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Electrosynthesis and characterization of molecularly imprinted polymers for peptides and proteins
(2019)
This thesis covers the synthesis of conjugates of 2-Deoxy-D-ribose-5-phosphate aldolase (DERA) with suitable polymers and the subsequent immobilization of these conjugates in thin films via two different approaches.
2-Deoxy-D-ribose-5-phosphate aldolase (DERA) is a biocatalyst that is capable of converting acetaldehyde and a second aldehyde as acceptor into enantiomerically pure mono- and diyhydroxyaldehydes, which are important structural motifs in a number of pharmaceutically active compounds. Conjugation and immobilization renders the enzyme applicable for utilization in a continuously run biocatalytic process which avoids the common problem of product inhibition. Within this thesis, conjugates of DERA and poly(N-isopropylacrylamide) (PNIPAm) for immobilization via a self-assembly approach were synthesized and isolated, as well as conjugates with poly(N,N-dimethylacrylamide) (PDMAA) for a simplified and scalable spray-coating approach. For the DERA/PNIPAm-conjugates different synthesis routes were tested, including grafting-from and grafting-to, both being common methods for the conjugation. Furthermore, both lysines and cysteines were addressed for the conjugation in order to find optimum conjugation conditions. It turned out that conjugation via lysine causes severe activity loss as one lysine plays a key role in the catalyzing mechanism. The conjugation via the cysteines by a grafting-to approach using pyridyl disulfide (PDS) end-group functionalized polymers led to high conjugation efficiencies in the presence of polymer solubilizing NaSCN. The resulting conjugates maintained enzymatic activity and also gained high acetaldehyde tolerance which is necessary for their use later on in an industrial relevant process after their immobilization.
The resulting DERA/PNIPAm conjugates exhibited enhanced interfacial activity at the air/water interface compared to the single components, which is an important pre-requisite for the immobilization via the self-assembly approach. Conjugates with longer polymer chains formed homogeneous films on silicon wafers and glass slides while the ones with short chains could only form isolated aggregates. On top of that, long chain conjugates showed better activity maintenance upon the immobilization.
The crosslinking of conjugates, as well as their fixation on the support materials, are important for the mechanical stability of the films obtained from the self-assembly process. Therefore, in a second step, we introduced the UV-crosslinkable monomer DMMIBA to the PNIPAm polymers to be used for conjugation. The introduction of DMMIBA reduced the lower critical solution temperature (LCST) of the polymer and thus the water solubility at ambient conditions, resulting in lower conjugation efficiencies and in turn slightly poorer acetaldehyde tolerance of the resulting conjugates. Unlike the DERA/PNIPAm, the conjugates from the copolymer P(NIPAM-co-DMMIBA) formed continuous, homogenous films only after the crosslinking step via UV-treatment. For a firm binding of the crosslinked films, a functionalization protocol for the model support material cyclic olefin copolymer (COC) and the final target support, PAN based membranes, was developed that introduces analogue UV-reactive groups to the support surface. The conjugates immobilized on the modified COC films maintained enzymatic activity and showed good mechanical stability after several cycles of activity assessment. Conjugates with longer polymer chains, however, showed a higher degree of crosslinking after the UV-treatment leading to a pronounced loss of activity. A porous PAN membrane onto which the conjugates were immobilized as well, was finally transferred to a dead end filtration membrane module to catalyze the aldol reaction of the industrially relevant mixture of acetaldehyde and hexanal in a continuous mode. Mono aldol product was detectable, but yields were comparably low and the operational stability needs to be further improved
Another approach towards immobilization of DERA conjugates that was followed, was to generate the conjugates in situ by simply mixing enzyme and polymer and spray coat the mixture onto the membrane support. Compared to the previous approach, the focus was more put on simplicity and a possible scalability of the immobilization. Conjugates were thus only generated in-situ and not further isolated and characterized. For the conjugation, PDMAA equipped with N-2-thiolactone acrylamide (TlaAm) side chains was used, an amine-reactive comonomer that can react with the lysine residues of DERA, as well as with amino groups introduced to a desired support surface. Furthermore disulfide formation after hydrolysis of the Tla groups causes a crosslinking effect. The synthesized copolymer poly(N,N-Dimethylacrylamide-co-N-2-thiolactone acrylamide) (P(DMAA-co-TlaAm)) thus serves a multiple purpose including protein binding, crosslinking and binding to support materials. The mixture of DERA and polymer could be immobilized on the PAN support by spray-coating under partial maintenance of enzymatic activity. To improve the acetaldehyde tolerance, the polymer in used was further equipped with cysteine reactive PDS end-groups that had been used for the conjugation as described in the first part of the thesis. The generated conjugates indeed showed good acetaldehyde tolerance and were thus used to be coated onto PAN membrane supports. Post treatment with a basic aqueous solution of H2O2 was supposed to further crosslink the spray-coated film hydrolysis and oxidation of the thiolactone groups. However, a washing off of the material was observed. Optimization is thus still necessary.
The present dissertation investigates profit-maximizing behavior in different phases of the negotiation process. Over the last decades, research dealt in detail with behavior of negotiation actors with the aim of identifying performance enhancing factors. The majority of those studies focused on behavior within the main negotiation phase. This work, however, considers phases which are, so far, underrepresented in research but show an impact on the negotiation process and outcome. Those phases are the pre-negotiation, the first offer, and the main negotiation phase which is further divided by breaks into several rounds. Within these phases, traits of behavior are analyzed that can be used strategically in order to impact the negotiation outcome. The dissertation contains three papers, each one dealing with a specific strategy within one phase. The first paper investigates communication behavior in the pre-negotiation phase. Content analysis of a negotiation experiment shows that the employment of positive communication elements such as the generation of enthusiasm for an upcoming project results in an increase of agreements on entering a negotiation and also leads to a higher willingness to make concessions. The second paper explores the impact of a semantic first anchor, which does not contain a specific number but only gives a numerical direction, on the opponent’s concession behavior and the final outcome. By means of two scenario-based questionnaires and a negotiation experiment it is demonstrated that semantic offers reveal an anchoring effect and lead to better negotiation outcomes. The third paper deals with the introduction of breaks and their effect on the following negotiation process. Therefore, content and outcome of another negotiation experiment are investigated. The analysis shows that breaks evoke a dominant impression but can negatively impact the atmosphere and thereby also the outcome. Finally, the gathered insights are brought together and discussed. The dissertation closes with implications for practice, limitations of the work, and ideas for future research.
Determining the relationship between genotype and phenotype is the key to understand the plasticity and robustness of phenotypes in nature. While the directly observable plant phenotypes (e.g. agronomic, yield and stress resistance traits) have been well-investigated, there is still a lack in our knowledge about the genetic basis of intermediate phenotypes, such as metabolic phenotypes. Dissecting the links between genotype and phenotype depends on suitable statistical models. The state-of-the-art models are developed for directly observable phenotypes, regardless the characteristics of intermediate phenotypes. This thesis aims to fill the gaps in understanding genetic architecture of intermediate phenotypes, and how they tie to composite traits, namely plant growth. The metabolite levels and reaction fluxes, as two aspects of metabolic phenotypes, are shaped by the interrelated chemical reactions formed in genome-scale metabolic network. Here, I attempt to answer the question: Can the knowledge of underlying genome-scale metabolic network improve the model performance for prediction of metabolic phenotypes and associated plant growth? To this end, two projects are investigated in this thesis. Firstly, we propose an approach that couples genomic selection with genome-scale metabolic network and metabolic profiles in Arabidopsis thaliana to predict growth. This project is the first integration of genomic data with fluxes predicted based on constraint-based modeling framework and data on biomass composition. We demonstrate that our approach leads to a considerable increase of prediction accuracy in comparison to the state-of-the-art methods in both within and across environment predictions. Therefore, our work paves the way for combining knowledge on metabolic mechanisms in the statistical approach underlying genomic selection to increase the efficiency of future plant breeding approaches. Secondly, we investigate how reliable is genomic selection for metabolite levels, and which single nucleotide polymorphisms (SNPs), obtained from different neighborhoods of a given metabolic network, contribute most to the accuracy of prediction. The results show that the local structure of first and second neighborhoods are not sufficient for predicting the genetic basis of metabolite levels in Zea mays. Furthermore, we find that the enzymatic SNPs can capture most the genetic variance and the contribution of non-enzymatic SNPs is in fact small. To comprehensively understand the genetic architecture of metabolic phenotypes, I extend my study to a local Arabidopsis thaliana population and their hybrids. We analyze the genetic architecture in primary and secondary metabolism as well as in growth. In comparison to primary metabolites, compounds from secondary metabolism were more variable and show more non-additive inheritance patterns which could be attributed to epistasis. Therefore, our study demonstrates that heterozygosity in local Arabidopsis thaliana population generates metabolic variation and may impact several tasks directly linked to metabolism. The studies in this thesis improve the knowledge of genetic architecture of metabolic phenotypes in both inbreed and hybrid population. The approaches I proposed to integrate genome-scale metabolic network with genomic data provide the opportunity to obtain mechanistic insights about the determinants of agronomically important polygenic traits.
The usage of mobile devices is rapidly growing with Android being the most prevalent mobile operating system. Thanks to the vast variety of mobile applications, users are preferring smartphones over desktops for day to day tasks like Internet surfing. Consequently, smartphones store a plenitude of sensitive data. This data together with the high values of smartphones make them an attractive target for device/data theft (thieves/malicious applications).
Unfortunately, state-of-the-art anti-theft solutions do not work if they do not have an active network connection, e.g., if the SIM card was removed from the device. In the majority of these cases, device owners permanently lose their smartphone together with their personal data, which is even worse.
Apart from that malevolent applications perform malicious activities to steal sensitive information from smartphones. Recent research considered static program analysis to detect dangerous data leaks. These analyses work well for data leaks due to inter-component communication, but suffer from shortcomings for inter-app communication with respect to precision, soundness, and scalability.
This thesis focuses on enhancing users' privacy on Android against physical device loss/theft and (un)intentional data leaks. It presents three novel frameworks: (1) ThiefTrap, an anti-theft framework for Android, (2) IIFA, a modular inter-app intent information flow analysis of Android applications, and (3) PIAnalyzer, a precise approach for PendingIntent vulnerability analysis.
ThiefTrap is based on a novel concept of an anti-theft honeypot account that protects the owner's data while preventing a thief from resetting the device.
We implemented the proposed scheme and evaluated it through an empirical user study with 35 participants. In this study, the owner's data could be protected, recovered, and anti-theft functionality could be performed unnoticed from the thief in all cases.
IIFA proposes a novel approach for Android's inter-component/inter-app communication (ICC/IAC) analysis. Our main contribution is the first fully automatic, sound, and precise ICC/IAC information flow analysis that is scalable for realistic apps due to modularity, avoiding combinatorial explosion: Our approach determines communicating apps using short summaries rather than inlining intent calls between components and apps, which requires simultaneously analyzing all apps installed on a device.
We evaluate IIFA in terms of precision, recall, and demonstrate its scalability to a large corpus of real-world apps. IIFA reports 62 problematic ICC-/IAC-related information flows via two or more apps/components.
PIAnalyzer proposes a novel approach to analyze PendingIntent related vulnerabilities. PendingIntents are a powerful and universal feature of Android for inter-component communication. We empirically evaluate PIAnalyzer on a set of 1000 randomly selected applications and find 1358 insecure usages of PendingIntents, including 70 severe vulnerabilities.
The Italian Army’s participation in Hitler’s war against the Soviet Union has remained unrecognized and understudied. Bastian Matteo Scianna offers a wide-ranging, in-depth corrective. Mining Italian, German and Russian sources, he examines the history of the Italian campaign in the East between 1941 and 1943, as well as how the campaign was remembered and memorialized in the domestic and international arena during the Cold War. Linking operational military history with memory studies, this book revises our understanding of the Italian Army in the Second World War.
STERILE APETALA (SAP) is known to be an essential regulator of flower development for over 20 years. Loss of SAP function in the model plant Arabidopsis thaliana is associated with a reduction of floral organ number, size and fertility. In accordance with the function of SAP during early flower development, its spatial expression in flowers is confined to meristematic stages and to developing ovules. However, to date, despite extensive research, the molecular function of SAP and the regulation of its spatio-temporal expression still remain elusive.
In this work, amino acid sequence analysis and homology modeling revealed that SAP belongs to the rare class of plant F-box proteins with C-terminal WD40 repeats. In opisthokonts, this type of F-box proteins constitutes the substrate binding subunit of SCF complexes, which catalyze the ubiquitination of proteins to initiate their proteasomal degradation. With LC-MS/MS-based protein complex isolation, the interaction of SAP with major SCF complex subunits was confirmed. Additionally, candidate substrate proteins, such as the growth repressor PEAPOD 1 and 2 (PPD1/2), could be revealed during early stages of flower development. Also INDOLE-3-BUTYRIC ACID RESPONSE 5 (IBR5) was identified among putative interactors. Genetic analyses indicated that, different from substrate proteins, IBR5 is required for SAP function. Protein complex isolation together with transcriptome profiling emphasized that the SCFSAP complex integrates multiple biological processes, such as proliferative growth, vascular development, hormonal signaling and reproduction. Phenotypic analysis of sap mutant and SAP overexpressing plants positively correlated SAP function with plant growth during reproductive and vegetative development.
Furthermore, to elaborate on the transcriptional regulation of SAP, publicly available ChIP-seq data of key floral homeotic proteins were reanalyzed. Here, it was shown that the MADS-domain transcription factors APETALA 1 (AP1), APETALA 3 (AP3), PISTILLATA (PI), AGAMOUS (AG) and SEPALLATA 3 (SEP3) bind to the SAP locus, which indicates that SAP is expressed in a floral organ-specific manner. Reporter gene analyses in combination with CRISPR/Cas9-mediated deletion of putative regulatory regions further demonstrated that the intron contains major regulatory elements of SAP in Arabidopsis thaliana.
In conclusion, these data indicate that SAP is a pleiotropic developmental regulator that acts through tissue-specific destabilization of proteins. The presumed transcriptional regulation of SAP by the floral MADS-domain transcription factors could provide a missing link between the specification of floral organ identity and floral organ growth pathways.