04 Fakultät Energie-, Verfahrens- und Biotechnik
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/5
Browse
296 results
Search Results
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 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 Strukturelle und funktionelle Charakterisierung von RERE, einem Gen mit möglicher Relevanz bei der Tumorentstehung(2001) Wärner, Thomas Michael; Pfizenmaier, Klaus (Prof.)RERE (RE repeats encoded) ist ein kürzlich beschriebenes Gen welches in der distalen Region von Chromosom 1p lokalisiert ist. Für diese genomische Region wurde durch molekularbiologische und zytogenetische Studien eine konsistente strukturelle Veränderung in verschiedenen menschlichen Tumoren nachgewiesen. Die Neuroblastom Zelllinie NGP enthält eine reziproke chromosomale Translokation/Duplikation in dieser genomischen Region. Die genomische Sequenz von RERE wurde als die den Bruchpunkt überlagernde Sequenz in der Zelllinie NGP nachgewiesen. In dieser Arbeit wurde die genomische Struktur von RERE beschrieben und die cDNA einer neuen RERE Splicevariante isoliert. In allen untersuchten humanen Geweben wurden mittels Northern blotting zwei dominante RERE-Transkripte nachgewiesen und diese als mögliche Splice Varianten identifiziert. Darüber hinaus wurde in allen untersuchten Tumorzelllinien mittels Western blotting zwei dominante Proteinbanden mit einem RERE Immunserum nachgewiesen. In 2 von 18 untersuchten Tumorzelllinien wurde zusätzlich jeweils eine kleinere dominante Proteinbande detektiert. Weiterhin konnte in dieser Arbeit gezeigt werden, daß überexprimiertes RERE in PML Oncogenic Domains (PODs) lokalisiert ist und mit den pro-apoptotischen Proteinen PML, BAX und mit Mitochondrien kolokalisiert. Bei RERE transfizierten Zellen wurde durch unterschiedliche Methoden Apoptose nachgewiesen. Durch die Untersuchung verschiedener RERE Proteinfragmente (gesamtes RERE und N- oder C-terminale Deletionsmutanten von RERE) konnte die Region beschrieben werden, die eine Kolokalisierung von RERE und PODs unterstützt und nachdem sie in verschiedene Zelllinien transfiziert wurde, mit dem Nachweis von Apoptose korreliert. Die Ergebnisse dieser Arbeit geben einen ersten Hinweis auf die Funktion von RERE. RERE könnte eine Verbindung zwischen PODs und der Kontrolle von Apoptose darstellen und somit eine wichtige Rolle bei der Tumorentstehung spielen.Item Open Access Transformation von B. subtilis 168 : Optimierung und Regulation des Transkriptionsfaktors ComK(2017) Franzen, Regine; Mattes, Ralf (Prof. Dr. rer. nat.)Item Open Access Lokalisation, Speicherung und Synthese von Polyphosphat in Agrobacterium tumefaciens C58(2021) Hellenbroich, Celina; Jendrossek, Dieter (apl. Prof. Dr. rer. nat.)Polyphosphat (PolyP) besitzt eine ubiquitäre Verbreitung und erfüllt, je nach Organismus, unterschiedliche und extrem vielfältige Aufgaben. In Prokaryonten liegt PolyP in sogenannten Granula vor, während in einzelligen Eukaryonten, eine Membran das PolyP von dem Cytoplasma abtrennt. Vorangegangene Arbeiten weisen darauf hin, dass sogenannte Acidocalcisomen, eben jene membranumschlossene PolyP-Speicher aus Eukaryonten, auch in dem Bodenbakterium Agrobacterium tumefaciens vorhanden sein könnten. Die vorliegende Arbeit zeigt jedoch, dass sich in A. tumefaciens, wie in Bakterien übliche, PolyP-Granula befinden, die nicht von einer Membran umschlossen sind. Im weiteren Verlauf wurde die Synthese von PolyP sowie die Lokalisation der Polyphosphatkinasen (PPKs) und anderer aus der Literatur bekannter, PolyP-assoziierter Proteine untersucht. Die PPK1At stellte sich hierbei als PolyP-Syntheseenzym heraus. Es folgte eine biochemische Charakterisierung der PPKs in vitro, bei der für die PPK2At, neben der Bildung von NDP und NTP, eine oligophosphorylierende Funktion bis hin zu nonaphosphorylierten Nukleosiden entdeckt wurde. Außerdem stellte sich heraus, dass das PolyP-Granulum während des Zellzyklus wanderte und vielleicht durch die PPK1At mit der DNA assoziiert sein könnte. Aufgrund dieser Erkenntnisse konnte ein Modell des PolyP-Granulums und den in dieser Arbeit identifizierten, assoziierten Proteinen erstellt werden. Eine Deletion der ppk1 hatte zudem Auswirkungen auf die Zellmorphologie, die Infektionsrate von Pflanzenzellen und die Generationszeit von A. tumefaciens.Item Open Access Studien zur biotechnologischen Anwendung und ökologischen Funktion von Pyrrolochinolinchinon(PQQ)-abhängigen Alkoholdehydrogenasen(2021) Wehrmann, Matthias; Hauer, Bernhard (Prof. Dr.)Item Open Access Molecular dynamics simulations of the substrate- and product specificity and mechanism of DNA- and protein lysine methyltransferases(2024) Schnee, Philipp; Jeltsch, Albert (Prof. Dr.)Protein Lysine Methyltransferases (PKMTs) regulate the epigenetic code of cells and their alteration via somatic mutations are often associated with cancer. The aim of this project is to rationalize the product and substrate specificity of this enzyme family by a combination of biochemical experiments and molecular dynamics simulations. Based on this, a detailed view of the underlying mechanism behind the disease associated mutations shall be gained, which may provide new possibilities for personalized cancer therapies.Item Open Access Overcoming glioblastoma intractability : pre-clinical characterisation of TRAIL sensitisation by marizomib and novel treatment perspectives(2022) Boccellato, Chiara; Morrison, Markus (Prof. Dr.)Glioblastoma (GBM) is the most aggressive cancer of the central nervous system (CNS). Surgical resection, adjuvant temozolomide-based chemotherapy and radiation are the primary treatments, yet the outcome of GBM patients remains poor with a median life expectancy of 15 to 17 months. Therefore, novel and effective treatment options are required, as are reliable pre-clinical experimental models that are suitable for exploratory studies on novel drugs and drug combinations. In this work, patient-derived cell line models (PDCL), generated from fresh primary or recurrent glioblastoma tumours, have been examined to assess prevalence of responsiveness to a highly stable hexavalent format of TRAIL receptor agonist (IZI1551) and to the blood brain barrier (BBB)-permeant proteasome inhibitor marizomib (MRZ). Serum-free medium and limited cultivation times of both 2D and 3D cancer cell cultures were adopted to maintain the characteristics of primary tumour cells. The degree of BBB permeability of marizomib was evaluated in the human hCMEC/D3 cell line model, which was also employed to test the efficacy of the IZI1551+MRZ combination in pre-clinical settings. It was found that IZI1551 and marizomib acted synergistically to induce apoptosis in the majority of low-passage PDCLs, both under 2D and 3D cultivation conditions. Altering the relative timing of drug exposure, specifically marizomib pre-treatment, led to even enhanced responses and allowed to lower drug concentrations without losing treatment efficacy. Importantly, the amount of marizomib that can cross the simple BBB model was sufficient to confer sensitisation to IZI1551. In cases of treatment resistance against IZI1551 and marizomib, lowering the mitochondrial apoptosis threshold with BH3 mimetics appeared sufficient to restore apoptosis sensitivity. Taken together, these results demonstrated that marizomib is a potent sensitiser of apoptosis induced by a 2nd generation TRAIL receptor agonist in glioblastoma. The optimized synergism between marizomib and IZI1551 in time-shifted treatment schedules, together with the ability of marizomib to cross the BBB, suggests this combination as a promising strategy to be tested in clinical settings. In the second part of this work, an alternative cell death pathway, namely ferroptosis, has been investigated as a strategy to bypass the obstacle of the apoptosis refractory state of highly resistant cancers such as glioblastoma. Ferroptosis is a recently identified form of iron-dependent regulated cell death that presents distinct features compared to apoptosis and that is characterised by the accumulation of toxic lipid peroxides. Here it was shown that the U-87 MG, a bona-fide glioblastoma cell line that was reported to be TRAIL resistant, displays a dose-dependent cell death response to the ferroptosis inducer RSL3. Surprisingly, it was found that BH3 mimetics antagonised this cytotoxicity. The unexpected consequences of combining these agents highlight the need to better understand the interactions between these drugs in order to advance their use as cancer therapeutics. Overall, this thesis presents diverse treatment options against glioblastoma that exploit either drugs classically inducing apoptosis or the alternative cell death modality of ferroptosis. Considering the limited availability of approved treatments, studies aiming at expanding the choice of glioblastoma therapeutics, such as those conducted in this work, are of particular importance and pave the way for their implementation at a clinical and pre-clinical level.Item Open Access Molekulare Mechanismen der Antiöstrogenwirkung beim Mammakarzinom(2002) Buck, Miriam; Pfizenmaier, Klaus (Prof. Dr.)Antiöstrogene haben sich als sehr effektiv in der Behandlung hormon-responsiver Mammakarzinome erwiesen. Im Verlauf der Therapie kommt es jedoch in der Regel zu einem Verlust der anithormonellen Wirkung. Die an der Entstehung der Antihormonresistenz beteiligten Mechanismen sind weitgehend ungeklärt. Ein entscheidender Schritt zur Aufklärung der antihormonellen Wirkung war die Beobachtung, dass die Behandlung hormonsensitiver Mammakarzinomzellen zu einer Aktivierung des inhibitorischen Wachstumsfaktors TGFb führt. Am Modell-System hormon-sensitiver MCF-7 Mammakarzinomzellen wurde im Rahmen dieser Arbeit die Beteiligung des TGFb-Systems an der Wirkung des nicht-steroidalen, partiellen Antiöstrogens 4-Hydroxytamoxifen und des steroidalen, reinen Antiöstrogens ICI 182.780 untersucht. Die Ergebnisse sollen zum besseren Verständnis der Interaktionen zwischen Hormonen und Wachstumsfaktoren und den damit im Zusammenhang stehenden Mechanismen der Antiöstrogenresistenz beitragen. Im Mittelpunkt des ersten Teils der Arbeit stand die antiöstrogene Regulation des TGFb-Systems. Untersucht wurde die Expression der Liganden TGFb1 und TGFb2, der Rezeptoren TbRI und TbRII und von Smad7. Zur genauen Quantifizierung der mRNA-Expression dieser Gene wurden spezifische LightCycler RT-PCRs etabliert. Es konnte gezeigt werden, dass nicht nur TGFb2 sondern auch TbRII einer antihormonellen Regulation unterliegt. Die Stärke der Induktion beider Gene korrelierte mit der wachstumsinhibitorischen Wirkung der Antiöstrogene. Smad7 wurde nur schwach durch Antiöstrogen induziert, TGFb1 und TbRI gar nicht. Im zweiten Teil der Arbeit wurde versucht Aufschluss darüber zu erhalten, welche Zweige des komplexen TGFb Signaltransduktionssystems an der antiöstrogenen Wirkung beteiligt sind. Als Endpunkt wurde u.a. die Aktivierung TGFb-sensitiver Promotoren untersucht. Das Reporterplasmid p3TP-lux wurde durch Antiöstrogene ca. 2fach stärker aktiviert als durch TGFb. Über Koexpression dominant-negativer TGFb-Rezeptoren und dominant-negativer Smad4-Proteine konnte gezeigt werden, dass die anitöstrogene Aktivierung von p3TP-lux TGFb vermittelt ist und über den Smad-Signaltransduktionsweg verläuft. Obwohl das steroidale Antiöstrogen ICI 182.780 einen deutlich stärkeren Effekt auf das TGFb-System hat als das partielle Antiöstrogen 4-Hydroxytamoxifen, war die Aktivierung von p3TP-lux durch beide Antiöstrogene annähernd gleich stark. Für eine vollständige Aktivierung von p3TP-lux ist eine Kooperation zwischen einem TGFb aktivierten Smad-Komplex und c-Jun/c-fos notwendig. Die Induktion von c-fos wird jedoch durch steroidale Antiöstrogene blockiert und kommt daher als Ursache der geringen Induktion von p3TP-lux durch ICI 182.780 in Frage. Da TGFb seine Wirkung neben dem Smad-Signaltransduktionsweg auch über MAP-Kinase-Wege entfalten kann, wurde mit Hilfe spezifischer pharmakologischer Inhibitoren die Beteiligung des MEK/Erk- und des p38-MAP-Kinase-Weges an der antihormonellen Wachstumsinhibition untersucht. Es konnte gezeigt werden, dass der p38-Weg über die Induktion von TGFb2 und TbRII an der antihormonellen Wachstumsinhibition beteiligt ist. Zusammenfassend wurde im Rahmen dieser Arbeit gezeigt, dass Antiöstrogene verschiedene Gene des TGFb-Signaltransduktionssystems differenziell regulieren. Die Untersuchungen zur Beteiligung der TGFb-Signaltransduktionswege an der antihormonellen Wirkung weisen darauf hin, dass diese differenzielle Regulation, durch die spezifische Aktivierung einiger TGFb-Signaltransduktionswege (Smad, p38) und gleichzeitige Inhibition anderer (MEK/Erk) erreicht wird. Da die Signaltransduktionswege in unterschiedlichem Ausmaß zur Aktivierung der verschiedenen Promotoren beitragen, kommt es unter Antiöstrogeneinfluss zu einer Modifizierung des TGFb induzierten Genexpressionsmusters und der spezifisch antiöstrogenen Wirkung.