotes and their viruses, the rapid evolution of CRISPR–Cas systems has resulted in extreme structural and functional diversity. As a result, a plethora of distinct CRISPR–Cas systems are represented in genomes of most archaea and almost half of the bacteria. The key players of this system are the crRNA binding effector complexes, and the associated nuclease domains. CRISPR–Cas systems are currently grouped into two classes each of which is subdivided into three types. Class 1 systems (consisting of types I, III, and IV) use a multi-subunit protein complex to achieve interference, and class 2 systems (consisting of types II, V, and VI) utilize a single multi-domain protein, that have been repurposed for genome editing applications in a wide range of organisms. The mechanism of crRNA maturation in CRISPR–Cas12a systems was unravelled during this thesis. Unlike the type II nuclease Cas9, which utilizes a tracrRNA as well as endogenous RNaseIII for maturation of its dual crRNA/tracrRNA guides, pre-crRNA processing in the Cas12a system proceeds in the absence of tracrRNA or other Cas proteins. It was demonstrated that Cas12a nucleases possess a previously unknown RNase domain that is responsible for cleaving the pre-crRNA to generate the mature crRNAs. The typical cleavage pattern revealed that Cas12a recognizes specific secondary structures and/or motifs on its direct repeats. Furthermore, the ability to autonomously process crRNA has significant implications from a genome editing standpoint, as it provides a simple route to editing multiple genomic loci at a time (multiplex editing). Using a single customized CRISPR array up to four genes in mammalian cells ex vivo and up to three genes in mouse brain cells in vivo were shown to be edited simultaneously.
The characterisation of a novel, diminutive type V-U1 Cas protein from Mycolicibacterium mucogenicum (MmuC2c4) was described in this thesis. Type V-U proteins are highly similar to the typical transposon-encoded TnpB-like proteins and each of them (type VU-1 to type VU-5) appear to have originated independently from distinct TnpB families. Akin to most type V proteins, MmuC2c4 was shown to recognize a 5’-TTN-3’ PAM on a double-stranded target DNA.
The characterisation of a type II-C Cas9 orthologue of the thermophilic bacterium Geobacillus thermodenitrificans T12, ThermoCas9 is described. This is one of the first reports that provides fundamental insights into a thermophilic CRISPR–Cas9 family member. It was demonstrated that ThermoCas9 is active in vitro between 20 and 70 ℃, that the structure of its sgRNA influences its activity at elevated temperatures, it has a more stringent PAM-preference at lower temperatures, it does not tolerate extensive spacer-protospacer mismatches, and it preferentially cleaves plasmid DNA compared to linear DNA. Furthermore, ThermoCas9 was employed for pyrF gene deletion and transcriptional silencing of ldhL gene at 55 ℃ in Bacillus smithii ET 138 and for pyrF gene deletion at 37 ℃ in Pseudomonas putida. This is the first time Cas9-based bacterial genome editing and silencing tools were used at temperatures above 42 ℃.
Four Cas12a orthologues were assessed for their salt tolerance as well as pH- and temperature stability using biochemical assays as described. Subsequently, Francisella tularensis subsp. novicida (FnCas12a) and Eubacterium eligens (EeCas12a) were applied for genome editing in a moderate thermophilic bacterium, Bacillus smithii. It is demonstrated that FnCas12a and EeCas12a are sub-optimally active in vivo at temperatures above 45 ℃. The wide growth temperature range of B. smithii ET 138 was employed for the controllable induction of Cas12a expression at temperatures below the 45 ℃ threshold. It was demonstrated that a mutant can be generated within a short span of 2-3 days. This process can be easily adapted for gene editing applications in a wide variety of both mesophilic and moderate thermophilic organisms. potential to harness the activity of anti-CRISPR (Acr) proteins for controllable bacterial genome engineering was also investigated. The Acr protein from Neisseria meningitidis (AcrIIC1Nme) was employed as an “on/off-switch” to control the activity of thermostable Cas9 orthologues from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9). Initially, it was proven that both ThermoCas9 and GeoCas9 can introduce lethal dsDNA breaks in E. coli at 37 ℃ in a tuneable manner. Next, it was demonstrated that AcrIIC1Nme traps both tested Cas9 orthologues in a DNA-bound, catalytically inactive state. The Cas9/AcrIIC1Nme complexes can promote a transcriptional silencing effect with efficiency comparable to the catalytically “dead” ThermodCas9 and GeodCas9 variants. Finally, a single-vector, tightly controllable and highly efficient Cas9/AcrIIC1Nme-based tool for coupled silencing and targeting in E. coli was developed. This tool may serve as a basis for further developing a controllable genome editing and transcriptional regulation in model as well as non-model microorganisms.
Furthermore, a novel biological role and mechanism for the CRISPR–Cas9 system of the pathogen Campylobacter jejuni (CjeCas9) was uncovered. It was demonstrated that upon C. jejuni infection of human cells, CjeCas9 is secreted into the cytoplasm of the infected cells and it can autonomously enter the nucleus. Inside the nucleus, it catalyses metal-dependent and sequence-independent nicking of double stranded DNA, eventually leading to cell death. Genome editing using CjeCas9 was compared with the commonly used Cas9 from Streptococcus pyogenes (SpyCas9), and the latter was shown to be superior in creating indels. It was concluded that the unique catalytic features make CjeCas9 nuclease less suitable for genome editing applications.
In conclusion, the research described in this PhD thesis has uncovered novel molecular requirements and mechanisms of several unique Class 2 CRISPR–Cas nucleases. Besides gaining insights into their biochemical mechanism, the potential of Class 2 nucleases has been harnessed for biotechnological applications. Additionally, a unique role and mechanism of CRISPR–Cas in virulence has been elucidated. The characterisation of nucleases such as FnCas12a, EeCas12a, MmuC2c4 and ThermoCas9 opens up exciting possibilities of utilizing them as genome and transcriptome engineering tools.
Fostering oral presentation competence in higher education
Ginkel, Stan van - \ 2019
Wageningen University. Promotor(en): M. Mulder, co-promotor(en): H.J.A. Biemans; J.T.M. Gulikers. - Wageningen : Wageningen University - ISBN 9789463439633 - 172
Presenting is considered as a core competence of the higher educated professional. However, it remains questionable how effective learning environments fostering presentation competence should be constructed. This thesis focuses on formulating evidence-based educational design principles. Further, research questions explore effective feedback processes within the context of realistic presentation skills courses. These studies verifies the potential impact of feedback sources, such as teachers, peers and the self, on developing students’ oral presentation competence. Besides studying the quality of differing feedback sources, Virtual Reality is studied as an alternative feedback mode encouraging students’ cognition, skills and attitudes towards presenting. The constructed VR-tool, based on the principles of this thesis, is already implemented in secondary education, higher education and the corporate sector.
The role of polycyclic aromatic hydrocarbons in developmental toxicity of petroleum substances
Kamelia, Lenny - \ 2019
Wageningen University. Promotor(en): I.M.C.M. Rietjens; P.J. Boogaard. - Wageningen : Wageningen University - ISBN 9789463950619 - 287
REACH requires prenatal developmental toxicity (PDT) testing for substances registered in the EU at a volume of ≥100 tonnes/year. One of the consequences is that many petroleum substances (PS) will need to be tested for their potential adverse effect on prenatal development according to the current OECD 414 testing guidelines. This will involve a huge number of experimental animals and a considerable amount of resources. Therefore, the application of in vitro alternative testing strategies may reduce the animal experimentation and resources needed to study PDT potencies of PS. Furthermore, since some PS with high concentrations of polycyclic aromatic hydrocarbons (PAH) may induce PDT whilst their gas-to-liquid (GTL) analogues, which are synthetic products completely devoid of aromatics, do not induce PDT, it was hypothesized that PDT observed for some PS is caused by certain types of PAH in these products. This hypothesis was tested in the present thesis using a battery of in vitro alternative assays.
Chapter 1 provided background information and presented the aim of the thesis. In addition, the selected test substances and in vitro alternative assays used in the present thesis were also introduced. In total, 19 samples derived from 6 PS and 2 GTL product categories were tested. These samples were selected because i) they represent a series with a systematic variation in PAH content, being substances containing a range of 3- to 7-ring PAHs including extremes regarding their PAH content (with and without PAHs) and ii) in vivo PDT data for these product categories were available, enabling in vitro-in vivo comparisons. The selected in vitro alternative assays were presented, including the embryonic stem cell test (EST), the zebrafish embryotoxicity test (ZET), and a panel of CALUX reporter gene assays. Finally, the general outline of the thesis was also provided.
Chapter 2 assessed the applicability of the EST to evaluate in vitro embryotoxic potencies of the DMSO extracts of 9 PS (varying in their PAH content, from 5 PS categories) and 2 GTL products (containing no PAHs) as compared to their in vivo potencies. All DMSO-extracts of PS induced a concentration-dependent inhibition of ES-D3 cell differentiation into beating cardiomyocytes at non-cytotoxic concentrations, and their potency was proportional to their 3- to 7-ring PAH content. In contrast, both GTL extracts, which are completely devoid of PAHs, tested negative in the EST. When the EST results were compared to in vivo PDT data of the corresponding PS, a good correlation was found between in vitro and in vivo results (R2: 0.97). Overall, the EST showed able to evaluate the in vitro embryotoxicity of PS, within and across categories, a result for the in vitro assay that was in line with the in vivo PDT data. The results also supported the hypothesis that PAHs are the primary inducers of the PDT resulting from PS exposure.
In Chapter 3, the role of endocrine- and dioxin-like activity in the developmental toxicity of PS extracts was investigated using a panel of Chemical Activated LUciferase gene eXpression (CALUX) assays. The same set of samples as in Chapter 2 was tested in the panel of CALUX assays that included agonist and antagonist assays for the androgen, estrogen-α, progesterone, and thyroid-β receptor, and also for the aryl hydrocarbon receptor (AhR). All DMSO-extracts of the PS showed strong AhR agonist activity and weak antiprogesterone, antiandrogen, and estrogenic activities. Only minor effects were seen for thyroid-related and antiestrogenic activity with some products. PS that are grouped in the same class induced similar luciferase expression profiles, suggesting a class specific signature of effects. None of the GTL products showed a meaningful interaction with the selected receptors, thus testing negative in all CALUX assays applied. The AhR-mediated activity of the PS correlated best (R2: 0.80) with the in vitro PDT potency of the corresponding PS as quantified previously in the EST, suggesting an important role of the AhR in mediating this effect. In conclusion, a high potential for endocrine and dioxin-like activity of some PS extracts was elucidated, which correlated with their in vitro PDT, and was driven by the type and level of PAHs present in the PS extracts. The prominent AhR-mediated activity as induced by the PS extracts tested could be one of the underlying mechanisms of PDT by these substances.
Chapter 4 investigated the usefulness of both the EST and the AhR CALUX assay to evaluate the in vitro PDT potency of an additional series of DMSO-extracts of HFOs, heavy PS containing mainly 3- to 7-ring PAHs, and one HRBO, a highly refined mineral oil that contains no aromatics and no PAHs. All DMSO-extracts of HFOs, but not of the HRBO, resulted in inhibition of ES-D3 cell differentiation in the EST and induced AhR-mediated activity in the AhR CALUX assay, and these potencies were was shown to be proportional to the amount of 3- to 7-ring PAHs they contain. Co-exposure of ES-D3 cells (EST) or H4IIE.luc cells (AhR CALUX assay) with the selected DMSO-extracts of PS and the AhR antagonist trimethoxyflavone (TMF), successfully counteracted the PS-induced inhibition of ES-D3 cell differentiation into cardiomyocytes as well as the AhR-mediated induction of gene expression by these substances. Moreover, also for this series of PS a good concordance was obtained when comparing the EST results with available in vivo PDT data. Altogether, the resulting data corroborate the hypothesis that PS-induced PDT is induced mainly by their 3- to 7-ring PAH content and that the observed PDT is partially mediated via the AhR.
In Chapter 5, the applicability of the ZET to evaluate developmental toxicity potency of the same set of samples as tested in Chapter 2 and 3 (DMSO-extracts of 9 PS and 2 GTL products) was investigated. All PS extracts, varying in PAH level and content, were able to inhibit the development of zebrafish embryos in a concentration-dependent manner and this potency could be associated with the amount of 3-5 ring PAHs they contain. On the contrary, DMSO-extracts of both GTL products, with no aromatics, showed no effect at all in the ZET. The potencies obtained in the ZET moderately correlated with those previously reported for the EST (R2: 0.61) and the AhR CALUX assay (R2: 0.66), while the correlation with potencies reported in in vivo studies were higher for the EST (R2: 0.85) than the ZET (R2: 0.69). Combining the results obtained from the EST (Chapter 2), AhR CALUX assay (Chapter 3), and ZET (Chapter 5) ranked and clustered the test substances in line with their in vivo potencies and chemical characteristics. It was concluded that the ZET did not outperform the EST as a stand-alone assay for testing PDT of PS but confirms the hypothesis that PAHs are the major inducers of PDT by some PS, and that the ZET is a useful addition to a battery of in vitro tests able to predict the in vivo PDT of PS.
In Chapter 6 we combined an exogenous biotransformation system, using hamster liver microsomes, with the EST to compare the in vitro PDT potency with and without bioactivation of two model 5-ring PAHs, benzo[a]pyrene (BaP) and dibenz[a,h]anthracene (DBA), and of PAH containing PS and GTL base oil (GTLb) extracts. In the absence of bioactivation, DBA, but not BaP, inhibited the differentiation of ES-D3 cells into beating cardiomyocytes. Upon bioactivation, BaP induced in vitro PDT, while its major metabolite 3-hydroxybenzo[a]pyrene was shown to be active in the EST as well. This indicates that BaP needs metabolic activation to exert its in vitro embryotoxic effect. The PS-induced PDT in the EST was not substantially changed following bioactivation, implying that metabolism may not play a crucial role for PS to exert their in vitro PDT effects. GTL extracts tested negative in the EST, with and without bioactivation. Altogether, although some PAH constituents require metabolic activation to be able to induce PDT, some do not and this latter also appeared to hold for the (majority of) the PS constituents responsible for the in vitro PDT of these complex substances.
Chapter 7, first presented an overview of the results and main findings, which was combined with a general discussion of the data obtained and with future perspectives for follow-up studies to be performed in the near future. It was concluded that PAHs present in PS are the major inducers of PDT caused by these substances and that this was successfully and adequately assessed using several in vitro alternative assays, including the EST, ZET, and AhR CALUX assay. The results obtained in Chapter 2, 4, and 5 of the thesis were used in a QSAR (quantitative structure activity relationship) approach to predict the in vivo PDT of a series of PS based on their PAH content. More PS extracts, ideally from different PS categories than those tested in the present thesis, should be tested to broaden the applicability domain of the proposed assay battery and the related QSAR approach for PDT testing of PS UVCBs in the future.
How fish larvae swim: from motion to mechanics
Voesenek, Cees J. - \ 2019
Wageningen University. Promotor(en): J.L. van Leeuwen, co-promotor(en): F.T. Muijres; G.J.F. van Heijst. - Wageningen : Wageningen University - ISBN 9789463439015 - 238
Most of the world's 34,000 known fish species are undulatory swimmers. Their body undulations are produced by fluid-structure interaction between water and the body of the fish, powered by its muscle system. Despite these complex physics, just-hatched fish larvae can already produce effective swimming motion. How they do this is not yet fully understood. With this thesis, we aim to contribute to answering this question by examining the biomechanics of swimming of early-development larval zebrafish. With novel experimental and computational techniques, we reconstructed the dynamics of the larvae from high-speed video. These analyses highlight the challenges that larval fish face during swimming, and how the larvae have evolved to solve these challenges.
In chapter 2 we reviewed the mechanics of swimming of larval fish. We examined the functional demands on the locomotory system of fish larvae: immediately after hatching, fish need to escape predators, search and hunt for food, and migrate and disperse. These demands need to be fulfilled by the larvae while undergoing large changes in their bodies, both internal and external. Furthermore, the swimming speed and size of many larvae causes them to be in the intermediate flow regime, where the nature of the flow changes considerably with changes in size or speed. In this chapter, we integrated previous literature to gain insight into how these functional demands on the locomotory system are met with the advantages and limitations of their developing bodies and the changing hydrodynamic regime.
In chapter 3, we analysed near-periodic swimming of zebrafish larvae with two-dimensional inverse dynamics from motion that was manually tracked from high-speed video images. We used these data to show how the intermediate flow regime affects the swimming dynamics of fish larvae. We used the Reynolds number, which indicates the relative importance of viscous forces to inertial forces, to characterise the flow regime that the larvae swim in. Furthermore, we applied the Strouhal number, a measure of the ratio of the approximate lateral tail speed to the forward swimming speed, to express changes in swimming kinematics. We found that the Strouhal number depends inversely on the Reynolds number. Fish swimming at low Reynolds numbers tend to use relatively high Strouhal numbers, indicating that their tail-beat amplitude and frequency are high. Even the larvae swimming at the highest Reynolds numbers still use relatively high Strouhal numbers (around 0.72) compared to adult fish (typically 0.2–0.3). Swimming at intermediate Reynolds numbers is associated with high drag, requiring the larvae to use high tail-beat amplitudes and frequencies (and therefore Strouhal number) to produce sufficient thrust. This mode of swimming requires relatively high-amplitude yaw torques, resulting in large angular amplitudes and an expected high energetic cost of transport: the small size of the larvae is a burden to their swimming.
Most of the previous research on fish swimming, including our chapter 3, has been done two-dimensionally. However, fish can perform complex, three-dimensional motions to escape predators, search or hunt for food, or manoeuvre through the environment. To expand our analyses to the third dimension, we developed a method to reconstruct the 3D motion of fish from multi-camera high-speed video, described in chapter 4. With an optimisation algorithm we find the 3D position, orientation, and body curvature that best fits the high-speed video frames. We demonstrated that the method allows us to reconstruct the swimming kinematics with high accuracy, while requiring minimal manual work. In addition, we developed a novel method to calculate resultant hydrodynamic forces and torques from the reconstructed motion. The described method is a valuable tool for analysing the biomechanics of swimming, providing data for future analyses of fish swimming.
In chapter 5, we apply this automated tracking method to analyse fast starts of zebrafish larvae five days after fertilisation. To be able to escape predators, the main functional demands on a fast start are producing sufficient speed within a narrow time frame and being able to generate a wide range of escape directions. To investigate how these demands are met, we used a five-camera high-speed video of fast-starting zebrafish larvae with unprecedented spatiotemporal resolution. From these videos, we reconstructed the 3D motion of the larvae and the resultant hydrodynamic forces and torques. Due to their undulatory swimming style, the larvae first need to bend into a C-shape before being able to produce a propulsive tail beat. For this reason, the first stage of the start is often considered ‘preparatory’. Based on the reconstructed forces and torques, we show that the first stage of the start, in addition to its preparatory role, also serves to provide most of the reorientation of the start. After this stage, the larvae unfold their bodies, moving their tails at high speeds and thus producing large propulsive forces. The turn angle produced during a start mostly depends on the amount of body curvature in the first stage, while the escape speed mainly depends on the duration of the start. This suggests that larvae are able to independently adjust the direction and speed of their escape.
Fish larvae are able to produce these escape responses and the subsequent swimming bout immediately after hatching, despite their bodies and brains still undergoing development. To understand how this is possible, we use an advanced inverse-dynamics approach, with computational fluid dynamics and a large-amplitude beam model, to reconstruct internal mechanics from the motion of the fish in chapter 6. We compute the internal bending moments from more than 100 3D-recordings of swimming over a range of developmental stages. We show that larvae use similar bending moment patterns across development, speeds and accelerations. By varying the amplitude and duration of this pattern, the larvae can adjust their swimming speed and/or acceleration. This similarity suggests that their muscle activation patterns are also similar, which would help to explain how just-hatched larvae with limited neural capacity can produce effective swimming motion across a range of speeds and accelerations.
In this thesis, we demonstrated that larval fish swim in a challenging hydrodynamic regime. Despite the relatively high drag, they can produce effective swimming motions to help them survive to adulthood. We developed novel methods to quantify this motion in 3D, and from it reconstructed the external and internal mechanics. With these inverse-dynamics approaches, we show that fish larvae can likely adjust their swimming in a relatively simple way, for both fast starts and continuous swimming. Thus, complex physics do not obstruct developing larvae from swimming effectively.
Getting a grip on tree frog attachment : structures, mechanisms, and biomimetic potential
Schöning-Langowski, Julian K.A. - \ 2019
Wageningen University. Promotor(en): J.L. van Leeuwen, co-promotor(en): D Dodou; M. Kamperman. - Wageningen : Wageningen University - ISBN 9789463431859 - 242
Tree frogs are versatile climbers that use their adhesive digital pads to attach to various substrates, even under challenging conditions. The pads are covered in mucus and can generate adhesive and frictional forces of up to several body weights per digit. For centuries, the remarkable attachment performance of tree frogs has fascinated scientists who aimed to unravel the fundamentals of tree frog attachment. Among the various mechanisms that have been discussed to explain tree frog attachment, capillary adhesion is the most commonly used one. Previous research was primarily directed at the role of the micropatterned pad surface in attachment, which resulted in the development of several tree-frog-inspired micropatterned adhesives. However, a comprehensive explanation of the mechanisms of tree frog attachment is still unavailable, and the role of internal pad structures in attachment is barely understood. In this thesis, I present an interdisciplinary analysis of the attachment apparatus of tree frogs with focus on subepidermal structures, hence contributing to the understanding of nature’s adhesive systems, and providing input for the design of biomimetic adhesives inspired by tree frogs.
In Chapter 2, we provided a synthesis of the status quo of knowledge on tree frog attachment—ranging from the functional pad morphology to experimental findings in support of the various hypothesised mechanisms of tree frog attachment—and identified gaps in the existing knowledge. An account of the functional morphology highlights the need for an exploration of the internal pad morphology and of the chemistry of the secreted mucus in order to deepen the understanding of tree frog attachment. By revisiting current analytical models, reported data, and observations, we show that the hypothesis of wet adhesion as primary mechanism in tree frog attachment is not sufficiently supported. Van der Waals forces, which were for a long time believed to play only a minor role due to mucus covering the pad surface, may well be involved in tree frog attachment.
In Chapter 3, we combined histochemistry and synchrotron micro-computer-tomography to characterise the internal morphology of the digital pads of the tree frog Hyla cinerea in 3D, with a focus on mechanical links between the adhesive pad surface and the internal digital skeleton. The ventral pad surface connects to the skeleton via several pathways of force transmission. A collagenous septum runs from the ventral cutis to the tip of the distal phalanx and compartmentalises the subcutaneous pad volume into a distal lymph space and a proximal space filled with mucus glands. Moreover, a collagen layer runs from the ventral cutis via interphalangeal ligaments to the middle phalanx. The fibres constituting this layer run in trajectories curved around the transverse pad-axis and form laterally separated ridges below the gland space. Using finite element analysis and numerical optimisation of a shear-loaded pad model, we show that the collagen fibres are presumably oriented along the trajectories of the maximum principal stresses, and the optimisation also results in ridge-formation, suggesting that the collagen layer is adapted towards a high stiffness during shear loading. Finally, immunohistochemical staining reveals the presence of several units of smooth muscle fibres in frog pads. These active structures may provide tree frogs with the ability to control the distribution of mechanical stresses across the ventral pad surface, hence facilitating modulation of the attachment strength.
Chapter 4 addresses the morphology of the digital mucus glands and the chemistry of the secreted mucus. In H. cinerea, the mucus glands opening to the ventral pad surface form a macrogland and thus differ significantly in their morphology from the ones opening to the dorsal (non-adhesive) pad surface. We identified a digital mucus macrogland in min. 10 neobatrachian families that are not exclusively arboreal, indicating that the macrogland morphology is determined by generic functional requirements arising from ‘ground contact’ (e.g. lubrication and compensation of mucus loss) as well as by specific requirements for climbing and attachment. Using cryo-histochemistry as well as infrared and sum frequency generation spectroscopy, we show that—in contrast to the gland morphology—the general mucus chemistry varies only little between different body locations in the tree frogs H. cinerea and Osteopilus septentrionalis, as well as between arboreal and non-arboreal (Pyxicephalus adspersus and Ceratophrys cranwelli) frog species. This observation suggests conservation of the mucus chemistry in the course of anuran evolution and does not support a specialisation of the mucus towards attachment.
In Chapter 5, we experimentally measured the attachment performance of the tree frogs H. cinerea and Litoria caerulea using a rotation platform setup. By analysing the effects of variations in nominal substrate roughness on attachment performance, we investigated the role of the previously proposed mechanism of mechanical interlocking in tree frog attachment. Tree frogs attach as well to nano- and microrough substrates as to a smooth substrate. Attachment performance only decreases above a nominal substrate roughness of 15 µm. These observations disagree with the expected variation of attachment performance with substrate roughness if interlocking were present, hence indicating that mechanical interlocking is negligible in tree frog attachment. Furthermore, observations of detaching frogs with our updated rotation platform setup show that tree frogs are able to withstand substantial inertial loads with only two attached limbs, suggesting that the maximum attachment performance of tree frogs is higher than reported previously.
Finally, I put in Chapter 6 the findings of this thesis in a wider scientific context. By integrating the outcomes of the review on tree frog attachment with the results of the experimental chapters and with insights obtained on tree-frog-inspired adhesives, I discuss the importance of the previously proposed mechanisms of tree frog attachment. I conclude that suction and mechanical interlocking are negligible. A contribution of hydrodynamic adhesion cannot be excluded, but seems to be a consequence of having a wet skin rather than a primary attachment mechanism. Whereas capillary adhesion was previously mentioned as primary adhesion mechanism, an integration of the available knowledge suggests that capillary adhesion acts together with van der Waals forces, with the latter as dominant attachment mechanism. To explore the hypothesised convergent evolution of the digital pads of tree frogs, I provide an account of what is known from this thesis and earlier works on the phylogeny of the adhesive pads. Overall, several morphological features (e.g. the micropatterned pad surface and the digital gland cluster) are widely distributed over anuran families, suggesting that the whole pad is a functional unit and raising the question if the pads evolved convergently or if they are derived from a common ancestor. In agreement with the antecedent sections, a tentative discussion of the evolutionary history of frog pads emphasises the importance of drainage, friction generation, and van der Waals forces in tree frog attachment. Based on the outcomes of this thesis, I provide suggestions for the design of tree-frog-inspired adhesives. The drainage-capabilities of frog pads render these a good model for the development of technical adhesives in a wet environment. Further promising trends are the design of fibre-reinforced micropatterned adhesives for heavy duty applications, and the creation of actuated adhesives with controllable adhesion inspired by the muscular structures found in the biological model. Finally, I present an outlook on how the field of tree frog attachment may develop in the coming years.
Quantifying evolution in wild populations
Ramakers, Jip Jacques Claudia - \ 2019
Wageningen University. Promotor(en): M.E. Visser, co-promotor(en): P. Gienapp. - Wageningen : Wageningen University - ISBN 9789463439206 - 277
The environment is changing and this is exerting selection pressures on wild populations. For example, the timing of phenological events such as reproduction and migration are driven by temperatures and climate change is leading to a differential shift in timing of phenology among trophic levels, in some cases leading to selection on consumer phenology. Individuals are often phenotypically plastic, meaning that they can change their phenotype (e.g. breeding time) in response to environmental conditions. This allows them to track the changing environment to some degree but ultimately a genetic change is necessary to safeguard populations from extinction in the long run. Many wild populations so far, however, could not be shown to be undergoing any genetic adaptation (e.g. a shift in their phenology) over time. Quantitative genetics, i.e. the study of the genetics of quantitative (polygenic) traits, is commonly used to identify the evolutionary parameters (genetic variation and selection) in wild populations to predict evolution, but for such predictions to be successful we need to understand the ecological factors underlying (constraints to) adaptation. In this thesis, I aimed to get an understanding of how populations are coping with environmental change and which ecological processes affect the rate of adaptation. I did this using a combined approach of field experiments to identify ecological constraints and long-term observations to make evolutionary predictions in the great tit (Parus major) and other vertebrate species.
In the first part of my thesis (Chapter 2), I provided a broad overview of what is known about the effects of climate change on the general biology of birds. Birds are affected by climate change in several ways: they may change (1) their geographical distribution due to a shifting ‘bioclimatic envelope’, (2) advance their timing of phenological events such as breeding and migration, (3) undergo morphological changes (e.g. in body size) as an energetic adaptation, and (4) undergo demographic changes as a direct result of changes in reproductive success or survival. There has been a strong bias in the literature on phenology and therefore we still have a lot to learn about the ecological and evolutionary consequences related to these other aspects of phenotypic change. Whether observed changes in phenology are due to plasticity or due to genetic change remains an open question.
Dutch great tits have been under increased selection for earlier laying due to increased mismatch with the caterpillar peak (the main food for their nestlings), but we see little (phenotypic) response. The lack of a response may be caused by an energetic constraint associated with breeding too early under harsh conditions, such that birds that do breed earlier may pay fitness costs. In the second part of my thesis I aimed to test whether birds were constrained to breed earlier. In Chapter 3, I used experimental food supplementation food prior to and during egg-laying to test whether females that were tricked into laying early would pay fitness costs (due to brood desertion or reduced chick-provisioning efforts) once food supplementation ceased upon the start of laying. Food supplementation was not effective at advancing laying, and any food increased, rather than decreased, fitness. Because food supplementation alone is not sufficient to test the constraints hypothesis (e.g. because it cannot distinguish whether birds are constrained by food or are just missing the essential cues to advance their breeding) we need an additional, clean manipulation of egg-laying date that does not affect the body condition of a female. In Chapter 4, I described the first results of a large-scale experiment in which great tits are genomically selected to breed early or late. Eggs produced by females from these selection lines were brought to the wild and raised by foster parents. I showed that selection lines (late vs early) did not differ in any aspect of early-life fitness (fledging success, nestling weight at fledging), but that the fitness parameters differed slightly between selection-line birds and their wild counterparts. Since only 11 birds from these fostered birds survived until breeding in 2018 (including 2 females from the early and 3 from the late selection line), we could not test whether these birds indeed bred respectively earlier or later, or whether earlier laying indeed led to higher fitness costs. I concluded that multiple years (with different environmental conditions and an increased sample size) would be needed to conclude whether birds are indeed constrained to breed earlier.
Ultimately, breeding success in great tits is largely determined by the match of the offspring needs with the caterpillar abundance. In Chapter 5, I explored the notion that to clearly understand phenological mismatch—and to determine whether birds really are mismatched—we need a thorough, temporal description of offspring needs and food availability to quantify the amount of temporal overlap between these distributions. I found that the classical way of defining mismatch, i.e. the difference in peak dates between great tit and caterpillar phenology, outperformed a more comprehensive measure that described the temporal overlap in a model explaining variation in offspring survival and selection for laying date. I concluded that a simple measure of mismatch in highly seasonal study systems is likely to be best for describing demographic processes, and that more complex measures are likely infeasible in most practical situations.
In the third part of my thesis, I deployed state-of-the-art quantitative genetic modelling approaches to unravel patterns of selection, genetic variation, and evolutionary response to selection in a reaction-norm context using long-term, pedigreed datasets of wild populations. Some methods to achieve this were explored in the preceding Intermezzo (Chapter 6 and 7). In Chapter 8, I investigated whether maternal effects as a form of transgenerational plasticity could affect the rate of adaptation in great tits. Using experimental and long-term data, I was able to show that the clutch size of a great tit is partly dependent on her body weight at fledgling and that it is negatively associated with the clutch size of her own mother. Such a negative maternal effect could constrain adaptation to a novel environment with selection for a larger of smaller clutch. We showed by simulation, however, that this negative maternal effect would likely have little impact on the rate of adaptation.
Phenological changes over time do not always match evolutionary predictions; one potential reason for this discrepancy is an unrecognised environmentally induced coupling between selection and the heritability of the trait. In Chapter 9, I investigated how general such a coupling is in wild vertebrate populations, and whether such a coupling affects the expected rate of adaptation. The expectation was that if heritability and selection are negatively associated, this constrains adaptation because little genetic variation is present under strong selection and vice versa. Making use of openly available datasets (see Chapter 7), we managed to estimate environment-specific heritability and selection in 50 traits from 16 populations of 10 species. We found that heritability and selection are only rarely associated and that this association is an unlikely explanation for apparent evolutionary stasis observed in wild populations.
Great tits respond strongly to temperature through phenotypic plasticity; this plasticity is described by a reaction norm, the linear function consisting of an elevation (the laying date in the average environment) and a slope (the sensitivity to the environment). Since different individuals have different reaction norms, selection on laying date may result in an evolutionary shift in the reaction norm. In Chapter 10, I found that individual great tits differ genetically in the elevation of the reaction norm, but not in its slope, and this reaction norm is under selection due to the advance in the caterpillar peak over time. I predicted quantitatively, however, that such evolution has been—and will be—too slow to be detected due to the high environmental variability in laying dates.
To conclude, I investigated the evolutionary potential of populations and aimed to identify ecological constraints in adaptation. I found that there is still a lot we need to learn about the ecological and evolutionary consequences of climate change beyond the few well-known study systems, including effects on demography and population viability. Experiments aimed at unravelling the fitness costs of breeding too early are inconclusive and warrant further investigation (with more samples and multiple environments). Powerful quantitative genetic tools are available to evolutionary ecologists to quantify evolutionary trajectories but these models must be based on reality to obtain reliable predictions. I have suggested in this thesis that realistic predictions could be benefited by the integration of multiple data sources (i.e. long-term observational and experimental data) and simulations. The use of open data can aid in achieving this through the answering of novel research questions at a broad taxonomic or geographic scale. Most importantly, we need a thorough understanding of the most important components of the ecosystem of our study species. Only then can we make sense of our evolutionary predictions.
Multi-scale monitoring and modelling of the Kapuas River Delta
Kästner, Karl - \ 2019
Wageningen University. Promotor(en): A.J.F. Hoitink; R. Uijlenhoet. - Wageningen : Wageningen University - ISBN 9789463434119 - 215
Rivers in the humid tropics are those with the largest discharges and sediment loads of the world. Their evergreen and ever wet catchments are hotspots of biodiversity and their fertile deltas are acres of plenty for the production of rice, palm oil and rubber. At present, tropical rivers, their catchments, and deltas face growing pressure from rapid economic development and climate change, which may permanently deteriorate their ecosystem services. Yet,despite their importance and advancing degradation, relatively little is known about their physiography. This thesis reduces this gap by contributing to our fundamental understanding of tropical rivers.This thesis in particular addresses fundamental questions regarding the hydro- and morphodynamics of large sand-bedded rivers and their tidally influenced deltas: How can river and tidal discharge be effectively measured? How do the cross-section geometry and bed material change along the fluvial-tidal transition? How do these trends differ between the distributaries? How does the tide propagate up-river river? How can sediment transport be efficiently measured with acoustic instruments? How are the discharge and the sediment divided at river bifurcations? To address these questions, the author undertook a year-long journey to West Kalimantan, during which he surveyed and monitored the Kapuas River. The Kapuas River is nearly pristine and thus gives a rare insight into the hydro- and morphodynamics of a river that has not yet been restricted by either dams, dykes or groins. Findings from the survey of the Kapuas River are generalized with idealized models.
Feel the burn: a collection of stories on hot’n’sharp DNA engineering
Mougiakos, Ioannis - \ 2019
Wageningen University. Promotor(en): J. van der Oost; R. van Kranenburg. - Wageningen : Wageningen University - ISBN 9789463434096 - 301
The global demand on chemicals and fuels is exponentially increasing. At the same time, the excessive exploitation of fossil-based resources for the coverage of this demand has a high environmental impact, motivating the production of green chemicals and biofuels from renewable resources. Nowadays, the microbial production of green chemicals and fuels gains increasing attention, especially due to the ease in the construction of metabolically engineered microorganisms with high production capacities. This ease was achieved via the development of efficient genome engineering tools. This thesis describes the development of novel genetic tools for mesophilic and thermophilic bacteria, for metabolic engineering purposes. More specifically, here is reported: 1) the development of the first CRISPR-SpCas9-based genome engineering tools for the mesophilic bacteria Rhodobacter sphaeroides and Pseudomonas putida, as well as for the moderate thermophilic bacterium Bacillus smithii, 2) the in vitro characterization of one of the first reported thermotolerant Cas9 homologs, denoted as ThermoCas9, as well the development of the first CRISPR-ThermoCas9-based genome engineering tools for strictly thermophilic bacteria, and 3) the use of the developed tools for the metabolic exploration and exploitation of the moderate thermophilic bacterium B. smithii, towards the characterization of its acetate production pathway and the enhancement of its dicarboxylic acids productivity.
Injecting spatial priors in Earth observation with machine vision
Gonzalez, Diego - \ 2019
Wageningen University. Promotor(en): D. Tuia. - Wageningen : Wageningen University - ISBN 9789463435604 - 130
Remote Sensing (RS) imagery with submeter resolution is becoming ubiquitous. Be it from satellites, aerial campaigns or Unmanned Aerial Vehicles, this spatial resolution allows to recognize individual objects and their parts from above.
This has driven, during the last few years, a big interest in the RS community on Computer Vision (CV) methods developed for the automated understanding of natural images.
A central element to the success of \CV is the use of prior information about the image generation process and the objects these images contain: neighboring pixels are likely to belong to the same object; objects of the same nature tend to look similar with independence of their location in the image; certain objects tend to occur in particular geometric configurations; etc.
When using RS imagery, additional prior knowledge exists on how the images were formed, since we know roughly the geographical location of the objects, the geospatial prior, and the direction they were observed from, the overhead-view prior.
This thesis explores ways of encoding these priors in CV models to improve their performance on RS imagery, with a focus on land-cover and land-use mapping.
Exploration of microbial systems as biocatalysts for conversion of synthesis gas to bio-based chemicals
Diender, Martijn - \ 2019
Wageningen University. Promotor(en): A.J.M. Stams, co-promotor(en): D.Z. Machado de Sousa. - Wageningen : Wageningen University - ISBN 9789463433914 - 204
Synthesis gas (syngas) fermentation is a process capable of processing a gaseous substrate via fermentation into commodity chemicals and fuels. Gas (mainly consisting of hydrogen, carbon monoxide and carbon dioxide) fed to the fermentation process can be obtained from a wide variety of sources, including off-gases from industry, gasification of solid carbon wastes (e.g. municipal waste, lignocellulosic biomass) or gas derived from electrochemical reduction/physicochemical reduction processes.
Current limitations of the fermentation process are the relatively poorly understood physiology and genetics of the biocatalysts involved. Therefore the work described in this thesis aimed at unravelling of the syngas metabolism of acetogenic and methanogenic strains, with main focus on carbon monoxide metabolism. In addition, the application of synthetic co-cultures for syngas fermentation was explored in order to assess if such cultivation approach could lead to broadening of the syngas fermentation product spectrum. In addition to co-cultivation proof-of-concept studies for application, new fundamental insights on the metabolism of the involved biocatalysts were obtained.
A FAIR approach to genomics
Koehorst, Jasper Jan - \ 2019
Wageningen University. Promotor(en): V.A.P. Martins dos Santos, co-promotor(en): P.J. Schaap; E. Saccenti. - Wageningen : Wageningen University - ISBN 9789463433693 - 245
The aim of this thesis was to increase our understanding on how genome information leads to function and phenotype. To address these questions, I developed a semantic systems biology framework capable of extracting knowledge, biological concepts and emergent system properties, from a vast array of publicly available genome information. In chapter 2, Empusa is described as an infrastructure that bridges the gap between the intended and actual content of a database. This infrastructure was used in chapters 3 and 4 to develop the framework. Chapter 3 describes the development of the Genome Biology Ontology Language and the GBOL stack of supporting tools enforcing consistency within and between the GBOL definitions in the ontology (OWL) and the Shape Expressions (ShEx) language describing the graph structure. A practical implementation of a semantic systems biology framework for FAIR (de novo) genome annotation is provided in chapter 4. The semantic framework and genome annotation tool described in this chapter has been used throughout this thesis to consistently, structurally and functionally annotate and mine microbial genomes used in chapter 5-10. In chapter 5, we introduced how the concept of protein domains and corresponding architectures can be used in comparative functional genomics to provide for a fast, efficient and scalable alternative to sequence-based methods. This allowed us to effectively compare and identify functional variations between hundreds to thousands of genomes. In chapter 6, we used 432 available complete Pseudomonas genomes to study the relationship between domain essentiality and persistence. In this chapter the focus was mainly on domains involved in metabolic functions. The metabolic domain space was explored for domain essentiality and persistence through the integration of heterogeneous data sources including six published metabolic models, a vast gene expression repository and transposon data. In chapter 7, the correlation between the expected and observed genotypes was explored using 16S-rRNA phylogeny and protein domain class content as input. In this chapter it was shown that domain class content yields a higher resolution in comparison to 16S-rRNA when analysing evolutionary distances. Using protein domain classes, we also were able to identify signifying domains, which may have important roles in shaping a species. To demonstrate the use of semantic systems biology workflows in a biotechnological setting we expanded the resource with more than 80.000 bacterial genomes. The genomic information of this resource was mined using a top down approach to identify strains having the trait for 1,3-propanediol production. This resulted in the molecular identification of 49 new species. In addition, we also experimentally verified that 4 species were capable of producing 1,3-propanediol.
As discussed in chapter 10, the here developed semantic systems biology workflows were successfully applied in the discovery of key elements in symbiotic relationships, to improve functional genome annotation and in comparative genomics studies. Wet/dry-lab collaboration was often at the basis of the obtained results.
The success of the collaboration between the wet and dry field, prompted me to develop an undergraduate course in which the concept of the “Moist” workflow was introduced (Chapter 9).
Vision principles for harvest robotics : sowing artificial intelligence in agriculture
Barth, Ruud - \ 2018
Wageningen University. Promotor(en): E.J. van Henten, co-promotor(en): J. Hemming. - Wageningen : Wageningen University - ISBN 9789463433181 - 325
The objective of this work was to further advance technology in agriculture, specifically by pursuing the research direction of agricultural robotics for harvesting in greenhouses, with the specific use-case of Capsicum annuum, also known as sweet or bell pepper. Within this scope, it was previously determined that the primary cause of agricultural robotics not yet maturing was the complexity of the tasks due to inherent variations of the crops, in turn limiting performance in harvest success and time. As a solution, it was suggested to further enhance robotic systems with sensing, world modelling and reasoning, for example by pursuing approaches like machine learning and visual servo control. In this work, we have followed this suggestion. It was identified that facilitating new levels of artificial intelligence in the domains of sensing and motion control would be one of the ways to improve upon classical mechanization. Specifically, we investigated the means of using machine learning based computer vision guided manipulation towards a basic form of world representation and autonomy. For this, in Chapter 2 we developed an eye-in-hand sensing and visual control framework for dense crops with the goal to overcome issues of occlusion and image registration that were previously introduced when sensing was performed externally from the robot manipulator. Additionally, simultaneous localization and mapping was investigated to aid in forming a world model. In Chapter 3 we aimed to reduce the requirement of annotating empirical images by providing a method to synthetically generate large sets of automatically annotated images as input for convolutional neural network (CNN) based segmentation models. An annotated dataset was created of 10,500 synthetic and 50 empirical images. In Chapter 4 we further investigated how synthetic images can be used to bootstrap CNNs for successful learning of empirical images. We provided computer vision in agriculture a pioneering machine learning based methodology for state-of-the-art plant part segmentation performance, whilst simultaneously reducing the reliance on labor intensive manual annotations. Chapter 5 explored applying a cycle consistent generative adversarial network to our dataset with the objective to generate more realistic synthetic images by translating them to the feature distribution of the empirical domain. We show that this approach can further improve segmentation performance whilst further reducing the requirement of annotated empirical images. In Chapter 6 we aimed to bring all previous chapters into practice. The objective was to estimate angles between fruit and stems from image segmentations to support visual servo control grasping in a sweet-pepper harvesting robot. Our approach calculated angles under unmodified greenhouse conditions that met the accuracy requirement of 25 degrees for 73% of the cases. Combined, the work shows a promising stepping stone towards agricultural robotics which could ensure the quality of meals and nourishment of a growing population. Furthermore, it can become an important technology for societal issues in developed nations, e.g. by solving current labor problems. It can further improve upon the quality of life and contribute to reaching an exemplary equilibrium of sustainable agricultural production.
Creation of fibrous plant protein foods
Dekkers, Birgit L. - \ 2018
Wageningen University. Promotor(en): A.J. van der Goot; R.M. Boom. - Wageningen : Wageningen University - ISBN 9789463433198 - 204
A transition from animal to plant-based protein is required to produce sufficient protein for the growing world population, while at the same time mitigates climate change. Especially the production of meat imposes a burden on the environment. Meat analogues, which are products that are similar to meat in its functionality, can help consumers to lower their meat consumption. The anisotropic, fibrous nature of meat is perhaps the most important characteristic of meat, which can be mimicked by structuring biopolymers, such as proteins and polysaccharides with the shear cell technology. The aim of this thesis is to obtain insight in the key mechanisms that play a role in the transformation of plant-based biopolymer blends into anisotropic/fibrous structures with shear cell technology. These two key mechanisms are the deformation of the two phases present in biopolymer blends, and the subsequent entrapment of this deformation during solidification. It was concluded that successful structure formation requires matching of the properties of the two phases. During structuring at elevated temperature, the two phases are deformed, while subsequent cooling ensures entrapment of the deformed dispersed phase(s) in the (continuous) phase. Ideally, the continuous and dispersed phase have different strength in the final product,.
Chapter 2 presents a method to determine the water distribution in soy protein isolate (SPI) – wheat gluten (WG) blends. The concentration of water in each separate phase was directly determined with time-domain nuclear magnetic resonance relaxometry (TD-NMR), and oscillatory rheology was used to indirectly asses the water distribution by determining the viscoelastic properties of the separate phases and the blend. It was shown that water distributes unevenly in SPI-WG blends: more water was absorbed by the SPI as compared to the WG phase. This methodology was developed for SPI-WG blends at room temperature and subsequently also applied to heated and sheared samples in Chapter 3. First, water distribution in the blend after a heat and/or shear treatment was assessed with TD-NMR and the outcomes were then used to predict the viscoelastic properties of the SPI and WG phase in the blend. This yielded insight in the deformability of the two phases in the blend. The viscoelastic properties were measured under conditions that are relevant for structure formation, i.e. during and after heating and shearing. It was shown that the water distribution was hardly affected by a heat or shear treatment, whereas the viscoelastic properties of the two phases changed significantly. The viscoelastic properties of SPI and WG became more similar due to water redistribution in the blend, which allows deformation and alignment of the dispersed phase during structuring.
Chapter 4 describes a study using a model blend that mimics soy protein concentrate (SPC). It consists of a relatively pure protein phase, soy protein isolate (SPI), and a soluble, more or less pure polysaccharide phase, pectin. This SPI-pectin blend formed fibrous materials at a similar heating temperature as SPC, being 140°C. Pectin formed the dispersed phase and was deformed when heated and sheared at optimal conditions. Chapter 5 extends the study on structure formation with SPI-pectin blends. Here, the deformation of the dispersed pectin phase and the influence of incorporated air were considered. The fibrous nature of these products appears upon tearing, and originates from detachment through or along the long side of the weak dispersed phase(s), being pectin and/or air. A model based on the rule of mixing was used to predict the mechanical anisotropy based on the volume fraction and the deformation of the weak, dispersed phase. The size and orientation of the dispersed phases, tailored by using different shear rates, were related to differences in fracture behavior when deforming the structures. Besides deformation, the strength and volume fraction of the weak phase(s) were important when composing a blend for fibrous structure formation. In Chapter 6, the behavior of the SPI and pectin phases in a blend was investigated by determining the viscoelastic properties while shearing and heating over time. A closed cavity rheometer (CCR) was used to determine these properties under similar conditions as used during fibrous structure formation. The addition of a small amount of pectin (2.2 wt.%) to a SPI dispersion (41.8 wt.%) resulted in viscoelastic behavior that changed in time during a shear treatment at elevated temperatures. Although one can clearly discern two distinct phases with SEM, the viscoelastic behavior of the SPI-pectin blend is more complex than that of a simple composite material.
Chapter 7 demonstrates the importance of the fractionation process on the structuring potential of soy proteins. An enriched soy protein fraction was obtained through an aqueous fractionation process. Those fractions could be used to make fibrous structures when: i) the soy protein fractions were toasted, which is a dry heating step, and ii) when a concentrate (75% protein) was combined with full fat flour, in such a ratio that the protein content was similar to commercial SPC. Toasting results in decreased protein solubility, increased water holding capacity and increased viscosity of the fractions, and these changes turned out to be important for fibrous structure formation.
Lastly, literature was reviewed to put all findings in perspective (Chapter 8). An overview is presented of all techniques that are commercially used and currently investigated to create meat-like structures. Structuring techniques are compared in their approach, being either bottom-up, which refers to assembly of structural elements that are then combined, or top-down, which refers to structuring of biopolymer blends using an overall force field. A bottom-up strategy has the potential to resemble the structure of meat most closely, by structuring the molecules including proteins into structural components (e.g. muscle cells) followed by assembly of individual structural components. A top-down strategy is more efficient in its use of resources and is better scalable, but can only create the desired structure on larger length scales. The techniques with a top-down strategy were further investigated by reviewing literature on similar processes outside this particular field of application, i.e. not meant to create fibrous structures. These insights were subsequently translated to the conditions as used in structure formation for meat analogues.
Chapter 9 concludes with a general discussion of all results presented in this thesis. The different chapters are integrated in design rules for fibrous structure formation. Furthermore, the complexity encountered when studying material and conditions during fibrous structure formation are discussed. Then, the potential and the challenges for understanding and applying fibrous structure formation with simple shear flow are summarized.
The overall societal goal of developing meat analogue food products is to help consumers in the transition from animal-based to a more plant-based diet. The scientific goal to obtain insight in fibrous structure formation with the shear technology as developed in this thesis is of importance, and can be the basis for developing the technology for the next generation meat analogues.
Encapsulation of lipids to delay lipolysis and reduce food intake
Corstens, Meinou N. - \ 2018
Wageningen University. Promotor(en): C.G.P.H. Schroën; A.A.M. Masclee, co-promotor(en): C.C. Berton-Carabin; F.J. Troost. - Wageningen : Wageningen University - ISBN 9789463432382 - 200
My PhD project aimed at developing an edible product for non-invasive weight management, and targeted a natural feedback mechanism to induce satiety: the ileal brake. In order to trigger the ileal brake mechanism, lipids and their non-absorbed metabolites need to be sensed in the ileum. We aimed at inducing this mechanism through targeted release of lipids after oral intake, for which we developed multi-layered emulsions and emulsion-alginate beads. We showed that emulsion-alginate beads control in vitro lipolysis as a function of bead size and alginate concentration, and confirmed these findings under dynamic in vitro gastrointestinal conditions (DIDGI). Moreover, ingestion of yoghurt with emulsion-alginate beads significantly reduced food intake by 6% in overweight volunteers compared to a control group, suggesting that activation of the ileal brake was achieved. These findings have important implications for the development of weight management strategies, and understanding satiety in general.
Cryptosporidium in rivers of the world: the GloWPa-Crypto model
Vermeulen, Lucie C. - \ 2018
Wageningen University. Promotor(en): C. Kroeze; G.J. Medema, co-promotor(en): N. Hofstra. - Wageningen : Wageningen University - ISBN 9789463438209 - 236
Diarrhoeal disease is very common around the world. Knowing more about the global burden of diarrhoeal disease and about the geographical distribution of pathogen pollution is important for decision making and water and sanitation planning. The objective of this thesis is to increase knowledge on the sources, fate and transport of Cryptosporidium in rivers worldwide using spatially explicit modelling. I present the Global Waterborne Pathogen model for Cryptosporidium (GloWPa-Crypto), the first global model of waterborne pathogen emissions to and concentrations in rivers. The model is used to provide information on pathogen concentrations in data-sparse regions, identify hotspot regions, identify the relative contribution of different sources, and in scenario analysis to study the impacts of global change or management strategies. Furthermore, the model can be applied in the analysis of risk, burden of disease and health-based treatment targets, and make a valuable contribution in meeting the Sustainable Development Goals.
The art of being small : brain-body size scaling in minute parasitic wasps
Woude, Emma van der - \ 2017
Wageningen University. Promotor(en): M. Dicke, co-promotor(en): H.M. Smid. - Wageningen : Wageningen University - ISBN 9789463436564 - 231
brain - insects - neurons - scaling - cognitive development - vespidae - parasitoid wasps - cum laude - hersenen - insecten - neuronen - schaalverandering - cognitieve ontwikkeling - vespidae - sluipwespen
Haller’s rule states that small animals have relatively larger brains than large animals. This brain-body size relationship may enable small animals to maintain similar levels of brain performance as large animals. However, it also causes small animals to spend an exceptionally large proportion of energy on the development and maintenance of energetically expensive brain tissue. The work that is presented in this thesis reveals how the smallest animals face the challenge to maintain ecologically required levels of cognitive performance, while being limited by small numbers of neurons and a restricted energy balance. Developing into a small adult has cognitive costs for the parasitic wasp Nasonia vitripennis, and relative brain size is strongly constrained in this species. The extremely small parasitic wasp Trichogramma evanescens forms an exception to Haller’s rule by showing isometric brain-body size scaling. Miniaturized insect species may apply this strategy to avoid the excessive energetic costs of relatively large brains, thereby achieving smaller brain and body sizes than would be possible in the situation that is described by Haller’s rule. This brain-scaling strategy does not result in affected memory performance of small T. evanescens compared to larger individuals, and appears to be facilitated by a large flexibility in the size of neural components, rather than in their number or structural complexity. Maintaining neural complexity may the underlying mechanism that maintains the cognitive abilities of the smallest brains, possibly at the cost of reduced longevity as a consequence of the small size of neuronal cell bodies. This strategy could form the art of being small.
Rural livelihoods and agricultural commercialization in colonial Uganda: conjunctures of external influences and local realities
Haas, Michiel A. de - \ 2017
Wageningen University. Promotor(en): E.H.P. Frankema, co-promotor(en): N.B.J. Koning. - Wageningen : Wageningen University - ISBN 9789463436281 - 250
cum laude - livelihoods - livelihood strategies - communities - rural areas - farmers - history - colonies - colonialism - income - gender - social inequalities - food crops - cash crops - uganda - east africa - middelen van bestaan - strategieën voor levensonderhoud - gemeenschappen - platteland - boeren - geschiedenis - kolonies - kolonialisme - inkomen - geslacht (gender) - sociale ongelijkheden - voedselgewassen - marktgewassen - uganda - oost-afrika
The economic history of Sub-Saharan Africa is characterized by geographically and temporally dispersed booms and busts. The export-led ‘cash-crop revolution’ in parts of Sub-Saharan Africa during the colonial era is a key example of an economic boom. This thesis examines how external influences and local realities shaped the nature, extent and impact of the ‘cash-crop revolution’ in colonial Uganda, a landlocked country in central east Africa, where cotton and coffee production for global markets took off following completion of a railway to the coast. The thesis consists of five targeted ‘interventions’ into contemporary debates of comparative African development. Each of these five interventions is grounded in the understanding that the ability of rural Africans to respond to and benefit from trade integration during the colonial era was mediated by colonial policies, resource endowments and local institutions.
The first chapter reconstructs welfare development of Ugandan cash-crop farmers. Recent scholarship on historical welfare development in Sub-Saharan Africa has uncovered long-term trends in standards of living. How the majority of rural dwellers fared, however, remains largely elusive. This chapter presents a new approach to reconstructing rural living standards in a historical context, building upon the well-established real wage literature, but moving beyond it to capture rural realities, employing sub-national rural survey, census, and price data. The approach is applied to colonial and early post-colonial Uganda (1915–70), and yields a number of findings. While an expanding smallholder-based cash-crop sector established itself as the backbone of Uganda’s colonial economy, farm characteristics remained largely stagnant after the initial adoption of cash crops. Smallholders maintained living standards well above subsistence level, and while the profitability of cash crops was low, their cultivation provided a reliable source of cash income. At the same time, there were pronounced limits to rural welfare expansion. Around the time of decolonization, unskilled wages rose rapidly while farm incomes lagged behind. As a result, an urban–rural income reversal took place. The study also reveals considerable differences within Uganda, which were mediated to an important extent by differential resource endowments. Smallholders in Uganda’s banana regions required fewer labour inputs to maintain a farm income than their grain-farming counterparts, creating opportunities for additional income generation and livelihood diversification.
The second chapter zooms in on labour migration which connected Belgian-controlled Ruanda-Urundi to British-controlled Buganda, the central province of Uganda on the shores of Lake Victoria. The emergence of new labour mobility patterns was a key aspect of economic change in colonial Africa. Under conditions of land abundance and labour scarcity, the supply of wage labour required either the ‘pull’ forces of attractive working conditions and high wages, or the ‘push’ forces of taxation and other deliberate colonial interventions. Building upon primary sources, I show that this case diverges from the ‘conventional’ narrative of labour scarcity in colonial Africa. I argue that Ruanda-Urundi should be regarded as labour abundant and that migrants were not primarily ‘pushed’ by colonial labour policies, but rather by poverty and limited access to agricultural resources. This explains why they were willing to work for low wages in Buganda. I show that African rural employers were the primary beneficiaries of migrant labour, while colonial governments on both sides of the border were unable to control the course of the flow. As in the first chapter, this chapter highlights that the effects of trade integration on African rural development were uneven, and mediated by differences in resource endowments, local institutions and colonial policies.
The third chapter zooms out of the rural economy, evaluating the broader opportunity structures faced by African men and women in Uganda, and discussing the interaction of local institutions and colonial policies as drivers of uneven educational and occupational opportunities. The chapter engages with a recent article by Meier zu Selhausen and Weisdorf (2016) to show how selection biases in, and Eurocentric interpretations of, parish registers have provoked an overly optimistic account of European influences on the educational and occupational opportunities of African men and women. We confront their dataset, drawn from the marriage registers of the Anglican Cathedral in Kampala, with Uganda’s 1991 census, and show that trends in literacy and numeracy of men and women born in Kampala lagged half a century behind those who wedded in Namirembe Cathedral. We run a regression analysis showing that access to schooling during the colonial era was unequal along lines of gender and ethnicity. We foreground the role of Africans in the spread of education, argue that European influences were not just diffusive but also divisive, and that gender inequality was reconfigured rather than eliminated under colonial rule. This chapter also makes a methodological contribution. The renaissance of African economic history in the past decade has opened up new research avenues to study the long-term social and economic development of Africa. We show that a sensitive treatment of African realities in the evaluation of European colonial legacies, and a critical stance towards the use of new sources and approaches, is crucial.
The fourth chapter singles out the role of resource endowments in explaining Uganda’s ‘cotton revolution’ in a comparative African perspective. Why did some African smallholders adopt cash crops on a considerable scale, while most others were hesitant to do so? The chapter sets out to explore the importance of factor endowments in shaping the degrees to which cash crops were adopted in colonial tropical Africa. We conduct an in-depth case study of the ‘cotton revolution’ in colonial Uganda to put the factor endowments perspective to the test. Our empirical findings, based on an annual panel data analysis at the district-level from 1925 until 1960, underscore the importance of Uganda’s equatorial bimodal rainfall distribution as an enabling factor for its ‘cotton revolution’. Evidence is provided at a unique spatial micro-level, capitalizing on detailed household surveys from the same period. We demonstrate that previous explanations associating the variegated responses of African farmers to cash crops with, either the role of colonial coercion, or the distinction between ‘forest/banana’ and ‘savannah/grain’ zones, cannot explain the widespread adoption of cotton in Uganda. We argue, instead, that the key to the cotton revolution were Uganda’s two rainy seasons, which enabled farmers to grow cotton while simultaneously pursuing food security. Our study highlights the importance of food security and labour seasonality as important determinants of uneven agricultural commercialization in colonial tropical Africa.
The fifth and final chapter further investigates the experience of African smallholders with cotton cultivation, providing a comparative explanatory analysis of variegated cotton outcomes, focusing in particular on the role of colonial and post-colonial policies. The chapter challenges the widely accepted view that (i) African colonial cotton projects consistently failed, that (ii) this failure should be attributed to conditions particular to Africa, which made export cotton inherently unviable and unprofitable to farmers, and that (iii) the repression and resistance often associated with cotton, all resulted from the stubborn and overbearing insistence of colonial governments on the crop per se. I argue along three lines. Firstly, to show that cotton outcomes were diverse, I compare cases of cotton production in Sub-Saharan Africa across time and space. Secondly, to refute the idea that cotton was a priori unattractive, I argue that the crop had substantial potential to connect farmers to markets and contribute to poverty alleviation, particularly in vulnerable, marginal and landlocked areas. Thirdly, to illustrate how an interaction between local conditions and government policies created conducive conditions for cotton adoption, I zoom in on the few yet significant ‘cotton success stories’ in twentieth century Africa. Smallholders in colonial Uganda adopted cotton because of favourable ecological and marketing conditions, and policies had an auxiliary positive effect. Smallholders in post-colonial Francophone West Africa faced much more challenging local conditions, but benefitted from effective external intervention and coordinated policy. On a more general level, this chapter demonstrates that, from a perspective of rural development, colonial policies should not only be seen as overbearing and interventionist, but also as inadequate, failing to aid rural Africans to benefit from new opportunities created by trade integration.
Amphibious anthropology : engaging with maritime worlds in Indonesia
Pauwelussen, Annet P. - \ 2017
Wageningen University. Promotor(en): Leontine Visser, co-promotor(en): Gerard Verschoor. - Wageningen : Wageningen University - ISBN 9789463430654 - 208
This thesis explores how people live amphibiously in dynamic land-sea environments. It is based on eighteen months of ethnographic fieldwork (2011 – 2013) in the Makassar Strait maritime region in Indonesia: a complex and amphibious land-sea interface. In Western science little attention is given to how people live at sea. There is a general land bias, by which people are seen as primarily belonging to the land. Yet people do live at sea, or rather: in these dynamic land-sea environments. Engaging with these mobile and sea-based ways of life of maritime people provides not only a fuller understanding of how people relate to their environment, but , essentially, it also enables a critical reflection of land-biased assumptions in science and society.
Anthropology, with its qualitative research methods is particularly suitable to do such in-depth and long-term engagement with other worlds. The research on which the thesis is based was carried out as a mobile ethnography, following people seawards, travelling with them for days to visit close family on faraway islands or joining them on their fishing and diving trips, including illegal fishers using bombs and cyanide poison on coral reefs. Also followed were the practices of marine conservation staff, as they organised field trips to fishing communities. These travels – described in the thesis – show how islands and marine spaces that are remote from the land, turn out to be regional hubs of oversees trade and family relations. From a sea-based perspective the Makassar Strait is a continuity of relations and movements flanked by land masses. This inverts the land-based perspective of the sea as an extension of the land by putting the sea centre stage.
The Makassar Strait figures in this thesis as an active and moving world – or worlds – of human-marine relations to learn from and theorise about the notion of ontological flow: fluidity of being and moving in relation. Flow is both movement as a pattern of activity – the flowing – and that what flows; elements, matter and meaning in motion. The notion of worlds in flow has infused recent ontological debates in anthropological theory in which reality is assumed contingent, fluid and multiple – thereby revitalising the philosophical work of earlier thinkers, among whom Michel Serres and Gilles Deleuze. This way of thinking complexity and ontological fluidity is central to literature that has emerged out of the cross-fertilisation of Science and Technology Studies (STS), anthropology and philosophy. Despite differences, these studies share the objective to follow, engage with and translate how, in practice, material and semiotic realities come to be and matter – instead of developing a way to ‘access reality better’.
The concept of ‘amphibiousness’ is mobilised to refer to living in and moving between different worlds that can intermingle but that cannot be reduced to each other. The concept is used to describe the human capacity to live in different worlds at the same time. This amphibious capacity is further elaborated 1) in terms of living in a hybrid land-water interface, 2) in terms of being able to move along with different understandings of the world, of reality, and 3) It refers to the methodology of the anthropologist who also needs to move in these worlds bodily and cognitively, to develop a sensitivity to and understanding of these different worlds. Amphibiousness captures the anthropological engagement with flow, multiplicity and otherness by way of moving between worlds in order to explore the moving interface between worlds, realities or ways of life that partly interact. The research question: How to grasp flow – the fluctuations of and between bodies, things or worlds in the making - conceptually and methodologically without reducing its vital mobility and fluidity? is elaborated in a methodological Chapter 2, and three research chapters (Chapter 3, Chapter 4, and Chapter 5) that each focus from a different angle on human-marine relations.
The research exposes fundamentally different, and sometimes conflicting, ways in which people understand and experience their relation to the sea. These were not just different perspectives on one maritime reality, or world. These were inherently different understandings of reality, and different ways in which this reality is put into practice, in which worlds – plural – are being created and sustained. In anthropology we speak of ontological difference, because it concerns with (radically) different notions of wat exists, what is real, what matters and what entities participate in the reproduction of the world.
Although these worlds are different – they cannot be reduced to each another - they are also not separated in any clear-cut way. They do flow into each other, as people, objects and ideas can amphibiously move in between. It is argued in this thesis that such amphibious translation is essential for more effective and equal collaboration in marine conservation. International environmental organisations insufficiently acknowledge (radically) different ways of doing and thinking human-marine relations. Disregarding these undermines the viability of conservation programs as it repeatedly leads to clashes between different ways in which maritime worlds are understood and organised in practice. To be effective, marine conservation needs to become amphibious; attentive to fundamentally different ways of understanding and experiencing the relationship between people and the sea, as well as the mobile practices of trade, fishing, travel and family affiliations through which these worlds are shaped beyond the borders of marine reserves.
Chapter 2 intends to answer the question how to grasp environmental otherness – radically different ways of understanding and experiencing human-marine relations – in and through ethnography. Chapter 3 serves to provide some empirical grounding to show the relevancy and urgency of a paradigmatic shift in conservation thinking, finding ways to engaging mobile maritime people like the Bajau. The solution to the ‘participation problem’ in conservation will not lie in developing ways to make local people participate more in Western conservation schemes. What is needed is an ontological shift in conservation thinking itself. Chapter 4 describes a conservation outreach project that attempts to educate and convert local people into coral protectors. Both coral and the sea-dwelling Bajau people appear to be amphibious beings, moving between a changeable land-water interface, and between different, fluidly interwoven ontological constellations. Failure of conservation organisations to recognise the ontologically ambiguous nature of ‘coral’ and ‘people’ translates to a breakdown of outreach goals. Chapter 5 provides a case study of a dangerous and destructive fishing practice (cyanide fishing) by which fishers dive beyond the limits of what their body can take – and spirits allow, a practice that generates feeling of both fear and enjoyment as they experience a process of becoming permeable to fluids, spirits and currents penetrating or leaking out of their bodies. This chapter exposes how cyanide fishing sustains as a way of life, involving and producing affective relations.
In Chapter 6 it is concluded how ontological multiplicity is of a heuristic and political relevance to social science, and anthropology in particular because it allows us to engage with radical difference – or the real on different terms – instead of explaining it away in our own terms. Engaging with such radical different is important because it allows to see the realities that systematically escape (scholarly) attention, yet affect the world nonetheless. This requires translation – the practice of relating different worlds, reals, repertoires or ways of life and bringing them into interaction – which is a process of, and a condition for, dialogue. The notion of amphibiousness has practical and political value, in particular for reconsidering conservation and development outreach and how it may be reframed as a process involving ontological dialogue. Providing room for ambiguity, thinking with amphibiousness furthermore encourages suspension of the (Western) tendency to explain the Other, to fix what does not add up.
River export of nutrients to the coastal waters of China: the MARINA model to assess sources, effects and solutions
Strokal, Maryna - \ 2016
Wageningen University. Promotor(en): Carolien Kroeze, co-promotor(en): S. Luan; Lin Ma. - Wageningen : Wageningen University - ISBN 9789462579729 - 226
cum laude - nutrients - rivers - coastal water - models - eutrophication - coastal areas - water pollution - china - voedingsstoffen - rivieren - kustwateren - modellen - eutrofiëring - kustgebieden - waterverontreiniging - china
Rivers export increasing amounts of nitrogen (N) and phosphorus (P) to the coastal waters of China. This causes eutrophication problems that can damage living organisms when oxygen levels drop and threaten human health through toxic algae. We know that these problems result from human activities on land such as agriculture and urbanization. However, the relative importance of these human activities for river export of nutrients to Chinese seas is not well studied. There are two important issues that need further investigation: the relative importance of upstream pollution on downstream impacts and the relative importance of typical sources of nutrients in Chinese rivers that are often ignored in existing modeling studies.
My PhD thesis, therefore, aims to better understand trends in river export of nutrients to the coastal waters of China by source from sub-basins, and the associated coastal eutrophication. To this end, I developed the MARINA model: Model to Assess River Inputs of Nutrients to seAs. For this, I used the existing Global NEWS-2 model (Nutrient Export from WaterSheds) as a starting point.
I formulated five sub-objectives to achieve the main objective:
To analyze the original Global NEWS-2 model for river export of nutrients and the associated coastal eutrophication (Chapter 2);
To develop a sub-basin scale modeling approach to account for impacts of upstream human activities on downstream water pollution, taking the Pearl River as an example (Chapter 3);
To quantify the relative share of manure point sources to nutrient inputs to rivers at the sub-basin scale (Chapter 4);
To quantify the relative share of sources to river export of nutrients at the sub-basin scale (Chapter 5);
To explore optimistic futures to reduce river export of nutrients and coastal eutrophication in China (Chapter 6).
The study area includes rivers draining roughly 40% of China. This includes the most densely populated areas, and areas with intensive economic activities. The rivers include the Yangtze (Changjiang), Yellow (Huanghe), Pearl, Huai, Hai and Liao. In the MARINA model, the drainage areas of the large Yangtze, Yellow and Pearl rivers are divided into up-, middle- and downstream sub-basins. The principle of the sub-basin approach of MARINA is that nutrients from human activities are transported by tributaries to outlets of sub-basins and then to the river mouth (coastal waters) through the main channel. The model takes into account nutrients that are partly lost or retained during transport towards the river mouth. The model quantifies river export of nutrients by source from sub-basins for 1970, 2000 and 2050.
The main six findings of the MARINA results for China are:
Finding 1: Dissolved N and P export by Chinese rivers increased by a factor of 2-8 between 1970 and 2000;
Finding 2: The potential for coastal eutrophication was low in 1970 and high in 2000 in China;
Finding 3: Most dissolved N and P in Chinese seas is from middlestream and downstream human activities;
Finding 4: Manure point sources are responsible for 20-80% of dissolved N and P in Chinese rivers;
Finding 5: In the future, river export of nutrients may increase in the Global Orchestration (GO) scenario of the Millennium Ecosystem Assessment. Current policy plans (CP scenario) may not sufficient to avoid this increase;
Finding 6: In optimistic scenarios (OPT-1 and OPT-2), the potential for coastal eutrophication is low in 2050, mainly as a result of assumed full implementation of: (1) high recycling rates of animal manure (OPT-1 and OPT-2), and (2) high efficiencies of nutrient removal in sewage systems (OPT-2, see Figure 1).
Figure 1. Illustration of future scenarios for coastal water quality in China. GO is Global Orchestration of the Millennium Ecosystem Assessment and assumes environmental actions that are either absent or ineffective in reducing water pollution. CP is based on GO, but incorporates the “Zero Growth in Synthetic Fertilizers after 2020” policy. OPT-1 and OP-2 are optimistic scenarios that assume high nutrient use efficiencies in agriculture (OPT-1, OPT-2) and sewage (OPT-2).
My PhD thesis reveals novel insights for effective environmental policies in China. It shows the importance of manure point sources in water pollution by nutrients. Clearly, managing this source will likely reduce coastal eutrophication in the future. Furthermore, the implementation of advanced technologies is essential when dealing with urban pollution. My PhD thesis may also be useful for other world regions with similar environmental problems as in China. The new, sub-basin scale MARINA model is rather transparent and thus can be applied to other large, data-poor basins that may benefit from the allocation of effective management options. With this I hope to contribute to future availability of sufficiently clean water for next generations, not only in China, but also in other world regions.