Chemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments, a better understanding of biochemical changes in the resistant tumors is needed. Therefore, especially in vivo models are very important tools to generate standardized cell-material, which can be examined by high throughput techniques. Thus, it should be possible to find new targets for therapy or even for diagnostic. This thesis focusses on the characterization of the in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft mouse model. First step of the work was the establishment of the xenograft mouse model. SCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. After 10 weeks of response to the therapy, the tumor growth relapsed and tissue grew with very fast doubling time again, despite of ongoing treatment. This aggressive manner of growth under therapy could be also observed in a re-implantation study where the reisolated CPA chemoresistant HUH-REISO tumors grew without a lag phase, indicating an endogenous imprinted component. To evaluate this, tumors were examined by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Histochemical analysis of HUH-REISO tumors revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO in comparison to parental HUH-7 cells and in vivo passaged HUH-PAS cells (in vivo grown without chemotherapeutic CPA pressure). The pronounced network of host murine vascularization in parental HUH-7 tumors was completely substituted by a network of human and murine vessel-like structures in HUH-REISO tumors under therapy. In addition, cell lines of these tumors were analyzed in endothelial trans-differentiation studies on matrigel. In those studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. Such a trans-differentiation capacity requires a lineage of cells with pluripotent capacities like so called tumor stem cells. Indeed, I could show in a comparative study on stemness and plasticity markers that Thy-1, Oct-4, Sox-2 and Nanog were upregulated in resistant xenografts. Furthermore, under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression, which I could also show by qRT-PCR. Notch-1 raised in HUH-PAS under therapeutic pressure, meanwhile it was conversely regulated in comparison to Thy-1, Oct-4, Sox-2 and Nanog in HUH-REISO. In both groups Notch-3 was inducible by 2 times CPA treatment and fell back on base level after further four therapeutic cycles in HUH-REISO. To conclude all these finding: chemoresistance of HUH-REISO was not manifested under standard in vitro, but only under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of trans-differentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.

To study the mechanisms of membrane protein insertion we established a protocol that allows isolation of in vivo assembled ribosome nascent chain complexes (RNCs) from E. coli in high yield and quality. To investigate the interaction of SecY with a translating ribosome, model membrane proteins of different length and topology were over-expressed and the respective RNCs were isolated under mild conditions to allow co-purification of the SecY complex. Analysis of the interaction of RNCs with SecY in vivo suggested that, as expected, a tight engagement of the ribosome and SecY is only established for nascent chains that are translocated co-translationally. We observed that SecY and the RNC do not form a stable complex at the moment of hydrophobic transmembrane segments inserting in the translocon. However, a stable engagement of the RNC with SecY was observed, when inserting a transmembrane segment with a type II topology into SecY followed by a hydrophilic loop of a certain length which allows the isolation of this complex. That suggested a dual binding mode of tight and loose coupling of SecY to the translating ribosome dependent on the nature of the nascent substrate. We present the first three dimensional structure of an in vivo assembled, tightly coupled polytopic RNC-SecYE complex at 7.2 Å solved by cryo-EM and single particle reconstruction. A molecular model based on the cryo-EM structure reveals that SecYE could be trapped in a post-insertion state, with the two substrate helices interacting with the periphery of SecY, while still translocating the hydrophilic loop. The lateral gate of SecY remains in a ‘pre-opened’ conformation during the translocation of the hydrophilic loop. The interaction sites of SecY with the ribosome were found as described. Remarkably, we could also reveal an interaction of helix 59 in the ribosome with nascent membrane protein via positively charged residues in the first cytoplasmic loop of the substrate. It is tempting to speculate that this interaction contributes to the positive inside rule. Though, we provided an unprecedented snapshot of an inserting polytopic membrane protein, the exact path of the nascent chain and the molecular mechanism of the actual insertion could not be solved so far. Expression of the E. coli tryptophanase (TnaA) operon is triggered by ribosome stalling during translation of the upstream TnaC leader peptide. Notably, this stalling is strictly dependent on the presence of tryptophan that acts in a hitherto unknown way. Here, we present a cryo-EM reconstruction of the stalled nascent TnaC leader peptide in the ribosomal exit tunnel. The structure of the TnaC-stalled ribosome was solved to an average resolution of 3.8 Å by cryo-EM and single particle analysis. It reveals the conformation of the silenced peptidyl-transferase center as well as the exact path of the stalled nascent peptide and its contacts in detail. Furthermore, we clearly resolve not a single but two free tryptophan molecules in the ribosomal exit tunnel. The nascent TnaC peptide chain together with distinct rRNA bases in the ribosomal exit tunnel creates two hydrophobic binding pockets for the tryptophan coordination. One tryptophan molecule is coordinated by V20 and I19 of TnaC and interacts with U2586 of the rRNA, the second tryptophan is bound between I19 and I15 in the area of A2058 and A2059 of the rRNA. Interestingly, the latter is also the binding platform for macrolide antibiotics. Engagement of L-Trp in these composite binding pockets leads to subtle conformational changes in residues of the ribosomal tunnel wall that are translated to the PTC eventually resulting in silencing by stabilizing the conformations of the conserved nucleotides A2602 and U2585. These conformations of the two nucleotides in the PTC are incompatible with the correct accommodation of the GGQ motive of release factor 2, thus inhibiting the peptide release.

No description available.

No description available.

Covalent Organic Frameworks (COFs) are a novel class of highly stable, purely organic crystalline frameworks made of molecular building blocks. For example, the condensation of boronic acids with appropriate polyols in principle allows the design of precisely controllable structures since their chemical and physical properties can be easily tuned through the selection of the building blocks. The young research field of COFs has attracted scientists due to their extraordinary and versatile properties, however, strategies to control the topology and the properties of the backbone as well as the inner surface are still not well established. With support of Prof. Knochel and his group, who contributed numerous new organic COF linkers, this thesis aims to extend the functionalization strategies for the design of Covalent Organic Frameworks. Investigation of the structural modification and the associated change in physical and chemical properties should lead to progress regarding the applicability of these materials. Employing the concept of reticular chemistry in combination with High Throughput Synthesis Techniques, the formation of a very large Covalent Organic Framework BTP-COF with 4 nm open pores was successfully carried out. The solvothermal co-condensation of 1,3,5-benzenetris(4-phenylboronic acid) (BTPA) and 2,3,6,7-tetrahydroxy-9,10-dimethyl-anthracene (THDMA) was carried out using microwave irradiation instead of conventional synthesis in an oven, thus synthesis time of BTP-COF was reduced from initially 72 h to 5 min. Extending the open pore diameter of a crystalline material to 4 nm, in combination with the resulting high accessible surface area of 2000 m2/g offers great potential to exploit organic reactions in the pores and enables the incorporation of large functional guests, such as polymers or dyes. Bearing these results in mind the scope of functionalization possibilities was expanded from the geometric extension to the chemical modification of the inner surface of COFs. Decorating the organic building blocks with small functional active groups, such as methyl-, -methoxy- and hydroxy- allowed for the successful synthesis of several organic frameworks. Chemical and physical properties of the backbone and the inner surface can be precisely tailored by chemical modification of the building blocks. In order to investigate post-synthetic modification strategies, the methyl- and hydroxy-groups were used as reaction anchor points to covalently attach molecules after framework formation. The co-condensation of benzene-1,3,5-triyltriboronic acid (BTBA) and the 9,10-dimethyl-anthracene-2,3,6,7-tetraol (DMAT) succeeded in the formation of AT-COF-Me. In a radical bromination reaction the methyl groups of an anthracene linker were successfully brominated giving AT-COF-Br without degrading the crystalline framework of AT-COF-Me. The formation of the resulting benzylic bromine was monitored with IR spectroscopy and solid state NMR, respectively. Elemental analysis results correspond to the bromination of half the -CH3 groups. Reaction of (2',5'-dihydroxy-[1,1':4',1''-terphenyl]-4,4''-diyl)diboronic acid (HTDBA) and 2,3,6,7,10,11-hexahydroxytri-phenylene (HHTP) The terphenyl-based hydroxyl substituted T-COF-OH, formed by (2',5'-dihydroxy-[1,1':4',1''-terphenyl]-4,4''-diyl)diboronic acid (HTDBA) and 2,3,6,7,10,11-hexahydroxytri-phenylene (HHTP), was tested in several nucleophilic substitution reactions. Esterification of the –OH group was achieved with either acetylchloride or in a Steglich type reaction with 4-pentynoic acid. X-ray diffraction analysis after the post-synthetic modification shows that the crystallinity of the framework was preserved. This indicates that T-COF-OH is compatible with the reaction conditions. The detection of the newly formed ester moieties in IR and in solid state NMR spectra proves the successful post-synthetic esterification of the –OH groups. Another approach to tailor functionality in COFs is to assemble monomers with distinct properties in COF synthesis. Modification of the backbone of the framework was realized with two heterocyclic building blocks. Benzothiadiazole (BTD) and thienothiophene (TT) monomers are known as building blocks of semiconducting polymers. These molecules were equipped with boronic acid or boronate ester moieties in para position. The linkers were then used in co-condensation reactions with HHTP. The synthesis of BTD-COF was carried out in a two step microwave synthesis procedure: first the pinacolboronate 4,7-Bis(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)benzo[c][1,2,5]thiadiazole (BTDA) was cleaved with HCl, in a second step addition of HHTP resulted in the crystalline product in only 60 min. TT-COF was synthesized in a conventional co-condensation reaction of thieno[3,2-b]thiophene-2,5-diyldiboronic acid (TTBA) with HHTP; the black TT-COF showed aborbance over the whole spectrum of the visible light. Upon irradiation with light the system showed significant photoconductivity. The 3 nm pores of the hole-transporting TT-COF offer enough space to incorporate large fullerene-based electron-transporting materials such as PCBM. This inclusion leads to a significant quenching of the luminescence of TT-COF, indicating light-induced charge transfer at the interface of these two materials. The oriented growth of thin films of porous COF-10, a product of the condensation of 4,4’-biphenyldiboronic acid(BPBA) and HHTP, and TT-COF on self-assembled monomer (SAM)-functionalized gold surfaces is shown. Films grown on boronic acid terminated SAMs result in a parallel orientation of the pores along the substrate. Scanning electron microscopy was used to investigate the morphology of the films. Homogenous films with thicknesses of around 150 nm and a total coverage of the substrates were obtained. In summary, several functionalization strategies are shown to control or tune the topology and properties of Covalent Organic Frameworks. Tuning the topology and functionality to large open pore systems or intrinsic semiconductivity allows incorporation of large functional molecules and study the host-guest interactions. The post-synthetic modification of COFs offers a synthetic pathway to integrate organic functionalities, which cannot be synthesized directly by co-condensation. These strategies provide the means necessary for a precise control of the pore environment and design a porous material for specific applications. A facile and rapid method to produce thin oriented COF films will pave the way for this material to fabricate technological devices, such as photovoltaic devices, sensors of OFETs.

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No description available.

All proteins are synthesized in the cytoplasm. However some proteins function outside of cells, in the cell membrane, in the endoplasmic reticulum or one of the connected vesicular systems and need to be sorted and accordingly transported. Consequently, the secretory pathway, a highly conserved protein targeting, translocation and membrane insertion system has evolved to cope with this task. The central actor in the secretory pathway is the heterotrimeric Sec61αβγ protein complex also known as the translocon. This protein complex has been shown to act as a transmembrane channel facilitating protein translocation into the lumen of the endoplasmic reticulum, a starting point in protein secretion. The complex also contains a lateral opening, termed ‘lateral gate’ through which proteins have been suggested to insert into the membrane of the endoplasmic reticulum. The mechanics of translocon opening for protein secretion or membrane insertion has been a matter of debate. The results presented here show secondary structure level resolution cryo-electron microscopic structures of the translocon in complex with stalled ribosome-nascent chain complexes carrying substrates with propensity for either secretion or membrane insertion as well as ribosome-bound inactive translocon molecules. The observed differences in secondary structure suggest a model for translocon activity in protein sorting.

No description available.

No description available.