Podcasts about ddc:540

In order to understand in more depth and on a genome wide scale the behavior of transcription factors (TFs), novel quantitative experiments with high-throughput are needed. Recently, HiTS-FLIP (High-Throughput Sequencing-Fluorescent Ligand Interaction Profiling) was invented by the Burge lab at the MIT (Nutiu et al. (2011)). Based on an Illumina GA-IIx machine for next-generation sequencing, HiTS-FLIP allows to measure the affinity of fluorescent labeled proteins to millions of DNA clusters at equilibrium in an unbiased and untargeted way examining the entire sequence space by Determination of dissociation constants (Kds) for all 12-mer DNA motifs. During my PhD I helped to improve the experimental design of this method to allow measuring the protein-DNA binding events at equilibrium omitting any washing step by utilizing the TIRF (Total Internal Reflection Fluorescence) based optics of the GA-IIx. In addition, I developed the first versions of XML based controlling software that automates the measurement procedure. Meeting the needs for processing the vast amount of data produced by each run, I developed a sophisticated, high performance software pipeline that locates DNA clusters, normalizes and extracts the fluorescent signals. Moreover, cluster contained k-mer motifs are ranked and their DNA binding affinities are quantified with high accuracy. My approach of applying phase-correlation to estimate the relative translative Offset between the observed tile images and the template images omits resequencing and thus allows to reuse the flow cell for several HiTS-FLIP experiments, which greatly reduces cost and time. Instead of using information from the sequencing images like Nutiu et al. (2011) for normalizing the cluster intensities which introduces a nucleotide specific bias, I estimate the cluster related normalization factors directly from the protein Images which captures the non-even illumination bias more accurately and leads to an improved correction for each tile image. My analysis of the ranking algorithm by Nutiu et al. (2011) has revealed that it is unable to rank all measured k-mers. Discarding all the clusters related to previously ranked k-mers has the side effect of eliminating any clusters on which k-mers could be ranked that share submotifs with previously ranked k-mers. This shortcoming affects even strong binding k-mers with only one mutation away from the top ranked k-mer. My findings show that omitting the cluster deletion step in the ranking process overcomes this limitation and allows to rank the full spectrum of all possible k-mers. In addition, the performance of the ranking algorithm is drastically reduced by my insight from a quadratic to a linear run time. The experimental improvements combined with the sophisticated processing of the data has led to a very high accuracy of the HiTS-FLIP dissociation constants (Kds) comparable to the Kds measured by the very sensitive HiP-FA assay (Jung et al. (2015)). However, experimentally HiTS-FLIP is a very challenging assay. In total, eight HiTS-FLIP experiments were performed but only one showed saturation, the others exhibited Protein aggregation occurring at the amplified DNA clusters. This biochemical issue could not be remedied. As example TF for studying the details of HiTS-FLIP, GCN4 was chosen which is a dimeric, basic leucine zipper TF and which acts as the master regulator of the amino acid starvation Response in Saccharomyces cerevisiae (Natarajan et al. (2001)). The fluorescent dye was mOrange. The HiTS-FLIP Kds for the TF GCN4 were validated by the HiP-FA assay and a Pearson correlation coefficient of R=0.99 and a relative error of delta=30.91% was achieved. Thus, a unique and comprehensive data set of utmost quantitative precision was obtained that allowed to study the complex binding behavior of GCN4 in a new way. My Downstream analyses reveal that the known 7-mer consensus motif of GCN4, which is TGACTCA, is modulated by its 2-mer neighboring flanking regions spanning an affinity range over two orders of magnitude from a Kd=1.56 nM to Kd=552.51 nM. These results suggest that the common 9-mer PWM (Position Weight Matrix) for GCN4 is insufficient to describe the binding behavior of GCN4. Rather, an additional left and right flanking nucleotide is required to extend the 9-mer to an 11-mer. My analyses regarding mutations and related delta delta G values suggest long-range interdependencies between nucleotides of the two dimeric half-sites of GCN4. Consequently, models assuming positional independence, such as a PWM, are insufficient to explain these interdependencies. Instead, the full spectrum of affinity values for all k-mers of appropriate size should be measured and applied in further analyses as proposed by Nutiu et al. (2011). Another discovery were new binding motifs of GCN4, which can only be detected with a method like HiTS-FLIP that examines the entire sequence space and allows for unbiased, de-novo motif discovery. All These new motifs contain GTGT as a submotif and the data collected suggests that GCN4 binds as monomer to these new motifs. Therefore, it might be even possible to detect different binding modes with HiTS-FLIP. My results emphasize the binding complexity of GCN4 and demonstrate the advantage of HiTS-FLIP for investigating the complexity of regulative processes.

In the present study, the structure and mechanism of two assembly chaperones of Rubisco, Raf1 and RbcX, were investigated. The role of Raf1 in Rubisco assembly was elucidated by analyzing cyanobacterial and plant Raf1 with a vast array of biochemical and biophysical techniques. Raf1 is a dimeric protein. The subunits have a two-domain structure. The crystal structures of two separate domains of Arabidopsis thaliana (At) Raf1 were solved at resolutions of 1.95 Å and 2.6–2.8 Å, respectively. The oligomeric state of Raf1 proteins was investigated by size exclusion chromatography connected to multi angle light scattering (SEC-MALS) and native mass spectrometry (MS). Both cyanobacterial and plant Raf1 are dimeric with an N-terminal domain that is connected via a flexible linker to the C-terminal dimerization domain. Both Raf1 poteins were able to promote assembly of cyanobacterial Rubisco in an in vitro reconstitution system. The homologous cyanobacterial system resulted in very high yields of active Rubisco (>90%), showing the great efficiency of Raf1 mediated Rubisco assembly. Two distinct oligomeric complex assemblies in the assembly reaction could be identified via native PAGE immunoblot analyses as well as SEC-MALS and native MS. Furthermore, a structure-guided mutational analysis of Raf1 conserved residues in both domains was performed and residues crucial for Raf1 function were identified. A new model of Raf1 mediated Rubisco-assembly could be proposed by analyzing the Raf1-Rubisco oligomeric complex with negative stain electron microscopy. The final model was validated by determining Raf1-Rubisco interaction sites using chemical crosslinking in combination with mass spectrometry. Taken together, Raf1 acts downstream of chaperonin-assisted Rubisco large subunit (RbcL) folding by stabilizing RbcL antiparallel dimers for assembly into RbcL8 complexes with four Raf1 dimers bound. Raf1 displacement by Rubisco small subunit (RbcS) results in holoenzyme formation. In the second part of this thesis, the role of eukaryotic RbcX proteins in Rubisco assembly was investigated. Eukaryots have two distinct homologs of RbcX, RbcX-I and RbcX-II. Both, plant and algal RbcX proteins were found to promote cyanobacterial Rubisco assembly in an in vitro reconstitution system. Mutation of a conserved residue important for Rubisco assembly in cyanobacterial RbcX also abolished assembly by eukaryotic RbcX, underlining functional similarities among RbcX proteins from different species. The crystal structure of Chlamydomonas reinhardtii (Cr) RbcX was solved at a resolution of 2.0 Å. RbcX forms an arc-shaped dimer with a central hydrophobic cleft for binding the C-terminal sequence of RbcL. Structural analysis of a fusion protein of CrRbcX and the C-terminal peptide of RbcL suggests that the peptide binding mode of CrRbcX may differ from that of cyanobacterial RbcX. RbcX homologs appear to have adapted to their cognate Rubisco clients as a result of co-evolution. Preliminary analysis of RbcX in Chlamydomonas indicated that the protein functions as a Rubisco assembly chaperone in vivo. Therefore, RbcX was silenced using RNAi in Chlamydomonas which resulted in a photosynthetic growth defect in several transformants when grown under light. RbcX mRNA levels were highly decreased in these transformants which resulted in a concomitant decrease of Rubisco large subunit levels. Biochemical and structural analysis from both independent studies in this thesis show that Raf1 and RbcX fulfill similar roles in Rubisco assembly, thus suggesting that functionally redundant factors ensure efficient Rubisco biogenesis.

Deutsche Übersetzung des Titels: Chemische Synthese und enzymatischer Einbau von künstlichen Nukleotiden

Fri, 15 Apr 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19393/ https://edoc.ub.uni-muenchen.de/19393/1/Popp_Tobias_Alexander.pdf Popp, Tobias Alexander ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Mon, 11 Apr 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19353/ https://edoc.ub.uni-muenchen.de/19353/1/Pobel_Roman.pdf Pobel, Roman ddc:540, ddc:500, Fakultät für Chemie und Pharmaz

Mon, 11 Apr 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19343/ https://edoc.ub.uni-muenchen.de/19343/1/Marchuk_Alexey.pdf Marchuk, Alexey

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism (IEM) caused by mutations in the phenylalanine hydroxylase (PAH) gene. The molecular mechanism underlying deficiency of the PAH protein is, in most of the cases, loss of function due to protein misfolding. PAH mutations induce disturbed oligomerisation, decreased stability and accelerated degradation of hepatic PAH, a key enzyme in phenylalanine metabolism. Since the development of a phenylalanine-restricted diet in the 1950ies, PKU is a prototype for treatable inherited diseases. About 60 years later, the natural PAH cofactor tetrahydrobiopterin (BH4) was shown to act as a pharmacological chaperone stabilising the misfolded PAH protein. In consequence, BH4 (KUVAN®) was introduced to the pharmaceutical market as an alternative treatment for BH4-responsive PAH deficiency. Therefore, PKU is also regarded as a prototype for a pharmacologically treatable protein misfolding disease. Despite the progress in PKU therapy, knowledge on the molecular basis of PKU and the BH4 mode of action was still incomplete. Biochemical and biophysical characterisation of purified variant PAH proteins, which were derived from patient’s mutations, aimed at a better understanding of the molecular mechanisms of PAH loss of function. We showed that local side-chain replacements induce global conformational changes with negative impact on molecular motions that are essential for physiological enzyme function. The development of a continuous real-time fluorescence-based assay of PAH activity allowed for robust analysis of steady state kinetics and allosteric behaviour of recombinantly expressed PAH proteins. We identified positive cooperativity of the PAH enzyme towards BH4, where cooperativity does not rely on the presence of phenylalanine but is determined by activating conformational rearrangements. In vivo investigations on the mode-of-action of BH4 revealed differences in pharmacodynamics but not in pharmacokinetics between different strains of PAH-deficient mice (wild-type, Pahenu1/1 and Pahenu1/2). These observations pointed to a significant impact of the genotype on responsiveness to BH4. The available database information on PAH function associated with PAH mutations was based on non-standardised enzyme activity assays performed in different cellular systems and under different conditions usually focusing on single PAH mutations. These inconsistent data on PAH enzyme activity hindered robust prediction of the patient’s phenotype. Furthermore, assays on single PAH mutations do not reflect the high allelic and phenotypic heterogeneity of PKU with 89 % of patients being compound heterozygotes. In addition, the knowledge on enzyme function and regulation in the therapeutic and pathologic metabolic context was still scarce. In order to get more insight into the interplay of the PAH genotype, the phenylalanine concentration and BH4 treatment, we performed functional analyses of both, single, purified PAH variants as well as PAH full genotypes in the physiological, pathological and therapeutic context. The analysis of PAH activity as a function of phenylalanine and BH4 concentrations enabled determination of the optimal working ranges of the enzyme and visualisation of differences in the regulation of PAH activity by BH4 and phenylalanine depending on the underlying genotype. Moreover, these PAH activity landscapes allowed for setting rules for dietary regimens and pharmacological treatment based on the genotype of the patient. Taken together, precise knowledge on the mechanism of the misfolding-induced loss of function in PAH deficiency enabled a better understanding of the molecular mode of action of pharmacological rescue of enzyme function by BH4. We implemented the combination of genotype-specific functional analyses together with biochemical, clinical and therapeutic data of individual patients as a powerful tool for phenotype prediction and paved the way for personalised medicine strategies in phenylketonuria.

FK506 binding protein 5 (FKBP5) has been linked to stress related diseases and treatment response in depression (Binder et al., 2004). The corresponding protein FKBP51 was first identified as co-chaperone of HSP90 in a complex with steroid hormone receptors, where it diminishes hormone affinity and nuclear translocation efficiency of the receptors (Pratt and Toft, 1997; Wochnik et al., 2005). With FKBP5 transcription being induced by glucocorticoid signalling, an ultra-short feedback loop is provided for regulation and termination of GR activity. Dysregulation of this ultra-short feedback loop interferes with the stress hormone regulation and likely contributes to the association of FKBP5 with stress-related psychiatric disorders. Recently, important actions of FKBP51 beyond glucocorticoid signalling have been characterised in shaping the posttranslational regulation of certain molecular pathways in response to treatment with particular psychopharmaca (Gassen et al., 2014, 2015). As a contribution to elucidating the role of FKBP5 in stress related diseases, a two-sided approach was taken in this study by analysing the role of FKBP5 in regulation of transcription and in calibrating the responsiveness of these pathways to psychopharmacological treatment. To elucidate the transcriptional effects of FKBP5 in an unbiased approach, the expression profile of mice with deleted FKBP5 and their litter mates with functional FKBP5 were compared. A marked difference in glyoxalase-1 (GLO1) transcription was observed with higher GLO1 transcription in mice with deleted FKBP5, which was reflected by about two-fold more GLO1 protein in these mice. The efforts in deciphering the role of FKBP5 in elevation of GLO1 expression led to the identification of a duplication of the GLO1 gene inherent to mice with deleted FKBP5; this likely explains the enhanced GLO1 expression in these mice. This observation exemplifies the flanking gene problem and is a note of caution for interpreting data from conventionally generated knock-out mice. Overall, deletion of FKBP5 did not markedly change gene expression. In the second part of this thesis, the molecular effects of psychopharmacologic drugs were profiled for their dependency on FKBP51 function to modulate intracellular pathways relevant for treatment outcome in a cellular FKBP5 knockout model. For this purpose, psychopharmaca from the classes of SSRIs, SSNRIs, TCAs, atypical antidepressants, mood stabilisers, and NMDA receptor antagonists were analysed. In addition to GSK3β and AKT, which were reported to interact with and be targeted by FKBP51 recently (Gassen et al., 2015; Pei et al., 2009), ERK was identified as a novel kinase interacting with and being targeted by FKBP51 in this work. With GSK3β, AKT, and ERK, three major kinases were observed to be regulated by psychopharmaca. The effects were not homogeneous across all psychopharmaca and only loosely followed drug classes. Moreover, regulation of these kinases as well as their downstream targets was non-uniformly influenced by FKBP51. With FKBP51 being a stress induced gene, this transcriptional mechanism efficiently links the stress response to the regulation of the targets analysed in this work. Moreover, markers of autophagy, a cellular degradation process which has been linked to neurotransmission, were detected to be regulated by valproic acid (VPA), a mood stabiliser with HDAC inhibitory activity. VPA, as well as a second HDAC inhibitor butyric acid (BUT) enhanced the transcription of late and delayed autophagy markers controlled by FOXO3 signalling. Considering the versatile action of FKBP51 on targets analysed in this work, the list of proteins modulated by FKBP5 seems by far not complete. The diversity of effects evoked by different psychopharmaca hints to superimposed molecular effects underlying treatment outcome. Better understanding of pathway responsiveness could yield molecular markers for personalised medication that could be utilised to improve treatment outcome in stress related psychiatric diseases.

Fri, 4 Mar 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19229/ https://edoc.ub.uni-muenchen.de/19229/1/Ratzke_Elfrun_A.pdf Ratzke, Elfrun Almuth

The tetraspanin peripherin-2 is a glyco-membrane protein exclusively expressed in the outer segments of rod and cone photoreceptors. Mutations in peripherin-2 are associated with retinal disorders characterized by Degeneration of rod or cone cells. Previous unpublished work identified peripherin-2 as a potential novel part of the protein complex comprising the B-subunit of the cyclic nucleotide-gated channel (CNGB1a and the light detector rhodopsin. In the first part of this study, using a combination of protein biochemical and FRET approaches in transfected HEK293 cells and in virally transduced murine rod outer segments, it could be demonstrated that peripherin-2 simultaneously binds to both, CNGB1a and rhodopsin. The interaction between peripherin-2 and rhodopsin was not described in previous studies. The binding domain mediating the peripherin-2/rhodopsin interaction could be narrowed down to the fourth transmembrane domain (TM4) of peripherin-2. Finally, the data revealed that the G266D point mutation in TM4 of peripherin-2 that is linked to a rod degenerative disease selectively disrupts the peripherin-2/rhodopsin interaction. To analyze if peripherin-2 also binds to cone opsins in the second part of this study, a similar experimental approach was conducted as used for the investigation of the peripherin-2/rhodopsin interaction. In this context, it was unveiled that peripherin-2 binds to both, short wavelength-and medium wavelength-sensitive cone opsin (S-opsin and M-opsin, respectively) in transfected HEK293 cells and in outer segments of transduced murine cones. Co-immunoprecipitation and quantitative FRET analysis revealed that binding of peripherin-2 to M-opsin was stronger than the peripherin-2/S-opsin interaction. This result was supported by transmission electron microscopy studies using gold particles coupled to opsin- and peripherin-2-specific antibodies. Finally, quantitative FRET analysis in transfected HEK293 cells and in transduced cone outer segments demonstrated that the V268I Point mutation in TM4 of peripherin-2 associated with a degenerative cone disease significantly attenuates the peripherin-2/M-opsin interaction. Taken together, this study provides a proof-of-principle for FRET-based analysis of protein-protein interactions in the outer segments of rod and cone photoreceptors. This approach led to the identification of hitherto unknown Protein complexes between peripherin-2 and opsins suggesting a novel physiological role of peripherin-2 in rods and cones. Finally, Analysis of disease-linked point mutations unveiled the molecular determinants of the peripherin-2/opsin interaction. These results might contribute to understanding the differential penetrance of certain point mutations in rods and cones.

Fluorescence microscopy is a widely used technique for imaging of biological structures due to its noninvasiveness although resolution of conventional fluorescence microscopes is limited to about 200-300 nm due to the diffraction limit of light. Super-resolution fluorescence microscopy offers an extension of the original method that allows optical imaging below the diffraction limit. In this thesis, a microscope for localization-based super-resolution fluorescence microscopy techniques such as Stochastic Optical Reconstruction Microscopy (STORM) or Photoactivated Localization Microscopy (PALM) was established. An epifluorescence microscope was built for this purpose that provides both widefield and Total Internal Reflection Fluorescence (TIRF) excitation modalities and focus was put on the special requirements of localization-based super-resolution methods. This included a high mechanical and optical stability realized by a compact design and implementation of a home-built perfect focus system. The setup was further designed to allow both two- and three-dimensional imaging. The work also included both the development of a setup control software and a software for the analysis of the required data. Different analysis methods and parameters were tested on simulated data before the performance of the microscope was demonstrated in two and three dimensions at appropriate test samples such as the cellular microtubule network. These experiments showed the capability of super-resolution microscopy to reveal underlying structures that cannot be resolved by conventional fluorescence microscopy. Resolutions could be achieved down to approximately 30 nm in the lateral and 115 nm in the axial dimension. Subsequently, the established method was applied to two biological systems. The first is a study of the budding of the human immunodeficiency virus type 1 (HIV-1) from the host cell. In this step of the viral reproduction cycle, the virus hijacks the cellular endosomal sorting complex required for transport (ESCRT) machinery to achieve membrane fission. ESCRT consists of the subcomplexes ESCRT-0, -I, -II and -III and additional related proteins, from which HIV-1 recruits certain components. The fission process is initiated by the HIV-1 Gag protein, which recruits the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX to the virus assembly site. Subsequently, ESCRT-III proteins CHMP4 and CHMP2 form transient lattices at the membrane, which are actively involved in membrane fission. However, the actual geometry of the ESCRT machinery assembling at the HIV-1 budding site that is driving the fission process is still not fully understood. Different models proposed either constriction of the budding neck by lattices surrounding the neck, by ESCRT structures within the neck or within the bud itself. A problematic aspect in previous studies was the usage of modified, tagged versions of the involved proteins for visualization. In this study, super-resolution microscopy was therefore applied to endogenous Tsg101, ALIX and CHMP2 isoform CHMP2A and to a version of CHMP4 isoform CHMP4B with a small HA-tag to elucidate the size and the distribution of the structure relative to the HIV-1 assembly sites. ESCRT structures colocalizing with HIV-1 exhibited closed, circular structures with an average size restricted to 45 and 60 nm in diameter. This size was significantly smaller than found for HIV-1 assembly sites and the constriction of the size, which was not observed for non-colocalizing ESCRT structures at the cell membrane, ruled out an external restriction model. Super-resolution imaging of ALIX often revealed an additional cloud-like structure of individual molecules surrounding the central clusters. This was attributed to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Together with experiments that confirmed the non-physiological behavior of tagged Tsg101 and a relative orientation of ESCRT clusters towards the edge of the assembly site, the results strongly point toward a within-neck model. A second project focused on the influence of external constriction on cell migration. The latter plays an important role in various processes in the human body ranging from wound healing to metastasis formation by cancer cells. Migration is driven by the lamellipodium, which is a meshwork of fine actin filaments that drive membrane protrusion. Endothelial cells were grown on micropatterns that confined the freedom of movement of the cells. Three-dimensional super-resolution imaging revealed that the lamellipodia of these cells showed a much broader axial extension than was the case for control cells that grew without confinement of migration. The different organization of the actin filament network showed a clear effect of environmental conditions on cellular migration. Overall, it was possible to build a super-resolution fluorescence microscope over the course of this study and establish the required analysis methods to allow STORM and PALM imaging below the diffraction limit of light. Two applications further showed that these tools are capable of answering currently discussed questions in the biological sciences.

Wed, 17 Feb 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19241/ https://edoc.ub.uni-muenchen.de/19241/1/Kick_Kerstin.pdf Kick, Kerstin ddc:540, ddc:500, Fakultät für Chemie und Pharmazie 0

Endothelial cell (EC) migration is an essential process in angiogenesis as ECs sprout from preexisting vessels, following chemotactic gradients. However, most of the data obtained about EC migration has been acquired in artificial two dimensional (2D) cell culture environments. Recent reports showed that migration in fibrillary environments can be mimicked by spatial confinement, achieved by micro patterning techniques (Doyle et al. 2009). In the first part of this work it was investigated whether a model system based on linearly structured surfaces allows to draw conclusions about the migration of ECs in fibrillary 3D collagen matrices. In order to estimate the cellular behavior of ECs on linearly structured surfaces, a comprehensive cell biological analysis was performed. ECs on narrow 3 µm wide tracks (also termed 1D in the following) migrated less efficient in comparison to ECs on broader tracks in regard to mean velocity, persistence, and run velocity. Additionally, ECs in 1D displayed a distinct actin cytoskeleton architecture, compressed nuclei, and different orientation of the centrosome in comparison to ECs on wider tracks. The frequent directional changes of ECs on narrow tracks were accompanied by pronounced membrane blebbing, while migrating and elongated cells displayed a lamellipodium as cellular protrusion. This behavior was contractility-dependent as both modes were provoked by using Blebbistatin or Calyculin A, respectively. The comparison between 1D and 3D migrating cells revealed a striking similarity in actin cytoskeleton architecture and in switching between two morphological modes. Cells migrating in 3D moved slower but more persistent after Blebbistatin treatment, which was likewise the case for cells migrating in 1D. In contrast to this, cells in the 2D system migrated faster but less persistent after Blebbistatin treatment. A Rac1 inhibitor used in this study showed the tendency to influence the migratory potential similarly in 1D and 3D, in contrast to 2D. However, a microtubule disrupting agent displayed different effects in 1D and 3D. These experiments demonstrated that the 1D system allows to draw conclusions about certain aspects of 3D migration. Thus, using this 1D migration system, important aspects of 3D migration can be mimicked in a highly controlled setting. In the second part of this work, a system for artificial tip cell formation was investigated. For the analysis of tip and stalk cells specifically structured surfaces were designed. These structures provided areas allowing only a restricted number of cell-cell contacts and areas allowing a high number of cell-cell contacts. ECs with a low number of cell-cell contacts displayed increased VEGFR2 expression levels in comparison to cells with a high number of cell-cell contacts, a phenomenon which was inhibited by using a Notch signaling inhibitor. This system will be a useful tool in the future to decipher tip and stalk cell competition within a defined cellular population and a defined microscopic frame

Eukaryotic genomes are organized inside the cell nucleus in a structured macromolecular DNA-protein polymer named chromatin, formed by single discrete unites called Nucleosomes. The packing of the genetic information into chromatin allows the efficient regulation of several nuclear processes, such as gene expression and transcription, DNA replication, cell cycle progression, chromosome segregation and DNA damage repair. Chromatin comes in two flavors: a transcriptionally active, more loosened state, called euchromatin and a transcriptionally silent or low expressed, more compact state, called heterochromatin. The assembly of silent chromatin or heterochromatin is fundamental for the regulation of every nuclear process and it is driven in most Eukaryotes by the deposition and the read-out of the histone H3 lysine 9 methylation (H3K9me) post-translational modification (PTM). H3K9me on the nucleosome is specifically bound by chromatin readers called chromodomains (CD) and this recognition is fundamental for the downstream processes that lead to the formation of heterochromatin and shut down the expression of single genes or entire gene clusters. Despite several studies have been done on different chromodomains binding to H3K9me histone tail peptides, to date there was no structural information on how chromodomains interact with their natural binding partners, the H3K9me3 Nucleosomes. In a preliminary structural study carried out in our laboratory we solved the cryo-electron microscopy (Cryo-EM) structure of the chromodomain of the fission yeast Chp1 protein (Chp1CD) in complex with an H3K9me nucleosome. The structure showed that the Chp1CD interacts not only with the histone H3 tail but also with the histone globular domains in the Nucleosome core, primarily with histone H3. Mutations in the residues of Chp1CD that form the binding interface with the Nucleosome core (two loops in the β-sheet of the domain) caused a drop of the affinity in vitro for the H3K9me Nucleosome, which was independent from the histone H3K9me tail interaction. Cells harboring the same Chp1CD loop mutations were defective in silencing centromeric transcripts and maintain the deposition of the H3K9me mark for heterochromatin formation. This indicated that Chp1CD-nucleosome core interaction is fundamental for heterochromatin formation in fission yeast and opened up to the possibility that chromodomains could read multiple histone PTMs, on both the recruiting histone tail and on the nucleosome core. This study substantially contributes to understand how chromodomains interact with chromatin, how much the nucleosome core interaction is conserved among different CDs and how different chromodomain proteins are regulated at the same loci. Understanding how chromodomain readers recognize nucleosomes is fundamental to uncover the basics of gene silencing and heterochromatin formation.

Mon, 1 Feb 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19111/ https://edoc.ub.uni-muenchen.de/19111/1/Hiekel_Anian.pdf Hiekel, Anian

Tue, 26 Jan 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19146/ https://edoc.ub.uni-muenchen.de/19146/1/Preimesser_Andreas.pdf Preimesser, Andreas ddc:540, ddc:500, Fakultät für Chemie und P

Tue, 26 Jan 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19146/ https://edoc.ub.uni-muenchen.de/19146/1/Preimesser_Andreas.pdf Preimesser, Andreas ddc:540, ddc:500, Fakultät für Chemie und P

Thu, 14 Jan 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19267/ https://edoc.ub.uni-muenchen.de/19267/2/Gnerlich_Felix.pdf Gnerlich, Felix ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Mon, 21 Dec 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19020/ https://edoc.ub.uni-muenchen.de/19020/1/Saller_Verena.pdf Saller, Verena ddc:540, ddc:500, Fakultät für

Tue, 15 Dec 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19287/ https://edoc.ub.uni-muenchen.de/19287/1/Ranft_Annekathrin.pdf Ranft, Annekathrin ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Die Gegenwart von Kohlenhydraten in biologischen Systemen ist von großer Bedeutung für eine Reihe von zellulären Zyklen, so z.B. die Zell-Zell-Erkennung, die Regulation von Proteinaktivitäten oder die Funktion von Kohlenhydraten als Liganden für die Zelladhäsion. Ferner besitzen eine Reihe von mucinartigen O-Glycanen vielfältige Funktionen in immunologischen Prozessen, welche mit unterschiedlichsten Krankheitsbildern, wie z.B. der Karzinogenese sowie Autoimmun- und Infektionskrankheiten einhergehen. Im Besonderen nehmen viele mucinartige Glycane mit funktionellen Oligosaccharidketten, einschließlich Sialinsäure-tragenden Strukturen wie z.B. Glycophorin, Leukosialin (CD 43 oder auch Sialophorin), Sialyl-Lewis x und Sialyl-Lewis a eine entscheidende Rolle als Liganden in Zell-Zell-Erkennungsmechanismen bei inflammatorischen Prozessen, T-Zell-Differenzierung und Krebsmetastasierung ein. Jüngst wurde eine Reihe innovativer Ansätze für die Immuntherapie von Krebs durch ein besseres Verständnis der abweichenden Glycosylierungsmuster von Glycoproteinen und Glycolipiden auf der Oberfläche von Krebszellen entwickelt, wobei ein Schwerpunkt der Arbeiten auf der Herstellung von kohlenhydrat-basierten Krebsvakzinen ruht. Vor diesem Hintergrund beschäftigt sich die folgende Arbeit mit der Synthese von Epitopen des Sialophorin (CD 43), einem bedeutenden O-Glycan-enthaltenden Sialoglycoprotein, welches auf Leukozyten exprimiert vorliegt und mit einer Reihe von Krankheitsbildern, wie rheumatoider Arthritis, Leukämie, dem Wiskott-Aldrich-Syndrom (WAS) und dem erworbenen Immunschwächesyndrom (AIDS), einhergeht. Der Aufbau komplexer Hexasaccharid-tragender Glycokonjugate konnte hierbei ausgehend von kommerziell erhältlichen Startmaterialien erfolgen. Im Vordergrund stand dabei die Entwicklung eines biomimetischen Synthesewegs, mit dessen Hilfe neben der natürlichen Struktur auch eine Reihe fluorierter Sialophorin-Derivate mit unterschiedlich fluorierten D-Galactose-Bausteinen in den beiden Trisaccharid-Einheiten hergestellt werden können. Dabei sollten sämtliche Glycosylaminosäuren mit einem zur Festphasensynthese kompatiblen Schutzgruppenmuster ausgestattet vorliegen, um einen Einbau in die N-terminale Partialsequenz des Glycoproteins Sialophorin (CD 43) zu erlauben. Beginnend mit der Synthese der unterschiedlich fluorierten D-Galactose-Derivate, der Glucosamin- und der Sialinsäure-Bausteine sollten die regio- und stereoselektiv notwendigen Di- und Trisaccharid-Bausteine chemisch verknüpft werden. Zu diesem Zweck wurden literaturbekannte Königs-Knorr- und Schmidt-Glycosylierungsreaktionen sowie stereoselektive Sialylierungsreaktionen unter Ausnutzung des Nitrileffekts genutzt. Ein weiterer Meilenstein war die regioselektive Glycosylierung der beiden Trisaccharid-Bausteine zu den geschützten Hexasaccharid-Threonin-Konjugaten, die damit für den Einbau in die N-terminale Partialsequenz des Sialoglycoproteins Sialophorin zur Verfügung stehen. Zudem wurden fluorierte sialylierte Trisaccharid-Threonin-Konjugate in die tandem repeat-Sequenz des epithelialen Mucins MUC1 eingebaut, die als potentielle Kandidaten neuer Antitumorvakzine eingesetzt werden können.

Thu, 3 Dec 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19027/ https://edoc.ub.uni-muenchen.de/19027/1/Dittrich_Sebastian.pdf Dittrich, Sebastian ddc:540, ddc:500, Fakultät für Ch

Mon, 30 Nov 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18948/ https://edoc.ub.uni-muenchen.de/18948/1/Neuhofer_Christian.pdf Neuhofer, Christian ddc:540, ddc:500, Fakultät für Ch

Most renewable energy sources suffer from intermittency and have to be coupled with sophisticated energy conversion and storage technologies. An elegant solution is offered by photoelectrochemical water splitting, where solar energy is directly converted into chemical energy by splitting water into oxygen and the energy carrier hydrogen. Photoelectrochemical water splitting requires two photoelectrodes which are immersed in an aqueous electrolyte. These photoelectrodes are semiconductors with valence and conduction bands straddling the redox potential of water. Upon illumination, electrons and holes are produced, separated and transferred to the electrolyte, leading to the evolution of oxygen at the photoanode and the evolution of hydrogen at the photocathode. The resulting hydrogen can be stored, transported and then either burnt in fuel cells to regain electrical energy or used for industrial applications like the Haber-Bosch process. The photoelectrodes are often nanostructured to increase the surface area, at which the reaction takes place. This strategy has been realized with several morphologies such as nanotubes, inverse opals, etc. and has often lead to performance increases of several hundred percent. Therefore, detailed knowledge of the morphology is important and can be obtained by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TEM is a powerful technique that allows imaging samples with a resolution down to the sub-Ångstrom scale. In addition, TEM can be combined with spectroscopic methods such as electron energyloss spectroscopy (EELS) and energy-dispersive X-ray spectroscopy (EDX) to quantify the chemical composition. In this thesis, three different materials systems were studied by TEM: noble metal nanoparticles on TiO2 for hydrogen evolution with the sacrificial agent MeOH, Fe2O3/WO3 dual absorber photoanodes and photocathodes out of the novel material FeCrAl oxide. Titania is one of the most researched photoanode materials. However, it only absorbs UV light. Au and Au/Ag core-shell nanoparticles were deposited by the project partners Michael Karnahl and Sandra Peglow of the LIKAT and the INP Greifswald, respectively, on anatase thin films by photodeposition and radio frequency magnetron sputtering. These noble metal nanoparticles absorb visible light by surface plasmon resonance and also act as co-catalysts for electrons excited in the titania and injected into them. Cross-section were prepared for a detailed TEM investigation of the microstructure. The distribution of the nanoparticles varied greatly with the synthesis method, as photodeposited particles grew in and on top of the titania, whereas the plasma-deposited nanoparticles only grew on top. Different growth and coarsening mechanisms could be identified and correlated to the synthesis conditions by careful particle size distribution determination. In addition to defect-free nanoparticles, several defects such as five-fold twinning, grain boundaries and stacking faults were found. The TEM analysis was complemented by optical absorption and photocatalysis measurements, and the synthesis as well as the properties could be correlated to microstructural features. Due to its narrow band gap, hematite is a popular photoanode material. However, it also has several disadvantages, which were addressed by several studies. Tin-doping increased the transfer efficiency and therefore the photocurrent, with the tin being enriched at the surface of the hematite nanoparticles and hinting at a structure-function relationship. Deposition of a Co3O4 co-catalyst and the introduction of a conductive scaffold all further increased the photocurrent. Another performance-increasing approach, combining multiple photocatalytically active materials, was tested with Fe2O3/WO3 dual absorbers prepared by Ilina Kondofersky of the group of Prof. Thomas Bein. WO3 was systematically applied as a scaffold and/or as a surface treatment. The arrangement of the different materials and the interfaces between them was studied in detail by TEM. Both the host-guest approach and the surface treatment strongly increased the performance compared to the pure materials and several beneficial interactions could be identified. For example, WO3 strongly scatters visible light, resulting in increased absorption by Fe2O3 and higher current densities. We also determined a cathodic shift in the onset potential to 0.8 V and, compared to pure Fe2O3, increased transfer rates of up to 88 %, and can therefore conclude that the Fe2O3/WO3 dual absorbers are a very promising system. In spite of all the different performance-enhancing strategies developed so far, it is becoming apparent that all currently available materials, regardless of how heavily they are improved, will not reach sufficient performances. This has led to the search for novel materials and in this thesis, meso- and macroporous photocathodes with the overall stoichiometry Fe0.84Cr1.0Al0.16O3 were investigated in close cooperation with Ilina Kondofersky. Using TEM cross-sections, a phase separation into Fe- and Cr-rich phases was observed for both morphologies and could be correlated to the precursor stabilities. In comparison to the mesoporous layer, the macroporous photocathode had a significantly increased charge collection efficiency and therefore performance, proving the benefits of tuning the morphology. In all studies, performance-increasing strategies were successfully applied and we found the performance to depend heavily on the morphologies. By combining the results of all techniques, insight into the complex interplay between synthesis conditions, morphology and properties could be achieved and the gained knowledge is expected to benefit future work.

Fri, 13 Nov 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18951/ https://edoc.ub.uni-muenchen.de/18951/1/Matthias_Philipp.pdf Matthias, Philipp ddc:540, ddc:500, Fakultät für Chemie und Ph

Mon, 26 Oct 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18820/ https://edoc.ub.uni-muenchen.de/18820/1/Anamur_Cihad.pdf Anamur, Cihad ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Tue, 20 Oct 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18862/ https://edoc.ub.uni-muenchen.de/18862/1/Birk_Gudrun.pdf Birk, Gudrun ddc:540, ddc:500, Fakultät für Chemie und Pharma

Today’s carbon-based economy will not be sustainable in the future. Not only will the known reserves of fossil fuels, like oil, natural gas or coal, be significantly reduced within the next 100 years, but the continued burning of fossil fuels also emits greenhouse gases, which have led to a global increase in temperature, called global warming. To preserve the environment for future generations and to prepare for the time when we will inevitably run out of fossil fuel, we have to change the way we produce our primary energy and focus research and investments on renewable energy sources. While energy from wind and water is already harvested with very high efficiencies, the utilization of solar energy still offers big room for improvements. Although conventional crystalline silicon cells achieve efficiencies around 25 %, their production is very energy intensive and relies on advanced production technologies, which makes them still rather expensive. To make photovoltaics a major part of our energy landscape, an easily prepared type of solar cell consisting of cheap and abundant materials is required. Novel organometal halide perovskite-type materials fulfill these requirements and have proven to be serious competitors for conventional photovoltaics. After only four years of research they already achieve power conversion efficiencies above 20 %. This thesis introduces a fast and easy way to prepare planar heterojunction solar cells based on methylammonium lead iodide (MAPbI3). The photoactive layer is deposited in a 2-step deposition approach, where a thin film of the lead precursor is converted into the final perovskite simply by immersing it into a solution of the other component. The resulting films consist of individual crystals sizes a few 100 nm and covering the whole substrate without significant gaps or holes. Solar cells prepared by this method achieve power conversion efficiencies of 15 %. Furthermore, by adjusting the temperature of the immersion bath, the orientation of the perovskite crystals can be controlled. The orientation, together with the resulting change in efficiency and resistance, gives interesting insights into the anisotropic charge transport properties of this class of materials. Additionally, the conventionally used hole blocking layer, titanium dioxide, was replaced by one made of fullerene molecules. The efficiencies achieved by solar cells employing this kind of electron selective contact reached almost 10 %, although the reproducibility was initially very low. This was attributed to a partial dissolution of the fullerene film during the subsequent preparation steps. To increase the stability of the layer, it was photo-polymerized using UV radiation. This not only reduces the solubility and therefore increases the fraction of solar cells achieving high efficiencies; it also changed the energy levels close to the bandgap. The bandgap energy of organic lead halide perovskite materials is strongly dependent on the composition. By exchanging some or all of the iodide in MAPbI3 with bromide, the difference between valence and conduction band can be changed from 1.5 eV (pure iodide) to 2.25 eV (pure bromide). This substitution can be performed gradually, so that phase pure materials with properties in between the two extremes are obtained. The pure bromide MAPbBr3 perovskite, however, does not perform efficiently in a planar heterojunction solar cell. Its close relative based on formamidinium FAPbBr3 has also been investigated for its suitability as active solar cell material. Although it is structurally very similar to MAPbBr3, with equivalent light absorption and emission properties, a 10 fold higher efficiency was observed for the FA-based compound. This striking difference is mainly attributed to an increased photoluminescence lifetime, resulting in an increased diffusion length of the free charge carriers. Apart from their application as light absorbing materials in solar cells, perovskites have also been investigated for their application as light emitters. Depending on the perovskite used, it was possible to demonstrate red light emission (MAPbI3) or green emission (MAPbBr3).

Wed, 14 Oct 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18795/ https://edoc.ub.uni-muenchen.de/18795/1/Rest_Sebastian_F.pdf Rest, Sebastian Franz ddc:540, ddc:500, Fakultät für Chemie und P

Mon, 12 Oct 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18907/ https://edoc.ub.uni-muenchen.de/18907/1/Barth_Florian.pdf Barth, Florian ddc:540, ddc:500, Fakultät für Chemie und Phar

Idiopathic pulmonary fibrosis (IPF) is an irreversible and progressive disease of the lungs, which is characterized by aberrant tissue remodeling and massive deposition of extracellular matrix proteins. This process is mainly conducted by myofibroblasts, an activated fibroblast phenotype. During the pathogenesis of IPF, the fine alveolar structure is destroyed and gas exchange declines, finally resulting in organ failure. So far, pharmacological treatment options are very limited and lung transplantation still remains the only curative therapy. Pathologic tissue remodeling in IPF is closely connected to altered cell and protein homeostasis. The ubiquitin-proteasome system is critical for degradation of polyubiquitinated proteins in a spatially and timely controlled manner, thereby regulating protein levels. The proteasome is a multicatalytic enzyme complex consisting of a barrel shaped 20S catalytic core particle (CP) and one or two 19S regulatory particles (RP), thus forming active 26S/30S proteasomes. Dysregulation of the proteasome has been reported for several chronic diseases of the heart, brain, and also lung. Furthermore, inhibition of the proteasome has been shown to provide antifibrotic effects in different organs, including the lung. As nothing is known about proteasome function in the pathogenesis of IPF, the first aim of the present study was to analyze proteasomal regulation during tissue remodeling and myofibroblast differentiation. For that, lung fibroblasts were treated with transforming growth factor-β (TGF-β) and proteasome activity as well as composition was examined. For in vivo testing, the bleomycin mouse model of lung fibrosis was used and human lung tissue of IPF patients was analyzed. It was found that induction of myofibroblast differentiation by TGF-β mediated assembly of 19S RPs with 20S CPs, thereby forming 26S/30S complexes, which was critically dependent on the regulatory particle non ATPase 6 subunit (Rpn6). In addition, silencing of Rpn6 in primary human lung fibroblasts counteracted TGF β induced myofibroblast differentiation. During bleomycin-induced fibrotic remodeling of mouse lungs, increased formation of 26S/30S proteasomes was accompanied by augmented expression of Rpn6 in fibrotic lungs. Here, Rpn6 was highly expressed in hyperplastic alveolar epithelial cells and Clara cells. Overexpression of Rpn6 was also observed in myofibroblasts and hyperplastic bronchiolar basal cells of fibrotic lung tissue of IPF patients and accompanied by enhanced polyubiquitination of proteins. As therapeutic application of proteasome inhibitors in pulmonary fibrosis showed controversial results including beneficial antifibrotic effects but also toxicity, the second aim of this study was to test whether site specific inhibition of the proteasome, using the novel second generation inhibitor oprozomib, provides antifibrotic effects in the absence of systemic side effects after local pulmonary application. Oprozomib was compared to the FDA-approved proteasome inhibitor bortezomib and tested on the human alveolar epithelial cancer cell line A549 and on primary mouse alveolar epithelial type II cells regarding its cytotoxic effects. Oprozomib was less toxic than bortezomib and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, oprozomib showed significant antifibrotic effects like reduction of collagen I and α-smooth muscle actin expression at non-toxic doses. When applied locally into the lungs of healthy mice via instillation, oprozomib was well tolerated and effectively reduced pulmonary proteasome activity. In bleomycin-challenged mice, however, locally applied oprozomib resulted in accelerated weight loss and increased mortality. Furthermore, oprozomib failed to reduce fibrosis in these mice, but rather augmented fibrotic lung remodeling in bleomycin-challenged animals. To conclude, this study identified a novel mechanism for fibrotic remodeling of the lungs involving 26S/30S proteasome activation via Rpn6 upon TGF-β-mediated myofibroblast differentiation. Increased levels of Rpn6 and polyubiquitinated proteins in IPF lungs further suggest an important contribution of the ubiquitin-proteasome system to the pathogenesis of this disease. Inhibition of the proteasome with the novel site-specific proteasome inhibitor oprozomib provided low toxicity and antifibrotic effects in alveolar epithelial cells and pulmonary fibroblasts. These results could not be confirmed in pulmonary fibrosis of bleomycin-treated mice, as oprozomib treatment showed high toxicity in fibrotic animals. In light of these data, current proteasome inhibitors, which block the catalytic core, might be too toxic as therapeutic agents for the treatment of fibrotic lung diseases. However, interference with the formation of 26S/30S proteasomes, as shown by Rpn6 knockdown, might provide a novel concept for therapeutic regulation of proteasome activity in lung fibrosis.

Wed, 30 Sep 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19068/ https://edoc.ub.uni-muenchen.de/19068/1/Niesser_Juergen_G.pdf Niesser, Jürgen Gerd ddc:540, ddc:500, Fakult

Fri, 18 Sep 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18799/ https://edoc.ub.uni-muenchen.de/18799/1/Moriya_Kohei.pdf Moriya, Kohei ddc:540, ddc:500, Fakultät für Chemie und Phar

Mon, 7 Sep 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18665/ https://edoc.ub.uni-muenchen.de/18665/1/Stuerzer_Christine.pdf Stürzer, Christine ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Porous materials play an important role in numerous environmental applications including energy storage, energy conversion and environmental remediation systems. Reducing structural features down to the nanoscale drastically alters materials properties and leads to the enhancement of materials performance. The successful fabrication of efficient functional materials requires a high degree of control over their morphology addressing the needs of target applications. The goal of this work was to develop a versatile general approach towards the synthesis of nanoporous metal oxides by using biogenic cellulose nanocrystals. Nanocrystalline cellulose (NCC) is an abundant biological nanomaterial that can be extracted from natural bulk celluloses. The present thesis demonstrates that the unique properties of NCC enable the efficient synthesis of porous titania and iron oxide (hematite) thin films by using sacrificial templating with cellulose nanocrystals. In particular, this study reveals the mechanism of metal oxide formation in the presence of cellulose, as well as the effect of NCC-templated porous scaffolds on titania performance in photocatalysis and dye sensitized solar cells. Chapter 1 provides general information about properties, application areas and common synthesis methods of nanoporous metal oxides, with an emphasis put on titanium oxide materials and biotemplating approaches. Chapter 2 discusses the basic principles of analytical methods employed to characterize porous nanomaterials. Chapters 3‒6 reveal the experimental procedures towards NCC-templated porous titania and hematite thin films, their characterization and their applications. First, the extraction of cellulose crystals from bulk celluloses is discussed. Different cellulose sources, as well as variable hydrolysis parameters have been employed to define the optimal procedure for the NCC preparation. Cotton fibers have provided the best results regarding the crystallinity, purity and shape of extracted cellulose crystals. Furthermore, repeated washings have been shown to narrow down the size distribution and to improve the crystallinity of cotton NCC. Chapter 4 focuses on the synthesis of porous titania thin films assisted by nanocrystalline cellulose. The tunable porosity of titania thin films is a key factor for successful applications in photovoltaics, sensing and photocatalysis. To synthesize NCC-templated titania, the cellulose nanocrystals are introduced to a titania precursor solution. The colloidal mixtures can be directly spin- or dip- coated on glass, silicon and transparent conducting oxide (TCO) substrates and then calcined to remove the template and to crystallize the titania porous network. The obtained structures are highly porous anatase morphologies having well-defined, narrow pore size distribution. We show that by varying the titania-to-template ratio it is possible to tune the surface area, pore size, pore anisotropy and dimensions of titania crystallites in the films. Post-treatment at high humidity and subsequent slow template removal promote pore widening; this treatment is also beneficial for the multilayer deposition of thick films. The NCC-templated mesoporous titania films show very high activity in the photocatalytic NO (nitrogen(II) oxide) conversion and in the degradation of 4-chlorophenol. Furthermore, the films are successfully applied as anodes in dye-sensitized solar cells. Chapter 5 presents a strategy toward enhancement of the photocatalytic activity of NCC-templated titania thin films by introducing solvothermally synthesized preformed anatase nanoparticles into a sol-gel based biotemplated titania scaffold. The synthesis is based on the self-assembly of two types of precursors, namely crystalline and sol-gel titania, directed by the biogenic NCC template. Due to the shape persistence of the template, the NCC-templated titania scaffolds can accommodate large amounts of preformed titania without a significant reduction of the film porosity. The resulting dual source titania thin films containing different amounts of preformed crystalline species were investigated with time resolved microwave conductivity (TRMC) measurements and tested in the photocatalytic conversion of 4-chlorophenol. The gradual addition of preformed nanoparticles leads to a consistent increase of the mean size of titania crystalline domains, whereas the porosity of the composite is well-preserved due to the rigid nature of the NCC template. The microwave conductivity studies establish increased photoconductivity of the films containing preformed anatase nanoparticles, in comparison to that of films made without the nanoparticles. The synergistic features of the dual source titania, namely the improved crystalline properties brought by the preformed nanocrystals in combination with the high surface area provided by the NCC-templated sol-gel titania, result in a very high photocatalytic activity of the films in the photocatalytic decomposition of 4-chlorophenol. In quantitative terms, the dual source titania films prepared with 75% nanoparticles exhibit a first order degradation rate constant of 0.53 h-1, strongly outperforming the activity of commercial P90 nanopowder showing a rate constant of 0.17 h-1 under the same conditions. We have also adapted the NCC templating protocol for the fabrication of porous iron oxide (hematite) thin films. Chapter 6 discusses the formation of porous iron oxide nanostructures via sol-gel transformations of molecular precursors in the confined space of self-organized cellulose nanocrystals used as a shape-persistent template. The obtained structures are highly porous hematite morphologies featuring pronounced anisotropic porosity. The character of the porous nanostructure depends on the iron salt used as precursor and on the heat treatment, respectively. Moreover, a post-synthetic hydrothermal treatment of the NCC/iron salt composites strongly affects the crystal growth, as well as the porous nanomorphology of the obtained hematite scaffolds. We demonstrate that the hydrothermal treatment alters the crystallization mechanism of the molecular iron precursors, which proceeds via the formation of anisotropic iron oxyhydroxide species. The present study reveals that the nanocellulose templating technique enables a straightforward fabrication of a variety of porous crystalline scaffolds with well-defined mesoporous structure. For the first time the NCC has been used for the fabrication of homogeneous porous metal oxide films on different substrates, in contrast to the previously reported powders or free-standing membranes. The versatility and flexibility of the NCC templating approach offers broad perspectives towards the generalization of this method for the fabrication of different types of nanoporous metal oxides.

Thu, 13 Aug 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18637/ https://edoc.ub.uni-muenchen.de/18637/1/Sohmen_Daniel.pdf Sohmen, Daniel ddc:540, ddc:500, Fakultät f

Neurodegenerative disorders such as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis or Prion diseases are chronic, incurable and often fatal. A cardinal feature of all neurodegenerative disorders is the accumulation of misfolded and aggregated proteins. Depending on the disease, these aggregated proteins are cell type specific and have distinct cellular localizations, compositions and structures. Despite intensive research, the contribution of protein misfolding and aggregation to the cell type specific toxicity is not completely understood. In recent years, quantitative proteomics has matured into an exceptionally powerful technology providing accurate quantitative information on almost all cellular proteins as well as protein interactions, modifications, and subcellular localizations, which cannot be addressed by other omics technologies. The aim of this thesis is to investigate key features of neurodegeneration such as misfolded proteins and toxic protein aggregates with cutting edge proteomics, presenting a technological “proof of concept” and novel insights into the (patho)physiology of neurodegeneration.

Wed, 5 Aug 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18537/ https://edoc.ub.uni-muenchen.de/18537/1/Kukushkin_Yury.pdf Kukushkin, Yury

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli perform an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations to exclude aggregation, in combination with highly sensitive spectroscopic methods, such as single-molecule Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS), we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP), and a natural GroEL substrate - dihydrodipicolinate synthase (DapA). We show that both proteins form highly flexible, kinetically trapped folding intermediates, when folding in free solution and do not engage in inter-molecular interactions, such as aggregation, at sufficiently low concentration. We find that in the absence of aggregation, GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. The folding of DapA could be measured at physiological temperature and was found to be ~130-fold accelerated by GroEL/ES. As accelerated folding was independent of repetitive cycles of protein binding and release from GroEL, we demonstrate that iterative annealing does not significantly contribute to chaperonin assisted substrate folding. With a single molecule FRET based approach, we show that a given substrate molecule spends most of the time (~80%) during the GroEL reaction cycle inside the GroEL central cavity, in line with the inner GroEL cage being the active principle in folding catalysis. Moreover, photoinduced electron transfer experiments on DM-MBP provided direct experimental evidence that the confining environment of the chaperonin cage restricts polypeptide chain dynamics. This effect is mainly mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. Taken together, we were able to develop novel approaches, based on single molecule spectroscopy and making use of GroEL as a single molecule sorting machine, to measure GroEL substrate folding rates at sub-nanomolar concentrations. We also, for the first time, provide direct experimental evidence of conformational restriction of an encapsulated polypeptide in a chaperonin cage. Our findings suggest that global encapsulation inside the GroEL/ES cavity, not iterative cycles of annealing and forced unfolding, can accelerate substrate folding by reduction of an entropic energy barrier to the folded state, in strong support of an active chaperonin mechanism. Accelerated folding is biologically significant as it adjusts folding rates relative to the rate of protein synthesis.

Tue, 4 Aug 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18533/ https://edoc.ub.uni-muenchen.de/18533/1/Izsak_Daniel.pdf Izsák, Dániel ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Thu, 30 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18685/ https://edoc.ub.uni-muenchen.de/18685/1/Liebner_Robert.pdf Liebner, Robert ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Mon, 27 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18504/ https://edoc.ub.uni-muenchen.de/18504/1/Patschinski_Pascal.pdf Patschinski, Pascal ddc:540, ddc:500, Fakultät für Chemie und Phar

Wed, 22 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18742/ https://edoc.ub.uni-muenchen.de/18742/1/Hellenbrand_Tim.pdf Hellenbrand, Tim ddc:540, ddc:500, Fak

Bond cleavage and formation are key steps in chemistry and biochemistry. The present work investigates the generation of diphenylmethyl cations (Ph2CH+) via photoinduced bond cleavage of diphenylmethyl derivatives with a cationic or neutral leaving group. The resulting Ph2CH+ cations and its numerous derivatives serve as reference electrophiles for one of the most extensive reactivity scales covering 40 orders of magnitude. In chapter 1, the focus is on the initial bond cleavage of diphenylmethyltriphenylphosphonium ions (Ph2CH−PPh3+) exhibiting a cationic leaving group. With the help of state-of-the-art quantum chemical and quantum dynamical methods, the reaction mechanism of the bond cleavage is revealed. Using a reduced model system, the potential energy surfaces can be calculated at the ONIOM level of theory along specially designed reactive coordinates. Two competing reaction channels emerge: a homolytic one in the S1 state and a heterolytic one in the ground state. They are connected via an energetically accessible conical intersection which makes an efficient generation of the observed Ph2CH+ cations feasible. In contradiction with the experiment in polar or moderately polar solvents, quantum dynamical calculations for the isolated molecule reveal the formation of Ph2CH• radicals. While electrostatic solvent effects are negligible in this system, dynamic solvent effects emerge as being essential to explain the molecular mechanism. Two methods with increasing complexity to describe the dynamic impact of the solvent environment are developed. The first approach, the dynamic continuum ansatz, treats the environment implicitly. It uses Stokes’ law and the dynamic viscosity of the solvent in combination with quantum chemically and dynamically evaluated quantities to obtain the decelerating force exerted on the dissociating fragments. The ansatz does not require any fitting of parameters. The second method, the QD/MD approach, is based on an explicit treatment of the solvent surrounding. It combines molecular dynamics (MD) simulations of the reactant in a box of solvent molecules with quantum dynamics (QD) calculations of the reactant’s dynamics. In this way, a more detailed microscopic picture of the molecular process can be derived taking into account individual arrangements of the solvent. Both methods unveil the crucial impact of the solvent cage on the bond cleavage mechanism. It hinders the free dissociation in the S1 state and guides the molecular system to the conical intersection. QD simulations including the non-adiabatic coupling around the conical intersection show the formation of Ph2CH+ within ∼400 fs which compares well with the initial rise of the cation absorption in the experiment. Chapter 2 deals with the position of the counterion X– in the ion pairs Ph2CH−PPh3+ X–, PhCH2−PPh3+ X–, and (p-CF3-C6H4)CH2−PPh3+ X– in solution with X– being Cl–, Br–, BF4–, and SbF6–. These structures are essential to clarify the role of oxidizable counterions like e.g. Cl– during the initial bond cleavage in dichloromethane. The structures determined quantum chemically in dichloromethane show a similar counterion position than in the crystal. They are confirmed by the good accordance of the calculated and measured 1H NMR shifts. The C(α)–H···X– hydrogen bonds account for the pronounced counterion-dependent 1H NMR shifts of the C(α)–H in CD2Cl2. The strong downfield shift of the signals increases according to SbF6– < BF4–

Tue, 21 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18520/ https://edoc.ub.uni-muenchen.de/18520/1/Muehlbacher_Wolfgang.pdf Mühlbacher, Wolfgang ddc:54

Fri, 17 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18511/ https://edoc.ub.uni-muenchen.de/18511/1/Mathaes_Roman_Willi.pdf Mathäs, Roman Willi ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Thu, 16 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18492/ https://edoc.ub.uni-muenchen.de/18492/1/Hermawan_Adam.pdf Hermawan, Adam ddc:540, ddc:500, Fakultät für Chemie und

In recent years there has been a considerable focus on the development of subunit vaccines, preferred over traditional vaccines for reasons of safety and purity. However, subunit vaccines are less immunogenic than attenuated vaccines and need therefor multiple administrations in combination with immunostimulatory adjuvants, in order to induce immunity. The sustained release of a vaccine together with the release of an adjuvant is a potential alternative to giving multiple doses. The aim of this thesis was to manufacture lipid implants for vaccine delivery by twin-screw (tsc) extrusion and evaluate the potency of these lipid systems to stimulate an immune response in vivo. To accomplish this, lipid implants consisting of cholesterol, soybean lecithin, and Dynasan 114 (D114) were prepared. Different formulations were evaluated for their extrudability before adding the model antigen ovalbumin (OVA) and the adjuvant Quil-A (QA) to the formulation. Investigating the release behaviour of OVA and QA showed that mainly cholesterol influences the release behaviour of OVA, increasing the fraction of cholesterol slows down the release of OVA. To further slow down the release of OVA from the implants, they were cured at different temperature resulting in an even longer OVA release. Furthermore, the addition of QA to the implants influenced the release behaviour of OVA and vice versa. The investigation of the implant polymorphism after the extrusion process as well as during storage showed good stability. To combine the advantage of particulate delivery and sustained release, preformed liposomes were incorporated into the implants prior to extrusion. For the analysis of the immune response, two sets of animal experiments in mice were performed, one evaluating the kinetics of the release of the model antigen in vivo, a second one to evaluate the immune response in vivo. Evaluation of these data indicated a correlation between the in vitro and in vivo release behaviour of OVA. Furthermore, immune responses similar to those induced by two booster injections, consisting of OVA and alum could be achieved using implant formulations containing QA. These results further emphasized the importance of adjuvant in the formulation. The incorporation of preformed liposomes into the implants on the other hand did not lead to an improved outcome. In a second part of this work, an in vivo tumour study was prepared, using the TRP2 peptide as active ingredient. Due to the use of this expensive peptide, a transfer to a different extruder was necessary. The influence that a change of the production device has on the implants characteristics was investigated. Once the formulation was adapted to the new extruder, implants containing TRP2 and QA were produced. The in vitro release of TRP2 proved to be very slow, much different from the OVA release. Furthermore, the preparation of vesicular phospholipid gels (VPGs) as an alternative lipid delivery system for TRP2 was investigated. The TRP2 release from the VPGs was also slow and incomplete. Both formulations were used in an in vivo tumour growth study. Mice were injected with B16F10luc2 melanoma cells, 6 days later formulations were administered. VPGs showed adverse reactions in the mouse and are therefore not s suitable delivery system. TRP2 implants showed a slow delay in the start of tumour growth, but were not more potent that TRP2 in PBS injections given to the mice. The very slow in vitro release data of TRP2 brought up the question about interactions between the lipid implants and the peptide influencing the release. Choosing peptides of different size and hydropathy, an investigation of their release behaviour and interaction with the implants was conducted. In conclusion, lipid implants were well tolerated and offer a great potential as sustained release delivery system for vaccines. They allow releasing the active component and the adjuvant together, enabling to achieve a strong immune response.

Tue, 14 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18623/ https://edoc.ub.uni-muenchen.de/18623/1/Zgela_Dominik.pdf Zgela, Dominik ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Tue, 14 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18439/ https://edoc.ub.uni-muenchen.de/18439/1/Antipova_Anna.pdf Antipova, Anna ddc:54