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In this thesis entitled “Saccharide Recognition - Boronic acids as Receptors in Polymeric Networks” different aspects of boronic acid synthesis, their analysis and incorporation or attachment to different polymeric networks and characterisation thereof were investigated. The following key aspects were considered: • Provision of a variety of different characterised arylboronic acids and benzoboroxoles • Attachment of certain derivatives to nanoparticles and the characterisation of saccharide binding by means of isothermal titration calorimetry and displacement assay (ARS) to enhance the association constant to saccharides at pH 7.4 • Enhancement of selectivity in polymeric systems by means of molecular imprinting using fructose as template and a polymerisable benzoboroxole as functional monomer for the recognition at pH 7.4 (Joined by a diploma thesis of F. Grüneberger) • Development of biomimetic saccharide structures and the development of saccharide (especially glucose and fructose) binding MIPs by using these structures as template molecules. In the first part of the thesis different arylboronic acid derivatives were synthesised and their binding to glucose or fructose was investigated by means of isothermal titration calorimetry (ITC). It could be derived, which is in parallel to the literature, that derivatives bearing a methylhydroxyl-group in ortho-position to the boron (benzoboroxole) exhibit in most cases a two-fold higher association constant compared to the corresponding phenylboronic acid derivative. To gain a deeper understanding NMR spectroscopy and mass spectrometry with the benzoboroxole and glucose or fructose was performed. It could be shown that the exchange rate in terms of NMR time scale is quite slow since in titration experiments new peaks appeared. Via mass spectrometry of a mixture between benzoboroxole and glucose or fructose, different binding stoichiometries could be detected showing that the binding of saccharides is comparable with their binding to phenylboronic acid. In addition, the use of Alizarin Red S as an electrochemical reporter was described for the first time to monitor the saccharide binding to arylboronic acids not only with spectroscopy. Here, the redox behaviour and the displacement were recorded by cyclic voltammograms. In the second part different applications of boronic acids in polymeric networks were investigated. The attachment of benzoboroxoles to nanoparticles was investigated and monitored by means of isothermal titration calorimetry and a colourimetric assay with Alizarin Red S as the report dye. The investigations by isothermal titration calorimetry compared the fructose binding of arylboronic acids and benzoboroxoles coupled to these nanoparticles and “free” in solution. It could be shown that the attached derivatives showed a higher binding constant due to an increasing entropy term. This states for possible multivalent binding combined with a higher water release. Since ITC could not characterise the binding of glucose to these nanoparticles due to experimental restrictions the glucose binding at pH 7.4 was shown with ARS. Here, the displacement of ARS by fructose and also glucose could be followed and consequently these nanoparticles can be used for saccharide determination. Within this investigation also the temperature stability of these nanoparticles was examined and after normal sterilisation procedures (121°C, 20 min.) the binding behaviour was still unchanged. To target the selectivity of the used polymeric networks, molecular imprinting was used as a technique for creating artificial binding pockets on a molecular scale. As functional monomer 3-methacrylamidobenzoboroxole was introduced for the recognition of fructose. In comparison to polymers prepared with vinylphenylboronic acid the benzoboroxole containing polymer had a stronger binding at pH 7.4 which was shown for the first time. In addition, another imprinted polymer was synthesised especially for the recognition of glucose and fructose employing biomimetic saccharide analogues as template molecule. The advantage to use the saccharide analogues is the defined template-functional monomer complex during the polymerisation which is not the case, for example, for glucose-boronic acid interaction. The biomimetic character was proven through structural superimposition of crystal structures of the analogues with already described crystal structures of boronic acid esters of glucose and fructose. A molecularly imprinted polymer was synthesised with vinylphenylboronic acid as the functional monomer to show that both glucose and fructose are able to bind to the polymer which was predicted by the structural similarity of the analogues. The major scientific contributions of this thesis are • the determination of binding constants for some, not yet reported saccharide – boronic acid / benzoboroxole pairs, • the use of ARS as electrochemical reporter for saccharide detection, • the thermodynamic characterisation of a saccharide binding nanoparticle system containing benzoboroxole and functioning at pH 7.4, • the use of a polymerisable benzoboroxole as functional monomer for saccharide recognition in neutral, aqueous environments • and the synthesis and utilisation of biomimetic saccharide analogues as template molecules especially for the development of a glucose binding MIP.
Receptors predominantly expressed on tumor cells represent one of the key prerequisites of targeted radiotherapy. The gastric inhibitory polypeptide receptor (GIPR) has emerged as a promising target due to its substantial overexpression in neuroendocrine neoplasms (NENs) and virtual absence in healthy tissues (Waser 2012). So far, only radiolabeled peptides targeting the somatostatin receptor 2 (SSTR2) are approved for targeted radiotherapy of inoperable, metastatic NENs.
The aim of this thesis was to develop highly affine GIPR tracers for targeted radiotherapy by continuous in vitro and in vivo characterization of peptide sequence modifications. It was hypothesized that a GIPR antagonist might increase the sensitivity to detect GIPR-positive tumors relative to the agonist GIP(1-30), as shown for SSTR2 tracers (Reubi 2017). Further comparison to the SSTR2 agonist and antagonist (DOTATATE, JR11) should allow compound ranking regarding their ability to detect NENs.
The novel GIPR-targeting antagonists were conjugated to DOTA, enabling complexation of diagnostic (e. g. 111In) and therapeutic radionuclides (e. g. 177Lu). Among the high number of compounds screened, 3BP-3775 proved to be the most promising candidate for further preclinical and clinical development. High GIPR affinity and long receptor residence time in vitro were reflected in strong tumor uptake and retention in vivo. Low initial kidney accumulation and fast subsequent clearance represented a major advantage relative to previously described GIPR-targeting molecules (Gourni 2014). Furthermore, administration of 177Lu-3BP-3775 demonstrated for the first time strong therapeutic efficacy of a GIPR-targeting compound. In vitro receptor autoradiography with 111In-3BP-3626 (GIPR antagonist) demonstrated up to 6-fold higher signals in gastroenteropancreatic and bronchial NENs, relative to the clinically utilized SSTR2 agonist 111In-DOTATATE. Both receptor antagonists, however, revealed similar signal strength. In contrast to 111In-JR11, 111In-3BP-3626 showed no binding to non-target tissues, which led to higher tumor-to-background ratios for 111In-3BP-3626. Signal strength of the GIPR agonist 111In-GIP(1-30) was close to background in all investigated tumor samples. The ranking of compounds according to their ability to detect NENs based on autoradiographic signal intensities was determined to be: 111In-3BP-3626 ~ 111In-JR11> 111In-DOTATATE > 111In GIP(1-30).
In summary, this thesis proposes the application of the GIPR antagonist 3BP-3775 for a targeted radiotherapy in GEP- and bronchial NENs.
Der Fokusmarker 'so'
(2021)
Background: Increased numbers of intestinal E. coli are observed in inflammatory bowel disease, but the reasons for this proliferation and it exact role in intestinal inflammation are unknown. Aim of this PhD-project was to identify E. coli proteins involved in E. coli’s adaptation to the inflammatory conditions in the gut and to investigate whether these factors affect the host. Furthermore, the molecular basis for strain-specific differences between probiotic and harmful E. coli in their response to intestinal inflammation was investigated. Methods: Using mice monoassociated either with the adherent-invasive E. coli (AIEC) strain UNC or the probiotic E. coli Nissle, two different mouse models of intestinal inflammation were analysed: On the one hand, severe inflammation was induced by treating mice with 3.5% dextran sodium sulphate (DSS). On the other hand, a very mild intestinal inflammation was generated by associating interleukin 10-deficient (IL-10-/-) mice with E. coli. Differentially expressed proteins in the E. coli strains collected from caecal contents of these mice were identified by two-dimensional fluorescence difference gel electrophoresis. Results DSS-experiment: All DSS-treated mice revealed signs of a moderate caecal and a severe colonic inflammation. However, mice monoassociated with E. coli Nissle were less affected. In both E. coli strains, acute inflammation led to a downregulation of pathways involved in carbohydrate breakdown and energy generation. Accordingly, DSS-treated mice had lower caecal concentrations of bacterial fermentation products than the control mice. Differentially expressed proteins also included the Fe-S cluster repair protein NfuA, the tryptophanase TnaA, and the uncharacterised protein YggE. NfuA was upregulated nearly 3-fold in both E. coli strains after DSS administration. Reactive oxygen species produced during intestinal inflammation damage Fe-S clusters and thereby lead to an inactivation of Fe-S proteins. In vitro data indicated that the repair of Fe-S proteins by NfuA is a central mechanism in E. coli to survive oxidative stress. Expression of YggE, which has been reported to reduce the intracellular level of reactive oxygen species, was 4- to 8-fold higher in E. coli Nissle than in E. coli UNC under control and inflammatory conditions. In vitro growth experiments confirmed these results, indicating that E. coli Nissle is better equipped to cope with oxidative stress than E. coli UNC. Additionally, E. coli Nissle isolated from DSS-treated and control mice had TnaA levels 4- to 7-fold higher than E. coli UNC. In turn, caecal indole concentrations resulting from cleavage of tryptophan by TnaA were higher in E. coli Nissle- associated control mice than in the respective mice associated with E. coli UNC. Because of its anti-inflammatory effect, indole is hypothesised to be involved in the extension of the remission phase in ulcerative colitis described for E. coli Nissle. Results IL-10-/--experiment: Only IL-10-/- mice monoassociated with E. coli UNC for 8 weeks exhibited signs of a very mild caecal inflammation. In agreement with this weak inflammation, the variations in the bacterial proteome were small. Similar to the DSS-experiment, proteins downregulated by inflammation belong mainly to the central energy metabolism. In contrast to the DSS-experiment, no upregulation of chaperone proteins and NfuA were observed, indicating that these are strategies to overcome adverse effects of strong intestinal inflammation. The inhibitor of vertebrate C-type lysozyme, Ivy, was 2- to 3-fold upregulated on mRNA and protein level in E. coli Nissle in comparison to E. coli UNC isolated from IL-10-/- mice. By overexpressing ivy, it was demonstrated in vitro that Ivy contributes to a higher lysozyme resistance observed for E. coli Nissle, supporting the role of Ivy as a potential fitness factor in this E. coli strain. Conclusions: The results of this PhD-study demonstrate that intestinal bacteria sense even minimal changes in the health status of the host. While some bacterial adaptations to the inflammatory conditions are equal in response to strong and mild intestinal inflammation, other reactions are unique to a specific disease state. In addition, probiotic and colitogenic E. coli differ in their response to the intestinal inflammation and thereby may influence the host in different ways.
Die Xanthin-Dehydrogenase aus Rhodobacter capsulatus ist ein cytoplasmatisches Enzym, welches ein (αβ)₂ Heterotetramer mit einer Größe von 275 kDa bildet. Die drei Kofaktoren (Moco, 2[2Fe2S], FAD) sind auf zwei unterschiedlichen Polypeptidketten gebunden. So sind die beiden spektroskopisch unterscheidbaren Eisen-Schwefel-Zentren und das FAD in der XdhA-Untereinheit und der Moco in der XdhB-Untereinheit gebunden. Im ersten Teil dieser Arbeit sollte untersucht werden, warum die R. capsulatus XDH ein Dimer bildet und ob ein intramolekularer Elektronentransfer existiert. Dafür wurde eine chimäre XDH-Variante [(α)₂(β₁wt/β₂E730A)] erzeugt, welche eine aktive und eine inaktive XdhB-Untereinheit trägt. Mit Hilfe von Reduktionsspektren sowie mit der Bestimmung der kinetischen Parameter für die Substrate Xanthin und NAD+ konnte gezeigt werden, dass die chimäre XDH-Variante katalytisch halb so aktiv war, wie der auf gleiche Weise gereinigte XDH-Wildtyp. Dies verdeutlicht, dass die noch aktive Untereinheit der Chimären selbstständig und unabhängig Substrat binden und hydroxylieren kann und ein intramolekularer Elektronentransfer zwischen den beiden XdhB-Untereinheiten nicht stattfindet. Ein weiteres Ziel war die funktionelle Charakterisierung der Mus musculus AOX1 sowie der humanen AOX1 hinsichtlich ihrer Substratspezifitäten und ihrer biophysikalischen Eigenschaften sowie der Charakterisierung der konservierten Aminosäuren im aktiven Zentrum der mAOX1. Da bislang noch kein heterologes Expressionssystem für ein aktives und stabiles rekombinantes AO-Protein existierte, wurde ein E. coli Expressionssystem mit der gleichzeitigen Expression der entsprechenden Mocosulfurase für mAOX1 und hAOX1 in dieser Arbeit etabliert. Mit Hilfe dieser Koexpression konnte die Aktivität der rekombinanten mAOX1 um 50 % gesteigert werden, wenn gleich auch der sulfurierte Moco-Anteil nur 20 % betrug. Um die konservierten Aminosäuren im aktiven Zentrum hinsichtlich ihrer Funktion der Substratbindung zu charakterisieren, wurden folgende Varianten erzeugt: V806E, M884R, V806/M884R sowie E1265Q. Mit Hilfe von kinetischen Substratuntersuchungen konnte gezeigt werden, dass die beiden Aminosäuren Val806 und Met884 für die Erkennung und die Stabilisierung von Aldehyden und N-Heterozyklen essentiell sind. Ein Austausch dieser beiden gegen Glutamat bzw. Arginin (wie bei R. capsulatus XDH) zeigte jedoch keine Xanthin- oder Hypoxanthinumsetzung. Für das Glu1265 wurde ebenfalls die Rolle als die Katalyse initiierende Aminosäure belegt.
This thesis investigates the processing of non-canonical word orders and whether non-canonical orders involving object topicalizations, midfield scrambling and particle verbs are treated the same by native (L1) and non-native (L2) speakers. The two languages investigated are Norwegian and German.
32 L1 Norwegian and 32 L1 German advanced learners of Norwegian were tested in two experiments on object topicalization in Norwegian. The results from the online self-paced reading task and the offline agent identification task show that both groups are able to identify the non-canonical word order and show a facilitatory effect of animate subjects in their reanalysis. Similarly high error rates in the agent identification task suggest that globally unambiguous object topicalizations are a challenging structure for L1 and L2 speakers alike.
The same participants were also tested in two experiments on particle placement in Norwegian, again using a self-paced reading task, this time combined with an acceptability rating task. In the acceptability rating L1 and L2 speakers show the same preference for the verb-adjacent placement of the particle over the non-adjacent placement after the direct object. However, this preference for adjacency is only found in the L1 group during online processing, whereas the L2 group shows no preference for either order.
Another set of experiments tested 33 L1 German and 39 L1 Slavic advanced learners of German on object scrambling in ditransitive clauses in German. Non-native speakers accept both object orders and show neither a preference for either order nor a processing advantage for the canonical order. The L1 group, in contrast, shows a small, but significant preference for the canonical dative-first order in the judgment and the reading task.
The same participants were also tested in two experiments on the application of the split rule in German particle verbs. Advanced L2 speakers of German are able to identify particle verbs and can apply the split rule in V2 contexts in an acceptability judgment task in the same way as L1 speakers. However, unlike the L1 group, the L2 group is not sensitive to the grammaticality manipulation during online processing. They seem to be sensitive to the additional lexical information provided by the particle, but are unable to relate the split particle to the preceding verb and recognize the ungrammaticality in non-V2 contexts.
Taken together, my findings suggest that non-canonical word orders are not per se more difficult to identify for L2 speakers than L1 speakers and can trigger the same reanalysis processes as in L1 speakers. I argue that L2 speakers’ ability to identify a non-canonical word order depends on how the non-canonicity is signaled (case marking vs. surface word order), on the constituents involved (identical vs. different word types), and on the impact of the word order change on sentence meaning. Non-canonical word orders that are signaled by morphological case marking and cause no change to the sentence’s content are hard to detect for L2 speakers.