Advances in protein topology mapping methods are urgently needed to complement the wealth of interactome data that is presently being generated at a rapid pace. Chemical crosslinking followed by mass spectrometry (MS) has evolved over the last decade as an attractive method for protein topology and interface mapping, and holds great promise as a counterpart to modern interactome studies in the field of proteomics. Furthermore, stabilization of proteins and protein complexes with crosslinking offers many advantages over high-resolution structural mapping methods, including the ability to study protein topologies in vivo. The reliance on direct detection of crosslinked peptides, however, continues to pose challenges to protein topology and interface mapping with chemical crosslinking plus MS. The present body of work aimed to develop a novel generic methodology that utilizes chemical crosslinking, cyanogen bromide (CNBr) cleavage and MS for the low-resolution mapping of protein topologies and interfaces. Through such low-resolution mapping of crosslinked regions, this novel strategy overcomes limitations associated with the direct detection of crosslinked peptides. Following optimization of various steps, the present method was validated with the bacterial DNA-directed RNA polymerase core complex and was subsequently applied to probe the tetrameric assembly of yeast Skp1p-Cdc4p heterodimers. Further improvements were made through the enrichment of crosslinked CNBr-cleaved protein fragments prior to their identification via MS. Two enrichment strategies were developed which depended upon the conjugation of tags to CNBr-cleaved peptide C-termini followed by either tandem affinity purification or tandem reversed-phase HPLC purification. These strategies were successfully applied for the efficient purification of disulfide-linked peptides from peptide mixtures. It is expected that the potential to achieve sensitive mapping of topologies and interfaces of multi-subunit protein complexes in vivo, in combination with further enhancements to permit studies on complex protein samples, will extend the utility of this method to complement large-scale interactome studies.

In the development of turbo machinery components it's desirable to not spend more time than necessary when setting up aero-thermal calculations to investigate uncertainties in the design. This report aims to describe general thoughts used in the development of an ICEM-mesh script and the possible configurations in the script file which enables the user to build mesh-grids with/without clearance gap at the hub and/or shroud for different blade geometries. It also aims to illustrate the performance analysis made on the Vinci LH2 turbine, a next generation upper stage engine to the Ariane 5 rocket, in which the effect of the tip gap size on the efficiency has been studied. The calculations made have shown good agreement with experimental data. The efficiency loss due to the mixing of fluid where leakage flow passes the tip gap, which results in growth of a strong vortex, and the fluid passing the blade tip, with almost no work extracted from it, has shown a quite linear efficiency dependence depending on the tip gap size.

Per- and polyfluoroalkyl substances are a large group of global environmental contaminants. They can be divided into two sub-groups, 1) perfluoroalkyl acids (PFAAs) and 2) so called precursors, i.e. compounds that can potentially be transformed to form PFAAs. PFAAs are today ubiquitous in wildlife and humans. Food and drinking water are assumed to be the dominant human exposure pathways for PFAAs. The main aim of this doctoral thesis was to develop highly sensitive and fully validated analytical methods for the determination of a range of PFAAs and selected precursors in dietary samples. The methods were based on liquid chromatography coupled to mass spectrometry. Samples were extracted by solvent extraction followed by a cleanup step employing solid phase extraction. The cleanup step could at the same time be used as a fractionation of ionic PFAAs and neutral precursors. Paper I and II describe the development of methods for simultaneous analysis of three groups of PFAAs including perfluoroalkyl phosphonic acids (PFPAs) in drinking water and food. Methyl piperidine was used as ion pairing agent, leading to highly sensitive analysis of PFPAs. A first screening of tap water samples and different food items revealed that human dietary exposure to PFPAs in Europe is currently not of concern. A novel method for simultaneous analysis of perfluoroalkyl carboxylic acids (PFCAs) and polyfluoroalkyl phosphate esters (PAPs) in food and packaging materials is described in paper III. Targeted food samples and their packaging were analyzed. The results showed that PAPs may contribute to human exposure to PFCAs. In paper IV temporal trends (1991-2011) of perfluorooctane sulfonic acid (PFOS) and its precursors in herring were investigated. Rapidly decreasing trends were found for precursors, whereas PFOS did not show a significant change over time. Precursors in fish may have played an important role for human exposure to PFOS in the 1990s but are probably negligible today. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p> / PERFOOD project (KBBE-227525)

Thesis (MSc Food Sc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Rooibos tea, produced from the endemic South African fynbos plant Aspalathus linearis, has various healthpromoting benefits which can largely be attributed to its phenolic composition. In this study, the reversedphase liquid chromatographic (RP-LC) separation of the principal phenolic constituents of aqueous rooibos infusions was optimised on conventional high-performance liquid chromatography (HPLC) instrumentation. The phenolic constituents comprised dihydrochalcones (aspalathin and nothofagin), flavones (orientin, isorientin, vitexin, isovitexin, luteolin, luteolin-7-O-glucoside and chrysoeriol), flavonols (quercetin, isoquercitrin, hyperoside and rutin), a hydroxycinnamic acid (ferulic acid) and a phenylpropanoid (phenylpyruvic acid glucoside, PPAG). A systematic approach towards method development was adopted: resolution was optimised by simultaneous optimisation of gradient conditions and temperature on a sub-2 6m phase to exploit the benefits of this phase for fast routine analyses. The developed HPLC method, utilising photodiode-array (PDA) detection, yielded complete separation of the 15 target analytes on the 1.8 µm C18 column, thermostatted to 37°C, within 37 min (total analysis time: 50 min). The method was successfully validated and demonstrated its suitability for the fast, quantitative analysis of aqueous infusions of unfermented and fermented rooibos. Mass spectrometric (MS) and tandem MS detection confirmed peak purity and permitted the tentative identification of 13 additional phenolic compounds, including a flavonol Odiglycoside (quercetin-3-O-robinobioside), a luteolin-6-C-pentoside-8-C-hexoside and a novel C-8-hexosyl derivative of aspalathin reported here for the first time. The HPLC-PDA method was subsequently applied to a large number of fermented rooibos samples representative of different production seasons (2009, 2010 and 2011) and quality grades (grades A, B, C and D) to capture as much potential variation in the phenolic composition as possible. Production season had no clear effect on the levels of the individual phenolic compounds in ‘cup-of-tea’ rooibos infusions, whilst high quality tea (grades A and B) was associated with higher levels of phenolic compounds and soluble solids than low quality tea (grades C and D). Steam-pasteurisation of the plant material, required to obtain a product of high microbiological quality, induced significant reductions in the mean values of most of the phenolic compounds in rooibos infusions. The major phenolic constituents of steam-pasteurised, fermented rooibos were isoorientin and orientin, whilst quercetin-3-O-robinobioside, PPAG and aspalathin were also present in high concentrations. Representative content values of the major phenolic compounds present in a typical ‘cup-of-tea’ rooibos infusion were thus obtained and the generated data are suitable for inclusion in food composition databases. The application of comprehensive two-dimensional liquid chromatography (LCxLC) was investigated as an alternative approach for the detailed investigation of rooibos phenolics. The combination of hydrophilic interaction chromatography (HILIC) in the first dimension and RP-LC in the second dimension offered different separation selectivities and hence a high degree of orthogonality. HILICxRP-LC provided a significant improvement in resolution, as is evident from practical peak capacities in excess of 2000 and 800 for the off-line and on-line methods, respectively. Further optimisation, particularly of the first dimension separation, is however required to improve the LCxLC separation of complex rooibos phenolic fractions. / AFRIKAANSE OPSOMMING: Gebruik van rooibostee, berei vanaf die eg Suid-Afrikaanse fynbosplant Aspalathus linearis, hou verskeie gesondheidsvoordele in wat grootliks toegeskryf kan word aan sy fenoliese samestelling. Die skeiding van die hoof fenoliese verbindings van ’n koppie rooibos is in hierdie studie deur middel van omgekeerde-fase vloeistof chromatografie (RP-LC) op konvensionele hoë-druk vloeistof chromatografiese (HPLC) toerusting geoptimiseer. Die fenoliese verbindings was verteenwoordigend van dihidrogalkone (aspalatien en notofagien), flavone (orientien, isoorientien, viteksien, isoviteksien, luteolien, luteolien-7-O-glukosied en krisoeriol), flavonole (kwersetien, isokwersetrien, hiperosied en rutien), ‘n hidroksiekaneulsuur (ferulasuur) en ‘n fenielpropanoied (fenielpirodruiwesuurglukosied, PPAG). Die ontwikkeling van die metode was sistematies benader: resolusie is op ‘n geselekteerde 1.8 6m stationêre fase met welombekende kinetiese voordele geoptimiseer deur die gradiëntkondisies en kolomtemperatuur gelyktydig te optimiseer. Die ontwikkelde HPLC metode, gekoppel aan ultraviolet-fotodiode deteksie (PDA), het binne 37 min (totale analiese tyd: 50 min) volledige skeiding van die 15 standaard verbindings op die 1.8 µm C18 kolom teen 37°C bewerkstellig. Die metode is suksesvol gevalideer en het sy toepaslikheid vir vinnige, kwantitatiewe analiese van ongefermenteerde en gefermenteerde rooibos gedemonstreer. Piek suiwerheid is deur middel van massa spektrometrie (MS) en tandem MS bevestig, wat ook die identifikasie van 13 addisionele verbindings toegelaat het, insluitende ‘n flavonol O-diglukosied (kwersetien-3-O-robinobiosied), ‘n luteolien-6-Cpentosied- 8-C-heksosied en ‘n unieke C-8-heksosiel afgeleide van aspalatien wat vir die eerste keer hier gemeld is. Die geoptimiseerde HPLC-PDA metode is gevolglik toegepas vir die analiese van ‘n groot aantal gefermenteerde rooibos monsters, verteenwoordigend van verskillende produksie seisoene (2009, 2010 en 2011) en kwaliteitsgrade (A, B, C en D). Hierdie lukraak-geselekteerde monsters het soveel as moontlik potensiële variasie in die fenoliese samestelling verseker. Produksie seisoen het geen definitiewe effek op die vlakke van die individuele fenoliese verbindings in ’n koppie rooibos gehad nie, terwyl hoë kwaliteit rooibos (grade A en B) geassosieër was met hoër vlakke van die individuele fenoliese verbindings en oplosbare vastestowwe in vergelyking met lae kwaliteit rooibos (grade C en D). Stoompasteurisasie van rooibos plantmateriaal, noodsaaklik om ‘n produk van hoë mikrobiologiese gehalte te verseker, het gelei tot ‘n betekenisvolle afname in meeste fenoliese verbindings in ’n koppie rooibos. Die hoof fenoliese verbindings van ‘n koppie stoom-gepasteuriseerde, gefermenteerde rooibos was orientien en isoorientien, terwyl kwersetien-3-O-robinobiosied, PPAG en aspalatien ook in noemenswaardige hoeveelhede aanwesig was. Verteenwoordigende waardes van die hoof fenoliese verbindings aanwesig in ‘n tipiese koppie rooibos is derhalwe verkry en die data is geskik vir insluiting in voedsel-samestelling databasisse. Die analiese van rooibos fenole met omvattende twee-dimensionele vloeistof chromatografie (LCxLC) is bestudeer as ‘n alternatiewe metode om verdere insig tot hierdie komplekse fenoliese fraksie te verkry. Die kombinasie van hidrofiliese interaksie chromatografie (HILIC) in die eerste dimensie en RP-LC in die tweede dimensie het ‘n uiters gesogte lae graad van korrelasie verskaf. HILICxRP-LC het ‘n besonderse toename in resolusie teweeg gebring, gekenmerk deur praktiese piek kapasiteite hoër as 2000 en 800 vir die af-lyn en aan-lyn metodes, onderskeidelik. Verdere optimisering, veral van die eerste dimensie skeiding, is egter nodig om die LCxLC skeiding van rooibos fenole te verbeter.

Submitted by LILIAN CHERUBIN CORREIA null (lilian_cherubin@hotmail.com) on 2016-09-02T14:12:24Z No. of bitstreams: 1 Dissertação_Lilian_Cherubin_Correia_2016_ficha e aprovação.pdf: 3233262 bytes, checksum: 3421e95ec88996d4b21b559ade72faf5 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-09-05T21:08:41Z (GMT) No. of bitstreams: 1 correia_lc_me_araiq.pdf: 3233262 bytes, checksum: 3421e95ec88996d4b21b559ade72faf5 (MD5) / Made available in DSpace on 2016-09-05T21:08:41Z (GMT). No. of bitstreams: 1 correia_lc_me_araiq.pdf: 3233262 bytes, checksum: 3421e95ec88996d4b21b559ade72faf5 (MD5) Previous issue date: 2016-08-12 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Separações utilizando cromatografia líquida bidimensional têm atraído grande interesse nas últimas décadas devido ao seu elevado potencial no poder de separação para análises de misturas complexas. Apesar do uso de colunas ortogonais e da multiplicação teórica da capacidade de picos quando essa técnica é utilizada, uma questão permanece: "As técnicas bidimensionais são realmente melhores do que as monodimensionais para análises de extratos vegetais?". Esta questão motivou o presente trabalho, que consistiu em comparar o desempenho entre LC e LC×LC, utilizando amostras de folhas de Casearia sylvestris, também conhecida como "guaçatonga", uma árvore amplamente utilizada na medicina popular brasileira por apresentar diversas atividades biológicas. Além disso, devido ao grande volume de resíduos gerados em LC, a substituição de solventes convencionais por etanol (EtOH), um candidato menos tóxico e biodegradável, foi implementada. A parte mais desafiadora de qualquer cromatografia multidimensional é o desenvolvimento do método cromatográfico, pelo grande número de variáveis que devem atuar de forma integrada em uma única análise. Duas estratégias de desenvolvimento foram propostas neste trabalho. A primeira foi dividida em 4 etapas, que consistiram em selecionar o par de colunas mais ortogonal dentre as combinações disponíveis; o desenvolvimento de um método para a 1D utilizando planejamento experimental; seguido do estudo das melhores condições para a 2D (2tc, tempo de modulação e tre, o tempo de reequilíbrio após cada modulação) e por último, a escolha da melhor forma de gradiente para a 2D. A segunda estratégia consistiu em primeiramente, selecionar o par de colunas mais ortogonais dentre as combinações disponíveis; depois otimizar por planejamento experimental as duas dimensões simultaneamente e finalmente a otimização da melhor forma de gradiente para a 2D. As duas estratégias se mostraram eficazes para desenvolvimento de método bidimensional abrangente, porém a segunda estratégia mostrou-se mais rápida, devido ao menor número de experimentos necessários. Porém, o uso de etanol mostrou-se inviável para as análises rápidas e com altas vazões da 2D, devido à sua elevada viscosidade e seu uso foi descontinuado. Finalmente, com o método bidimensional completamente otimizado, este foi comparado com dois métodos em 1D-LC previamente descritos na literatura para C. sylvestris e utilizando-se a métrica de capacidade de picos, um melhor desempenho na técnica de LC×LC pôde ser observado. Concluiu-se, portanto, que a performance observada em LC×LC é significantemente melhor do que foi alcançada em 1D-LC, porém seu uso dependerá do objetivo do estudo, pois essa melhoria foi alcançada em tempos de análise muito mais longos (aproximadamente 4x) e com maior geração de resíduos (aproximadamente 30x), ou de avanços tecnológicos na técnica. / Bidimensional liquid chromatographic separations have attracted intense interest over the past decade based on the potential improvements in resolving power they offer for analysis of complex samples. Despite the use of orthogonal columns and the theoretical multiplication of peak capacity from both dimensions, one question remains: “Are bidimensional techniques superior to monodimensional ones for analysis of vegetal extracts?”. This question led to the purpose of the present work, that was to compare the performance between LC and LC×LC, using leaves from Casearia sylvestris, a widely used tree in Brazilian folk medicine by its biological activities. Furthermore, due to the large volume of waste generated by LC, the replacement of conventional solvents by ethanol (EtOH), a less toxic and biodegradable alternative solvent was chosen. The most challenging issue of any multidimensional chromatography is the method development, since the large number of interacting parameters that must work together in a single run. Two strategies for method development were used in this work. The first one was divided into 4 steps: experiments were carried out to select the most orthogonal pair of columns, 1D method development using design of experiments, another design of experiments for 2D conditions was proposed (2tc, modulation time and tre, re-equilibration time after each modulation) and aware of the best conditions for both dimensions, 2D gradient types were evaluated. The second strategy consisted of selecting the most orthogonal pair of columns, optimization of both dimensions simultaneously by design of experiments and finally evaluation of 2D gradient type. Both strategies were efficient for comprehensive bidimensional method development, but the second one was found to be faster, due to the lesser number of required experiments. Nevertheless, using ethanol as mobile phase proved to be impractical for fast and high flow rates analysis of 2D due to its high viscosity and its use as mobile phase was rejected. Finally, a comparison between the fully optimized bidimensional chromatographic method and two 1D-LC methods previously described in the literature for C. sylvestris were made. Using peak capacity as a metric of comparison, a higher performance was achieved for LC×LC. This study concluded that LC×LC provides a higher performance than 1D-LC, however, its use will depend of the analysis purpose because this gain in performance was achieved with longer analysis times (approximately 4x) and greater generation of residues (approximately 30x), or technological improvements.

Understanding the goal and behaviour of end users is difficult. Moreover, silo thinking is common in large corporations. Needed are methods that support an understanding of the users’ needs as well as improve communication among development departments that require different needs and information. A Persona is a fictional person based on interviews or other data collecting methods, that describes the users’ needs, goals and issues with the product. This thesis describes a Persona method specifically designed for development processes in large corporations. Twenty-nine interviews were made with a standard Persona method in several countries in Europe with end users for a large forklift truck company. Subsequent analysis with a focus to make a method more affective was performed. The resulting method, Quick Persona Method (QPM) presents an affective process involving knowledge sharing among departments. Furthermore, the method is expected to break a silo thinking culture, to be time efficient, usable, understandable and improve communication among departments.

MSc (Chemistry) / Department of Chemistry

The goal of this thesis was to develop a high-throughput shotgun-MS lipidomics method to qualitatively and quantitively analyze the major mammalian brain ganglioside classes: GM1, GD1, GT1 and GQ1. As a starting point for the method to be developed, a modified ganglioside extraction method from Svennerholm and Ladisch was used (Svennerholm and Fredman, 1980; Ladisch and Gillard, 1985). The efficiencies and the impact of different extraction procedures to the overall performance were evaluated with a software called OptiVal™. The evaluation showed that the most important steps of the protocol are the salt concentration of the water phase during the 2-phase extraction, and 10 mM NaCl yielded the best sensitivity. Also, the number of washing steps with water during reverse solid phase extraction using C18 resin has a significant effect. The next step was to find suitable standards for quantification of the individual ganglioside classes. Since deuterated and alike ganglioside standards were commercially not available, we initially used a deuterated PE standard with limited success. A collaboration with the Ludger Johannes lab provided us with modified C17-ganglioside standards. The term “modified” describes the enzymatic exchange of the fatty acid in the hydrophobic tail by a 17-carbon atom long fatty acid. Since odd numbered fatty acids occur very rarely in nature, it is possible to use the measured intensity of the modified ceramide headgroup of 35:1 (Sphingosine C18:1 + Fatty Acid C17:0) to quantify natural gangliosides. Ideally, we would need to have a fitting modified C17-ganglioside standard for each class to be quantified. Since first only GM1 as a modified standard was available, it was necessary to determine response factors (RFs) for the ganglioside classes GD1, GT1 and GQ1. RFs were assessed empirically by titrating a variety of equimolar concentrations of the modified C17-GM1 standard versus wildtype standards of the other ganglioside classes. After establishment of the RFs it was possible to determine the limits of detection (LOD) and quantification (LOQ) for the ganglioside classes GD1, GT1 and GQ1 - with regard to the modified C17-GM1 standard. When the modified C17-standards for GD1 and GT1 became available, I was able to find out whether the correct internal standards are superior to the proxy method via response factors. The results clearly showed that the use of a correct class standards is preferable. For GQ1 no modified C17-standard was obtainable, therefore this class still has to be quantified via RFs. Experiments showed that the modified C17-GT1 standard is best suited for that. Another major goal was to integrate the ganglioside method into the general lipid analysis workflow of the high-throughput shotgun mass spectrometry platform that we were using. To achieve these goals adjustments on the evaluated (=old) protocol had to be done. These adjustments included changes in the extraction steps from the Svennerholm & Ladisch more into the direction of a Bligh & Dyer based extraction method. This meant abandoning the 2-phase extraction step as well as the chloroform/methanol/water (C/M/W) 4:8:3 extraction, in favor of a C/M 10:1 followed by a C/M 2:1 extraction of 150 mM ammonium-bicarbonate water solution. The goal behind this was to enable a combination of the global lipidome extraction (Surma et al., 2015) with the ganglioside extraction. Another important improvement was scaling up the extraction process. The use of standard single solid phase extraction (SPE) cartridges was limiting the extraction throughput to only 24 samples at a time, therefore the single SPE cartridges were replaced with the 96-well SPE SOLA™ plates. To process the SOLA™ plates it was necessary to establish the usage of a vacuum manifold. Combined, these changes lowered the overall process time of the protocol from nearly two working days to one working day, without significant loss of sensitivity regarding the measured sample concentrations. This was assessed by performing the mouse brain tissue titration experiment, with all three modified C17-ganglioside class standards GM1, GD1 and GT1. Finally, the established method was applied to investigate the difference in ganglioside levels in the cerebellum compared to the brain hemispheres in mice of different age. First the C/M 10:1 and 2:1 extraction was done for the analysis of all non-ganglioside lipids in the sample. The leftover water phase was then loaded onto the SOLA™ plates and processed with the new protocol. The results matched the given goals - to establish a protocol to measure and quantify the four major brain ganglioside classes in combination with the global lipidomics in a high-throughput manner - and thus were a success. To the best of our knowledge, this was the first time such a broad lipidomic measurement has been performed, hence no other studies exist to which the outcome could be compared.

Augmented reality is an old technology that is still far away from being perfect. It is also quickly being improved upon and the state of AR today has come a long way from AR just a couple of years ago. New big players have recently introduced their tools and have made it easier than ever to develop AR applications. In this study we look at what established methods (if any) there are for AR evaluation, develop AR evaluation methods that fit our needs, carry out the evaluation and analyze the collected data. We also note some important things to think about when working with AR to increase tracking and recognition stability. The recommendations are: try to have reference images with high scores, have reference objects that are distinct enough from one another to not be mixed up and make sure that the visual for the reference image matches the visual for the reference object in its intended viewing environment.

Gatumiljön som ett offentligt rum har möjligheter att utvecklas till utrymmen med flera samverkande funktioner. En av funktionerna är att gaturummet ska fungera som en social plats, eftersom den offentliga miljön tillhör alla invånare och ska utformas efter allas behov. Denna studie tar sin utgångspunkt i en tidigare studie som är utförd i USA, som tillämpar en forskningsmetod som kvantifierar socialt beteende genom observationer i gatumiljö. Utgångspunktens metod söker sambandet mellan gatumiljöns platsegenskaper och social aktivitet via en multipel regressionsanalys. Syftet är att besvara vilka fysiska funktioner och attribut som påverkar gatumiljöns livlighet, det vill sägaden sociala aktiviteten. Målet med denna studie är att anpassa den befintliga forskningsmetoden till en svensk kontext och applicera den på ett studieområde. Utgångspunktens tillvägagångssätt av observationer och inventering har därför anpassats och tillämpats genom empirin av pilotstudier. Resultatet visar att ett statistiskt samband mellan platsegenskaper och gatumiljöns livlighet inte kan säkerställas i en multipel regressionsanalys. Det går däremot att visa på statiskt samband mellan gatumiljöns livlighet och tiden på dygnet. I sin helhet visar studien att metodanpassning är komplext att genomföra avseende tid och erfarenhet, men även valideringen av den. Denna studie kan därför betraktas som en pilotstudie, och lämnar alternativa tillvägagångsätt för framtida forskning. / Streets as a public space have opportunities to become spaces with several interacting functions. One of these functions includes streets as a social place since the public environment belongs to all residents and should be designed for all needs. This study is based on a former study conducted in the U.S.A.that quantifies social behavior through observations in the street environment. The applied method seeks the association between the physical characteristics of the street environment and social activity by using multiple regression analysis. The purpose was to answer which physical functions and attributes affect the liveliness of the street environment, i.e., the social activity in the U.S.A. This study aims to adapt the existing research method into a Swedish context and apply it within a study area in Gävle. The results show that a statistical association between physical characteristics and the liveliness of the street cannot be ensured by a multiple regression analysis. On the other hand, it is possible to show a static association between the liveliness of the street and a time period of the day. This study shows that method adaption is complex in terms of time and experience, but also in terms of validation. Thisstudy can therefore be considered a pilot study and provides alternative approaches for future research.