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The food systems in developed societies face a host of challenges in the early 21st century; increasing urbanisation, supply chain shocks due to extreme climate events and pandemics, high impacts of air freight, nutrient run-off and animal farming, and tremendous amounts of wasted food and nutrients. A transformation to a more sustainable and resilient food system includes, among other pathways, increased consumption of fruits and vegetables, a localisation of food production and the recovery of nutrients in urban organic waste. Urban agriculture thus receives emerging research interest, ranging from community gardens to high-tech indoor farming. Efforts so far have focused on socio-economic aspects and life-cycle assessments of individual case studies or have described the qualitative potential of integrating urban waste streams into urban farming. Significant gaps remain in the literature about the carbon and land intensity of different growing methods when scaled-up and the quantification of productivity and nutrient recovery potentials. On the city and regional level, the design of productive food systems integrated with biowaste management and renewable energy systems has found little attention so far. These gaps are addressed in this thesis through mathematical modelling and geospatial analysis at multiple scales, including the facility, the urban area, a metropolitan area, and the regional level. The match of supply and demand of a scaled-up urban food production system and their organic waste flow utilisation potential was assessed and compared for two urban areas, Glasgow and Lyon. Potentially convertible land and rooftop space were determined with geospatial processing, and monthly biowaste utilisation in those new farms was optimised based on nutrient requirements and CO2 emission savings. The results showed that high self-sufficiency came with significantly increased CO2 emissions for the productive urban food system in the colder climate due to high demand and carbon intensity of electricity for greenhouses, which also negated the benefits of utilising a large share of the biowaste. The next project thus investigated the interplay and logistics of local composting treatment combined with low-input local farming for 76 districts in Porto from waste system perspective. Existing fields, convertible greenspaces, and compost island requirements were obtained from geospatial analysis and waste collection pilots and distance scripts informed collection and transport requirements to central facilities. The results showed that more remote districts could benefit both in cost and climate impacts from having their own decentralised biowaste systems, although mainly from collection route reduction rather than a conversion of greenspaces. Focusing more on the contextual sustainability of high-intensive growing methods, the energy consumption of electrified greenhouses and vertical farm facilities was assessed for ten regions across the globe. A rigorous thermodynamic model for lighting and air conditioning requirements was optimised for minimum specific energy in relation to the outside climate on an hourly basis. The outcomes showed that significant energy reductions can be achieved in greenhouses with optimised climate control, while local climate conditions and design choices still have a strong impact. Vertical farms, or plant factories, had significantly higher energy consumptions, even in extreme climates. The final project applied the learnings from the energy optimisation to a land-use based assessment of regionalised vegetable supply with renewable energy generation for nine city-regions. Potentially available land for energy generation was assessed, and the selection and sizing of solar PV and wind farms were economically optimised for the required electrical load. The results show that although greenhouse production requires oversized renewable energy systems due to higher load fluctuations, its land footprint is lower than that of open-field and vertical farming. The land use relative to existing and available land contextualised this finding, but a full regionalised vegetable supply was feasible and desirable for most of the regions. The implications from the work are that to achieve increased food self-sufficiency and climate change mitigation, energy-efficient and low-carbon urban agriculture approaches are indispensable. Recommendations for specific urban growing systems are strongly context-dependent and many transferable aspects of relevance to decision-makers have been quantified in this work. Further, the integration of biowaste mainly provides benefits from a holistic waste management perspective and thus a multi-stakeholder approach and commitments will always be required.

Oncolytic viruses (including measles virus) offer an alternative approach to reduce the high mortality rate of late-stage cancer. Several measles virus strains infect and lyse cancer cells efficiently, but the broad application of this therapeutic concept is hindered by the large number of infectious particles required (108-1012 TCID50 per dose). The manufacturing process must, therefore, achieve high titers of oncolytic measles virus (OMV) during upstream production and ensure that the virus product is not damaged during purification by applying appropriate downstream processing (DSP) unit operations. DSP is currently a production bottleneck because there are no specific platforms for OMV. Infectious OMV must be recovered as intact, enveloped particles, and host cell proteins and DNA must be reduced to acceptable levels to meet regulatory guidelines that were developed for virus-based vaccines and gene therapy vectors. Handling such high viral titers and process volumes is technologically challenging and expensive. This review considers the state of the art in OMV purification and looks at promising DSP technologies. We discuss here the purification of other enveloped viruses where such technologies could also be applied to OMV. The development of DSP technologies tailored for enveloped viruses is necessary to produce sufficient titers for virotherapy, which could offer hope to millions of patients suffering from incurable cancer.

High-resolution structural information on membrane proteins is essential for understanding cell biology and for structure-based design of new medical drugs and drug delivery strategies. X-ray diffraction (XRD) can provide {\AA}ngstrom-level information about the structure of membrane proteins. Ideally protein structures should be solved in environments as close to the original biological context as possible. However, it is virtually impossible to crystallize proteins within the complex environment of a biological membrane. Instead, membrane proteins are typically transferred from their native membrane environment into detergent micelles, chemically stabilized and crystallized, all of which can compromise the conformation. This makes it imperative to develop alternative high-resolution techniques which are compatible with biological conditions. Here, we describe how a combina-tion of surface-sensitive vibrational spectroscopy in model membranes and molecular dynamics simulations can account for the native membrane environment. We observe the structure of glycerol facilitator channel (GlpF), an aquaporin membrane channel finely tuned to selectively transport water and glycerol molecules across the membrane barrier. We find subtle but significant differences between the XRD structure and the in-ferred in situ structure of GlpF., Comment: 18 pages, 8 figures

The optimization of recombinant protein production in bacteria is an important stage of process development, especially for difficult-to-express proteins that are particularly sensitive or recalcitrant. The optimal expression level must be neither too low, which would limit yields, nor too high, which would promote the formation of insoluble inclusion bodies. Expression can be optimized by testing different combinations of elements such as ribosome binding sites and N-terminal affinity tags, but the rate of protein synthesis is strongly dependent on mRNA secondary structures so the combined effects of these elements must be taken into account. This substantially increases the complexity of high-throughput expression screening. To address this limitation, we generated libraries of constructs systematically combining different ribosome binding sites, N-terminal affinity tags, and periplasmic translocation sequences representing two secretion pathways. Each construct also contained a green fluorescent protein (GFP) tag to allow the identification of high producers and a thrombin cleavage site enabling the removal of fusion tags. To achieve proof of principle, we generated libraries of 200 different combinations of elements for the expression of an antimicrobial peptide (AMPs), an antifungal peptide, and the enzyme urate oxidase (uricase) in Escherichia coli and Vibrio natriegens. High producers for all three difficult-to-express products were enriched by fluorescence-activated cell sorting. Our results indicated that the E. coli ssYahJ secretion signal is recognized in V. natriegens and efficiently mediates translocation to the periplasm. Our combinatorial library approach therefore allows the cross-species direct selection of high-producer clones for difficult-to-express proteins by systematically evaluating the combined impact of multiple construct elements.

The therapeutic use of oncolytic measles virus (MV) for cancer treatment requires >108 infectious MV particles per dose in a highly pure form. The concentration/purification of viruses is typically achieved by tangential flow filtration (TFF) but the efficiency of this process for the preparation of MV has not been tested in detail. We therefore investigated the influence of membrane material, feed composition, and pore size or molecular weight cut-off (MWCO) on the recovery of MV by TFF in concentration mode. We achieved the recovery of infectious MV particles using membranes with a MWCO < 300 kDa regardless of the membrane material and whether or not serum was present in the feed. However, serum proteins in the medium affected membrane flux and promoted fouling. The severity of fouling was dependent on the membrane material, with the cellulose-based membrane showing the lowest susceptibility. We found that impurities such as proteins and host cell DNA were best depleted using membranes with a MWCO > 300 kDa. We conclude that TFF in concentration mode is a robust unit operation to concentrate infectious MV particles while depleting impurities such as non-infectious MV particles, proteins, and host cell DNA.

Oncolytic viruses offer new hope to millions of patients with incurable cancer. One promising class of oncolytic viruses is Measles virus, but its broad administration to cancer patients is currently hampered by the inability to produce the large amounts of virus needed for treatment (1010-1012 virus particles per dose). Measles virus is unstable, leading to very low virus titers during production. The time of infection and time of harvest are therefore critical parameters in a Measles virus production process, and their optimization requires an accurate online monitoring system. We integrated a probe based on dielectric spectroscopy (DS) into a stirred tank reactor to characterize the Measles virus production process in adherent growing Vero cells. We found that DS could be used to monitor cell adhesion on the microcarrier and that the optimal virus harvest time correlated with the global maximum permittivity signal. In 16 independent bioreactor runs, the maximum Measles virus titer was achieved approximately 40 hr after the permittivity maximum. Compared to an uncontrolled Measles virus production process, the integration of DS increased the maximum virus concentration by more than three orders of magnitude. This was sufficient to achieve an active Measles virus concentration of > 1010 TCID50 ml−1.

Measles virus (MV) has a natural affinity for cancer cells and oncolytic MV preparations have therefore been investigated in several clinical trials as a potential treatment for cancer. The main bottleneck in the administration of oncolytic MV to cancer patients is the production process, because very large doses of virus particles are required for each treatment. Here, we investigated the productivity of different host cells and found that a high infection efficiency did not necessarily result in high virus yields because virus release is also dependent on the host cell. As well as producing large numbers of active MV particles, host cells must perform well in dynamic cultivation systems. In screening experiments, the highest productivity was achieved by Vero and BJAB cells, but only the Vero cells maintained their high virus productivity when transferred to a stirred tank reactor. We used dielectric spectroscopy as an online monitoring system to control the infection and harvest times, which are known to be critical process parameters. The precise control of these parameters allowed us to achieve higher virus titers with Vero cells in a stirred tank reactor than in a static cultivation system based on T‐flasks, with maximum titers of up to 1011 TCID50 ml−1.

Insect-based expression platforms such as the baculovirus expression vector system (BEVS) are widely used for the laboratory- and industrial-scale production of recombinant proteins. Thereby, major drawbacks to gain high-quality proteins are the lytic infection cycle and the shear sensitivity of infected insect cells due to turbulence and aeration. Smaller bubbles were formerly assumed to be more harmful than larger ones, but we found that cell damage is also dependent on the concentration of protective agents such as Pluronic®. At the appropriate concentration, Pluronic forms a layer around air bubbles and hinders the attachment of cells, thus limiting the damage. In this context, we used microaeration to vary bubble sizes and confirmed that size is not the most important factor, but the total gas surface area in the reactor is. If the surface area exceeds a certain threshold, the concentration of Pluronic is no longer sufficient for cell protection. To investigate the significance of shear forces, a second study was carried out in which infected insect cells were cultivated in a hollow fiber module to protect them from shear forces. Both model studies revealed important aspects of the design and scale-up of BEVS processes for the production of recombinant proteins

The knowledge about the oncolytic activity of viruses like measles opens up new possibilities in the effective treatment of incurable cancer. However, the successful therapy, requires high doses of oncolytic virus. In clinical trials the effective treatment dose was estimated with over 1011 TCID50 (50% tissue culture infectious dose) [Russel et al.]. Imagining the demand for cancer patients, currently used virus production systems like T-flasks or cell factories cannot provide for the required high virus doses. However, besides the virus amount, also the stability has to be considered, as only active virus particles are suitable for cancer treatment. The measles virus is very sensitive towards temperature at cultivation conditions (half life approx. 1 h at 37 °C). Regarding the batch production process in cell factories, the virus particle can be harvest only once and, as the virus is released over a length of time, inactivation of virus particles will occur. To ensure future therapy, this calls for an innovative bioprocess development that includes the integration of both efficient upstream and downstream processes with a suitable in-process control system. Present study shows a new bioprocess concept for production and purification of oncolytic measles viruses for the use in cancer therapy. This approach not only increases virus yield by integration of a two-step virus purification system and online sensors, it also leads to decreased product contamination by e.g. host cell proteins and free DNA.

It is shown that the self-assembly of diamagnetic molecule submonolayers on a surface can be influenced by magnetic stray field landscapes emerging from artificially fabricated magnetic domains and domain walls. The directed local chemisorption of diamagnetic subphthalocyaninatoboron molecules in relation to the artificially created domain pattern is proved by a combination of surface analytical methods: ToF-SIMS, X-PEEM, and NEXAFS imaging. Thereby, a new method to influence self-assembly processes and to produce patterned submonolayers is presented.

Large-scale human mesenchymal stem cell expansion calls for a bioreaction system, that provides a sufficient growth surface. An alternative to static cultivations systems like cell factories are disposable stirred tank reactors. Here, microcarriers provide the required growth surface, but these make it difficult to achieve a complete homogenization in the bioreactor, while avoiding shear stress. To gain insight into this process, we investigated the impact of different power inputs (0.02-2.6 W m−3) on the mixing time (tm). Whereas tm was inversely proportional to agitation in a one-phase-system, aeration resulted in a constant mixing time at 30-70 rpm. A high microcarrier concentration (30 g L−1) and low stirrer speed (30 rpm) in the liquid-solid system caused a 50-fold increase in tm and the formation of a discrete non-mixed upper zone. The effect of the microcarrier concentration on tm became negligible at higher stirrer speeds. In the three-phase system, microcarrier settling was prevented by aeration and a minimal specific power input of 0.6 W m−3 was sufficient for complete homogenization. We confirmed that a low power input during stem cell expansion leads to inhomogeneity, which has not been investigated in the three-phase system up to date.

To further improve the coverage of organic monolayers on hydrogen-terminated silicon (H–Si) surfaces with respect to the hitherto best agents (1-alkynes), it was hypothesized that enynes (H–C=C–HC-CH–R) would be even better reagents for dense monolayer formation. To investigate whether the increased delocalization of ß-carbon radicals by the enyne functionality indeed lowers the activation barrier, the kinetics of monolayer formation by hexadec-3-en-1-yne and 1-hexadecyne on H–Si(111) were followed by studying partially incomplete monolayers. Ellipsometry and static contact angle measurements indeed showed a faster increase of layer thickness and hydrophobicity for the hexadec-3-en-1-yne-derived monolayers. This more rapid monolayer formation was supported by IRRAS and XPS measurements that for the enyne show a faster increase of the CH2 stretching bands and the amount of carbon at the surface (C/Si ratio), respectively. Monolayer formation at room temperature yielded plateau values for hexadec-3-en-1-yne and 1-hexadecyne after 8 and 16 h, respectively. Additional experiments were performed for 16 h at 80° to ensure full completion of the layers, which allows comparison of the quality of both layers. Ellipsometry thicknesses (2.0 nm) and contact angles (111–112°) indicated a high quality of both layers. XPS, in combination with DFT calculations, revealed terminal attachment of hexadec-3-en-1-yne to the H–Si surface, leading to dienyl monolayers. Moreover, analysis of the Si2p region showed no surface oxidation. Quantitative XPS measurements, obtained via rotating Si samples, showed a higher surface coverage for C16 dienyl layers than for C16 alkenyl layers (63% vs 59%). The dense packing of the layers was confirmed by IRRAS and NEXAFS results. Molecular mechanics simulations were undertaken to understand the differences in reactivity and surface coverage. Alkenyl layers show more favorable packing energies for surface coverages up to 50–55%. At higher coverages, this packing energy rises quickly, and there the dienyl packing becomes more favorable. When the binding energies are included the difference becomes more pronounced, and dense packing of dienyl layers becomes more favorable by 2–3 kcal/mol. These combined data show that enynes provide the highest-quality organic monolayers reported on H–Si up to now.

The effect of low‐energy (50 eV) electron irradiation on self‐assembled monolayers (SAMs) of aromatic selenolates on polycrystalline Au{111} was studied by synchrotron‐based X‐ray photoelectron spectroscopy and near‐edge X‐ray absorption fine structure spectroscopy. As a test system, SAMs of biphenylselenolate (BPSe) were used, and analogous biphenylthiolate (BPT) SAMs were taken as a reference. The BPSe films were found to exhibit the expected high stability of aromatic SAMs toward electron irradiation, primarily mediated by the dominance of cross‐linking between the individual molecular species. The cross‐sections of the most prominent irradiation‐induced processes in the BPSe SAMs were obtained and found to be in a range of 0.07–0.2 × 10–16 cm2. Comparison of the BPSe and BPT SAMs showed a higher stability of the former system toward electron irradiation. This phenomenon was explained by a combined effect of a higher degree of crystallinity of the BPSe SAMs and a higher strength of the selenolate‐gold as compared to thiolate‐gold bond. The latter aspect is an important result of the present study, which may help to rationalize the current controversy regarding the relative strength of these two anchor groups. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)