@phdthesis{Collenburg2018,
  author    = {Collenburg, Lena},
  title     = {The Role of Ceramides and Sphingomyelinases for Dynamic Membrane Processes in T Cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151161},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Previous work of our group has established a role of sphingomyelinases in the regulation of T cell responses to TCR or pathogen stimulation, and this became particularly evident at the level of actin cytoskeletal dynamics. The formation of lipid membrane microdomains is crucial for receptor clustering and signal induction, and therefore, ceramide accumulation by membrane sphingomyelin breakdown is needed for signalling- complex-assembly. Pathogen-induced overshooting of SMase activation substantially impacted the formation of membrane protrusions, with T cell spreading as well as a front/rear polarisation upon CD3/CD28 co-stimulation [103]. On the other hand, NSM activation is part of the physiological TCR signal [67], indicating that a spatiotemporally balanced NSM activation is crucial for its physiological function. It involves actin cytoskeletal reorganisation and T cell polarisation. These two functions are also of central importance in directional T cell migration and motility in tissues. This thesis aims on defining the role of NSM in compartmentalisation of the T cell membrane in polarisation and migration. Therefore, functional studies on the impact of NSM activity in these processes had to be complemented by the development of tools to study ceramide compartmentalisation in living T cells.},
  subject      = {Ceramides},
  language  = {en}
}
@phdthesis{Candemir2018,
  author    = {Candemir, Esin},
  title     = {Involvement of neuronal nitric oxide synthase (NOS-I) PDZ interactions in neuropsychiatric disorders},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151194},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Neuronal nitric oxide (NO) synthase (NOS-I) and its adaptor protein (NOS1AP) have been repeatedly and consistently associated with neuropsychiatric disorders in several genetic association and linkage studies, as well as functional studies. NOS-I has an extended PDZ domain which enables it to interact with postsynaptic density protein 95 (PSD-95) bringing NOS-I in close proximity to NMDA receptors. This interaction allows NMDA receptor activity dependent calcium-influx to activate NOS-I, linking NO synthesis to regulation of glutamatergic signaling pathways. NOS1AP is a PDZ-domain ligand of NOS-I and has been proposed to compete with PSD-95 for NOS-I interaction. Studies performed on post-mortem brain tissues have shown increased expression of NOS1AP in patients with schizophrenia and bipolar disorder, suggesting that increased NOS-I/NOS1AP interactions might be involved in neuropsychiatric disorders possibly through disruption of NOS-I PDZ interactions. Therefore, I have investigated the involvement of NOS-I in different endophenotypes of neuropsychiatric disorders by targeting its specific PDZ interactions in vitro and in vivo. To this end, I used recombinant adeno-associated virus (rAAV) vectors expressing NOS1AP isoforms/domains (NOS1AP-L: full length NOS1AP; NOS1AP-LC20: the last 20 amino acids of NOS1AP-L, containing the PDZ interaction motif suggested to stabilize interaction with NOS-I; NOS1AP-LΔC20: NOS1AP-L lacking the last 20 amino acids; NOS1AP-S: the short isoform of NOS1AP), residues 396-503 of NOS1AP-L (NOS1AP396-503) encoding the full NOS-I interaction domain, and N-terminal 133 amino acids of NOS-I (NOS-I1-133) encoding for the extended PDZ-domain. Neuropsychiatric disorders involve morphological brain changes including altered dendritic development and spine plasticity. Hence, I have examined dendritic morphology in primary cultured hippocampal and cortical neurons upon overexpression of constructed rAAV vectors. Sholl analysis revealed that overexpression of NOS1AP-L and NOS1AP-LΔC20 mildly reduced dendritic length/branching. Moreover, overexpression of all NOS1AP isoforms/domains resulted in highly altered spine plasticity including significant reduction in the number of mature spines and increased growth of filopodia. These findings suggest that NOS1AP affects dendritic growth and development of dendritic spines, which may involve both, increased NOS-I/NOS1AP interaction as well as interaction of NOS1AP with proteins other than NOS-I. Interestingly, the observed alterations in dendritic morphology were reminiscent of those observed in post-mortem brains of patients with neuropsychiatric disorders. Given the dendritic alterations in vitro, I have examined, whether disruption of NOS-I PDZ interaction would also result in behavioral deficits associated with neuropsychiatric disorders. To this end, rAAV vectors expressing NOS1AP-L, NOS1AP396-503, NOS-I1-133, and mCherry were stereotaxically delivered to the dorsal hippocampus of 6-week-old male C57Bl/6J mice. One week after surgery, mice were randomly separated into two groups. One of those groups underwent three weeks of chronic mild stress (CMS). Afterwards all mice were subjected to a comprehensive behavioral analysis. The findings revealed that overexpression of the constructs did not result in phenotypes related to anxiety or depression, though CMS had an anxiolytic effect independent of the injected construct. Mice overexpressing NOS-I1-133, previously shown to disrupt NOS-I/PSD-95 interaction, showed impaired spatial memory, sensorimotor gating, social interaction, and increased locomotor activity. NOS1AP overexpressing mice showed mild impairments in sensorimotor gating and spatial working memory and severely impaired social interaction. NOS1AP396-503 overexpressing mice also showed impaired social interaction but enhanced sensorimotor gating and reduced locomotor activity. Taken together, these behavioral findings indicate an involvement of NOS-I PDZ interactions in phenotypes associated with positive symptoms and cognitive deficits of psychotic disorders. In summary, this study revealed an important contribution of NOS-I protein interactions in the development of endophenotypic traits of neuropsychiatric disorders, in particular schizophrenia, at morphological and behavioral levels. These findings might eventually aid to a better understanding of NOS-I-dependent psychopathogenesis, and to develop pharmacologically relevant treatment strategies.},
  subject      = {Stickstoffmonoxid-Synthase},
  language  = {en}
}
@phdthesis{Kleffel2018,
  author    = {Kleffel, Sonja Beate},
  title     = {The role of cancer cell-expressed PD-1 in tumorigenesis and tumor immune evasion},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151205},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Melanoma and Merkel cell carcinoma (MCC) are highly aggressive cancers of the skin that frequently escape immune recognition and acquire resistance to chemotherapeutic agents, which poses a major obstacle to successful cancer treatment. Recently, a new class of therapeutics targeting the programmed cell death-1 (PD-1) immune checkpoint receptor has shown remarkable efficacy in the treatment of both cancers. Blockade of PD-1 on T cells activates cancer-specific immune responses that can mediate tumor regression. The data presented in this Ph.D. thesis demonstrates that PD-1 is also expressed by subsets of cancer cells in melanoma and MCC. Moreover, this work identifies PD-1 as a novel tumor cell-intrinsic growth receptor, even in the absence of T cell immunity. PD-1 is expressed by tumorigenic cell subsets in melanoma patient samples and established human and murine cell lines that also co-express ABCB5, a marker of immunoregulatory tumor- initiating cells in melanoma. Consistently, melanoma-expressed PD-1 downmodulates T effector cell functions and increases the intratumoral frequency of tolerogenic myeloid- derived suppressor cells. PD-1 inhibition on melanoma cells by RNA interference, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, including in mice lacking adaptive immunity. Engagement of melanoma- PD-1 by its ligand PD-L1 promotes tumor growth, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuates growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor activates mTOR signaling mediators, including ribosomal protein S6. In a proof-of-concept study, tumoral expression of phospho-S6 in pretreatment tumor biopsies correlated with clinical responses to anti-PD-1 therapy in melanoma patients. In MCC, PD-1 is similarly co-expressed by ABCB5+ cancer cell subsets in clinical tumor specimens and established human cell lines. ABCB5 renders MCC cells resistant to the standard-of-care chemotherapeutic agents, carboplatin and etoposide. Antibody-mediated ABCB5 blockade reverses chemotherapy resistance and inhibits tumor xenograft growth by enhancing chemotherapy-induced tumor cell killing. Furthermore, engagement of MCC-expressed PD-1 by its ligands, PD-L1 and PD-L2, promotes proliferation and activates MCC-intrinsic mTOR signaling. Consistently, antibody- mediated PD-1 blockade inhibits MCC tumor xenograft growth and phosphorylation of mTOR effectors in immunocompromised mice. In summary, these findings identify cancer cell-intrinsic functions of the PD-1 pathway in tumorigenesis and suggest that blocking melanoma- and MCC-expressed PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy. Additionally, these results establish ABCB5 as a previously unrecognized chemoresistance mechanism in MCC.},
  subject      = {Melanom},
  language  = {en}
}
@phdthesis{Lerch2018,
  author    = {Lerch, Maike Franziska},
  title     = {Characterisation of a novel non-coding RNA and its involvement in polysaccharide intercellular adhesin (PIA)-mediated biofilm formation of \(Staphylococcus\) \(epidermidis\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155777},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, have been recognised as an important cause of health care-associated infections due to catheterisation, and livestock-associated infections. The colonisation of indwelling medical devices is achieved by the formation of biofilms, which are large cell-clusters surrounded by an extracellular matrix. This extracellular matrix consists mainly of PIA (polysaccharide intercellular adhesin), which is encoded by the icaADBC-operon. The importance of icaADBC in clinical strains provoking severe infections initiated numerous investigations of this operon and its regulation within the last two decades. The discovery of a long transcript being located next to icaADBC, downstream of the regulator gene icaR, led to the hypothesis of a possible involvement of this transcript in the regulation of biofilm formation (Eckart, 2006). Goal of this work was to characterise this transcript, named ncRNA IcaZ, in molecular detail and to uncover its functional role in S. epidermidis. The ~400 nt long IcaZ is specific for ica-positive S. epidermidis and is transcribed in early- and mid-exponential growth phase as primary transcript. The promotor sequence and the first nucleotides of icaZ overlap with the 3' UTR of the preceding icaR gene, whereas the terminator sequence is shared by tRNAThr-4, being located convergently to icaZ. Deletion of icaZ resulted in a macroscopic biofilm-negative phenotype with highly diminished PIA-biofilm. Biofilm composition was analysed in vitro by classical crystal violet assays and in vivo by confocal laser scanning microscopy under flow conditions to display biofilm formation in real-time. The mutant showed clear defects in initial adherence and decreased cell-cell adherence, and was therefore not able to form a proper biofilm under flow in contrast to the wildtype. Restoration of PIA upon providing icaZ complementation from plasmids revealed inconsistent results in the various mutant backgrounds. To uncover the functional role of IcaZ, transcriptomic and proteomic analysis was carried out, providing some hints on candidate targets, but the varying biofilm phenotypes of wildtype and icaZ mutants made it difficult to identify direct IcaZ mRNA targets. Pulse expression of icaZ was then used as direct fishing method and computational target predictions were executed with candidate mRNAs from aforesaid approaches. The combined data of these analyses suggested an involvement of icaR in IcaZ-mediated biofilm control. Therefore, RNA binding assays were established for IcaZ and icaR mRNA. A positive gel shift was maintained with icaR 3' UTR and with 5'/3' icaR mRNA fusion product, whereas no gel shift was obtained with icaA mRNA. From these assays, it was assumed that IcaZ regulates icaR mRNA expression in S. epidermidis. S. aureus instead lacks ncRNA IcaZ and its icaR mRNA was shown to undergo autoregulation under so far unknown circumstances by intra- or intermolecular binding of 5' UTR and 3' UTR (Ruiz de los Mozos et al., 2013). Here, the Shine-Dalgarno sequence is blocked through 5'/3' UTR base pairing and RNase III, an endoribonuclease, degrades icaR mRNA, leading to translational blockade. In this work, icaR mRNA autoregulation was therefore analysed experimentally in S. epidermidis and results showed that this specific autoregulation does not take place in this organism. An involvement of RNase III in the degradation process could not be verified here. GFP-reporter plasmids were generated to visualise the interaction, but have to be improved for further investigations. In conclusion, IcaZ was found to interact with icaR mRNA, thereby conceivably interfering with translation initiation of repressor IcaR, and thus to promote PIA synthesis and biofilm formation. In addition, the environmental factor ethanol was found to induce icaZ expression, while only weak or no effects were obtained with NaCl and glucose. Ethanol, actually is an ingredient of disinfectants in hospital settings and known as efficient effector for biofilm induction. As biofilm formation on medical devices is a critical factor hampering treatment of S. epidermidis infections in clinical care, the results of this thesis do not only contribute to better understanding of the complex network of biofilm regulation in staphylococci, but may also have practical relevance in the future.},
  subject      = {Biofilm},
  language  = {en}
}
@phdthesis{Semeniak2018,
  author    = {Semeniak, Daniela},
  title     = {Role of bone marrow extracellular matrix proteins on platelet biogenesis and function},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155857},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Platelets, small anucleated blood cells responsible for hemostasis, interact at sights of injury with several exposed extracellular matrix (ECM) proteins through specific receptors. Ligand binding leads to activation, adhesion and aggregation of platelets. Already megakaryocytes (MKs), the immediate precursor cells in bone marrow (BM), are in constant contact to these ECM proteins (ECMP). The interaction of ECMP with MKs is, in contrast to platelets, less well understood. It is therefore important to study how MKs interact with sinusoids via the underlying ECMP. This thesis addresses three major topics to elucidate these interactions and their role in platelet biogenesis. First, we studied the topology of ECMP within BM and their impact on proplatelet formation (PPF) in vitro. By establishing a four-color immunofluorescence microscopy we localized collagens and other ECMP and determined their degree of contact towards vessels and megakaryocytes (MKs). In in vitro assays we could demonstrate that Col I mediates increased MK adhesion, but inhibits PPF by collagen receptor GPVI. By immunoblot analyses we identified that the signaling events underyling this inhibition are different from those in platelet activation at the Src family kinase level. Second, we determined the degree of MK-ECM interaction in situ using confocal laser scanning microscopy of four-color IF-stained femora and spleen sections. In transgenic mouse models lacking either of the two major collagen receptors we could show that these mice have an impaired association of MKs to collagens in the BM, while the MK count in spleen increased threefold. This might contribute to the overall unaltered platelet counts in collagen receptor-deficient mice. In a third approach, we studied how the equilibrium of ECMP within BM is altered after irradiation. Collagen type IV and laminin-α5 subunits were selectively degraded at the sinusoids, while the matrix degrading protease MMP9 was upregulated in MKs. Platelet numbers decreased and platelets became hyporesponsive towards agonists, especially those for GPVI activation. Taken together, the results indicate that MK-ECM interaction differs substantially from the well-known platelet-ECM signaling. Future work should further elucidate how ECMP can be targeted to ameliorate the platelet production and function defects, especially in patients after BM irradiation.},
  subject      = {Knochenmark},
  language  = {en}
}
@phdthesis{Boeck2018,
  author    = {B{\"o}ck, Thomas},
  title     = {Multifunctional Hyaluronic Acid / Poly(glycidol) Hydrogels for Cartilage Regeneration Using Mesenchymal Stromal Cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155345},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Improved treatment options for the degenerative joint disease osteoarthritis (OA) are of major interest, since OA is one of the main sources of disability, pain, and socioeconomic burden worldwide [202]. According to epidemiological data, already 27 million people suffer from OA in the US [23]. Moreover, the WHO expects OA to be the fourth most common cause of disability in 2020 [203], illustrating the need for effective and long-lasting therapy options of severe cartilage defects. Despite numerous clinically available products for the treatment of cartilage defects [62], the development of more cartilage-specific materials is still at the beginning. Hyaluronic acid (HA) is a major component of the cartilaginous extracellular matrix (ECM) and inherently creates a cell-friendly niche by providing cell attachment and migration sites. Furthermore, it is known that the functional groups of HA are well suited for chemical modification. These characteristics render HA an attractive material for hydrogel-based tissue engineering approaches. Poly(glycidol) (PG) as chemical crosslinker basically features similar chemical characteristics as the widely used poly(ethylene glycol) (PEG), but provides additional side groups at each repeating unit that can be further chemically functionalized. With the introduction of PG as multifunctional crosslinker for HA gels, a higher cross-linking density and, accordingly, a greater potential for biomimetic functionalization may be achieved. However, despite the mentioned potential benefits, PG has not been used for cartilage regeneration approaches so far. The initial aim of the study was to set up and optimize a HA-based hydrogel for the chondrogenic differentiation of mesenchymal stromal cells (MSCs), using different amounts and variations of cross-linkers. Therefore, the hydrogel composition was optimized by the utilization of different PEG diacrylate (PEGDA) concentrations to cross-link thiol-modified HA (Glycosil, HA-SH) via Michael addition. We aimed to generate volumestable scaffolds that simultaneously enable a maximum of ECM deposition. Histological and biochemical analysis showed 0.4\% PEGDA as the most suitable concentration for these requirements (Section 5.1.2). In order to evaluate the impact of a differently designed cross-linker on MSC chondrogenesis, HA-SH was cross-linked with PEGTA (0.6\%) and compared to PEGDA (0.4\%) in a next step. Following this, acrylated PG (PG-Acr) as multifunctional cross-linker alternative to acrylated PEG was evaluated. It provides around five times more functional groups when utilized in PG-Acr (0.6\%) HA-SH hydrogels compared to PEGTA (0.6\%) HA-SH hydrogels, thus enabling higher degrees of biomimetic functionalization. Determination of cartilage-specific ECM components showed no substantial differences between both cross-linkers while the deposition of cartilaginous matrix appeared more homogeneous in HA-SH PG-Acr gels. Taken together, we were able to successfully increase the possibilities for biomimetic functionalization in the developed HA-SH hydrogel system by the introduction of PG-Acr as cross-linker without negatively affecting MSC chondrogenesis (Section 5.1.3). The next part of this thesis focused extensively on the biomimetic functionalization of PG-Acr (0.6\%) cross-linked HA-SH hydrogels. Here, either biomimetic peptides or a chondrogenic growth factor were covalently bound into the hydrogels. Interestingly, the incorporation of a N-cadherin mimetic (HAV), a collagen type II binding (KLER), or a cell adhesion-mediating peptide (RGD) yielded no improvement of MSC chondrogenesis. For instance, the covalent binding of 2.5mM HAV changed morphology of cell nuclei and reduced GAG production while the incorporation of 1.0mM RGD impaired collagen production. These findings may be attributed to the already supportive conditions of the employed HA-based hydrogels for chondrogenic differentiation. Most of the previous studies reporting positive peptide effects on chondrogenesis have been carried out in less supportive PEG hydrogels or in significantly stiffer MeHA-based hydrogels [99, 101, 160]. Thus, the incorporation of peptides may be more important under unfavorable conditions while inert gel systems may be useful for studying single peptide effects (Section 5.2.1). The chondrogenic factor transforming growth factor beta 1 (TGF-b1) served as an example for growth factor binding to PG-Acr. The utilization of covalently bound TGF-b1 may thereby help overcome the need for repeated administration of TGF-b1 in in vivo applications, which may be an advantage for potential clinical application. Thus, the effect of covalently incorporated TGF-b1 was compared to the effect of the same amount of TGF-b1 without covalent binding (100nM TGF-b1) on MSC chondrogenesis. It was successfully demonstrated that covalent incorporation of TGF-b1 had a significant positive effect in a dose-dependent manner. Chondrogenesis of MSCs in hydrogels with covalently bound TGF-b1 showed enhanced levels of chondrogenesis compared to hydrogels into which TGF-b1 was merely mixed, as shown by stronger staining for GAGs, total collagen, aggrecan and collagen type II. Biochemical evaluation of GAG and collagen amounts, as well as Western blot analysis confirmed the histological results. Furthermore, the positive effect of covalently bound TGF-b1 was shown by increased expression of chondrogenic marker genes COL2A1, ACAN and SOX9. In summary, covalent growth factor incorporation utilizing PG-Acr as cross-linker demonstrated significant positive effects on chondrogenic differentiation of MSCs (Section 5.2.2). In general, PG-Acr cross-linked HA hydrogels generated by Michael addition represent a versatile hydrogel platform due to their high degree of acrylate functionality. These hydrogels may further offer the opportunity to combine several biological modifications, such as the incorporation of biomimetic peptides together with growth factors, within one cell carrier. A proof-of-principle experiment demonstrated the suitability of pure PG gels for studying single peptide effects. Here, the hydrogels were generated by the utilization of thiol-ene-click reaction. In this setting, without the supportive background of hyaluronic acid, MSCs showed enhanced chondrogenic differentiation in response to the incorporation of 1.0mM HAV. This was demonstrated by staining for GAGs, the cartilage-specific ECM molecules aggrecan and type II collagen, and by increased GAG and total collagen amounts shown by biochemical analysis. Thus, pure PG gels exhibit the potential to study the effects and interplay of peptides and growth factors in a highly modifiable, bioinert hydrogel environment. The last section of the thesis was carried out as part of the EU project HydroZONES that aims to develop and generate zonal constructs. The importance of zonal organization has attracted increased attention in the last years [127, 128], however, it is still underrepresented in tissue engineering approaches so far. Thus, the feasibility of zonal distribution of cells in a scaffold combining two differently composed hydrogels was investigated. A HA-SH(FMZ) containing bottom layer was generated and a pure PG top layer was subsequently cast on top of it, utilizing both times thiol-ene-click reaction. Indeed, stable, hierarchical constructs were generated that allowed encapsulated MSCs to differentiate chondrogenically in both zones as shown by staining for GAGs and collagen type II, and by quantification of GAG amount. Thus, the feasibility of differently composed zonal hydrogels utilizing PG as a main component was successfully demonstrated (Section 5.4). With the first-time utilization and evaluation of PG-Acr as versatile multifunctional cross-linker for the preparation of Michael addition-generated HA-SH hydrogels in the context of cartilage tissue engineering, a highly modifiable HA-based hydrogel system was introduced. It may be used in future studies as an easily applicable and versatile toolbox for the generation of biomimetically functionalized hydrogels for cell-based cartilage regeneration. The introduction of reinforcement structures to enhance mechanical resistance may thereby further increase the potential of this system for clinical applications. Additionally, it was also demonstrated that thiol-ene clickable hydrogels can be used for the generation of cell-laden, pure PG gels or for the generation of more complex, coherent zonal constructs. Furthermore, thiol-ene clickable PG hydrogels have already been further modified and successfully been used in 3D bioprinting experiments [204]. 3D bioprinting, as part of the evolving biofabrication field [205], offers the possibilities to generate complex and hierarchical structures, and to exactly position defined layers, yet at the same time alters the requirements for the utilized hydrogels [159, 206-209]. Since a robust chondrogenesis of MSCs was demonstrated in the thiol-ene clickable hydrogel systems, they may serve as a basis for the development of hydrogels as so called bioinks which may be utilized in more sophisticated biofabrication processes.},
  subject      = {Hyalurons{\"a}ure},
  language  = {en}
}
@phdthesis{Ziegenhals2018,
  author    = {Ziegenhals, Thomas},
  title     = {The role of the miR-26 family in neurogenesis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156395},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {For the differentiation of a embryonic stem cells (ESCs) to neuronal cells (NCs) a complex and coordinated gene regulation program is needed. One important control element for neuronal differentiation is the repressor element 1 silencing transcription factor (REST) complex, which represses neuronal gene expression in non-neuronal cells. Crucial effector proteins of the REST complex are small phosphatases such as the CTDSPs (C-terminal domain small phosphatases) that regulate polymerase II activity by dephosphorylating the C-terminal domain of the polymerase, thereby repressing target genes. The stepwise inactivation of REST, including the CTDSPs, leads to the induction of a neuron-specific gene program, which ultimately induces the formation of neurons. The spatio-temporal control of REST and its effector components is therefore a crucial step for neurogenesis. In zebrafish it was shown that the REST-associated CTDSP2 is negatively regulated by the micro RNA (miR) -26b. Interestingly, the miR-26b is encoded in an intron of the primary transcript of CTDSP2. This gives the fundament of an intrinsic regulatory negative feedback loop, which is essential for the proceeding of neurogenesis. This feedback loop is active during neurogenesis, but inactive in non-neuronal cells. The reason for this is that the maturation of the precursor miR (pre-miR) to the mature miR-26 is arrested in non neuronal cells, but not in neurons. As only mature miRs are actively repressing genes, the regulation of miR-26 processing is an essential step in neurogenesis. In this study, the molecular basis of miR-26 processing regulation in the context of neurogenesis was addressed. The mature miR is processed from two larger precursors: First the primary transcript is cleaved by the enzyme DROSHA in the nucleus to form the pre-miR. The pre-miR is exported from the nucleus and processed further through the enzyme DICER to yield the mature miR. The mature miR can regulate gene expression in association with the RNA-induced silencing complex (RISC). Multiple different scenarios in which miR processing was regulated were proposed and experimentally tested. Microinjection studies using Xenopus leavis oocytes showed that slowdown or blockage of the nucleo-cytoplasmic transport are not the reason for delayed pre-miR-26 processing. Moreover, in vitro and in vivo miR-processing assays showed that maturation is most likely regulated through a in trans acting factor, which blocks processing in non neuronal cells. Through RNA affinity chromatographic assays using zebrafish and murine lysates I was able to isolate and identify proteins that interact specifically with pre-miR-26 and could by this influence its biogenesis. Potential candidates are FMRP/FXR1/2, ZNF346 and Eral1, whose functional characterisation in the context of miR-biogenesis could now be addressed. The second part of my thesis was executed in close colaboration with the laboratory of Prof. Albrecht M{\"u}ller. The principal question was addressed how miR-26 influences neuronal gene expression and which genes are primarily affected. This research question could be addressed by using a cell culture model system, which mimics ex vivo the differentiation of ESCs to NCs via neuronal progenitor. For the functional analysis of miR-26 knock out cell lines were generated by the CRISPR/Cas9 technology. miR-26 deficient ESC keep their pluripotent state and are able to develop NPC, but show major impairment in differentiating to NCs. Through RNA deep sequencing the miR-26 induced transcriptome differences could be analysed. On the level of mRNAs it could be shown, that the expression of neuronal gene is downregulated in miR-26 deficient NCs. Interestingly, the deletion of miR-26 leads to selectively decreased levels of miRs, which on one hand regulate the REST complex and on the other hand are under transcriptional control by REST themself. This data and the discovery that induction of miR-26 leads to enrichment of other REST regulating miRs indicates that miR-26 initiates neurogenesis through stepwise inactivation of the REST complex.},
  subject      = {miRNS},
  language  = {en}
}
@phdthesis{Chen2018,
  author    = {Chen, Jiangtian},
  title     = {Functions of allatostatin A (AstA) and myoinhibitory peptides (MIPs) in the regulation of food intake and sleep in Drosophila},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156838},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Neuropeptides and peptide hormones carrying neural or physiological information are intercellular signalling substances. They control most if not all biological processes in vertebrates and invertebrates by acting on specific receptors on the target cell. In mammals, many different neuropeptides and peptide hormones are involved in the regulation of feeding and sleep. In \textit{Drosophila}, allatostatin A (AstA) and myoinhibitory peptides (MIPs) are brain-gut peptides. The AstA receptors are homologues of the mammalian galanin receptors and the amino acid sequences of MIPs are similar to a part of galanin, which has an orexigenic effect and is implicated in the control of sleep behaviour in mammals. I am interested in dissecting pleiotropic functions of AstA and MIPs in the regulation of food intake and sleep in \textit{Drosophila}. \par In the first part of the dissertation the roles of brain-gut peptide allatostatin A are analysed. Due to the genetic and molecular tools available, the fruit fly \textit{Drosophila melanogaster} is chosen to investigate functions of AstA. The aims in this part are to identify pleiotropic functions of AstA and assign specific effects to the activity of certain subsets of AstA expressing cells in \textit{Drosophila} adults. A new and restricted \textit{AstA\textsuperscript{34}-Gal4} line was generated. The confocal imaging result showed that AstA neurons are located in the posterior lateral protocerebrum (PLP), the gnathal ganglia (GNG), the medullae, and thoracic-abdominal ganglion (TAG). AstA producing DLAa neurons in the TAG innervate hindgut and the poterior part of midgut. In addition, AstA are detected in the enteroendocrine cells (EECs).\par Thermogenetic activation and neurogenetic silencing tools with the aid of the \textit{UAS/Gal4} system were employed to manipulate the activity of all or individual subsets of AstA cells and investigate the effects on food intake, locomotor activity and sleep. Our experimental results showed that thermogenetic activation of two pairs of PLP neurons and/or AstA expressing EECs reduced food intake, which can be traced to AstA signalling by using \textit{AstA} mutants. In the locomotor activity, thermogenetic activation of two pairs of PLP neurons and/or AstA expressing EECs resulted in strongly inhibited locomotor activity and promoted sleep without sexual difference, which was most apparent during the morning and evening activity peaks. The experimental and control flies were not impaired in climbing ability. In contrast, conditional silencing of the PLP neurons and/or AstA expressing EECs reduced sleep specifically in the siesta. The arousal experiment was employed to test for the sleep intensity. Thermogenetically activated flies walked significantly slower and a shorter distance than controls for all arousal stimulus intensities. Furthermore, PDF receptor was detected in the PLP neurons and the PLP neurons reacted with an intracellular increase of cAMP upon PDF, only when PDF receptor was present. Constitutive activation of AstA cells by tethered PDF increased sleep and thermogenetic activation of the PDF producing sLNvs promoted sleep specifically in the morning and evening. \par The study shows that the PLP neurons and/or EECs vis AstA signalling subserve an anorexigenic and sleep-regulating function in \textit{Drosophila}. The PLP neurons arborise in the posterior superior protocerebrum, where the sleep relevant dopaminergic neurons are located, and EECs extend themselves to reach the gut lumen. Thus, the PLP neurons are well positioned to regulate sleep and EECs potentially modulate feeding and possibly locomotor activity and sleep during sending the nutritional information from the gut to the brain. The results of imaging, activation of the PDF signalling pathway by tethered PDF and thermoactivation of PDF expressing sLNvs suggest that the PLP neurons are modulated by PDF from sLNv clock neurons and AstA in PLP neurons is the downstream target of the central clock to modulate locomotor activity and sleep. AstA receptors are homologues of galanin receptors and both of them are involved in the regulation of feeding and sleep, which appears to be conserved in evolutionary aspect.\par In the second part of the dissertation, I analysed the role of myoinhibitory peptides. MIPs are brain-gut peptides in insects and polychaeta. Also in \textit{Drosophila}, MIPs are expressed in the CNS and EECs in the gut. Previous studies have demonstrated the functions of MIPs in the regulation of food intake, gut motility and ecdysis in moths and crickets. Yet, the functions of MIPs in the fruit fly are little known. To dissect effects of MIPs regarding feeding, locomotor activity and sleep in \textit{Drosophila melanogater}, I manipulated the activity of MIP\textsuperscript{W{\"U}} cells by using newly generated \textit{Mip\textsuperscript{W{\"U}}-Gal4} lines. Thermogenetical activation or genetical silencing of MIP\textsuperscript{W{\"U}} celles did not affect feeding behaviour and resulted in changes in the sleep status. \par My results are in contradiction to a recent research of Min Soohong and colleagues who demonstrated a role of MIPs in the regulation of food intake and body weight in \textit{Drosophila}. They showed that constitutive silencing of MIP\textsuperscript{KR} cells increased food intake and body weight, whereas thermogenetic activation of MIP\textsuperscript{KR} cells decreased food intake and body weight by using \textit{Mip\textsuperscript{KR}-Gal4} driver. Then I repeated the experiments with the \textit{Mip\textsuperscript{KR}-Gal4} driver, but could not reproduce the results. Interestingly, I just observed the opposite phenotype. When MIP\textsuperscript{KR} cells were silenced by expressing UAS-tetanus toxin (\textit{UAS-TNT}), the \textit{Mip\textsuperscript{KR}\$>\$TNT} flies showed reduced food intake. The thermogenetic activation of MIP\textsuperscript{KR} cells did not affect food intake. Furthermore, I observed that the thermogenetic activation of MIP\textsuperscript{KR} cells strongly reduced the sleep duration.\par In the third part of the dissertation, I adapted and improved a method for metabolic labelling for \textit{Drosophila} peptides to quantify the relative amount of peptides and the released peptides by mass spectrometry under different physiological and behavioural conditions. qRT-PCR is a practical technique to measure the transcription and the corresponding mRNA level of a given peptide. However, this is not the only way to measure the translation and production of peptides. Although the amount of peptides can be quantified by mass spectrometry, it is not possible to distinguish between peptides stored in vesicles and released peptides in CNS extracts. I construct an approach to assess the released peptides, which can be calculated by comparing the relative amount of peptides between two timepoints in combination with the mRNA levels which can be used as semiquantitative proxy reflecting the production of peptides during this period. \par After optimizing the protocol for metabolic labelling, I carried out a quantitative analysis of peptides before and after eclosion as a test. I was able to show that the EH- and SIFa-related peptides were strongly reduced after eclosion. This is in line with the known function and release of EH during eclosion. Since this test was positive, I next used the metabolic labelling in \textit{Drosophila} adult, which were either fed \textit{ad libitum} or starved for 24 hrs, and analysed the effects on the amount of AstA and MIPs. In the mRNA level, my results showed that in the brain \textit{AstA} mRNA level in the 24 hrs starved flies was increased compared to in the \textit{ad libitum} fed flies, whereas in the gut the \textit{AstA} mRNA level was decreased. Starvation induced the reduction of \textit{Mip} mRNA level in the brain and gut. Unfortunately, due to technical problems I was unable to analyse the metabolic labelled peptides during the course of this thesis.\par},
  subject      = {AstA},
  language  = {en}
}
@phdthesis{Balasubramanian2018,
  author    = {Balasubramanian, Srikkanth},
  title     = {Novel anti-infectives against pathogenic bacteria},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163882},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Marine sponge-associated actinomycetes are reservoirs of diverse natural products with novel biological activities. Their antibiotic potential has been well explored against a range of Gram positive and negative bacteria. However, not much is known about their anti-infective or anti-virulence potential against human pathogens. This Ph.D. project aimed to investigate the anti-infective (anti-Shiga toxin and anti-biofilm) potential of sponge-derived actinobacteria through identification and isolation of their bioactive metabolites produced and characterizing their mechanism of action by transcriptomics. This thesis is divided into three studies with the overall objective of exploring the anti-infective efficacy of actinomycetes-derived extracts and compound(s) that could possibly be used as future therapeutics. The first study deals with investigation on the anti-Shiga toxin effects of sponge-associated actinomycetes. Diarrheal infections pose a huge burden in several developing and developed countries. Diarrheal outbreaks caused by Enterohemorrhagic Escherichia coli (EHEC) could lead to life-threatening complications like gastroenteritis and haemolytic uremic syndrome (HUS) if left untreated. Shiga toxin (Stx) produced by EHEC is a major virulence factor that negatively affects the human cells, leading them to death via apoptosis. Antibiotics are not prescribed against EHEC infections since they may enhance the risk of development of HUS by inducing the production and release of Stx from disintegrating bacteria and thereby, worsening the complications. Therefore, an effective drug that blocks the Stx production without affecting the growth needs to be urgently developed. In this study, the inhibitory effects of 194 extracts and several compounds originating from a collection of marine sponge-derived actinomycetes were evaluated against the Stx production in EHEC strain EDL933 with the aid of Ridascreen® Verotoxin ELISA assay kit. It was found that treatment with the extracts did not lead to significant reduction in Stx production. However, strepthonium A isolated from the culture of Streptomyces sp. SBT345 (previously cultivated from the Mediterranean sponge Agelas oroides) reduced the Stx production (at 80 μM concentration) in EHEC strain EDL933 without affecting the bacterial growth. The structure of strepthonium A was resolved by spectroscopic analyses including 1D and 2D-NMR, as well as ESI-HRMS and ESI-HRMS2 experiments. This demonstrated the possible application of strepthonium A in restraining EHEC infections. VI In the second study, the effect of marine sponge-associated actinomycetes on biofilm formation of staphylococci was assessed. Medical devices such as contact lenses, metallic implants, catheters, pacemakers etc. are ideal ecological niches for formation of bacterial biofilms, which thereby lead to device-related infections. Bacteria in biofilms are multiple fold more tolerant to the host immune responses and conventional antibiotics, and hence are hard-to-treat. Here, the anti-biofilm potential of an organic extract derived from liquid fermentation of Streptomyces sp. SBT343 (previously cultivated from the Mediterranean sponge Petrosia ficiformis) was reported. Results obtained in vitro demonstrated its anti-biofilm (against staphylococci) and non-toxic nature (against mouse macrophage (J774.1), fibroblast (NIH/3T3) and human corneal epithelial cell lines). Interestingly, SBT343 extract could inhibit staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces without affecting the bacterial growth. High Resolution Fourier Transform Mass Spectrometry (HR-MS) analysis indicated the complexity and the chemical diversity of components present in the extract. Preliminary physio-chemical characterization unmasked the heat stable and non-proteinaceous nature of the active component(s) in the extract. Finally, fractionation experiments revealed that the biological activity was due to synergistic effects of multiple components present in the extract. In the third study, anti-biofilm screening of 50 organic extracts generated from solid and liquid fermentation of 25 different previously characterized sponge-derived actinomycetes was carried out. This led to identification of the anti-biofilm organic extract derived from the solid culture of Streptomyces sp. SBT348 (previously cultivated from the Mediterranean sponge Petrosia ficiformis). Bioassay-guided fractionation was employed to identify the active fraction Fr 7 in the SBT348 crude extract. Further purification with semi-preparative HPLC led to isolation of the bioactive SKC1, SKC2, SKC3, SKC4 and SKC5 sub-fractions. The most active sub-fraction SKC3 was found to be a pure compound having BIC90 and MIC values of 3.95 μg/ml and 31.25 μg/ml against S. epidermidis RP62A. SKC3 had no apparent toxicity in vitro on cell lines and in vivo on the greater wax moth Galleria melonella larvae. SKC3 was stable to heat and enzymatic treatments indicating its non-proteinaceous nature. HR-MS analysis revealed the mass of SKC3 to be 1258.3 Da. Structure elucidation of SKC3 with the aid of 1D and 2D-NMR data is currently under investigation. Further, to obtain insights into the mode of action of SKC3 on S. epidermidis RP62A, RNA sequencing was done. Transcriptome data revealed that SKC3 was recognized by RP62A at 20 min and SKC3 negatively interfered with the central metabolism of staphylococci at 3 h. Taken VII together, these findings suggest that SKC3 could be a lead structure for development of new anti-staphylococcal drugs. Overall, the results obtained from this work underscore the anti-infective attributes of actinomycetes consortia associated with marine sponges, and their applications in natural product drug discovery programs.},
  subject      = {Marine sponges},
  language  = {en}
}
@phdthesis{Bury2018,
  author    = {Bury, Susanne},
  title     = {Molekularbiologische Untersuchungen der antagonistischen Effekte des probiotischen \(Escherichia\) \(coli\) Stamms Nissle 1917 auf Shiga-Toxin produzierende \(Escherichia\) \(coli\) St{\"a}mme},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163401},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Shiga toxin produzierende E. coli (STEC) stellen mit einer Infektionsdosis von gerade einmal 100 Bakterien ein großes Risiko f{\"u}r unsere Gesundheit dar. Betroffene Patienten k{\"o}nnen milde Krankheitssymptome wie w{\"a}ssrigen Durchfall aufweisen, welcher sich allerdings zu blutigem Durchfall oder dem h{\"a}molytisch ur{\"a}mischen Syndrom (HUS) weiterentwickeln kann. Die Ursache f{\"u}r das Krankheitsbild ist das zytotoxische Protein Shiga-Toxin (Stx), welches von STEC St{\"a}mmen produziert wird, eukaryotischen Zellen angreift und den apoptotischen Zelltod induziert. Es konnte gezeigt werden, dass infizierte Patienten in ihrem Krankheitsverlauf stark variieren, was unter anderem auf die Zusammensetzung ihrer Mikrobiota zur{\"u}ckzuf{\"u}hren sein k{\"o}nnte. Diesbez{\"u}glich k{\"o}nnen zum Beispiel einige Bakterien bereits die Darmbesiedlung von STEC St{\"a}mmen unterbinden, wohingegen andere die Toxin Produktion der pathogenen St{\"a}mme beeinflussen und wieder andere von den stx tragenden Phagen infiziert werden k{\"o}nnen und daraufhin selbst zu Toxin produzierenden St{\"a}mmen werden. Da die genetischen Informationen f{\"u}r das Toxin auf einem Prophagen im Genom der STEC St{\"a}mme kodiert ist, f{\"u}hrt eine Antibiotika Behandlung von infizierten Patienten zwar zum Tod der Bakterien, hat allerdings auch einen Wechsel vom lysogenen zum lytischen Phagen Zyklus und damit einen enormen Anstieg an freigesetztem Stx zur Folge. In den letzten Jahrzehnten kam es immer wieder zu Epidemien mit STEC St{\"a}mmen, welche auch einige Todesopfer forderten. Die Behandlung von Patienten erfolgt auf Grund von mangelnden Behandlungsm{\"o}glichkeiten meist nur symptomatisch, weswegen neue Strategien f{\"u}r die Behandlung einer STEC Infektion dringend ben{\"o}tigt werden. Der probiotische E. coli Stamm Nissle 1917 (EcN) z{\"a}hlt bereits seit mehr als 100 Jahren als Medikament f{\"u}r Behandlungen von Darmentz{\"u}ndungen. In vitro und in vivo Studien mit dem probiotischen Stamm und STEC St{\"a}mmen konnten zeigen, dass EcN die Produktion von Stx unterdr{\"u}ckt und gleichzeitig die STEC Zellzahl reduziert. Diese Ergebnisse waren der Anlass f{\"u}r diese Studie in der die Auswirkungen von EcN auf STEC St{\"a}mme genauer untersucht wurden, um eine m{\"o}gliche Behandlung von STEC Infektionen mit dem Probiotikum zu gew{\"a}hrleisten. Eines der Hauptziele dieser Studie war es, herauszufinden, ob EcN von stx-Phagen infiziert werden kann und damit selbst zu einem Toxin Produzenten wird. In diesem Falle w{\"a}re eine Behandlung mit dem E. coli Stamm ausgeschlossen, da es den Krankheitsverlauf verschlimmern k{\"o}nnte. Verschiedene experimentelle Ans{\"a}tze in denen versucht wurde den YaeT stx-Phagen Rezeptor tragenden Stamm zu infizieren schlugen fehl. Weder mittels PCR Analysen, Phagen Plaque Assays oder der Phagen Anreicherung konnte eine Lyse oder eine Prophagen Integration nachgewiesen werden. Transkriptom Analysen konnten zeigen, dass Gene eines lambdoiden Prophagen in EcN in Anwesenheit von stx-Phagen stark reguliert sind. Auch andere E. coli St{\"a}mme, welche sich ebenfalls durch eine Resistenz gegen{\"u}ber einer stx-Phagen Infektion auswiesen, wurden positiv auf lambdoide Prophagen untersucht. Einzig dem stx-Phagen sensitiven K-12 Stamm MG1655 fehlt ein kompletter lambdoider Prophage, weswegen die Vermutung nahe liegt, dass ein intakter lambdoider Prophage vor der Superinfektion mit stx-Phagen sch{\"u}tzten kann. In weiteren Experimenten wurde der Einfluss der Mikrozin-negativen EcN Mutante SK22D auf STEC St{\"a}mme untersucht. Es konnte gezeigt werden, dass SK22D nicht nur die Produktion des zytotoxischen Proteins unterdr{\"u}ckt, sondern auch mit der Produktion der stx-Phagen von allen getesteten STEC St{\"a}mmen interferiert (O157:H7, O26:H11, O145:H25, O103:H2, O111:H- und zwei O104:H4 Isolate vom STEC Ausbruch in Deutschland im Jahr 2011). Transwell Studien konnten zeigen, dass der Faktor, welcher die Transkription des Prophagen unterdr{\"u}ckt, von SK22D sekretiert wird. Die Ergebnisse lassen vermuten, dass die Pr{\"a}senz von SK22D den lysogenen Zustand des Prophagen st{\"u}tzt und somit den lytischen Zyklus unterdr{\"u}ckt. Da stx-Phagen eine große Gefahr darstellen andere E. coli St{\"a}mme zu infizieren, haben wir uns in weiteren Studien dem Einfluss von EcN auf isolierte Phagen gewidmet. Die Kultivierungsexperimente von EcN mit Phagen zeigten, dass der probiotische Stamm in der Lage war die stx-Phagen in ihrer Effizienz der Lyse des K 12 Stammes MG1655 von~ 1e7 pfus/ml auf 0 pfus/ml nach einer 44 st{\"u}ndigen Inkubation zu inaktivieren. Diese Inaktivierung konnte auf die Aktivit{\"a}t eines hitzestabilen Proteins, welches in der station{\"a}ren Wachstumsphase synthetisiert wird, zur{\"u}ckgef{\"u}hrt werden. Studien welche einen Anstieg der Biofilmmasse zur Folge hatten zeigten eine gesteigerte Effizienz in der Phagen Inaktivierung, weswegen Komponenten des Biofilms m{\"o}glicherweise die Phagen Inaktivierung herbeif{\"u}hren. Neben dem direkten Einfluss auf die Phagen wurde auch ein Schutzeffekt von SK22D gegen{\"u}ber dem stx-Phagen empf{\"a}nglichen K 12 St{\"a}mmen untersucht. Lysogene K 12 St{\"a}mme zeichneten sich durch eine enorme Stx und stx-Phagen Produktion aus. Die Pr{\"a}senz von SK22D konnte den K 12 vermittelten Anstieg der pathogenen Faktoren unterbinden. Transwell Ergebnisse und Kinetik Studien lassen vermuten, dass SK22D eher die Phagen Infektion von K-12 St{\"a}mmen unterbindet als die Lyse von lysogenen K-12 St{\"a}mmen zu st{\"o}ren. Eine m{\"o}gliche Erkl{\"a}rung f{\"u}r den Schutz der K-12 St{\"a}mme vor einer stx-Phagen Infektion k{\"o}nnte darin liegen, dass die K-12 St{\"a}mme innerhalb der SK22D Kultur wachsen und dadurch von den infekti{\"o}sen Phagen abgeschirmt werden. Zusammenfassend konnte in dieser Studie gezeigt werden, dass der probiotische Stamm EcN sowohl die Lyse von STEC St{\"a}mmen unterdr{\"u}ckt als auch die infekti{\"o}sen stx-Phagen inaktiviert und sensitive E. coli St{\"a}mme vor der Phagen Infektion sch{\"u}tzen kann. Diese Ergebnisse sollten als Grundlage f{\"u}r in vivo Studien herangezogen werden, um eine m{\"o}gliche Behandlung von STEC infizierten Patienten mit dem Probiotikum zu gew{\"a}hrleisten.},
  subject      = {EHEC},
  language  = {en}
}