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Aggressive Freeze-DryingBosch, Thomas 29 July 2014 (has links) (PDF)
No description available.
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Generierung und Screening von Oximbibliotheken zur Entwicklung neuer potenter und selektiver mGAT1-InhibitorenKern, Felix 11 July 2014 (has links) (PDF)
mGAT1 ist der im zentralen Nervensystem am weitesten verbreitete GABA-Transporter. In der vorliegenden Dissertation wird ein Screeningkonzept von Oximbibliotheken auf neue potente und selektive mGAT1-Inhibitoren vorgestellt. Die Bibliotheken wurden durch Umsetzung verschiedener Hydroxylaminderivate und jeweils einer Aldehydbibliothek bestehend aus vier verschiedenen Aldehyden unter geeigneten Bedingungen generiert. Diese eingestellten Bibliotheken wurden nach Verdünnung mittels MS-Bindungsassays gescreent und die potentesten Bibliotheken anschließend in Dekonvolutionsexperimenten untersucht, um herauszufinden, welche Oxime für die hohe Bindungsaffinität der Bibliothek gegenüber mGAT1 verantwortlich waren. Diese Oxime wurden nachsynthetisiert und deren Bindungsaffinitäten und inhibitorischen Potenzen gegenüber mGAT1 bestimmt. Auf diesem Weg konnte eine Serie neuer mGAT1-Inhibitoren mit Binungsaffinitäten im niedrigen nanomolaren Bereich ermittelt werden. Ein Oxim erzielte die höchste inhibitorische Potenz, die für einen mGAT1-Inhibitor bisher in einem GABA-Uptake-Assay ermittelt werden konnte.
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GroEL/ES modulates the mechanism and accelerates the rate of TIM-barrel domain foldingPopova, Kristina 10 December 2014 (has links) (PDF)
The interactome of GroEL/ES has been characterized extensively in several studies and substrates of the chaperonin have been classified (Kerner et al., 2005; Fijiwara et al., 2010). However, the question of what makes some proteins GroEL-dependent and how exactly the chaperonin system promotes their folding remained unresolved. Moreover, it has been unclear how the chaperonin acts on its substrates and whether the protein folding pathway is modified inside the cage as compared to free solution. The aim of this study, therefore, was to characterise and compare the spontaneous and chaperonin-assisted refolding pathway of an obligate substrate of GroEL/ES, in order to elucidate the mechanism of GroEL/ES action.
This study presents evidence that encapsulation in the GroEL/ES-cage accelerates the rate and modulates the mechanism of folding of its obligate TIM-barrel substrate, dihydrodipicolinate synthase. We found that the spontaneous refolding of DAPA is slow due to high cooperativity of the process, as it initiates from an ensemble of unstructured intermediates. We demonstrated that the confining environment of the chaperonin cage promotes formation of the TIM-barrel structure in a segmental manner, lowering the entropic component of the activation barrier and accelerating the rate of DAPA folding. Moreover, the spontaneous refolding pathway of a GroEL-independent homolog of DAPA, MsNANA, closely resembles that of DAPA inside the chaperonin cage. Thus, we conclude that GroEL/ES is a powerful folding catalyst for the substrates that otherwise fail to effectively reach their native state.
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Aryl-Aryl-Kopplungen, Decarbonylierungen und Decarboxylierungen von aromatischen Anhydriden und Carbonsäuren durch das Cu(0)/Cu(I)-PaarZwiener, Matthias 15 December 2014 (has links) (PDF)
No description available.
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Proton-sponge activity and receptor-targeting of sequence-defined nucleic acid carriersLächelt, Ulrich 04 December 2014 (has links) (PDF)
No description available.
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Correlation of structure and performance in high temperature polymer electrolyte membrane fuel cellsHeinzl, Christoph 27 October 2014 (has links) (PDF)
No description available.
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The total synthesis of (−)-nitidasin, synthetic studies toward astellatol and retigeranic acid B, and development of novel photochromic ligands for L-type voltage-gated calcium channelsHuber, Florian Mauritius Erasmus 12 November 2014 (has links) (PDF)
The first part of present doctoral thesis is dedicated to sesterterpenoid natural products, which constitute a relatively small subclass of the family of terpenoid compounds. In particular, a small number of biosynthetically closely related iso-propyl-substituted trans-hydrindane- based congeners were chosen as targets for a synthetic program that envisioned their chemical construction via a unified approach. In this context, the first total synthesis of the complex tetracarbocyclic sesterterpenoid (−)-nitidasin was disclosed. To this end, a highly convergent installation of its western cyclopentane moiety was demonstrated by use of an unprecedented Li-alkenyl addition to a versatile trans-hydrindanone building block. An efficient closure of the central 8-membered carbocycle was achieved by means of ring-closing metathesis that benefitted from finely tuned conformational parameters of the employed substrate. In summary, the developed protocol provides a scalable and continuously high yielding route for the assembly of the rare carbon skeleton of (−)-nitidasin. Furthermore, advanced synthetic studies toward the pentacyclic natural products retigeranic acid B and astellatol are described. Notably, the latter involved a biomimetic retrosynthetic proposal for the elaboration of its unique fused ring system. Two different strategies were followed in order to access viable precursors for the suggested cationic cascade, of which the latter allowed for the investigation of the mentioned key step experimentally and via computational methods.
In the second section of this manuscript, the design and synthesis of the first known photochromic antagonist for L-type voltage-gated calcium channels is presented. The ultimate goal of this project lies in the establishment of a photopharmacological tool that supports the further elucidation of Ca2+-dependent cellular events, especially in neurons. The central pharmacophore of the prepared compounds was derived from the benzothiazepine drug cis-(+)-diltiazem. This parent substance also served as the starting point for the developed short and convergent semisynthetic strategy. Thirteen analogues were synthesized in total and a preliminary lead structure was identified using electrophysiolocigal methods. Based on these findings, two compounds of increased solubility and desirable photochemical properties were prepared. A possible explanation for their photodependent mode of action was proposed, which is currently subject of our ongoing investigations.
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Involvement of latent TGF-beta binding protein 1 in CADASIL-relevant Notch3 aggregationKast, Jessica 08 December 2014 (has links) (PDF)
CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) represents the most prevalent hereditary form of cerebral small vessel disease (SVD) resulting in early-onset stroke and vascular dementia. It is caused by stereotyped missense mutations in the transmembrane receptor Notch3, which alter the number of cysteine residues in the extracellular domain (ECD). This leads to the abnormal multimerization and extracellular deposition of mutant Notch3-ECD at the plasma membrane of smooth muscle cells in small blood vessels. Notch3-ECD-containing aggregates are the earliest manifestation of the disease and excess Notch3-ECD is believed to recruit functionally important extracellular matrix proteins resulting in brain vessel dysfunction.
Biochemical and histological approaches on post-mortem brain tissue from CADASIL patients and control subjects as well as in vitro assays were used to study the consequences of Notch3-ECD deposition on the ECM components thrombospondin-2, fibrillin-1 and fibronectin and members of the latent transforming growth factor-β (TGF-β) binding protein (LTBP) family. It is demonstrated that the structural matrix components fibrillin-1 and fibronectin are enriched and contribute to the prominent thickening of CADASIL vessel walls without co-localizing with Notch3-ECD deposits, likely as a result of fibrotic adaptation secondary to aggregate formation. For LTBP-1, a key regulator of the TGF-β signaling pathway, an accumulation as well as a striking co-localization with Notch3-ECD deposits is shown suggesting specific recruitment into aggregates. Furthermore, increased levels of the TGF-β pro-domain (also known as latency-associated peptide, LAP) were found implying dysregulation of the TGF-β pathway in CADASIL development. Finally, a direct interaction of LTBP-1 with Notch3-ECD is demonstrated and evidence for a co aggregation with mutant Notch3 in vitro is provided. Conclusively, I propose LTBP-1 as a novel component of Notch3 deposits with a role in CADASIL pathogenesis.
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Proteomics and phosphoproteomics applied to cell signaling and cancerD'souza, Rochelle 28 November 2013 (has links) (PDF)
Signaling networks control and regulate outcomes in cells and organisms in both normal physiology and pathophysiological states. Signaling is traditionally represented and studied as a series of stepwise enzymatic events constituting a cascade. However, it is increasingly apparent that such representations limit understanding of signal transduction since these linear cascades function in an interconnected network that includes extensive cross talk among receptors and pathways. Mass spectrometry (MS)-based proteomics is a useful tool that allows a system-wide investigation of signaling events at the levels of post-translational modifications (PTMs), protein-protein interactions and changes in protein expression on a large scale. This technology now allows accurate quantification of thousands of proteins and their modifications in response to any perturbation.
This thesis work is dedicated to the optimization and employment of quantitative mass spectrometry to cellular signaling and an application to segregate two lymphoma subtypes at the levels of protein expression and phosphorylation, employing state of the art liquid chromatography (LC)-MS/MS technologies coupled with improved sample preparation techniques and data analysis algorithms.
In the first project I investigated the feasibility of a new, high accuracy fragmentation method called higher energy collisional dissociation (HCD) for the analysis of phospho-peptides. Using this method we were able to measure the phospho-proteome of a single cell line in 24h of measurement time which was a great improvement to previous capabilities. This fragmentation method that was originally thought to be slower and less sensitive than the standard method of low resolution collision induced dissociation (CID) fragmentation. However, our work proves this not to be the case and we showed that HCD outperformed the existing low resolution strategy [1].
In the second project I employed this HCD fragmentation technique on the LTQ-Orbitrap Velos for addressing the clinical question of segregating two subtypes of diffuse B-cell lymphoma (DLBCL). These subtypes are histologically indistinguishable but had been segregated on the basis of a gene expression signature. I employed the recently developed ‘super-SILAC’ approach with a ‘super-SILAC mix’ of multiple labeled cell lines. This heavy reference mix was spiked into several cell lines derived from the two DLBCL subtypes and analyzed LC-MS, resulting in successful segregation based on a distinct proteomic signature [2].
The third project deals with the in-depth analysis of the phospho-proteome of a human cancer cell line on a quadrupole-Orbitrap mass spectrometer using a label-free quantification approach. Our analysis uncovered about 50,000 distinct phosphorylated peptides in a single cell type across a number of cellular conditions allowing assessment of global properties of this large dataset. Strikingly, we found that at least three-quarters of the proteome can be phosphorylated which is much higher than current estimates. We also analyzed phosphotyrosine events using enrichment with anti-phospho-tyrosine antibodies to identify more than 1,500 site specific phosphorylation events. Unexpectedly tyrosine phosphorylated proteins were enriched among higher abundance proteins. The observed difference in phospho-protein abundance correlated with the substrate Km values of tyrosine kinases. For the first time we calculated site specific occupancies using label- free quantification and observed widespread full phosphorylation site occupancy during mitosis.
In the final and main project, I applied proteomics and phospho-proteomics to the study of signal transduction in response to transforming growth factor-beta (TGF-β), a multifunctional cytokine. TGF-β signaling regulates many biological outcomes including cell growth, differentiation, morphogenesis, tissue homeostasis and regeneration. The cellular responses to this multifunctional ligand are diverse and can even be opposed to each other, depending on the cell type and the conditions. To shed light on the reasons for the different outcomes, we analyzed the early phospho-proteome and ensuing proteome alterations in response to TGF-β treatment in a keratinocyte cell line. The early SILAC based phospho-proteome analysis uncovered over 20,000 phosphorylation events across five time points (0 to 20 min) of TGF-β treatment. Building on our recent advances in instrumentation, sample preparation, and data analysis algorithms we measured a deep TGF-β responsive proteome at six late time points (6h to 48h) with corresponding controls in only eight days of measurement time. Our label-free approach identified about 8,000 proteins and quantified more than 6,000 of them. This deep proteome covered well established pathways involved in TGF-β signaling, allowing global evaluation at the level of individual pathway members. Combining the TGF-β responsive proteome with an in-silico upstream regulator analysis, we correctly retrieved several known and predicted novel transcription factors driving TGF-β induced cytostasis, de-differentiation and epithelial to mesenchymal transition (EMT). The combined analysis of transcription factor regulation with early phosphorylation changes and proteome changes enabled visualization of the intricate interplay of key transcription factors, kinases and various pathways driving cytostatis, EMT and other processes induced by TGF-β.
In summary, my thesis developed a highly efficient phospho-proteomic workflow, which was applied to the measurement of a very deep phospho-proteome of a single cancer cell line allowing analysis of its global features. The main achievement was the first in-depth and combined study of the phospho-proteome and resulting proteome changes following a defined signaling event, in this case leading to a time-resolved view of TGF- β signaling events relevant in cancer.
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The role of branched actin networks in dendritic cell physiologyEichner, Alexander 31 October 2013 (has links) (PDF)
No description available.
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