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Item Open Access ROSIE : RObust Sparse ensemble for outlIEr detection and gene selection in cancer omics data(2022) Jensch, Antje; Lopes, Marta B.; Vinga, Susana; Radde, NicoleThe extraction of novel information from omics data is a challenging task, in particular, since the number of features (e.g. genes) often far exceeds the number of samples. In such a setting, conventional parameter estimation leads to ill-posed optimization problems, and regularization may be required. In addition, outliers can largely impact classification accuracy. Here we introduce ROSIE, an ensemble classification approach, which combines three sparse and robust classification methods for outlier detection and feature selection and further performs a bootstrap-based validity check. Outliers of ROSIE are determined by the rank product test using outlier rankings of all three methods, and important features are selected as features commonly selected by all methods. We apply ROSIE to RNA-Seq data from The Cancer Genome Atlas (TCGA) to classify observations into Triple-Negative Breast Cancer (TNBC) and non-TNBC tissue samples. The pre-processed dataset consists of 16,600 genes and more than 1,000 samples. We demonstrate that ROSIE selects important features and outliers in a robust way. Identified outliers are concordant with the distribution of the commonly selected genes by the three methods, and results are in line with other independent studies. Furthermore, we discuss the association of some of the selected genes with the TNBC subtype in other investigations. In summary, ROSIE constitutes a robust and sparse procedure to identify outliers and important genes through binary classification. Our approach is ad hoc applicable to other datasets, fulfilling the overall goal of simultaneously identifying outliers and candidate disease biomarkers to the targeted in therapy research and personalized medicine frameworks.Item Open Access Precision 3D‐printed cell scaffolds mimicking native tissue composition and mechanics(2020) Erben, Amelie; Hörning, Marcel; Hartmann, Bastian; Becke, Tanja; Eisler, Stephan A.; Southan, Alexander; Cranz, Séverine; Hayden, Oliver; Kneidinger, Nikolaus; Königshoff, Melanie; Lindner, Michael; Tovar, Günter E. M.; Burgstaller, Gerald; Clausen‐Schaumann, Hauke; Sudhop, Stefanie; Heymann, MichaelCellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell‐ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell‐ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two‐photon stereolithography is adopted to print up to mm‐sized high‐precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein‐based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two‐pass printing or post‐print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7‐300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D‐lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single‐cell and tissue dynamics in response to defined mechanical and bio‐molecular cues and is ultimately scalable to full organs.Item Open Access Construction of robust Escherichia coli strains for large-scale production(2022) Ziegler, Martin; Takors, Ralf (Prof. Dr.-Ing.)The biotechnical production of many fine chemicals, proteins or pharmaceuticals depends on large-scale microbial cultivations. Due to limited mixing, heterogeneities in process relevant parameters such as nutrient concentrations arise in such fermentations. Escherichia coli (E. coli) is a model organism frequently used in the biotechnological industry. If E. coli is cultivated under heterogeneous conditions, biological reactions of the microorganism result in reduced process performance. Since large-scale fermentations are not economically feasible in academic settings, scale-down reactors that mimic aforementioned heterogeneities are used to investigate heterogenous fermentations. Previous studies in scale-down reactors unraveled that, depending on the process strategy, the unstable supply of a limiting primary carbon or nitrogen source such as glucose or ammonium is one of the underlying causes of process performance loss. Low concentrations of glucose or ammonium elicit the stringent response as a biological starvation reaction which comprises extensive transcriptional reactions. In the first project that contributes to this thesis, the regulatory and transcriptional reactions of the strains E. coli MG1655 and E. coli SR to repeated exposure to ammonium starvation zones were examined in a scale-down reactor. The scale-down reactor followed a two-compartment approach and consisted of a stirred tank reactor and a plug-flow reactor simulating passage through a starvation zone. E. coli SR is a strain with modulated stringent response. It was observed that short-term starvation stimuli do not trigger this regulatory program in E. coli SR and the transcriptional reaction was noticeably reduced. Long-term adaptation of the strain to repeated cycles of limitation and starvation also clearly differed from E. coli MG1655. Despite lack of the stringent response, E. coli SR showed no deficits in the assimilation of the limiting ammonium or in biomass yield on ammonium. In the second project of this thesis, a series of deletion strains with robust phenotype against glucose starvation zones were constructed. Candidate genes were identified and successively removed from the genome of E. coli MG1655 by Recombineering. The fundamental growth parameters of the strains were determined in shaking flask fermentations and no noticeable differences compared to E. coli MG1655 were found. Chemostat cultivations in a scale-down reactor with glucose as the limiting nutrient source revealed that the final strain of the deletion series, E. coli RM214, had a significantly lower maintenance coefficient under heterogeneous conditions than E. coli MG1655. Moreover, in an exemplary heterologous protein productionscenario E. coli RM214 rhaB- pJOE4056.2_tetA proved to be more robust to heterogeneities and showed a significantly higher product yield than E. coli MG1655 rhaB- pJOE4056.2_tetA. In the third project of this thesis, the production of pyruvate in E. coli MG1655 by inhibition of pyruvate dehydrogenase through CRISPR interference was investigated. A central goal was to achieve the stable production in nitrogen-limited conditions. For this, different target sequences in the operon pdhR-aceEF-lpd were tested and the strains cultivated in shaking flask fermentations. All tested target sequences were generally suitable to trigger the accumulation of pyruvate. Combined CRISPR interference against two target sequences did not lead to an increased pyruvate yield in most cases. In addition, the strains E. coli MG1655 pdCas9 psgRNA_aceE_234 and E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 were characterized in two phase fermentations in lab-scale reactors. The initial phase was an unlimited exponential growth phase and was followed by an ammonium-limited production phase. E. coli MG1655 pdCas9 psgRNA_aceE_234 only produced pyruvate during the exponential phase, and reuptake of pyruvate occurred in the second phase. In contrast, E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 stably produced pyruvate during the exponential and the ammonium-limited phase and is a potential chassis strain for the growth-decoupled production of pyruvate derived bioproducts. The overarching research issues of the projects were the characterization of strains in heterogeneous conditions and the development of new strategies to improve their performance. The collected data leads me to conclude that the construction of robust microbial strains for large-scale applications is both expedient and feasible. Tailored genetic modifications are the method of choice to achieve this goal. Furthermore, suitable genetic constructs offer promising possibilities for the stable growth-decoupled production of chemicals in nitrogen-limited conditions.Item Open Access Regulation of the catalytic activity and specificity of DNA nucleotide methyltransferase 1(2014) Bashtrykov, Pavel; Jeltsch, Albert (Prof. Dr.)DNA nucleotide methyltransferase 1 (Dnmt1) is mainly responsible for the maintenance of DNA methylation in mammals and plays a crucial role in the epigenetic control of gene expression. Dnmt1 recognizes and methylates hemimethylated CpG sites formed during DNA replication. In the present work, the mechanistic details of the substrate recognition by the catalytic domain of Dnmt1, the possible role of the CXXC and RFTS domains of Dnmt1 in the regulation of specificity and activity of Dnmt1, and the influence of the Ubiquitin-like PHD and RING finger domain-containing 1 (Uhrf1) protein on the enzymatic properties of Dnmt1 was investigated. Using modified substrates, the functional roles of individual contacts of the Dnmt1 catalytic domain with the CpG site of the DNA substrate were analysed. The data show that the interaction with the 5-methylcytosine:guanine pair is required for the catalytic activity of Dnmt1, whereas the contacts to the non-target strand guanine are not important, since its replacement with adenine increased the activity of Dnmt1. It was proposed that the CXXC domain binding to unmethylated CpG sites increases the specificity of Dnmt1 for hemimethylated DNA. Our data showed that the CXXC domain does not influence the enzyme’s specificity in the full-length Dnmt1. In contrast, mutagenesis in the catalytic domain introducing an M1235S exchange resulted in a significant reduction in specificity. Therefore, the readout for the hemimethylated DNA occurs within its catalytic domain. It was observed in a crystal structure that the RFTS domain of Dnmt1 inhibits the activity of the enzyme by binding to the catalytic domain and blocking the entry of the DNA. By amino acid substitution in the RFTS domain its positioning within the catalytic domain was destabilized and a corresponding increase in the catalytic rate was observed, which supports this concept and suggests a possible mechanism to allosterically regulate the activity of Dnmt1 in cells. Uhrf1 has been shown to target Dnmt1 to replicated DNA, which is essential for DNA methylation. Here it is demonstrated that Uhrf1 as well as its isolated SRA domain increase the activity and specificity of Dnmt1 in an allosteric mechanism. The stimulatory effect was independent of the SRA domain’s ability to bind hemimethylated DNA. The RFTS domain of Dnmt1 is required for the stimulation, since its deletion or blocking of its interaction with the SRA domain, significantly reduced the ability of Uhrf1 to increase the activity and specificity of Dnmt1. Uhrf1, therefore, plays multiple roles that support DNA methylation including targeting of Dnmt1, its stimulation and an increase of its specificity.Item Open Access Development of novel bispecific antibodies for cancer therapy targeting the receptor tyrosine kinases HER4 and EGFR(2024) Kühl, Lennart; Kontermann, Roland E. (Prof. Dr.)In this study, novel mono- and bispecific antibodies targeting the ErbB receptor family members EGFR and HER4 were investigated. Dual targeting of EGFR and HER4 by a bispecific, tetravalent antibody comprising a novel, antagonistic HER4-targeting antibody showed inhibition of proliferation and migration for a HB-EGF-stimulated ovarian cancer cell line. No inhibitory effects in a breast cancer cell line expressing EGFR and HER4 indicated that successful dual targeting does not solely rely on target expression. The complexity of HER4 with its isoforms and their different signaling properties makes HER4 a challenging cancer target that needs further in-depth research. To overcome resistances based on escape mutations located in the epitopes of clinically approved antibodies, novel antagonistic EGFR-targeting antibodies binding to a different epitope were developed. This epitope was mapped to domain III of EGFR and binding to clinically relevant EGFR ectodomain mutations resulted in inhibition of EGFR signaling in stable cell lines used as test systems. Favorable activities in comparison to clinically approved antibodies regarding inhibition of EGFR signaling and proliferation were observed for cancer cell lines expressing the EGFR wildtype. Bispecific T-cell engagers can lead to a T-cell mediated target cell killing independent of intracellular downstream signaling in the cancer cell. One challenge for the applicability of T-cell engagers in solid tumors is to keep the balance between T-cell mediated tumor cell killing and severe side-effects caused by a systemic activation of the immune system. Studies on eleven different eIg-based formats for EGFR-binding T-cell engagers showed that valency, geometry, and size influenced their activity profile. Furthermore, one bivalent and one trivalent, bispecific format were investigated for two novel EGFR-targeting moieties. As these molecules bind to clinically relevant escape mutations located in the ectodomain of EGFR, they are expected to show activity in patients with an acquired resistance to approved EGFR-targeting antibodies. These molecules led to a robust T-cell mediated cytotoxicity of cancer cells expressing EGFR. Additionally, benefits regarding an EGFR-level dependent cytotoxicity were observed for reduced binding to EGFR. An initial in vivo study using surrogate molecules in a syngeneic mouse model showed reduction of tumor growth and prolonged survival for treatment with a trivalent, bispecific T-cell engager comprising a novel EGFR-binding moiety. Taken together, beneficial effects of the novel molecules may contribute to improved therapies for patients with both pre-existing and acquired resistances to EGFR-targeting antibodies.Item Open Access Insights into the structural and functional properties of the eukaryotic porin Tom40(2012) Gessmann, Dennis; Nußberger, Stephan (Prof. Dr.)Tom40 forms the preprotein conducting channel in the outer membrane of mitochondria enabling transport of up to 1500 different preproteins through an optimized pore environment. Moreover, Tom40 exhibits a voltage-dependent gating mechanism in terms of an ‘electrical switch’ making this eukaryotic beta-barrel a promising target for nanopore based applications. In this work, new bioinformatics methods were developed and verified through practical approaches to shed light on the structural elements of Tom40 facilitating its particular function in mitochondria. Based on these results, Tom40 proteins were designed with modified and optimized structural properties. TmSIP, a physical interaction model developed for TM beta-barrel proteins, was used to identify weakly stable regions in the TM domain of Tom40 from mammals and fungi. Three unfavorable beta-strands were determined for human Tom40A. Via CD and Trp-fluorescence spectroscopy it was shown that substitution of key amino acid residues in theses strands resulted in an improved resistance of the protein to chemical and thermal perturbations. Further, the mutated form of hTom40A was strictly found in its monomeric state. Equal improvements were gained for the apparent stability of Tom40 from Aspergillus fumigatus. Tom40 was isolated and purified in its native state from Neurospora crassa mitochondria. Time-limited proteolysis of native NcTom40 coupled to mass spectrometry revealed comparable protease-accessibility to VDAC isoform 1 from mammals suggesting a similar fold. Thus, a homology model of NcTom40 was developed on the basis of the solved mouse VDAC-1 crystal structure. It was found that Tom40 forms a 19-stranded beta-barrel with an N-terminal alpha-helix inside the pore. Further, a conserved ‘polar slide’ in the pore interior is possibly involved in preprotein translocation and a second conserved domain, termed ‘helix anchor region’, in arresting the helix inside the Tom40 pore. Based on the homology model of NcTom40, the structure and function of the N-terminal domain of Tom40 was addressed. Examination of the model structure revealed two different domains for the N-terminus, the inner-barrel and outer-barrel N-terminus. In vivo investigations showed that both parts prevent a heat-induced dysfunction of Tom40 in N. crassa mitochondria independently. By applying CD spectroscopy the predicted N-terminal alpha-helix could be allocated to the inner-barrel N-terminus. Further, in combination with Trp-fluorescence spectroscopy it was found that the N-terminal alpha-helix unfolds independently from the Tom40 beta-barrel, but is not necessary for pore stability or integrity. However, a conserved amino acid residue, Ile47 of NcTom40, in the inner-barrel N-terminus is essential for the structural integrity of the N-terminal alpha-helix. In conclusion, these results may offer a basis for future works on TM beta-barrel proteins with the aim to alter the structural properties in the absence of a high atomic resolution structure or an established knowledge of the biochemical and biophysical properties.Item Open Access The Bacteroidetes Aequorivita sp. and Kaistella jeonii produce promiscuous esterases with PET-hydrolyzing activity(2022) Zhang, Hongli; Perez-Garcia, Pablo; Dierkes, Robert F.; Applegate, Violetta; Schumacher, Julia; Chibani, Cynthia Maria; Sternagel, Stefanie; Preuss, Lena; Weigert, Sebastian; Schmeisser, Christel; Danso, Dominik; Pleiss, Juergen; Almeida, Alexandre; Höcker, Birte; Hallam, Steven J.; Schmitz, Ruth A.; Smits, Sander H. J.; Chow, Jennifer; Streit, Wolfgang R.Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.Item Open Access Mechanistic studies on the DNA methyltransferases DNMT3A and DNMT3B(2021) Dukatz, Michael; Jeltsch, Albert (Prof. Dr.)In this work, both regulatory and catalytic mechanisms of de novo methyltransferases were investigated, which include interactions with other proteins and the specific recognition of the substrate sequence. Another part of this work strived to elucidate how enzymatic generation of 3-methylcytosine by DNMT3A can occur.Item Open Access Construction of a super-competent Bacillus subtilis 168 using the PmtlA-comKS inducible cassette(2015) Rahmer, Regine; Morabbi Heravi, Kambiz; Altenbuchner, JosefCompetence is a physiological state that enables Bacillus subtilis 168 to take up and internalize extracellular DNA. In practice, only a small subpopulation of B. subtilis 168 cells becomes competent when they enter stationary phase. In this study, we developed a new transformation method to improve the transformation efficiency of B. subtilis 168, specially in rich media. At first, different competence genes, namely comK, comS, and dprA, were alone or together integrated into the chromosome of B. subtilis 168 under control of mannitol-inducible PmtlA promoter. Overexpression of both comK and comS increased the transformation efficiency of B. subtilis REG19 with plasmid DNA by 6.7-fold compared to the wild type strain 168. This transformation efficiency reached its maximal level after 1.5 h of induction by mannitol. Besides, transformability of the REG19 cells was saturated in the presence of 100 ng dimeric plasmid or 3000 ng chromosomal DNA. Studying the influence of global regulators on the development of competence pointed out that important competence development factors, such as Spo0A, ComQXPA, and DegU, could be removed in REG19. On the other hand, efficient REG19 transformation remained highly dependent on the original copies of comK and comS regardless of the presence of PmtlA-comKS. Finally, novel plasmid-free strategies were used for transformation of REG19 based on Gibson assembly.Item Open Access A timed off-switch for dynamic control of gene expression in Corynebacterium glutamicum(2021) Siebert, Daniel; Altenbuchner, Josef; Blombach, BastianDynamic control of gene expression mainly relies on inducible systems, which require supplementation of (costly) inducer molecules. In contrast, synthetic regulatory circuits, which allow the timed shutdown of gene expression, are rarely available and therefore represent highly attractive tools for metabolic engineering. To achieve this, we utilized the VanR/PvanABK* regulatory system of Corynebacterium glutamicum, which consists of the transcriptional repressor VanR and a modified promoter of the vanABK operon (PvanABK*). VanR activity is modulated by one of the phenolic compounds ferulic acid, vanillin or vanillic acid, which are co-metabolized with d-glucose. Thus, gene expression in the presence of d-glucose is turned off if one of the effector molecules is depleted from the medium. To dynamically control the expression of the aceE gene, encoding the E1 subunit of the pyruvate dehydrogenase complex that is essential for growth on d-glucose, we replaced the native promoter by vanR/PvanABK* yielding C. glutamicum ΔPaceE::vanR-PvanABK*. The biomass yield of this strain increased linearly with the supplemented amount of effector. After consumption of the phenolic compounds growth ceased, however, C. glutamicumΔPaceE::vanR-PvanABK* continued to utilize the residual d-glucose to produce significant amounts of pyruvate, l-alanine, and l-valine. Interestingly, equimolar concentrations of the three phenolic compounds resulted in different biomass yields; and with increasing effector concentration, the product spectrum shifted from pyruvate over l-alanine to l-valine. To further test the suitability of the VanR/PvanABK* system, we overexpressed the l-valine biosynthesis genes ilvBNCE in C. glutamicum ΔPaceE::vanR-PvanABK*, which resulted in efficient l-valine production with a yield of about 0.36 mol l-valine per mol d-glucose. These results demonstrate that the VanR/PvanABK* system is a valuable tool to control gene expression in C. glutamicum in a timed manner by the cheap and abundant phenolic compounds ferulic acid, vanillin, and vanillic acid.