@phdthesis{Hagen2017,
  author    = {Hagen, Franziska},
  title     = {Sphingolipids in gonococcal infection},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153852},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, has the potential to spread in the human host and cause a severe complication called disseminated gonococcal infection (DGI). The expression of the major outer membrane porin PorBIA is a characteristic of most gonococci associated with DGI. PorBIA binds to the scavenger receptor expressed on endothelial cells (SREC-I), which mediates the so-called low phosphate-dependent invasion (LPDI). This uptake mechanism enables N. gonorrhoeae to rapidly invade epithelial and endothelial cells in a phosphate-sensitive manner. We recently demonstrated that the neutral sphingomyelinase, which catalyses the hydrolysis of sphingomyelin to ceramide and phosphorylcholine, is required for the LPDI of gonococci in non-phagocytic cells. Neutral sphingomyelinase 2 (NSM2) plays a key role in the early PorBIA signaling by recruiting the PI3 kinase to caveolin. The following activation of the PI3 kinase-dependent downstream signaling leads to the engulfment of the bacteria. As a part of this work, I could confirm the involvement of the NSM2. The role of the enzyme was further elucidated by the generation of antibodies directed against NSM2 and the construction of an epithelium-based NSM2 knockout cell line using CRISPR/Cas9. The knockout of the NSM2 strongly inhibits the LPDI. The invasion could be, however, restored by the complementation of the knockout using an NSM2-GFP construct. However, the results could not be reproduced. In this work, I could show the involvement of further members of the sphingolipid pathway in the PorBIA-mediated invasion. Lipidome analysis revealed an increase of the bioactive molecules ceramide and sphingosine due to gonococcal infection. Both molecules do not only affect the host cell, but seem to influence the bacteria as well: while ceramide seems to be incorporated by the gonococci, sphingosine is toxic for the bacteria. Furthermore, the sphingosine kinase 2 (SPHK2) plays an important role in invasion, since the inhibition and knockdown of the enzyme revealed a negative effect on gonococcal invasion. To elucidate the role of the sphingosine kinases in invasion in more detail, an activity assay was established in this study. Additionally, the impact of the sphingosine-1-phosphate lyase (S1PL) on invasion was investigated. Inhibitor studies and infection experiments conducted with a CRISPR/Cas9 HeLa S1PL knockout cell line revealed a role of the enzyme not only in the PorBIA-mediated invasion, but also in the Opa50/HSPG-mediated gonococcal invasion. The signaling experiments allowed the categorization of the SPHK and S1PL activation in the context of infection. Like the NSM2, both enzymes play a role in the early PorBIA signaling events leading to the uptake of the bacteria. All those findings indicate an important role of sphingolipids in the invasion and survival of N. gonorrhoeae. In the last part of this work, the role of the NSM2 in the inhibition of apoptosis in neutrophils due to gonococcal infection was investigated. It could be demonstrated that the delayed onset of apoptosis is independent of neisserial porin and Opa proteins. Furthermore, the influence of neisserial peptidoglycan on PMN apoptosis was analysed using mutant strains, but no connection could be determined. Since the NSM2 is the most prominent sphingomyelinase in PMNs, fulfils manifold cell physiological functions and has already been connected to apoptosis, the impact of the enzyme on apoptosis inhibition due to gonococcal infection was investigated using inhibitors, with no positive results.},
  subject      = {gonococcal},
  language  = {en}
}
@phdthesis{Bruehlmann2017,
  author    = {Br{\"u}hlmann, David},
  title     = {Tailoring Recombinant Protein Quality by Rational Media Design},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147345},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Nowadays, more than half of the biotherapeutics are produced in mammalian cell lines as a result of correct protein folding and assembly as well as their faculty to bring about a variety of post-translational modifications. The widespread progression of biosimilars has moved the focus in mammalian cell-culture process development. Thereby, the modulation of quality attributes of recombinant therapeutic proteins has increasingly gained importance from early process development stages. Protein quality directly shapes the clinical efficacy and safety in vivo, and therefore, the control of the complex post-translational modifications, such as glycosylation (e.g. high mannose, fucosylation, galactosylation and sialylation), charge variants, aggregates and low-molecular-weight species formation, is pivotal for efficient receptor binding and for triggering the desired immune responses in patients. In the frame of biosimilar development, product quality modulation methods using the potential of the host cell line are particularly sought after to match the quality profile of the targeted reference medicinal product (RMP) as closely as possible. The environment the cell is dwelling in directly influences its metabolism and the resulting quality profile of the expressed protein. Thereby the cell culture medium plays a central role in upstream manufacturing. In this work, concentration adjustment of selected media components and supplementation with a variety of compounds was performed to alter various metabolic pathways, enzyme activities and in some cases the gene expression levels of Chinese Hamster Ovary (CHO) cells in culture. The supplementation of cell culture medium with the trisaccharide raffinose in fed-batch cultures entailed an increase of the abundance of high mannose glycans in two different CHO cell lines. Raffinose especially favored mannose 5 glycans. At the same time, it impaired cell culture performance, induced changes on the intracellular nucleotide levels and even varied the expression levels of glycosylation-related genes. Supplementation with a number of galactosyltransferase inhibiting compounds, in particular fluorinated galactose analogs (alpha- and beta-2F-peracetyl-galactose), consistently decreased the production of galactosylated monoclonal antibodies (mAb). By means of targeted addition during the culture rather than at the beginning, the inhibition was further increased, while limiting detrimental effects on both growth and productivity. High-throughput screening in 96-deepwell plates showed that spermine and L-ornithine also reduced the level of galactosylation. On the other hand, exploratory screening of a variety of potentially disulfide-bridge-reducing agents highlighted that the inherent low-molecular-species level of the proprietary platform cell culture process was likely due to favored reduction. This hypothesis was reinforced by the observation that supplementation of cysteine and N-acetylcysteine promoted fragmentation. Additionally, fragmentation decreased with higher protein expression. At that point, aiming to improve the efficiency in process development, a rational experimental design method was developed to identify and to define the optimal concentration range of quality modulating compounds by calling on a combination of high throughput fed-batch testing and multivariate data analysis. Seventeen medium supplements were tested in five parallel 96-deepwell plate experiments. The selection process of promising modulators for the follow-up experiment in shake tubes consisted in a three-step procedure, including principal component analysis, quantitative evaluation of their performance with respect to the specifications for biosimilarity and selection following a hierarchical order of decisions using a decision tree. The method resulted in a substantial improvement of the targeted glycosylation profile in only two experimental rounds. Subsequent development stages, namely validation and transfer to industrial-scale facilities require tight control of product quality. Accordingly, further mechanistic understanding of the underlying processes was acquired by non-targeted metabolomic profiling of a CHO cell line expressing a mAb cultured in four distinct process formats. Univariate analysis of intra- and extracellular metabolite and temporal glycosylation profiles provided insights in various pathways. The numerous of parameters were the main driver to carry out principal component analysis, and then, using the methodology of partial-least-square (PLS) projection on latent structures, a multivariate model was built to correlate the extracellular data with the distinct glycosylation profiles. The PLS observation model proved to be reliable and showed its great benefit for glycan pattern control in routine manufacturing, especially at large scale. Rather than relying on post-production interpretation of glycosylation results, glycosylation can be predicted in real-time based on the extracellular metabolite levels in the bioreactor. Finally, for the bioactivity assessment of the glycan differences between the biosimilar and the reference medicinal product (RMP), the health agencies may ask for in the drug registration process, extended ranges of glycan variants need to be generated so that the in vitro assays pick up the changes. The developed glycosylation modulator library enabled the generation of extreme glycosylation variants, including high mannose, afucosylated, galactosylated as well as sialic acid species of both a mAb and an antibody fusion molecule with three N-glycosylation sites. Moreover, to create increased variety, enzymatic glycoengineering was explored for galactosylation and sialylation. The glyco variants induced significant responses in the respective in vitro biological activity assays. The data of this work highlight the immense potential of cell culture medium optimization to adjust product quality. Medium and feed supplementation of a variety of compounds resulted in reproducible and important changes of the product quality profile of both mAbs and a fusion antibody. In addition to the intermediate modulation ranges that largely met the requirements for new-biological-entity and biosimilar development, medium supplementation even enabled quick and straightforward generation of extreme glycan variants suitable for biological activity testing.},
  subject      = {Zellkultur},
  language  = {en}
}
@phdthesis{Iltzsche2017,
  author    = {Iltzsche, Fabian},
  title     = {The Role of DREAM/MMB-mediated mitotic gene expression downstream of mutated K-Ras in lung cancer},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-154108},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The evolutionary conserved Myb-MuvB (MMB) multiprotein complex has an essential role in transcriptional activation of mitotic genes. MMB target genes as well as the MMB associated transcription factor B-Myb and FoxM1 are highly expressed in a range of different cancer types. The elevated expression of these genes correlates with an advanced tumor state and a poor prognosis. This suggests that MMB could contribute to tumorigenesis by mediating overexpression of mitotic genes. Although MMB has been extensively characterized biochemically, the requirement for MMB to tumorigenesis in vivo remains largely unknown and has not been tested directly so far. In this study, conditional knockout of the MMB core member Lin9 inhibits tumor formation in vivo in a mouse model of lung cancer driven by oncogenic K-Ras and loss of p53. The incomplete recombination observed within tumors points towards an enormous selection pressure against the complete loss of Lin9. RNA interference (RNAi)-mediated depletion of Lin9 or the MMB associated subunit B-Myb provides evidence that MMB is required for the expression of mitotic genes in lung cancer cells. Moreover, it was demonstrated that proliferation of lung cancer cells strongly depends on MMB. Furthermore, in this study, the relationship of MMB to the p53 tumor suppressor was investigated in a primary lung cancer cell line with restorable p53 function. Expression analysis revealed that mitotic genes are downregulated after p53 re-expression. Moreover, activation of p53 induces formation of the repressive DREAM complex and results in enrichment of DREAM at mitotic gene promoters. Conversely, MMB is displaced at these promoters. Based on these findings the following model is proposed: In p53-negative cells, mitogenic stimuli foster the switch from DREAM to MMB. Thus, mitotic genes are overexpressed and may promote chromosomal instability and tumorigenesis. This study provides evidence that MMB contributes to the upregulation of G2/M phase-specific genes in p53-negative cells and suggests that inhibition of MMB (or its target genes) might be a strategy for treatment of lung cancer.},
  subject      = {Nicht-kleinzelliges Bronchialkarzinom (NSCLC)},
  language  = {en}
}