@phdthesis{Klett2009,
  author    = {Klett, Sebastian},
  title     = {Untersuchungen zur Rolle der Proteinkinase B auf die Expression fibroserelevanter Gene},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-50178},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2009},
  abstract  = {Kardiovaskul{\"a}re Erkrankungen stellen im Alter f{\"u}hrende Todesursachen in der westlichen Welt dar. Der Proteinkinase B, auch AKT genannt, kommt am Herzen eine zentrale Rolle bez{\"u}glich der Organogenese zu. Ihre Funktion wird in Verbindung mit der Insulinwirkung, dem Einfluss auf den Kohlenhydrat-stoffwechsel sowie die Steuerung von Entwicklung und Differenzierung von Geweben durch Modulation des Zell{\"u}berlebens, des Zellwachstum und der Zellteilung diskutiert. Auch das postnatale Herzwachstum in physiologischer sowie pathologischer Auspr{\"a}gung wird durch den PKB/AKT-Signalweg reguliert. Diez et al.zeigten bei seneszenten Kardiofibroblasten der Ratte in vitro eine reduzierte Expression der PKB/AKT-1. Auch bei Myokard-Biopsaten von jungen und alten Patienten (ohne Myokardpathologie) konnte gezeigt werden, dass die Expressionst{\"a}rke der AKT-1/PKB mit dem Alter abnahm. Die vorliegende Arbeit sollte die Frage kl{\"a}ren ob AKT/PKB zu einer ver{\"a}nderten Expression von extrazellul{\"a}ren Matrix-Proteinen, die sie abbauenden Matrixmetalloproteinasen bzw. deren Inhibitoren f{\"u}hrt und damit zu einer Umstrukturierung der Extrazellul{\"a}ren Matrix im Sinne einer Fibrose des Myokards beitragen k{\"o}nnte. Nach adenoviraler Transfektion von Kardiofibroblasten mit einer konstitutiv aktiven bzw. inaktiven PKB/AKT-Mutanten, erfolgte die Analyse der differentiellen Genexpression fibroserelevanter Gene mittels RT-PCR und Agarosegelelektrophorese in drei verschiedenen Zellzust{\"a}nden. Die Auswertung der gewonnen Daten zeigte, dass von den untersuchten Genen lediglich die Matrix-Metalloproteinasen (MMPs).-2/-9/-13 einer deutlichen Regulation unterworfen sind. Die Darstellung auf Proteinebene gelang f{\"u}r die MMP-9 und MMP-2 letztendlich mittels Zymographie. Bezugnehmend auf die Fragestellung zeigte sich bei der durchgef{\"u}hrten Genexpressionsanalyse fibroserelevanter Gene eine Regulation der MMP-2/-9 und -13 durch die PKB/AKT auf m-RNS-Ebene, mit deutlichem Anstieg der f{\"u}r die MMP-9 und MMP-13 kodierenden m-RNS bei Inaktivierung der PKB/AKT. Auf Proteinebene besteht lediglich bei der MMP-9 eine entsprechende {\"A}nderung der Proteinmenge. Die MMP-2-Enzymmenge zeigte sich auf basalem Expressionsniveau als nicht reguliert. Die MMP-13 konnte zymographisch nicht nachgewiesen werden. Bezugnehmend auf die durch Diez et al. gefundene Herabregulation der PKB-Akt in seneszenten Kardiofibroblasten der Ratte k{\"o}nnte die in der Arbeit gezeigte Steigerung der Genexpression der MMP-2/-9 und 13 die Voraussetzung eines zur Fibrose f{\"u}hrenden Remodelings der kardialen extrazellul{\"a}ren Matrix im Sinne einer Degradation der EZM darstellen. Nach Schram und Sweeney et al. kann sich ein Remodeling der extrazellul{\"a}ren Matrix des Herzens schwerpunktm{\"a}ßig sowohl als Ver{\"a}nderungen in der Synthese von matrixbildenden Proteinen als auch in Ver{\"a}nderungen der Expression und Aktivit{\"a}t von MMPs und TIMPs darstellen. Neben der wichtigen Wirkung eines antiapoptotischen Zellschutzes k{\"o}nnte die Serin-Threonin-Kinase PKB/AKT mit an der Entstehung einer myokardialen Fibrose beteiligt sein. Weitere Untersuchungen sind aber n{\"o}tig, diese Frage eindeutig zu kl{\"a}ren.},
  subject      = {Proteinkinase B},
  language  = {de}
}
@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{Frischholz2021,
  author    = {Frischholz, Sebastian},
  title     = {Resveratrol Counteracts IL-1β-mediated Impairment of Extracellular Matrix Deposition in 3D Articular Chondrocyte Constructs},
  doi       = {10.25972/OPUS-23745},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-237453},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Articular cartilage is an exceptional connective tissue which by a network of fibrillar collagen and glycosaminoglycan (GAG) molecules allows both low- friction articulation and distribution of loads to the subchondral bone (Armiento et al., 2018, Ulrich-Vinther et al., 2003). Because of its very limited ability to self-repair, chondral defects following traumatic injury increase the risk for secondary osteoarthritis (OA) (Muthuri et al., 2011). Still, current OA treatments such as common nonsteroidal anti-inflammatory drugs (NSAIDs) and joint replacement primarily address end-stage symptoms (Tonge et al., 2014). As low-grade inflammation plays a pivotal role in the pathogenesis of OA (Robinson et al., 2016), there is a strong demand for novel therapeutic concepts, such as integrating application of anti-inflammatory agents into cartilage cell- based therapies in order to effectively treat OA affected joints in early disease stages. The polyphenolic phytoalexin resveratrol (RSV), found in the skin of red grapes, berries, and peanuts, has been shown to have effective anti-inflammatory properties (Shen et al., 2012). However, its long-term effects on 3D chondrocyte constructs cultured in an inflammatory environment with regard to tissue quality have remained unexplored so far. Therefore, in this study, pellets made from expanded porcine articular chondrocytes were cultured for 14 days with either the pro-inflammatory cytokine interleukin-1β (IL-1β) (1 - 10 ng/ml) or RSV (50 μM) alone, or a co-treatment with both agents. Constructs treated with chondrocyte medium only served as control. Treatment with IL-1β at 10 ng/ml resulted in a significantly smaller pellet size and reduced DNA content. However, RSV counteracted the IL-1β-induced decrease and significantly enhanced diameter and DNA content. Also, in terms of GAG deposition, treatment with IL-1β at 10 ng/ml resulted in a tremendous depletion of absolute GAG content and GAG/DNA. Again, RSV co-treatment counteracted the inflammatory stimulus and led to a partial recovery of GAG content. Histological analysis utilizing safranin-O staining confirmed these findings. Marked expression of the cartilage-degrading enzyme matrix metalloproteinase 13 (MMP13) was detected in IL-1β-treated pellets, but none upon RSV co- treatment. Moreover, co-treatment of IL-1β-challenged constructs with RSV significantly increased absolute collagen content. However, under non- inflammatory conditions, RSV induced gene expression and protein accumulation of collagen type X, a marker for undesirable hypertrophy. Taken together, in the present thesis, RSV was demonstrated to elicit marked beneficial effects on the extracellular matrix composition of 3D cartilaginous constructs in long-term inflammatory culture in vitro, but also induced hypertrophy under non-inflammatory conditions. Based on these findings, further experiments examining multiple concentrations of RSV under various inflammatory conditions appear desirable concerning potential therapeutic applicability in OA.},
  subject      = {Resveratrol},
  language  = {en}
}
@phdthesis{Weber2007,
  author    = {Weber, Martin V. R. H.},
  title     = {Populationsbiologische und pathogenetische Aspekte von Neisseria meningitidis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-25775},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2007},
  abstract  = {Meningokokken sind nach wie vor eine wichtige Ursache f{\"u}r Gehirnhautentz{\"u}ndungen und Sepsen weltweit, vor allem bei Kindern und Jugendlichen. Weil viele Pathomechanismen dieses Erregers bislang noch unvollst{\"a}ndig verstanden sind, wurden im Rahmen der vorliegenden Doktorarbeit populationsbiologische und pathogenetische Aspekte von Neisseria meningitidis untersucht. Die Kapsel ist der haupts{\"a}chliche Pathogenit{\"a}tsfaktor von Meningokokken und wichtig f{\"u}r die Besiedelung von neuen Wirten. Isolate von symptomfreien Tr{\"a}gern sind allerdings h{\"a}ufig unbekapselt. Um Ursachen oder Mechanismen f{\"u}r den Verlust der Kapselexpression aufzudecken, wurden insgesamt 166 Isolate der Bayerischen Meningokokkentr{\"a}gerstudie untersucht. Alle Isolate besaßen s{\"a}mtliche zur Kapselsynthese notwendigen Gene, exprimierten aber keine Kapsel. Bei 39 Isolaten fanden sich L{\"a}ngenvariationen in homopolymeren Sequenzen (slipped strand mispairing, SSM) in den Genen siaA und siaD. 46 Isolate enthielten Insertionselemente (IS1301, IS1016 und IS1106) in den Genen der Kapselsynthese. Irreversible Mutationen (Deletionen, Insertionen, Basensubstitutionen) wurden bei 47 Isolaten gefunden. Ver{\"a}nderungen der Promotorregion schienen keine Rolle zu spielen. Es wurden bei insgesamt sechs Isolaten zwei nicht-synonyme Mutationen in unmittelbarer N{\"a}he zum putativen aktiven Zentrum der UDP-N- Acetylglukosamin-2-Epimerase entdeckt, die einen Verlust der Kapselsynthese erkl{\"a}ren k{\"o}nnten. Insgesamt wurden keine Akkumulationen von Mutationen in defekten Genen gefunden und es gab auch keine Korrelationen zwischen den verschieden Ursachen und bestimmten klonalen Linien. Die erhaltenen Ergebnisse legen nahe, dass die meisten der zur Blockierung der Kapselexpression f{\"u}hrenden Ereignisse erst im aktuellen Wirt aufgetreten sind und dass zumindest bei bestimmten klonalen Linien die Verbreitung von der Expression einer Kapsel abh{\"a}ngig ist. Viele pathogene Bakterien nutzen zur Infektion des Menschen die ubiquit{\"a}r im K{\"o}rper vorkommende Protease Plasmin. Dazu binden diese Plasmin oder das Proenzym Plasminogen. Auch Meningokokken interagieren mit Plasmin und Plasminogen. Im Rahmen der vorliegenden Arbeit konnten drei Rezeptormolek{\"u}le f{\"u}r Plasminogen identifiziert werden. Die drei Proteine Enolase, DnaK und Peroxiredoxin konnten mit verschiedenen Methoden auf der Oberfl{\"a}che der Erreger nachgewiesen werden. Die Bindung des Plasminogens ist bei Meningokokken ausschließlich {\"u}ber Lysinreste der Rezeptoren vermittelt, die C-terminalen Lysinreste der hier identifizierten Rezeptormolek{\"u}le spielen aber, wenn {\"u}berhaupt, nur eine untergeordnete Rolle. Die Bindung von Plasminogen war durch rekombinante Rezeptorproteine konzentrationsabh{\"a}ngig inhibierbar. Plasminogen konnte von Meningokokken auch aus dem Serum rekrutiert werden. Gebundenes Plasminogen war mit uPA (Urokinase Plasminogen Aktivator) aktivierbar und physiologisch aktiv, was durch die Degradation von Fibrinogen nachgewiesen wurde. Das gebundene Plasmin wurde durch die Bakterien vor der Desaktivierung durch \&\#945;2- Antiplasmin gesch{\"u}tzt. Des Weiteren konnte gezeigt werden, dass Meningokokken auch mit weiteren Faktoren des Fibrinolyse-Systems (uPA) interagieren. Sie rekrutierten uPA an ihre Oberfl{\"a}che und gebundenes uPA war physiologisch aktiv. Die erhaltenen Ergebnisse best{\"a}rken die These, dass Meningokokken die Faktoren des Fibrinolyse-Systems f{\"u}r ihre Pathogenese nutzen.},
  subject      = {Neisseria meningitidis},
  language  = {de}
}
@phdthesis{Dodt2021,
  author    = {Dodt, Katharina Anna},
  title     = {Monitoring enzyme activity by using mass-encoded peptides and multiplexed detection},
  doi       = {10.25972/OPUS-22937},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229377},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Cell culture models are helpful tools to study inflammatory diseases, like rheumatoid arthritis (RA), osteoarthritis (OA), arteriosclerosis or asthma, which are linked to increased matrix metalloproteinase (MMP) activity. Such cell culture models often focus on the secretion of cytokines and growth factors or the direct effects of disease on tissue destruction. Even though the crucial role of MMPs in inflammatory diseases is known, the results of MMP studies are contradictious and the use of MMPs as biomarkers is inconsistent. MMPs play an important role in disease pathology, as they are involved in elastin degradation in the walls of alveoli in chronic obstructive pulmonary disease (COPD), tumor angiogenesis and metastasis and in cartilage and bone degradation in arthropathies. In RA and OA MMPs are secreted by osteocytes, synoviocytes, and by infiltrating immune cells in response to the increased concentration of inflammatory mediators, like growth factors and cytokines. MMPs are zinc and calcium-dependent proteinases and play an important role in physiological and pathological extracellular matrix (ECM) turn over. Their substrate specificity gives them the ability to degrade all major ECM components, like aggrecan, elastin, gelatin, fibronectin and all types of collagen even the triple helix of collagen monomers. The ECM consists of two large three-dimensional cross-linked macromolecule classes: one are fibrous proteins, like collagen and elastin fibers that are responsible for ECM's structure, tensile strength, resiliency, reversible extensibility, and deformability and the second class is comprised of proteoglycans composed of glycosaminoglycan (GAG) chains covalently attached to protein cores that are multifunctionally involved in signaling pathways and cell interactions. ECM is present within all tissues and organs and changes in ECM structure contribute to pathogenesis, e.g. wounded and fibrotic tissue, COPD or tumours. This thesis primarily focuses on the development of a diagnostic peptide system, that enables to gain information on MMP activity from ECM by deploying the isobaric mass encoding strategy. The core element of the developed system is an isotopically labelled peptide sequence (mass tag), that is released in response to elevated levels of MMPs and allows multiplexed detection in tandem mass spectrometry (LC-MS/MS). The mass reporters possess a modular structure with different functionalities. C-terminal either a transglutaminase (TG) recognition sequence or a high molecular weight polyethylene glycol (PEG) moiety was attached to immobilize the mass reporters covalently or physically at the injection site. The following matrix metalloproteinase substrate sequence (MSS) is incorporated in two different versions with different sensitivity to MMPs. The MSS were applied in pairs for relative quantification consisting of the cleavable version synthesized with natural L-amino acids and the non-cleavable D-amino acid variant. The mass tag was synthesized with isotopically labelled amino acids and is separated from the MSS by a UV light-sensitive molecule. N-terminal the mass tag is followed by a tobacco etch virus protease (TEV) sensitive sequence, that is responsible to separate the mass tag from the affinity tag, which was either the Strep-tag II sequence or biotin and were added for purification purposes. Chapter 1 presents a step-by-step protocol on how to design a mass tag family allowing for multiplexed analysis by LC-MS/MS. The multiplexing is achieved by developing an isobar mass tag family with four family members, which are chromatographically indistinguishable, but due to the mass encoding principles they fragment in distinct y-type ions with a mass difference of 1 or 2 Da each in MS2. Furthermore, it is explained how to covalently attach the mass reporter peptides onto ECM by the activated calcium-catalyzed blood coagulation transglutaminase factor XIII (FXIIIa). The lysine of mass reporter's TG sequence (D-domain of insulin-like growth factor-I (IGF-I)) and a glutamine in fibronectin are covalently crosslinked by FXIIIa and build an isopeptide bond. Elevated levels of MMP release the mass reporters from ECM by recognizing the inter-positioned MSS. The designed mass reporters were able to monitor enzyme activity in an in vitro setting with cell-derived ECM, which was shown in Chapter 2. The modular structured mass reporters were investigated in a proof of concept study. First, the different modules were characterized in terms of their MMP responsiveness and their sensitivity to TEV protease and UV light. Then the FXIIIa-mediated coupling reaction was detailed and the successful coupling on ECM was visualized by an immunosorbent assay or confocal laser scanning microscopy. Finally, the immobilized mass reporters on ECM were incubated with MMP-9 to investigate their multiplexing ability of MMP activity. The cleaved mass reporter fragments were purified in three steps and mass tags were analyzed as mix of all four in LC-MS/MS. Chapter 3 describes the change from an immobilizing system as seen in chapter 1 and 2 to a soluble enzyme activity monitoring system that was applied in an osteoarthritic mouse model. Instead of the immobilizing TG sequence the C-terminal MMS was extended with two amino acids where one holds an azide moiety to perform a strain-promoted azide-alkyne cycloaddition to a high molecular weight dibenzocyclooctyne-polyethylene glycol (DBCO-PEG), which was chosen to retain the mass reporters at the injection site. Furthermore, the N-terminal affinity tag was extended with a 2.5 kDa PEG chain to increase the half-life of the mass reporter peptides after MMP release. The systems biocompatibility was proved but its enzyme monitoring ability in an in vivo setting could not be analyzed as samples degraded during shipping resulting from the Chinese customs blocking transport to Germany. In summary the diagnostic peptide system was developed in two variants. The immobilized version one from chapter 1 and 2 was designed to be covalently attached to ECM by the transglutaminase-mediated cross-linking reaction. In an in vitro setting the functionality of the mass reporter system for the detection of MMP activity was successfully verified. The second variant comprises of a soluble mass reporter system that was tested in an OA mouse model and showed biocompatibility. With these two designed systems this thesis provides a flexible platform based on multiplexed analysis with mass-encoded peptides to characterize cell culture models regarding their MMP activity, to deploy cell-derived ECM as endogenous depot scaffold and to develop a mass tag family that enables simultaneous detection of at least four mass tags.},
  subject      = {Extrazellul{\"a}re Matrix},
  language  = {en}
}
@phdthesis{Schlegelmilch2012,
  author    = {Schlegelmilch, Katrin},
  title     = {Molecular function of WISP1/CCN4 in the musculoskeletal system with special reference to apoptosis and cell survival},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73430},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Human adult cartilage is an aneural and avascular type of connective tissue, which consequently reflects reduced growth and repair rates. The main cell type of cartilage are chondrocytes, previously derived from human mesenchymal stem cells (hMSCs). They are responsible for the production and maintainance of the cartilaginous extracellular matrix (ECM), which consists mainly of collagen and proteoglycans. Signal transmission to or from chondrocytes, generally occurs via interaction with signalling factors connected to the cartilaginous ECM. In this context, proteins of the CCN family were identified as important matricellular and multifunctional regulators with high significance during skeletal development and fracture repair. In this thesis, main focus lies on WISP1/CCN4, which is known as a general survival factor in a variety of cell types and seems to be crucial during lineage progression of hMSCs into chondrocytes. We intend to counter the lack of knowledge about the general importance of WISP1-signalling within the musculoskeletal system and especially regarding cell death and survival by a variety of molecular and cell biology methods. First, we established a successful down-regulation of endogenous WISP1 transcripts within different cell types of the human musculoskeletal system through gene-silencing. Interestingly, WISP1 seems to be crucial to the survival of all examined cell lines and primary hMSCs, since a loss of WISP1 resulted in cell death. Bioinformatical analyses of subsequent performed microarrays (WISP1 down-regulated vs. control samples) confirmed this observation in primary hMSCs and the chondrocyte cell line Tc28a2. Distinct clusters of regulated genes, closely related to apoptosis induction, could be identified. In this context, TRAIL induced apoptosis as well as p53 mediated cell death seem to play a crucial role during the absence of WISP1 in hMSCs. By contrast, microarray analysis of WISP1 down-regulated chondrocytes indicated rather apoptosis induction via MAPK-signalling. Despite apoptosis relevant gene regulations, microarray analyses also identified clusters of differentially expressed genes of other important cellular activities, e.g. a huge cluster of interferon-inducible genes in hMSCs or gene regulations affecting cartilage homeostasis in chondrocytes. Results of this thesis emphasize the importance of regulatory mechanisms that influence cell survival of primary hMSCs and chondrocytes in the enforced absence of WISP1. Moreover, findings intensified the assumed importance for WISP1-signalling in cartilage homeostasis. Thus, this thesis generated an essential fundament for further examinations to investigate the role of WISP1-signalling in cartilage homeostasis and cell death.},
  subject      = {Knorpelzelle},
  language  = {en}
}
@phdthesis{Hupp2007,
  author    = {Hupp, Markus},
  title     = {Inhibition von alpha V Integrinen vermindert die Migration von prim{\"a}ren glatten Gef{\"a}ssmuskelzellen und schw{\"a}cht die Tyrosinphosphorylierung der "focal adhesion kinase".},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-24731},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2007},
  abstract  = {Die isch{\"a}mische Herzerkrankung (Angina pectoris, Herzinfarkt), eine f{\"u}hrende Todesursache in den Industrienationen, wird haupts{\"a}chlich durch Intervention an den Koronararterien mittels Ballondilatation meist in Kombination mit einer Stentimplantation behandelt. Trotz aktueller Verbesserungen im Stentdesing und dem Einsatz von medikamente-freisetzenden Stents kommt es immer noch in etwa 10 - 20 \% der Interventionen, in Abh{\"a}ngigkeit des jeweiligen Risikoprofils, zu der Entwicklung einer Restenose. Der pathophysiologische Prozess der Neointimaentwicklung, welche der Restenoseentstehung nach Stentimplantation zu Grunde liegt, ist im Wesentlichen durch einwandernde glatte Gef{\"a}ssmuskelzellen (GMZ) bedingt. Eine zentrale Rolle bei der Zellwanderung nehmen alpha V Integrine ein. Um den Prozess der Restenose zu verhindern, stellen somit diese Rezeptoren und die durch sie ausgel{\"o}sten Signalkaskaden einen vielversprechenden Angriffspunkt dar. Wir konnten nach Stimulation von GMZ aus Schweinen mit den EZM Proteinen Vitronektin (VN), Fibronektin (FN) und Kollagen (CN) eine verst{\"a}rkte Tyrosinphosphorylierung (PTyr) v.a. eines etwa 116 kDa grossen Proteins zeigen, das mittels Immunpr{\"a}zipitation als "focal adhesion kinase" (FAK) identifiziert wurde. VN stellte hierbei den st{\"a}rksten Stimulus dar. Die erh{\"o}hte PTyr zeigte sich am Tyrosinrest 397 von FAK (PTyr-397), der Autophosphorylierungsstelle der Kinase, und deutet damit auf eine durch Proteinstimulation induzierte, erh{\"o}hte Aktivit{\"a}t von FAK hin. Die erh{\"o}hte PTyr von FAK war nicht zu beobachten, wenn die GMZ ohne spezifische Rezeptor-Ligand Interaktion auf Poly-L-Lysin adh{\"a}rierten. Die Aktivierung von FAK nach Stimulation mit VN, FN und CN war dabei abh{\"a}ngig von alpha V Integrinen und lies sich durch Zugabe eines kompetetiven alpha V Inhibitors in einer Dosis- abh{\"a}ngigen Weise unterdr{\"u}cken. Die Inhibition war am deutlichsten nach VN Stimulation zu beobachten. Bei Kultivierung der Zellen mit dem Inhibitor {\"u}ber 7 Tage in einer Konzentration von 1 µM war in der Durchlichtmikroskopie im Vergleich zu kultivierten Zellen ohne Inhibitor keine Ver{\"a}nderung zu erkennen. Bei 10 µM Cilengitide verkleinerte sich der Zelldurchmesser, die Zellen blieben jedoch an der Oberfl{\"a}che der Zellkulturschalen adh{\"a}rent. In der IF Mikroskopie zeigte sich nach 30 min eine Abnahme der intrazellul{\"a}ren PTyr und ein Abbau des Aktinzytoskeletts unter Einfluss von 10 µM des Inhibitors auf kultivierte adh{\"a}rente GMZ. Es gab keinen Hinweis auf Induktion einer Apoptose. Auf VN und CN konnten die zuvor suspendierten GMZ gut adh{\"a}rieren, w{\"a}hrend auf einer mit FN beschichteten Oberfl{\"a}che die Anzahl der angehefteten Zellen im Vergleich zu einer unbeschichteten Oberfl{\"a}che nicht anstieg. Die Adh{\"a}sion der GMZ auf VN konnte der Hemmstoff stark vermindern, w{\"a}hrend er auf die Adh{\"a}sion auf FN und CN keinen Einfluss hatte. Die Inhibition der Aktivierung von FAK durch den alpha V Integrininhibitor korrelierte mit der Reduktion der durch die Proteinen stimulierten Migration (Haptotaxis) der prim{\"a}ren GMZ. Der Inhibitor f{\"u}hrte bei einer Konzentration von 10 µM bei VN Stimulation zu einer fast vollst{\"a}ndigen Inhibition der Haptotaxis, w{\"a}hrend er nach FN bzw. CN Stimulation die Anzahl der gewanderten Zellen bei dieser Konzentration um etwa 30 \% verringerte. Die Migrationsrate wurde mit Hilfe eines modifizierten Boyden-Kammer Migrationsversuchs ermittelt. Unsere Ergebnisse unterstreichen die Schl{\"u}sselrolle der alpha V Integrine in der Motilit{\"a}t von GMZ. Sie nehmen Einfluss auf Signalkaskaden, die von FAK abh{\"a}ngig sind und die Haptotaxis zu regulieren scheinen. Diese Ergebnisse machen die alpha V Integrine zu einem vielversprechenden Angriffspunkt, um eine Restenose nach Stentimplantation zu verhindern. Der Einsatz des alpha V Integrininhibitors Cilengitide, der mit einem Medikamente-freisetzenden Stent lokal an dem Ort des pathologischen Geschehens appliziert werden kann, l{\"a}sst somit eine weitere Reduktion der Restenoserate erhoffen, was in einem n{\"a}chsten Schritt mittels eines in vivo Modells untersucht werden muss.},
  subject      = {Zellmigration},
  language  = {de}
}
@phdthesis{Hauptstein2022,
  author    = {Hauptstein, Julia},
  title     = {Hyaluronic Acid-based Multifunctional Bioinks for 3D Bioprinting of Mesenchymal Stromal Cells for Cartilage Regeneration},
  doi       = {10.25972/OPUS-26068},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-260681},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Articular cartilage is a highly specialized tissue which provides a lubricated gliding surface in joints and thereby enables low-friction movement. If damaged once it has a very low intrinsic healing capacity and there is still no treatment in the clinic which can restore healthy cartilage tissue. 3D biofabrication presents a promising perspective in the field by combining healthy cells and bioactive ink materials. Thereby, the composition of the applied bioink is crucial for defect restoration, as it needs to have the physical properties for the fabrication process and also suitable chemical cues to provide a supportive environment for embedded cells. In the last years, ink compositions with high polymer contents and crosslink densities were frequently used to provide 3D printability and construct stability. But these dense polymeric networks were often associated with restricted bioactivity and impaired cell processes like differentiation and the distribution of newly produced extracellular matrix (ECM), which is especially important in the field of cartilage engineering. Therefore, the aim of this thesis was the development of hyaluronic acid (HA)-based bioinks with a reduced polymer content which are 3D printable and additionally facilitate chondrogenic differentiation of mesenchymal stromal cells (MSCs) and the homogeneous distribution of newly produced ECM. Starting from not-printable hydrogels with high polymer contents and restricted bioactivity, distinct stepwise improvements were achieved regarding stand-alone 3D printability as well as MSC differentiation and homogeneous ECM distribution. All newly developed inks in this thesis made a valuable contribution in the field of cartilage regeneration and represent promising approaches for potential clinical applications. The underlying mechanisms and established ink design criteria can further be applied to other biofabricated tissues, emphasizing their importance also in a more general research setting.},
  subject      = {Hyalurons{\"a}ure},
  language  = {en}
}
@phdthesis{Reidl2009,
  author    = {Reidl, Sebastian},
  title     = {Funktionale Charakterisierung an der Biofilmbildung beteiligter Faktoren pathogener und kommensaler Escherichia coli},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-35684},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2009},
  abstract  = {Multizellul{\"a}re Gemeinschaften in Form bakterieller Biofilme stellen aus medizinischer Sicht ein großes klinisches Problem dar. H{\"a}ufig lassen sich chronische oder rezidivierende Erkrankungen aber auch nosokomiale Infektionen auf die multizellul{\"a}re Lebensweise von humanpathogenen Erregern zur{\"u}ckf{\"u}hren. Sowohl fakultativ als auch obligat pathogene Escherichia coli-St{\"a}mme besitzen eine Vielzahl unterschiedlicher Faktoren, die die Biofilmbildung beeinflussen. Daran beteiligt sind unter anderem Flagellen, extrazellul{\"a}re polymere Substanzen, Adh{\"a}sine oder Oberfl{\"a}chen-assoziierte Proteine wie Autotransporter. Gegenstand der vorliegenden Arbeit ist die funktionale Charakterisierung des Proteins Antigen 43 (Ag43). Aufgrund seiner Autoaggregation-vermittelnden Eigenschaft tr{\"a}gt Ag43 ebenfalls zur Mikrokoloniebildung und Biofilmreifung bei. Antigen 43 ist ein Autotransporterprotein, welches innerhalb der Bakterienspezies Escherichia coli weit verbreitet ist. Interessanterweise besitzen viele E. coli-Isolate gleich mehrere identische oder {\"a}hnliche Kopien von agn43, die in der Regel von variablen Genombereichen (genomische Inseln, Plasmide) kodiert werden. Am Beispiel der Antigen 43-Varianten des uropathogenen Escherichia coli (UPEC)-Stammes 536 (O6:K15:H31), des kommensalen E. coli Isolats Nissle 1917 (O6:K5:H1) sowie des E. coli K-12-Laborstammes MG1655 (OR:H48:K-) ist die Bedeutung des Autotransporterproteins im Rahmen dieser Arbeit n{\"a}her untersucht worden. Hierf{\"u}r wurden die verschiedenen agn43-Allele in ein geeignetes Vektorsystem kloniert und im Adh{\"a}sin-freien Escherichia coli K-12-Stamm MG1655 \&\#916;fim\&\#916;flu exprimiert. Da Antigen 43 in Wildtypst{\"a}mmen posttranslational glykosyliert vorliegt, sind die Experimente zus{\"a}tzlich unter Einfluß der heterologen, AIDA I-spezifischen Heptosyltransferase Aah ('Autotransporter Adhesin Heptosyltransferase') durchgef{\"u}hrt worden. Anhand von Bindungsstudien (intermolekulare Autoaggregation, Zelladh{\"a}sionstests) wurde gezeigt, daß sich einzelne Ag43-Varianten teilweise in ihren Eigenschaften unterscheiden. Im direkten Vergleich mit AIDA-I ('Adhesin Involved in Diffuse Adherence') enteropathogener Escherichia coli (EPEC) konnte f{\"u}r Ag43 nur eine Funktion als schwaches Adh{\"a}sin nachgewiesen werden. Die heterologe O-Glykosylierung beeinflußte die Funktionalit{\"a}t des Antigen 43 in unterschiedlichem Ausmaß. Je nach Autotransporter-Variante f{\"u}hrte die Heptosylierung entweder zu signifikant reduzierten Affinit{\"a}ten, oder sie hatte keinen Effekt auf die Bindungskapazit{\"a}t des Ag43. Antigen 43 weist zudem strukturelle Homologien zu vergleichbaren Dom{\"a}nen anderer Autotransporter auf. F{\"u}r viele dieser Proteine konnte bereits eine adh{\"a}sive oder invasive Funktion nachgewiesen werden. Eine m{\"o}gliche Interaktion von Ag43 mit eukaryontischen Rezeptoren ist hingegen noch nicht bzw. nur unvollst{\"a}ndig untersucht worden. In Overlay assays und ELISAs wurde f{\"u}r Antigen 43 hier erstmals die spezifische Bindung an die extrazellul{\"a}ren Matrixkomponenten Kollagen und Laminin gezeigt. Zusammenfassend deuten die Ergebnisse dieser Arbeit darauf hin, daß das Escherichia coli spezifische Autotransporterprotein Antigen 43 nicht nur an der bakteriellen Biofilmbildung, sondern auch an der Besiedlung epithelialer Gewebe beteiligt sein kann. Seine Expression verschafft Bakterien einen Kolonisationsvorteil, der mit erh{\"o}hter Fitneß einhergeht. Die Aah-vermittelte O-Glykosylierung scheint f{\"u}r die Funktionalit{\"a}t von Ag43 nicht zwingend erforderlich zu sein. Des weiteren ist im Rahmen der vorliegenden Arbeit ein Testsystem entwickelt worden, das auf der Basis von Fluoreszenz-in-situ-Hybridisierungen (FISH) die Differenzierung von verschiedenen (uro-)pathogenen Mikroorganismen erm{\"o}glicht. Das etablierte Protokoll eignet sich nicht nur f{\"u}r die diagnostische Erregeridentifizierung, sondern auch in Abh{\"a}ngigkeit des Probenmaterials zur Untersuchung von (Multispezies-)Biofilmen.},
  subject      = {Escherichia coli},
  language  = {de}
}
@phdthesis{Gutmann2019,
  author    = {Gutmann, Marcus},
  title     = {Functionalization of cells, extracellular matrix components and proteins for therapeutic application},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170602},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Glycosylation is a biochemical process leading to the formation of glycoconjugates by linking glycans (carbohydrates) to proteins, lipids and various small molecules. The glycans are formed by one or more monosaccharides that are covalently attached, thus offering a broad variety depending on their composition, site of glycan linkage, length and ramification. This special nature provides an exceptional and fine tunable possibility in fields of information transfer, recognition, stability and pharmacokinetic. Due to their intra- and extracellular omnipresence, glycans fulfill an essential role in the regulation of different endogenous processes (e.g. hormone action, immune surveillance, inflammatory response) and act as a key element for maintenance of homeostasis. The strategy of metabolic glycoengineering enables the integration of structural similar but chemically modified monosaccharide building blocks into the natural given glycosylation pathways, thereby anchoring them in the carbohydrate architecture of de novo synthesized glycoconjugates. The available unnatural sugar molecules which are similar to endogenous sugar molecules show minimal perturbation in cell function and - based on their multitude functional groups - offer the potential of side directed coupling with a target substance/structure as well as the development of new biological properties. The chemical-enzymatic strategy of glycoengineering provides a valuable complement to genetic approaches. This thesis primarily focuses on potential fields of application for glycoengineering and its further use in clinic and research. The last section of this work outlines a genetic approach, using special Escherichia coli systems, to integrate chemically tunable amino acids into the biosynthetic pathway of proteins, enabling specific and site-directed coupling with target substances. With the genetic information of the methanogen archaea, Methanosarcina barkeri, the E. coli. system is able to insert a further amino acid, the pyrrolysine, at the ribosomal site during translation of the protein. The natural stop-codon UAG (amber codon) is used for this newly obtained proteinogenic amino acid. Chapter I describes two systems for the integration of chemically tunable monosaccharides and presents methods for characterizing these systems. Moreover, it gives a general overview of the structure as well as intended use of glycans and illustrates different glycosylation pathways. Furthermore, the strategy of metabolic glycoengineering is demonstrated. In this context, the structure of basic building blocks and the epimerization of monosaccharides during their metabolic fate are discussed. Chapter II translates the concept of metabolic glycoengineering to the extracellular network produced by fibroblasts. The incorporation of chemically modified sugar components in the matrix provides an innovative, elegant and biocompatible method for site-directed coupling of target substances. Resident cells, which are involved in the de novo synthesis of matrices, as well as isolated matrices were characterized and compared to unmodified resident cells and matrices. The natural capacity of the matrix can be extended by metabolic glycoengineering and enables the selective immobilization of a variety of therapeutic substances by combining enzymatic and bioorthogonal reaction strategies. This approach expands the natural ability of extracellular matrix (ECM), like the storage of specific growth factors and the recruitment of surface receptors along with synergistic effects of bound substances. By the selection of the cell type, the production of a wide range of different matrices is possible. Chapter III focuses on the target-oriented modification of cell surface membranes of living fibroblast and human embryonic kidney cells. Chemically modified monosaccharides are inserted by means of metabolic glycoengineering and are then presented on the cell surface. These monosaccharides can later be covalently coupled, by "strain promoted azide-alkyne cycloaddition" (SPAAC) and/or "copper(I)-catalyzed azide-alkyne cycloaddition" (CuAAC), to the target substance. Due to the toxicity of the copper catalysator in the CuAAC, cytotoxicity analyses were conducted to determine the in vivo tolerable range for the use of CuAAC on living cell systems. Finally, the efficacy of both bioorthogonal reactions was compared. Chapter IV outlines two versatile carrier - spacer - payload delivery systems based on an enzymatic cleavable linker, triggered by disease associated protease. In the selection of carrier systems (i) polyethylene glycol (PEG), a well-studied, Food and Drug Administration approved substance and very common tool to increase the pharmacokinetic properties of therapeutic agents, was chosen as a carrier for non-targeting systems and (ii) Revacept, a human glycoprotein VI antibody, was chosen as a carrier for targeting systems. The protease sensitive cleavable linker was genetically inserted into the N-terminal region of fibroblast growth factor 2 (FGF-2) without jeopardizing protein activity. By exchanging the protease sensitive sequence or the therapeutic payload, both systems represent a promising and adaptable approach for establishing therapeutic systems with bioresponsive release, tailored to pre-existing conditions. In summary, by site-specific functionalization of various delivery platforms, this thesis establishes an essential cornerstone for promising strategies advancing clinical application. The outlined platforms ensure high flexibility due to exchanging single or multiple elements of the system, individually tailoring them to the respective disease or target site.},
  subject      = {Glykosylierung},
  language  = {en}
}