TY - THES A1 - Pickert, Julia Felicia T1 - Untersuchungen zum Einfluss des Insulin-like growth factor Rezeptors auf Signalnetzwerke im Multiplen Myelom T1 - Investigating the influence of the insulin-like growth factor receptor on signalling networks in multiple myeloma N2 - Das MM ist eine maligne Erkrankung, die von biologischer und klinischer Heterogenität geprägt ist. Sie ist durch die monoklonale Vermehrung von Plasmazellen charakterisiert. In vorangegangenen Studien wurde eine Häufung von Mutationen in RTK nachgewiesen. Diese gingen mit einem negativen Einfluss auf das Überleben von MM Patientinnen und Patienten einher. Im Rahmen dieser Arbeit wurde der Einfluss des IGF1R an HMZL mittels siRNA-vermitteltem IGF1R-Knockdown untersucht und dessen Effekt auf das Signalnetzwerk mittels Western Blot Analysen ermittelt. Um die Heterogenität des MM besser abzubilden, wurden sechs verschiedenen HMZL ausgewählt. Der IGF1R-Knockdown war in allen HMZL sowohl anhand der Reduktion der IGF1R-Expression als auch der IGF1R-Aktivierung deutlich nachweisbar. Stellvertretend für den PI3K/AKT Signalweg wurde die AKT-Aktivierung untersucht, welche nach IGF1R-Knockdown in allen Linien abnahm. Im Ras/Raf/MEK/ERK Signalweg fiel eine deutliche Reduktion der ERK1/2- und MEK-Aktivierung in den von PCL stammenden HMZL L-363 und MM.1S, sowie in JJN-3 mit der Hochrisikotranslokation t(14;16) auf. Entsprechend der Beobachtungen für die AKT-Aktivierung, nahm die PYK2-Aktivierung in allen HMZL nach IGF1R-Knockdown ab, was auf ein Zusammenspiel von IGF1R, PYK2 und AKT in allen HMZL hindeutet. Zukünftige Untersuchungen werden zeigen, ob IGF1R Inhibitoren alleine oder in Kombination mit z.B. AKT, PYK2 oder Proteasomen-Inhibitoren in bestimmten molekularen MM Subgruppen ein effektives therapeutisches Ziel sind. N2 - MM is a haematological malignancy of great biological and clinical heterogeneity. It is characterised by monoclonal proliferation of plasma cells. The accumulation of mutations in RTK has previously been reported and was associated with a negative impact on MM patient survival. The IGF1R influence on its downstream signaling in HMCL was investigated using a siRNA mediated IGF1R-knockdown and Western Blot analysis. Six different HMCL were chosen to reflect this heterogenous disease. The IGF1R-knockdown successfully reduced both expression and activation level of IGF1R in all HMCL. Furthermore, phosphorylation level of AKT, representing the PI3K/AKT pathway, decreased in all six HMCL following the IGF1R-knockdown. For the analysis of the Ras/Raf/MEK/ERK pathway both ERK1/2 and MEK were selected. Following the IGF1R-knockdown phosphorylation level of ERK1/2 and MEK were reduced in HMCL L-363 and MM.1S, both derived from patients with plasma call leukaemia and in JJN-3 which harbours t(14;16), a high risk translocation. In accordance with decreased activation level in AKT the IGF1R-knockdown resulted in reduced phosphorylation level of PYK2 in all six HMCL suggesting an interaction of IGF1R, PYK2 and AKT. Future research will reveal whether IGF1R inhibition by itself or in combination with e.g. AKT, PYK2 or proteasome inhibitors will be an effective therapeutic target in selected molecular MM subgroups. KW - Plasmozytom KW - Rezeptor KW - Insulin-like Growth Factor KW - Multiples Myelom KW - Insulin like-growth factor Rezeptor KW - Multiple Myeloma Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369815 ER - TY - THES A1 - Wucherpfennig, Sophia T1 - HTS (high-throughput drug screening) zur Untersuchung der Blut-Hirn-Schranken-Permeabilität in vitro beim zerebral metastasierten Mammakarzinom T1 - High-throughput drug screening to investigate blood-brain barrier permeability in vitro with a focus on breast cancer chemotherapeutic agents N2 - Die Blut-Hirn-Schranke (BHS) stellt eine selektiv durchlässige Barriere dar, die den Austausch von Stoffen zwischen Blut und ZNS kontrolliert und so neuroprotektiv wirkt. Sie verhindert allerdings nicht nur die Passage toxischer Metaboliten, sondern verwehrt auch vielen therapeutischen Wirkstoffen den Zugang zum Gehirn. Die Forschung an Methoden zum Erreichen höherer Arzneimittelkonzentrationen im Gehirn ist deshalb essenziell für die Behandlung zerebraler Erkrankungen wie dem zerebral metastasierten Mammakarzinom. Ziel dieser Arbeit war es deshalb, Wirkstoffe zu identifizieren, die die Permeabilität der BHS erhöhen. Die Substanzdatenbank LO1208 von Sigma-Aldrich wurde im Rahmen eines HTS auf ihre permeabilitätsbeeinflussenden Eigenschaften untersucht. Hierbei konnten 31 Substanzen identifiziert werden, welche die Permeabilität von BLECs um mindestens 50 % erhöhen. Aus diesen wurden 4-Amino-1,8-naphthalimid (PARP-Inhibitor) und GW2974 (TKI) für eine genauere Analyse ausgewählt. Als dritter Wirkstoff wurde Ibuilast (Inhibitor der PDE4, des MIF sowie des Toll-like-Rezeptor-4) untersucht, wobei dieser keine signifikante Veränderung der Permeabilität bewirkt. Die Messung des TEERs und der Permeabilität für Fluorescein bestätigten die Ergebnisse aus dem HTS, welches demnach zukünftig für Permeabilitätstests eingesetzt werden kann. Die Zellviabilität wird durch 4 Amino-1,8-naphthalmid nicht beeinflusst. GW2974 und Ibudilast zeigen bei 500 µM einen toxischen Einfluss auf MCF-7-Zellen. BLECs werden durch 100 µM GW2974 gehemmt. Es konnte gezeigt werden, dass die erhöhte Permeabilität mit einer Veränderung der TJ-Proteinexpression einhergeht. 4-Amino-1,8-naphthalimid senkt die Expression von Occludin auf mRNA- und Proteinebene. GW2974 vermindert zusätzlich die Expression von VE-Cadherin, Claudin-5 und ZO-1. Darüber hinaus wurde die Wirkung auf Effluxpumpen untersucht. Die Ergebnisse der mRNA- und Protein-expression weichen voneinander ab, weshalb eine genauere Untersuchung der Translationsvorgänge sinnvoll erscheint. Glut-1 wird in GW2974 behandelten Zellen überexprimiert, was auf eine erhöhte Aktivität der BLECs hinweist. GW2974 und 4-Amino-1,8-naphthalimid könnten durch ihre permeabilitätssteigernde Wirkung die Ansprechrate einer systemischen Behandlung von PatientInnen mit einem zerebral metastasierten Mammakarzinom erhöhen und somit ihre Prognose verbessern. Detaillierte Studien zu Kombinationstherapien, den notwendigen Wirkstoff-konzentrationen und eventuellen negativen neurologischen Wirkungen sollten erwogen werden. N2 - The Blood-Brain Barrier (BBB) represents a selectively permeable barrier that controls the exchange of substances between the blood and the brain and thus has a neuroprotective effect. However, it not only prevents the passage of toxic metabolites, but also limits the access of therapeutic agents to the brain. Further research into methods to achieve higher drug concentrations in the brain is essential for the treatment of cerebral diseases such as cerebral metastatic breast cancer. The goal of this study was to identify drugs that increase the permeability of the BBB. The substance database LO1208 from Sigma-Aldrich was examined for its permeability-influencing properties as part of a high throughput drug screening (HTS). 31 of the examined substances showed an increase of the permeability on brain-like endothelial cells (BLECs) by at least 50%. Thereof 4-amino-1,8-naphthalimide (PARP inhibitor) and GW2974 (TKI) were selected for a more detailed analysis. Ibudilast (inhibitor of PDE4, MIF and Toll-like receptor-4) was found to be the third most active substance, although it did not cause any significant change in permeability. The measurement of the trans endothelial electrical resistance (TEER) and the permeability for fluorescein confirmed the results from the HTS and therefore is suggested to be used in further permeability tests in the future. Cell viability is not affected by 4 amino-1,8-naphthalmide. GW2974 and Ibudilast have a toxic effect on MCF-7 cells at a concentration of 500 µM, whereas BLECs are inhibited at a concentration of 100 µM of GW2974. The results show that the increased permeability is associated with a change in tight junction protein expression. 4-Amino-1,8-naphthalimide decreases the expression of occludin at mRNA and protein level. GW2974 also reduces the expression of VE-cadherin, claudin-5 and ZO-1. In addition to the abovementioned analysis, also the effect on efflux pumps was investigated. As the results of the mRNA and protein expression differ from each other, a more detailed analysis will be necessary to investigate the translation process. Glut-1 is overexpressed in GW2974-treated cells, which indicates an increased activity of the BLECs. GW2974 and 4-amino-1,8-naphthalimide could increase the response rate to systemic therapy of patients with cerebral metastatic breast cancer through their permeability-enhancing effect and thereby improve their prognosis. Detailed studies on combination therapies, the necessary drug concentrations and possible negative neurological effects are recommended to gain further insight. KW - Blut-Hirn-Schranke KW - Brustkrebs KW - Hirnmetastase KW - zerebral matastasierte Mammakarzinom KW - High-throughput drug screening KW - Blut-Hirn-Schrankenpermeabilität KW - High throughput screening KW - Hochdurchsatzscreening Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369964 ER - TY - THES A1 - Brohm, Katharina Andrea T1 - (Differential-) Diagnostik bei primärem Hyperaldosteronismus: Ermittlung eines LC-MS/MS-spezifischen Aldosterongrenzwerts für den Kochsalzbelastungstest und Evaluation des Orthostasetests hinsichtlich der Differenzierung von Subgruppen T1 - (Differential) Diagnosis in Primary Aldosteronism: Determination of an LC-MS/MS-Specific Aldosterone Cut-Off Value for the Saline Infusion Test and Evaluation of the Postural Stimulation Test Regarding the Differentiation of Subtypes N2 - Der primäre Hyperaldosteronismus (PA) stellt aktuell den häufigsten Grund für das Vorliegen einer sekundären Hypertonie dar. Der in der Bestätigungsdiagnostik verwendete Kochsalzbelastungstest basiert dabei auf einem fehlenden Absinken der Aldosteronkonzentration im Testverlauf bei Patient:innen mit PA im Vergleich zu Patient:innen mit essentieller Hypertonie (EH). Die Konzentrationsbestimmung erfolgte bisher mittels Immunoassay. Mit der LC-MS/MS steht jedoch mittlerweile eine weitere wichtige analytische Methode in der quantitativen Bestimmung von Steroidhormonen zur Verfügung, welche in dieser Arbeit im Hinblick auf den Kochsalzbelastungstest untersucht wurde. Hohe Bedeutung kommt außerdem der Subtypdifferenzierung des PA zu, da die Ätiologie der Erkrankung wegweisend für die Art der Therapie ist. Das Ziel dieser Studie war einerseits die Ermittlung eines LC-MS/MS-spezifischen Aldosteron-Cut-off-Wertes im Kochsalzbelastungstest und die Evaluation des Nutzens der Bestimmung von Steroidprofilen in der Diagnostik des PA. Zum anderen wurde der diagnostische Nutzen des Orthostasetests zur Unterscheidung von unilateraler und bilateraler Genese bei vorliegendem PA untersucht. Im Rahmen dieser Studien wurden 187 bzw. 158 Patient:innen analysiert, die zwischen 2009 und 2019 bei Verdacht auf oder Vorliegen eines PA im Universitätsklinikum Würzburg vorstellig wurden. Die Diagnose wurde gemäß der aktuellen Leitlinie anhand der Ergebnisse des Kochsalzbelastungstests, NNVKs, Bildgebung und postoperativen Outcomes gestellt. Mithilfe der LC-MS/MS wurden erneut die Aldosteronkonzentrationen der aufbewahrten Serumproben des Kochsalzbelastungstests, sowie ein erweitertes Steroidpanel bestimmt. Unter Verwendung einer ROC-Analyse wurden die jeweils bestehenden Cut-off-Werte optimiert bzw. neu ermittelt. Die mittels Immunoassay bestimmten Aldosteronkonzentrationen lagen um 28 ng/L höher als die mittels LC-MS/MS bestimmten Konzentrationen. Trotzdem lag der neu ermittelte LC-MS/MS-spezifische Aldosteron-Cut-off-Wert für den Kochsalzbelastungstest bei 69 ng/L und damit höher als der für den Immunoassay geltende, optimierte Aldosteron-Cut-off von 54 ng/L. Unter Verwendung des LC-MS/MS- spezifischen Cut-off-Werts erreichte der Kochsalzbelastungstest eine Sensitivität von 78,6% bei einer Spezifität von 89,3%. Die Sensitivität des Immunoassay-spezifischen Cut-off-Werts betrug 95,2% bei einer Spezifität von 86,9%. Das Bestimmen des gesamten Steroidprofils führte zu keiner zusätzlichen diagnostischen Information bei Durchführung des Kochsalzbelastungstests. Bei Betrachtung der gesamten Patient:innenkohorte erreichte der Orthostasetest, basierend auf einem Absinken der Plasmaaldosteronkonzentration nach 4h in Orthostase um ≥ 28% eine Sensitivität von 36,7% bei einer Spezifität von 100%. Wurde das Vorliegen eines gültigen Tests (Cortisolabfall nach 4h ≥ 10%) oder das Vorliegen einer unilateralen Raumforderung in der Bildgebung vorausgesetzt, stieg die Sensitivität des Orthostasetests auf 51,4% bzw. 51,6% bei gleichbleibend hoher Spezifität von 100% an. Abschließend lässt sich sagen, dass der Orthostasetest keine Alternative zum NNVK darstellt, jedoch als einfache, nicht invasive Methode der zusätzlichen Orientierung zur Untersuchung der Ätiologie des PAs dienen kann. Eine prospektive Evaluation der jeweils neu ermittelten Cut-off-Werte wird notwendig sein, um deren Anwendbarkeit im klinischen Alltag zu überprüfen. Außerdem könnte die Bestimmung der Hybridsteroide 18-Oxocortisol und 18-Hydroxycortisol wegweisend für die Genese des PA sein. N2 - Primary aldosteronism (PA) is currently the most common cause of secondary hypertension. The saline infusion test used in confirmatory diagnostics is based on the lack of decrease in aldosterone concentration during the test in patients with PA compared to those with essential hypertension (EH). Until now, concentration determination has been performed using immunoassay. However, LC-MS/MS has now become an important analytical method for the quantitative determination of steroid hormones, which was investigated in this work in relation to the saline infusion test. Subtype differentiation of PA is also of great significance, as the subtype determines the therapy. The aim of this study was to determine an LC-MS/MS-specific aldosterone cut-off value in the saline infusion test and to evaluate the benefit of determining steroid profiles in the diagnosis of PA. Additionally, the diagnostic value of the postural stimulation test to differentiate between unilateral and bilateral disease in the presence of PA was investigated. In these studies, 187 and 158 patients, respectively, who presented with suspected or confirmed PA at the University Hospital Würzburg between 2009 and 2019 were analyzed. The diagnosis was made according to current guidelines based on the results of the saline infusion test, adrenal vein sampling, imaging, and postoperative outcomes. Using LC-MS/MS, aldosterone concentrations of the stored serum samples from the saline infusion test and an extended steroid panel were determined. ROC analysis was used to optimize or newly determine the existing cut-off values. Aldosterone concentrations determined by immunoassay were 28 ng/L higher than those determined by LC-MS/MS. Nevertheless, the newly determined LC-MS/MS-specific aldosterone cut-off value for the saline infusion test was 69 ng/L, which is higher than the optimized aldosterone cut-off of 54 ng/L for the immunoassay. Using the LC-MS/MS-specific cut-off value, the saline infusion test achieved a sensitivity of 78.6% with a specificity of 89.3%. The sensitivity of the immunoassay-specific cut-off value was 95.2% with a specificity of 86.9%. Determining the entire steroid profile did not provide any additional diagnostic information when performing the saline infusion test. Considering the entire patient cohort, the postural stimulation test, based on a decrease in plasma aldosterone concentration after 4 hours in an upright position by ≥ 28%, achieved a sensitivity of 36.7% with a specificity of 100%. When the test was considered valid (cortisol decrease after 4 hours ≥ 10%) or the presence of a unilateral mass on imaging was assumed, the sensitivity of the postural stimulation test increased to 51.4% and 51.6%, respectively, with a consistently high specificity of 100%. In conclusion, the postural stimulation test does not serve as an alternative to adrenal vein sampling but can provide additional information in investigating the subtype of PA as a simple, non-invasive method. A prospective evaluation of the newly determined cut-off values will be necessary to verify their applicability in clinical practice. Additionally, determining the hybrid steroids 18-oxocortisol and 18-hydroxycortisol could be crucial for understanding the subtype of PA. KW - Aldosteronismus KW - Aldosteron KW - primärer Hyperaldosteronismus KW - LC-MS/MS KW - Kochsalzbelastungstest KW - Orthostasetest Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369382 ER - TY - THES A1 - Jorgacevic, Ivana T1 - Elucidating the interconnection of GvHD and Western diet-induced atherosclerosis T1 - Aufklärung des Zusammenhangs von GvHD und durch westliche Ernährung induzierter Atherosklerose N2 - Allogeneic hematopoietic cell transplantation (Allo-HCT) is the main and only treatment for many malignant and non-malignant haematological disorders. Even though the treatment has improved through the years and patient life expectancy has increased, graft versus host disease (GvHD) is still considered the main obstacle and one of the main reasons for increased mortality. Furthermore, improved patient’s survival and life expectancy brought into question the late post-HCT complications. The leading cause of late death after allo-HCT is the relapse of primary disease as well as chronic GvHD (cGvHD). However, a clear relationship was also described with pulmonary complications, endocrine dysfunction and infertility, and cataracts in post-HCT patients. In the last years big concern regarding a cumulative cardiovascular incidence in long-term survivors has been raised. Severe cardiovascular disease (CVD) is caused by atherosclerosis which is considered a chronic inflammatory disease of blood vessels. As such, it takes a long time from endothelial damage, as the onset event, and followed plaque formation to a manifestation of severe consequences, such as stroke, coronary heart disease, or peripheral arterial disease. Endothelial damage is well documented in patients post-HCT. In the context of allo-HCT, the endothelial damage is induced by the conditioning regimen with or without total body irradiation (TBI). Furthermore, endothelial cells (ECs) have been documented as a target of GvHD and increased concentration of circulating endothelial cells (CEC) coinciding with an increase in the number of circulating alloreactive T cells. According to 2021 ESC Guidelines on CVD prevention, the main atherosclerotic CVD (ASCVD) risk factors are blood apolipoprotein B (ApoB)-containing lipoproteins (of which low-density lipoprotein (LDL) is the most abundant), high blood pressure, cigarette smoking and diabetes mellitus (DM). GvHD is considered a high-risk factor for the onset of dyslipidaemia, hypertension, and DM. Overall, the risk of premature cardiovascular death is 2.7 fold increased in comparison to the general population, while the cumulative incidence of cardiovascular complications was shown to be up to 47% at ten years after reduced intensity conditioning (RIC), post-HCT. However, up to date, there are no available studies elucidating the interconnection between GvHD and atherosclerosis. The goal of this study was, therefore, to investigate the involvement of GvHD in the progression of atherosclerosis as well as to elucidate whether cytotoxic, CD8+ T cells that were shown to play a significant role in endothelial damage during the course of skin GvHD on one hand, and inducers of formation of unstable plaque on the other, are involved in this interconnection. For that purpose we established a novel minor histocompatibility anti gens (miHAg) allo-HCT Western diet (WD)-induced atherosclerosis mouse model. We were able to show that GvHD has a significant impact on atherosclerosis development in B6.Ldlr−/− recipient mice even in the absence of overt clinical disease activity. It seems that the impact is at least partly induced by CD8+ T cells, that showed significantly increased infiltration of aortic lesions in mice facing subclinical GvHD. As studies have shown in regular atherosclerotic mouse models as well as in humans, these CD8+ T cells exhibited not only increased expression of genes involved in activation, survival and differentiation to cytotoxic phenotype, but also some genes pointing out their exhaustion, that were absent in CD4+ T cell cluster. When anti-CD8β antibody was applied once per week along with WD feeding for eight weeks, the plaque formation was significantly reduced in aorta and aortic root pointing out the importance of these cells in an alloreactivity induced lesion formation. Furthermore, anti-CD8β treatment led to significantly decreased necrotic core formation followed by overall increase in plaque stability. Strikingly, bone marrow plus T cells (BMT) recipients fed WD showed significantly increased serum cholesterol levels in comparison to bone marrow (BM) (a group lacking alloreactive T cells that induce GvHD). This effect was reversed when anti-CD8β treatment was applied, suggesting, at least partly, an impact of alloreactive CD8+ T cells on cholesterol levels. Expression of genes responsible for lipid metabolism pointed out the tendency of the liver to regulate the increased cholesterol levels, however, the mechanism behind this phenotype still remains to be revealed. On the other hand, the impact of obesity, induced by chronic high-fat diet (HFD) feeding, has been shown to be an independent risk factor for gastrointestinal GvHD. Similarly, in major histocompatibility complex (MHC) disparate allo-HCT mouse model, we have noticed that even short-term WD intake leads to a significant decrease in survival of mice post-HCT. When the concentration of transplanted alloreactive T cells was reduced, the survival was improved, pointing out the involvement of these cells in the pathogenesis. Additionally, bioluminescence imaging (BLI) during initiation and effector phase of acute GvHD (aGvHD) revealed increased infiltration of alloreactive T cells in mice fed WD. Studies in an obesity model, we could confirm the involvement of specifically CD4+ T cells in WD induced impact, as the relative number of these cells was significantly increased in small intestine on day six post-HCT in mice fed WD. This increased intestinal infiltration was preceded by increase in the number of alloreactive T cells expressing intestine homing receptor (α4β7 integrin) in peripheral lymph nodes (LNs). Even though the number of T cells was not changed in the spleen of WD fed mice, the subset of CD4+ and CD8+ T cells that were highly secreting TNFα was increased as well as the expression of genes regulating pro-inflammatory cytokines such as IL-6 and interferon (IFN)γ pointing out significant WD-induced inflammation. Moreover, slight tendency towards increased intestinal permeability and load of translocated luminal bacteria, that we observed, could induce severe endotoxemia and dysregulated systemic immune response that could lead to detrimental induction of cell death. Justifying our speculations, we noted increased levels of transaminases and an increase in lactate dehydrogenase (LDH) levels (pointing out significant tissue damages). However, the exact mechanism behind this detrimental WD impact still remains to be elucidated. N2 - Die allogene hämatopoetische Zelltransplantation (engl.: allogeneic hematopoietic cell transplantation; allo-HCT) ist die wichtigste und einzige Behandlung für viele bösartige und nicht bösartige hämatologische Erkrankungen. Auch wenn sich die Behandlung im Laufe der Jahre verbessert hat und die Lebenserwartung der Patienten gestiegen ist, gilt die Transplantat-gegen-Wirt-Krankheit (engl.: graft versus host disease; GvHD) nach wie vor als Haupthindernis und ist einer der Hauptgründe für die erhöhte Sterblichkeit. Darüber hinaus hat die Verbesserung der Überlebensrate und der Lebenserwartung der Patienten dazu geführt, dass die Spätkomplikationen nach der HCT in Frage gestellt wer den. Die Hauptursache für den späten Tod nach einer allo-HCT ist das Wiederauftreten der Primärerkrankung und die chronische GvHD (cGvHD). Es wurde jedoch auch ein ein deutiger Zusammenhang mit pulmonalen Komplikationen, endokriner Dysfunktion und Unfruchtbarkeit sowie Katarakten bei Patienten nach einer HCT beschrieben. In den letzten Jahren wurde große Besorgnis hinsichtlich einer kumulativen kardio vaskulären Inzidenz bei Langzeitüberlebenden geäußert. Schwere Herz-Kreislauf Erkrankungen werden durch Atherosklerose verursacht, die als chronische Entzündu ngserkrankung der Blutgefäße gilt. Von der Endothelschädigung als Beginn und der anschließenden Plaquebildung bis zur Manifestation schwerwiegender Folgen wie Schla ganfall, koronare Herzkrankheit oder periphere arterielle Verschlusskrankheit vergeht eine lange Zeit. Endothelschäden sind bei Patienten nach HCT gut dokumen tiert. Im Zusammenhang mit der allo-HCT wird die Endothelschädigung durch das Konditionierungsschema mit oder ohne TBI induziert. Darüber hinaus wurde dokumentiert, dass Endothelzellen ein Ziel der GvHD sind und dass eine erhöhte Konzentration zirkulierender Endothelzellen (engl: circulating endothelial cells; CEC) mit einem Anstieg der Anzahl zirkulierender alloreaktiver T-Zellen korreliert. Nach den ESC-Leitlinien 2021 zur Prävention von Herz-Kreislauf-Erkrankungen sind die wichtigsten Risikofaktoren für atherosklerotische Herz-Kreislauf-Erkrankungen (engl.: atherosclerotic cardiovascular disease; ASCVD) Apolipoprotein B (ApoB)-haltige Lipoproteine im Blut (von denen das Low-Density-Lipoprotein (LDL) am häufigsten vorkommt), Bluthochdruck, Zigarettenrauchen und Diabetes mellitus (DM). GvHD gilt als Hochrisikofaktor für das Auftreten von Dyslipidämie, Bluthochdruck und DM. Insgesamt ist das Risiko eines vorzeitigen kardiovaskulären Todes im Vergleich zur Allgemeinbevölkerung um das 2,7-fache erhöht, während die kumulative Inzidenz kardiovaskulärer Komp likationen zehn Jahre nach einer Konditionierung mit reduzierter Intensität (RIC) nach einer HCT bei bis zu 47% lag. Bislang gibt es jedoch keine Studien, die den Zusam menhang zwischen GvHD und Atherosklerose aufklären. Ziel dieser Studie war es daher, die Beteiligung der GvHD am Fortschreiten der Atherosklerose zu untersuchen und zu klären, ob zytotoxische CD8+ T-Zellen, die einerseits eine bedeutende Rolle bei der En dothelschädigung im Verlauf der Haut-GvHD spielen und andererseits die Bildung insta biler Plaques induzieren, an diesem Zusammenhang beteiligt sind. Zu diesem Zweck haben wir ein neuartiges miHAg-allo-HCT Atherosklerose-Mausmodell etabliert. Wir konnten zeigen, dass GvHD einen signifikanten Einfluss auf die Entwicklung von Atherosklerose in B6.Ldlr−/−-Empfängermäusen hat, selbst wenn keine klinische Krankheitsaktivität vor 3 Chapter 1. Summary liegt. Es scheint, dass dieser Einfluss zumindest teilweise durch CD8+ T-Zellen induziert wird, die bei Mäusen mit subklinischer GvHD eine signifikant erhöhte Infiltration von Aortenläsionen zeigten. Dies wurde auch in Studien in regulären Atherosklerose-Modellen sowie beim Menschen gezeigt. Diese CD8+-T-Zellen wiesen nicht nur eine erhöhte Expression von Genen auf, die an der Aktivierung, dem Überleben und der Differenzierung zum zytotoxischen Phänotyp beteiligt sind, sondern auch einige Gene, die auf zelluläre Erschöpfung hinweisen, die im CD4+-T-Zell-Cluster fehlten. Wurde ein Anti-CD8β-Antikörper einmal wöchentlich zusammen mit der Fütterung von WD acht Wochen lang verabreicht, so wurde die Plaquebildung in der Aorta und der Aortenwurzel signifikant reduziert, was auf die Bedeutung dieser Zellen bei der durch Alloreaktivität induzierten Läsionsbildung hinweist. Darüber hinaus führte eine Anti-CD8β-Behandlung zu einer signifikant verringerten Bildung eines nekrotischen Kerns, gefolgt von einer allge meinen Zunahme der Plaquestabilität. Auffallend ist, dass BMT-Empfänger, die mit WD gefüttert wurden, im Vergleich zu BM (einer Gruppe ohne alloreaktive T-Zellen, die GvHD induzieren) signifikant erhöhte Serumcholesterinwerte aufwiesen. Dieser Effekt kehrte sich um, wenn eine Anti-CD8β-Behandlung durchgeführt wurde, was zumindest teilweise auf einen Einfluss alloreaktiver CD8+-T-Zellen auf den Cholesterinspiegel schließen lässt. Die Expression von Genen, die für den Lipidstoffwechsel verantwortlich sind, wies auf die Tendenz der Leber hin, den erhöhten Cholesterinspiegel zu regulieren; der Mechanismus, der diesem Phänotyp zugrunde liegt, muss jedoch noch aufgeklärt werden. Andererseits hat sich gezeigt, dass die durch chronische Fütterung induzierte Fettleibigkeit ein un abhängiger Risikofaktor für gastrointestinale GvHD ist. In ähnlicher Weise haben wir in dem MHC disparaten allo-HCT-Mausmodell festgestellt, dass selbst eine kurzfristige WD-Zufuhr zu einer signifikanten Verringerung des Überlebens der Mäuse nach der HCT führte. Wenn die Konzentration der transplantierten alloreaktiven T-Zellen reduziert wurde, verbesserte sich die Überlebensrate, was auf die Beteiligung dieser Zellen an der Pathogenese hinweist. Darüber hinaus zeigte die Biolumineszenz-Bildgebung (engl.: bio luminiscence imaging; BLI) während der Initiations- und Effektorphase der aGvHD eine erhöhte Infiltration alloreaktiver T-Zellen bei Mäusen, die mit WD gefüttert wurden. Wie Studien gezeigt in einem Adipositasmodell vorgeschlagen haben, konnten wir die Beteili gung von spezifisch CD4+ T-Zellen an der WD-induzierten Wirkung bestätigen, da die relative Anzahl dieser Zellen im Dünndarm am sechsten Tag nach der HCT bei Mäusen, die mit WD gefüttert wurden, signifikant erhöht war. Dieser erhöhten Darminfiltration ging ein Anstieg der Zahl alloreaktiver T-Zellen voraus, die den Darm-Homing-Rezeptor (α4β7-Integrin) in den peripheren LNs exprimieren. Obwohl sich die Anzahl der T-Zellen in der Milz von mit WD gefütterten Mäusen nicht veränderte, war die Untergruppe der CD4+- und CD8+-T-Zellen, die in hohem Maße TNFα sezernierten, ebenso erhöht wie die Expression von Genen, die pro-inflammatorische Zytokine wie IL-6 und IFNγ reg ulieren, was auf eine signifikante WD-induzierte Entzündung hinweist. Darüber hinaus könnte die von uns beobachtete leichte Tendenz zu einer erhöhten intestinalen Perme abilität und Belastung mit translozierten luminalen Bakterien eine schwere Endotoxämie und eine dysregulierte systemische Immunantwort auslösen, die zu einer schädlichen In duktion des Zelltods führen könnte. Zur Untermauerung unserer Spekulationen stellten wir erhöhte Transaminasenwerte und einen Anstieg der LDH-Werte fest (was auf erhe bliche Gewebeschäden hinweist).Jedoch verbleibt der genaue Mechanismus, der zu den verheerenden Auswirkungen von WD führt, ungeklärt. KW - Periphere Stammzellentransplantation KW - Arteriosklerose KW - GvHD KW - HCT KW - Atherosclerosis Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325792 ER - TY - THES A1 - Adhikari, Bikash T1 - Targeted degradation of Myc-interacting oncoproteins T1 - Gezielte Degradation von mit Myc interagierenden Onkoproteinen N2 - The hallmark oncoprotein Myc is a major driver of tumorigenesis in various human cancer entities. However, Myc’s structural features make it challenging to develop small molecules against it. A promising strategy to indirectly inhibit the function of Myc is by targeting its interactors. Many Myc-interacting proteins have reported scaffolding functions which are difficult to target using conventional occupancy- driven inhibitors. Thus, in this thesis, the proteolysis targeting chimera (PROTAC) approach was used to target two oncoproteins interacting with Myc which promote the oncogenicity of Myc, Aurora-A and WDR5. PROTACs are bifunctional small molecules that bind to the target protein with one ligand and recruit a cellular E3- ligase with the other ligand to induce target degradation via the ubiquitin- proteasome system. So far, the most widely used E3-ligases for PROTAC development are Cereblon (CRBN) and von Hippel–Lindau tumor suppressor (VHL). Furthermore, there are cases of incompatibility between some E3-ligases and proteins to bring about degradation. Hence there is a need to explore new E3- ligases and a demand for a tool to predict degradative E3-ligases for the target protein in the PROTAC field. In the first part, a highly specific mitotic kinase Aurora-A degrader, JB170, was developed. This compound utilized Aurora-A inhibitor alisertib as the target ligand and thalidomide as the E3-ligase CRBN harness. The specificity of JB170 and the ternary complex formation was supported by the interactions between Aurora-A and CRBN. The PROTAC-mediated degradation of Aurora-A induced a distinct S- phase defect rather than mitotic arrest, shown by its catalytic inhibition. The finding demonstrates that Aurora-A has a non-catalytic role in the S-phase. Furthermore, the degradation of Aurora-A led to apoptosis in various cancer cell lines. In the second part, two different series of WDR5 PROTACs based on two protein- protein inhibitors of WDR5 were evaluated. The most efficient degraders from both series recruited VHL as a E3-ligase and showed partial degradation of WDR5. In addition, the degradation efficiency of the PROTACs was significantly affected by the linker nature and length, highlighting the importance of linker length and composition in PROTAC design. The degraders showed modest proliferation defects at best in cancer cell lines. However, overexpression of VHL increased the degradation efficiency and the antiproliferative effect of the PROTACs. In the last part, a rapamycin-based assay was developed to predict the degradative E3-ligase for a target. The assay was validated using the WDR5/VHL and Aurora- A/CRBN pairs. The result that WDR5 is degraded by VHL but not CRBN and Aurora-A is degraded by CRBN, matches observations made with PROTACs. This technique will be used in the future to find effective tissue-specific and essential E3-ligases for targeted degradation of oncoproteins using PROTACs. Collectively, the work presented here provides a strategy to improve PROTAC development and a starting point for developing Aurora-A and WDR5 PROTACs for cancer therapy. N2 - Das Onkoprotein Myc ist ein wichtiger Faktor bei der Tumorentstehung in verschiedenen menschlichen Krebsarten. Die strukturellen Merkmale von Myc machen es jedoch schwierig, kleine Moleküle gegen dieses Protein zu entwickeln. Eine vielversprechende Strategie zur indirekten Hemmung der Funktion von Myc besteht darin, auf seine Interaktoren abzuzielen. Viele Proteine, die mit Myc interagieren, haben Gerüstfunktionen, die mit herkömmlichen Inhibitoren nur schwer zu hemmen sind. Daher wurde in dieser Arbeit der PROTAC-Ansatz (Proteolysis Targeting Chimera) verwendet, um zwei Onkoproteine, die mit Myc interagieren und die Onkogenität von Myc fördern, ins Visier zu nehmen: Aurora-A und WDR5. PROTACs sind bifunktionale kleine Moleküle, die mit einem Liganden an das Zielprotein binden und mit dem anderen Liganden eine zelluläre E3-Ligase rekrutieren, um den Abbau des Zielproteins über das Ubiquitin-Proteasom-System einzuleiten. Die bisher am häufigsten verwendeten E3-Ligasen für die Entwicklung von PROTACs sind Cereblon (CRBN) und der von Hippel-Lindau-Tumorsuppressor (VHL). Außerdem gibt es Fälle von Inkompatibilität zwischen einigen E3-Ligasen und Proteinen, die abgebaut werden sollen. Daher besteht die Notwendigkeit, neue E3-Ligasen zu erforschen und Werkzeuge zur Vorhersage abbauender E3-Ligasen für das Zielprotein zu entwickeln. Im ersten Teil wurde ein hochspezifischer Degrader der mitotischen Kinase Aurora-A, JB170, entwickelt. Bei dieser Verbindung wurde der Aurora-A-Inhibitor Alisertib als Zielligand und Thalidomid als Binder für die E3-Ligase CRBN verwendet. Die Spezifität von JB170 und die ternäre Komplexbildung wurden durch die Wechselwirkungen zwischen Aurora-A und CRBN unterstützt. Der durch PROTAC vermittelte Abbau von Aurora-A führte zu einem deutlichen Defekt in der S-Phase und nicht zu einem mitotischen Stillstand, wie es für dessen katalytische Hemmung beobachtet wurde. Dies zeigt, dass Aurora-A eine nicht-katalytische Funktion in der S-Phase hat. Außerdem führte der Abbau von Aurora-A in verschiedenen Krebszelllinien zur Apoptose. Im zweiten Teil wurden zwei verschiedene Serien von WDR5 PROTACs auf der Grundlage von zwei Protein-Protein-Inhibitoren von WDR5 untersucht. Die effizientesten Degrader aus beiden Serien rekrutierten VHL als E3-Ligase und zeigten einen teilweisen Abbau von WDR5. Darüber hinaus wurde die Abbaueffizienz der PROTACs erheblich von der Art und Länge des Linkers beeinflusst, was die Bedeutung der Linkerlänge und -zusammensetzung bei der Entwicklung von PROTACs unterstreicht. Die Abbauprodukte zeigten bestenfalls bescheidene Proliferationsdefekte in Krebszelllinien. Eine Überexpression von VHL erhöhte jedoch die Abbaueffizienz und den antiproliferativen Effekt der PROTACs. Im letzten Teil wurde ein auf Rapamycin basierender Assay entwickelt, um die abbauende E3-Ligase für ein Target vorherzusagen. Der Assay wurde anhand der Paare WDR5/VHL und Aurora-A/CRBN validiert. Das Ergebnis, dass WDR5 von VHL, aber nicht von CRBN abgebaut wird und Aurora-A von CRBN abgebaut wird, stimmt mit den Beobachtungen überein, die mit PROTACs gemacht wurden. Diese Technik wird in Zukunft eingesetzt werden, um wirksame gewebespezifische und essentielle E3-Ligasen für den gezielten Abbau von Onkoproteinen mit Hilfe von PROTACs zu finden. Insgesamt bieten die hier vorgestellten Arbeiten eine Strategie zur Verbesserung der PROTAC-Entwicklung und einen Ausgangspunkt für die Entwicklung von Aurora-A- und WDR5-PROTACs für die Krebstherapie. KW - Degradation KW - PROTACs KW - Oncoprotein KW - Cancer KW - Onkoprotein Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-317326 ER - TY - THES A1 - Fleißner, Janik Frank Hans-Werner T1 - Die Bedeutung von Oncostatin M für die Lipidhomöostase Apoe- und Ldlr-deletierter Mäuse T1 - The Significance of Oncostatin M for the Lipid Homeostasis in Apoe and Ldlr Knockout Mice N2 - OSM, ein Vertreter der IL-6-Typ-Zytokine, ist nicht nur für entzündliche, sondern auch für metabolische Prozesse von Bedeutung. Vorarbeiten der Arbeitsgruppe GEIER/HERMANNS und Studien von KOMORI et al. legen protektive Eigenschaften des Zytokins nahe, da Mäuse, denen OSMR fehlte, Charakteristika des metabolischen Syndroms aufwiesen. Zur eingehenderen Untersuchung der von OSM vermittelten Wirkung auf den murinen Lipidstoffwechsel wurden zwei für die NAFLD und Atherosklerose anfällige Modelle herangezogen und jeweils in Gegenwart und Abwesenheit des Osmr studiert: Weibliche Apoe-/-(Osmr-/-) und Ldlr-/-(Osmr-/-) Mäuse wurden über einen Zeitraum von zwölf Wochen mit westlicher Diät gefüttert, wöchentlich gewogen, am Ende der Diät geopfert und geerntet. Wildtypische C57Bl/6-Mäuse erfuhren die gleiche Behandlung und dienten als Referenzgruppe. Im Rahmen des Promotionsprojektes wurden Leberfettgehalt, Serumlipidspiegel, Lipoproteinfraktionen und Stuhllipide von Apoe-deletierten Mäusen bestimmt und mit bereits vorhandenen Daten der Ldlr-/-(Osmr-/-) und wildtypischen Mäuse in Beziehung gesetzt. Expressionsanalysen von am Lipidstoffwechsel beteiligten Genen in Darm-, Leber- und Fettgewebe trugen dazu bei, OSM-abhängige Regulationen aufzudecken. Ldlr-/- Tiere nahmen unter der Diät exzessiv zu, hatten hohe Serumspiegel an Leptin, Gluco-se und Lipiden, eine Lebersteatose und, begleitet von einer Induktion des Vldlr, erhöhte inflammatorische Marker im visceralen Fettgewebe. Der zusätzliche Knockout des Osmr ging mit einer geringeren Vldlr-Expression im Fettgewebe und einer hepatozytären Induktion von Cyp7a1 einher und resultierte in einem metabolisch günstigeren Phänotyp. Apoe-defiziente Tiere unterschieden sich hinsichtlich ihrer Gewichtszunahme nicht von Ldlr-/-Osmr-/- und C57Bl/6-Mäusen. Überraschenderweise zeigten sich im Serum von Apoe-/-Osmr-/- jedoch gegenüber Apoe-/- Mäusen erhöhte Konzentrationen des Gesamt- und VLDL-Cholesterins, der Triglyceride und freien Fettsäuren. Obwohl Lebern der Apoe-/-Osmr-/- Mäuse geringere Ldlr- und Lrp1-mRNA-Spiegel als die der Apoe-/- Mäuse aufwiesen, hatten sie einen höheren hepatischen Cholesteringehalt. Bei gesteigerter Cpt1a-Expression fiel der hepatische Tri-glyceridgehalt Apoe-deletierter Mäuse geringer aus als in Ldlr-/-(Osmr-/-) und wildtypischen Tieren. Unter Umgehung einer Fettgewebsentzündung präsentierten Apoe-defiziente Mäuse Hinweise einer inflammatorischen Leberschädigung, die pathogenetisch am ehesten mit einer gestörten Cholesterinhomöostase in Verbindung zu bringen war. Abhängig vom genetischen Hintergrund des Mausmodells hatte OSM schützende oder schädliche Effekte auf den Lipidmetabolismus. Die Ergebnisse der vorliegenden Arbeit betonen die entscheidende Bedeutung entzündlicher, von OSM modulierter Prozesse für den Fettstoffwechsel in Leber- und Fettgewebe. Weiterführende Experimente sind nötig, um die den Beobachtungen zugrunde liegenden molekularen Mechanismen zu entschlüsseln. N2 - OSM, a member of the IL-6-type family, plays a pivotal role not only in inflammatory pro-cesses, but also in the regulation of metabolism. In line with studies conducted by KOMORI et al., findings obtained by GEIER/HERMANNS revealed characteristics of the metabolic syndrome in mice lacking the OSMR. Therefore, protective properties of OSM were suggested. In order to further investigate OSM-mediated effects on murine lipid metabolism, two models prone to NAFLD and atherosclerosis were employed and studied in the presence and absence of Osmr: Female Apoe-/-(Osmr-/-) and Ldlr-/-(Osmr-/-) mice were fed a Western-type diet for twelve weeks, weighed weekly, sacrificed and harvested at the end of the diet. Wild-type C57Bl/6 mice underwent the same procedure and were used as a reference group. Thereafter, lipid levels and lipoprotein fractions in the sera of Apoe-deleted mice were deter-mined. In addition, their lipid content in liver tissue and stool was measured. Findings were compared with data from Ldlr-/-(Osmr-/-) and wild-type mice. To reveal OSM-dependent regulations of genes playing a key role in lipid metabolism, gene expression analyses were performed in intestinal, liver, and adipose tissue samples from all mice groups. Ldlr-/- animals excessively gained weight during the diet, had high serum levels of leptin, glucose, and lipids, hepatic steatosis, and, accompanied by induction of Vldlr, increased inflammatory markers in visceral adipose tissue. The additional knockout of Osmr was accom-panied by a lower Vldlr expression in adipose tissue and an induction of liver Cyp7a1, resulting in a metabolically favorable phenotype. In terms of weight gain, Apoe-deficient animals were not different from Ldlr-/-Osmr-/- and C57Bl/6 mice. Surprisingly, however, serum from Apoe-/-Osmr-/- mice showed increased concentrations of total and VLDL cholesterol, triglyc-erides, and free fatty acids when compared to Apoe-/- animals. Despite lower hepatic Ldlr and Lrp1 mRNA levels, Apoe-/-Osmr-/- mice had a higher hepatic cholesterol content than Apoe-/- mice. Fitting to an increased Cpt1a expression, the hepatic triglyceride content of Apoe-deleted mice was lower than in Ldlr-/-(Osmr-/-) and wild-type mice. Most likely due to an impaired hepatic cholesterol homeostasis, liver sections of Apoe-deleted mice displayed features of inflammation, whereas the adipose tissues of these animals remained rather unscathed. Depending on the genetic background of the mouse model, OSM had protective or deleterious effects on lipid metabolism. The results of this project emphasize the significance of OSM regarding both inflammation and metabolism in liver and adipose tissue. Further ex-periments are needed to unravel the molecular mechanisms underlying these observations. KW - Apolipoprotein E KW - LDL-Rezeptor KW - Oncostatin M KW - Oncostatin-M-Rezeptor KW - Lipoprotein KW - Oncostatin M receptor KW - lipoprotein KW - Interleukin-6-Typ-Zytokine KW - Interleukin-6 type cytokines Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-280592 ER - TY - THES A1 - Bakirci, Ezgi T1 - Development of \(In\) \(vitro\) Models for Tissue Engineering Applications Using a High-Resolution 3D Printing Technology T1 - Entwicklung von \(In\) \(vitro\)-Modellen für Tissue-Engineering-Anwendungen mithilfe einer hochauflösenden 3D-Drucktechnologie N2 - In vitro models mimic the tissue-specific anatomy and play essential roles in personalized medicine and disease treatments. As a sophisticated manufacturing technology, 3D printing overcomes the limitations of traditional technologies and provides an excellent potential for developing in vitro models to mimic native tissue. This thesis aims to investigate the potential of a high-resolution 3D printing technology, melt electrowriting (MEW), for fabricating in vitro models. MEW has a distinct capacity for depositing micron size fibers with a defined design. In this thesis, three approaches were used, including 1) extending the MEW polymer library for different biomedical applications, 2) developing in vitro models for evaluation of cell growth and migration toward the different matrices, and 3) studying the effect of scaffold designs and biochemical cues of microenvironments on cells. First, we introduce the MEW processability of (AB)n and (ABAC)n segmented copolymers, which have thermally reversible network formulation based on physical crosslinks. Bisurea segments are combined with hydrophobic poly(dimethylsiloxane) (PDMS) or hydrophilic poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEG-PPO) segments to form the (AB)n segmented copolymers. (ABAC)n segmented copolymers contain all three segments: in addition to bisurea, both hydrophobic and hydrophilic segments are available in the same polymer chain, resulting in tunable mechanical and biological behaviors. MEW copolymers either support cells attachment or dissolve without cytotoxic side effects when in contact with the polymers at lower concentrations, indicating that this copolymer class has potential in biological applications. The unique biological and surface properties, transparency, adjustable hydrophilicity of these copolymers could be beneficial in several in vitro models. The second manuscript addresses the design and development of a melt electrowritten competitive 3D radial migration device. The approach differs from most of the previous literature, as MEW is not used here to produce cell invasive scaffolds but to fabricate an in vitro device. The device is utilized to systematically determine the matrix which promotes cell migration and growth of glioblastoma cells. The glioblastoma cell migration is tested on four different Matrigel concentrations using a melt electrowritten radial device. The glioblastoma U87 cell growth and migration increase at Matrigel concentrations 6 and 8 mg mL-1 In the development of this radial device, the accuracy, and precision of melt electrowritten circular shapes were investigated. The results show that the printing speed and design diameter are essential parameters for the accuracy of printed constructs. It is the first instance where MEW is used for the production of in vitro devices. The influence of biochemical cues and scaffold designs on astrocytes and glioblastoma is investigated in the last manuscript. A fiber comprising the box and triangle-shaped pores within MEW scaffolds are modified with biochemical cues, including RGD and IKVAV peptides using a reactive NCO-sP(EO-stat-PO) macromer. The results show that astrocytes and glioblastoma cells exhibit different phenotypes on scaffold designs and peptide-coated scaffolds. N2 - In-vitro-Modelle sind Werkzeuge, die die gewebespezifische Anatomie nachbilden und eine wesentliche Rolle in der personalisierten Medizin und bei der Behandlung von Krankheiten spielen. Als hochentwickelte, multifunktionale Fertigungstechnologie überwindet der 3D-Druck die Grenzen herkömmlicher Technologien und bietet ein hervorragendes Potenzial für die Herstellung von In-vitro-Modellen. Der 3D-Druck ist eine der vielversprechendsten Techniken, um biologische Materialien in einer komplexen Anordnung zusammenzusetzen, die das natürliche Gewebe nachahmt. In dieser Arbeit soll das Potenzial der hochauflösenden 3D-Drucktechnologie melt electrowriting (MEW), für die Herstellung von In-vitro-Modellen untersucht werden. Wir konzentrieren uns auf drei Ansätze: 1) die Erweiterung der MEW-Polymerbibliothek für verschiedene biomedizinische Anwendungen, 2) die Entwicklung von In-vitro-Modellen zur Bewertung des Zellwachstums und der Zellmigration in Richtung der verschiedenen Matrizes und 3) die Untersuchung der Auswirkungen von MEW-Gerüstdesigns und biochemischen Faktoren der Mikroumgebung auf Zellen. Zunächst haben wir die MEW-Verarbeitbarkeit von segmentierten (AB)n- und (ABAC)n-Copolymeren vorgestellt, die eine thermisch reversible Netzwerkformulierung auf der Grundlage physikalischer Vernetzungen aufweisen. Bisurea-Segmente werden mit hydrophoben hydrophobic poly(dimethyl siloxane) (PDMS) oder hydrophilen poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEG-PPO) Segmenten kombiniert, um die (AB)n segmentierten Copolymere zu bilden. Segmentierte (ABAC)n-Copolymere enthalten alle drei Segmente: Zusätzlich zu den Bisurea-Segmenten sind sowohl hydrophobe als auch hydrophile Segmente in derselben Polymerkette vorhanden, was den segmentierten (ABAC)n-Copolymeren abstimmbare mechanische und biologische Eigenschaften verleiht. MEW-Copolymere unterstützten entweder die Anhaftung an Zellen oder lösten sich ohne zytotoxische Nebenwirkungen auf, wenn sie in niedrigeren Konzentrationen mit ihnen in Berührung kamen, was darauf hindeutet, dass diese Copolymerklasse über umfassende biologische Eigenschaften verfügt. Die einzigartigen biologischen Eigenschaften und Oberflächeneigenschaften, die Transparenz und die einstellbare Hydrophilie dieser Copolymere könnten in verschiedenen In-vitro-Modellen von Vorteil sein. Das zweite Manuskript befasst sich mit einem durch MEW hergestellten wettbewerbsfähigen 3D-Radialmigrationsdesign. Der Ansatz unterscheidet sich vom Großteil der MEW-Literatur, da MEW nicht zur Herstellung von invasiven Zellgerüsten verwendet wurde, sondern zur Herstellung eines In-vitro-Designs diente. Das Design wurde verwendet, um systematisch die Matrix zu bestimmen, die die Zellmigration und das Wachstum von Glioblastomzellen fördert. Die Migration der Glioblastomzellen wurde auf vier verschiedenen Matrigel-Konzentrationen unter Verwendung einer durch MEW hergestellten Radialvorrichtung getestet. Das Wachstum und die Migration der Glioblastomzellen U87 nahmen bei Matrigelkonzentrationen von 6 und 8 mg mL-1 zu. Wir untersuchten auch die Genauigkeit und Präzision der durch MEW erzeugten Kreisformen. Die Ergebnisse zeigten, dass die Druckgeschwindigkeit und der Designdurchmesser wesentliche Parameter für die Genauigkeit der gedruckten Konstrukte sind. Die Arbeit ist die erste Studie, die MEW für die Herstellung von In-vitro-Modellen verwendet. Im letzten Manuskript wurde der Einfluss von biochemische Funktionalisierung in Kombination mit Gerüstdesigns auf Astrozyten und Glioblastome untersucht. Die kastenförmigen und achteckigen MEW-Gerüste wurden mit biochemischen Wirkstoffen modifiziert, darunter RGD- und IKVAV-Peptide unter Verwendung von reaktivem NCO-sP(EO-stat-PO). Wir fanden heraus, dass Astrozyten und Glioblastomzellen unterschiedliche Phänotypen auf den verschiedenen Designs und mit Peptiden beschichteten Gerüsten aufweisen. KW - Melt electrowriting KW - 3D-Druck KW - 3D printing KW - In vitro model Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-251645 ER - TY - THES A1 - Das [geb. Nitschke], Felix Marcel T1 - DNA-Methylierung und Genexpression von FKPB5 als Teil des Stresshormonsystems bei von Depressionen und Herzinsuffizienz Betroffenen sowie gesunden Kontrollen T1 - DNA methylation and gene expression of FKPB5 as part of the stress hormone system in people affected by depression and heart failure as well as healthy controls N2 - FKBP5 stellt im Stresssystem der HPA-Achse ein zentrales Gen bei der Regulation der Sensitivität des Glukokortikoidrezeptors und somit der Reaktion auf Stress dar. Zur Adaptation an Umwelteinflüsse ist es selbst in ein komplexes System von Regulationsmechanismen eingebettet, die unter anderem epigenetische Modifikationen in Form von DNA-Methylierung umfassen. Bisherige Studien legen eine starke Assoziation von FKBP5 zu stressinduzierten psychischen Erkrankungen nahe und weisen auf eine Dysregulation der HPA-Achse als möglichen Pathomechanismus hin. Für die enge klinische Interaktion von Depression und Herzinsuffizienz sowie eine ebenfalls vermutete Rolle der HPA-Achse in der Pathogenese letzterer, könnte FKBP5 daher ein entscheidendes Bindeglied darstellen. Gleichzeitig bietet die Identifikation einer über FKBP5 ausgedrückten Dysregulation der HPA-Achse einen biologischen Befund, der als Marker für das Ansprechen einer antidepressiven Therapie herangezogen werden könnte. Ziel dieser Arbeit war daher die Untersuchung eines möglichen Einflusses regulatorischer Parameter von FKBP5 auf die Herzinsuffizienz sowie eine Prüfung dieser als mögliche Biomarker für einen Erfolg der antidepressiven Therapie. Dazu wurden Blutproben von ProbandInnen der GEParD- bzw. DaCFail-Studie mit Depression, Herzinsuffizienz sowie gesunde Kontrollen untersucht. Durch Pyrosequenzierung bisulfitkonvertierter DNA erfolgte die Bestimmung der Methylierung regulatorischer CpGs. Die Messung der relativen mRNA-Expression erfolgte durch den Einsatz einer qPCR. In der Auswertung fand sich keine differentielle mRNA-Expression oder Methylierung zwischen den vier Untersuchungsgruppen. Allerdings reagierten depressive PatientInnen verglichen mit der Kontrollgruppe mit einer geringeren Zunahme der mRNA-Expression als Reaktion auf den mDST. Das Therapieansprechen in der Depressionsgruppe wiederum war mit einer niedrigeren Methylierung auf CpG7 sowie einer höheren mRNA-Expression zu Therapiebeginn assoziiert. Im Behandlungsverlauf führte eine Abnahme der mRNA-Expression bei den Respondern zu einer Annäherung beider Gruppen. Diese Arbeit konnte keine Hinweise für eine Rolle von FKBP5 in der Pathogenese der Herzinsuffizienz finden. Allerdings zeigten die Befunde zur Regulation des Gens bei Glukokortikoidstimulation eine hohe Konstanz zu vorherigen Ergebnissen. In diesen Kontext reihen sich auch die Ergebnisse für das Therapieansprechen ein, die aufgrund einer Herabregulation der HPA-Achse im Therapieverlauf die Idee einer ursächlichen HPA-Dysregulation in der Gruppe der Responder bekräftigen. Für sich allein genommen lassen sich mRNA-Expression und Methylierung aufgrund mangelnder Sensitivität und Spezifität nicht als Biomarker für das Therapieansprechen einsetzen. Die bisherigen Befunde bestärken aber eine mögliche Rolle in einer Batterie unterschiedlicher Biomarker auf verschiedenen Ebenen, wie Klinik, Psychometrie und Physiologie. N2 - FKBP5 represents a central gene in the stress system of the HPA axis in the regulation of the sensitivity of the glucocorticoid receptor and thus the reaction to stress. To adapt to environmental influences, it is itself embedded in a complex system of regulatory mechanisms, including epigenetic modifications in the form of DNA -Methylation. Previous studies suggest a strong association of FKBP5 with stress-induced mental illnesses and point to a dysregulation of the HPA axis as a possible pathomechanism. FKBP5 could therefore represent a crucial link for the close clinical interaction between depression and heart failure as well as a suspected role of the HPA axis in the pathogenesis of the latter. At the same time, the identification of HPA axis dysregulation expressed via FKBP5 provides a biological finding that could be used as a marker for the response to antidepressant therapy. The aim of this work was therefore to investigate a possible influence of regulatory parameters of FKBP5 on heart failure and to examine these as possible biomarkers for the success of the antidepressive therapy. For this purpose, blood samples from subjects of the GEParD or DaCFail study with depression, heart failure and healthy controls were examined. Pyrosequencing of bisulfite-converted DNA was used to determine the methylation of regulatory CpGs. The relative mRNA expression was measured using qPCR. The analysis found no differential mRNA expression or methylation between the four study groups. However, depressed patients responded with a smaller increase in mRNA expression in response to the mDST compared to the control group. The treatment response in the depression group was associated with lower methylation on CpG7 and higher mRNA expression at the start of therapy. Over the course of treatment, a decrease in mRNA expression in responders led to a convergence of both groups. This work did not find any evidence for a role for FKBP5 in the pathogenesis of heart failure. However, the findings on the regulation of the gene during glucocorticoid stimulation showed a high degree of consistency with previous results. The results for the treatment response also fit into this context, which strengthen the idea of a causal HPA dysregulation in the group of responders due to a downregulation of the HPA axis during the course of therapy. Taken alone, mRNA expression and methylation cannot be used as biomarkers of treatment response due to a lack of sensitivity and specificity. However, the findings so far support a possible role in a battery of different biomarkers at different levels, such as clinical, psychometrics and physiology. KW - Gen FKBP5 KW - Methylierung KW - Genexpression KW - Depression KW - Herzinsuffizienz KW - FKBP5 Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369730 ER - TY - THES A1 - Junghanns, Lara Madeleine T1 - Resistenzmechanismen gegen Amphotericin B in humanpathogenen Hefepilzen T1 - Resistance mechanism to amphotericin B in human pathogenic yeasts N2 - Die 2009 erstmals entdeckte Spezies C. auris erlangte binnen kürzester Zeit zunehmend weltweite Aufmerksamkeit. Vor allem die Tendenz der Multiresistenzentwicklung und das rasche Auslösen von nosokomialen Infektionen erschweren den Umgang und die Therapie von C. auris Infektionen im Vergleich zu anderen Candida Spezien. Diese Dissertationsarbeit umfasst eine systematische Resistenzanalyse der im NRZMyk vorhandenen Stammsammlung aus C. auris und C. parapsilosis Isolaten, um Aufschluss über den Wirkmechanismus von Amphotericin B in Hefepilzen zu erlangen. Anhand der zunächst durchgeführten Amphotericin B-Resistenztestungen kristallisierten sich CAU37 und CAU43 mit MHK-Werten bis zu 12 µg/ml als stark Amphotericin B-resistente Isolate heraus. Die Analyse der Sequenzierungsergebnisse zeigte bei beiden Stämmen eine Mutation im ERG4 Gen an Position 576, welche nicht eindeutig als alleinige Ursache für die verminderte Amphotericin B-Empfindlichkeit festgelegt werden konnte. Dennoch wurde im Rahmen eines Survival Assays bei beiden Amphotericin B-resistenten Isolaten anfänglich eine konzentrationsabhängige Aktivität gegenüber Amphotericin B festgestellt, bevor ein Nachwachsen der Kulturen beobachtet wurde. Somit wurde die Vermutung aufgestellt, dass lediglich ein Teil der aufgebrachten Candida-Zellen abgetötet wird und dies in einer Vermehrung der überlebenden Zellen resultiert. Des Weiteren konnte im Rahmen von Resistenztestungen mit dem Sphingolipidinhibitor Myriocin nachgewiesen werden, dass vor allem in Amphotericin B-resistenten Isolaten eine deutliche Wirkungsverstärkung des Polyens hervorgerufen wird. Diese Sensitivitätssteigerung ist allgemein bei allen C. auris Isolaten zu beobachten, fällt bei resistenten Stämmen jedoch deutlich stärker aus. Hierdurch kam die Annahme auf, dass Amphotericin B-Resistenzen auch in möglichen Veränderungen des Sphingolipid-Haushaltes begründet sein könnten. Darüber hinaus scheint Myriocin keinen Einfluss auf Fluconazol-resistente oder FKS-mutierte Echinocandin-resistente C. auris Stämme zu haben. Das ebenfalls untersuchte und von Myriocin abgeleitete Medikament Fingolimod hatte jedoch ebenfalls keinen wirkungsverstärkenden Effekt. Allerdings reagierte ein Großteil der C. auris Isolate (57,6 %) sensitiv gegenüber dem neusten medizinisch bekannten Triazol Isavuconazol und es konnte erstmalig ein ECV-Wert von 0,03125 µg/ml festgelegt werden. Ein valider Vergleich von C. auris zu C. parapsilosis war aufgrund der mangelnden Anzahl an C. parapsilosis Isolaten jedoch nicht möglich N2 - The species C. auris, which was first discovered in 2009, quickly attracted worldwide attention. In particular, the development of multidrug resistance and the rapid onset of nosocomial infections complicate the management and treatment of C. auris infections compared to other Candida species. This dissertation comprises a systematic resistance analysis of the strain collection available at the NRZMyk from C. auris and C. parapsilosis isolates in order to shed light on the mechanism of action of amphotericin B in yeast fungi. CAU37 and CAU43 ermerged as highly amphotericin B-resistant isolates in the initially performed amphotericin B resistance tests, with MIC values up to 12 µg/ml. Sequencing results showed a mutation in the ERG4 gene at position 576 in both strains, which can`t be clearly identified as the main cause of the reduced susceptibility to amphotericin B. Nevertheless both amphotericin B-resistant isolates initially showed a concentration dependent activity against amphotericin B, followed by a regrowth of the cultures. The hypothesis is, that only some of the applied Candida cells are killed, resulting in a proliferation of the surviving cells. Furthermore the resistance tests with the sphingolipid inhibitor Myriocin in combination with amphotericin B showed that sublethal myriocin concentrations increased the C. auris susceptibility to amphotericin B. This increase in sensitivity is generally observed in all C. auris isolates, but is significantly stronger in resistant strains. This leeds to the assumption that amphotericin B resistance can also be due to possible changes in the sphingolipid balance. Furthermore, myriocin does not appear to have any influence on fluconazole-resistant or FKS-mutated echinocandin-resistant C. auris strains. Fingolimod, a drug also investigated and derived from Myriocin, doesn`t have any enhancing effect either. However the majority of C. auris isolates (57.6 %) reacted sensitively to the latest medically known triazole isavuconazole and for the first time an ECV value of of 0.03125 µg/ml could be determined. A valid comparison of C. auris to C. parapsilosis was not possible due to the lack of C. parapsilosis isolates. KW - Candida KW - antifungal susceptibility KW - Multidrug-Resistenz KW - Amphotericin B KW - Sphingolipide KW - Fingolimod KW - Candida auris KW - Multiresistenz KW - Myriocin KW - Isavuconazol KW - Antimykotikaresistenz KW - C. auris KW - Empdindlichkeitsprüfung Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369861 ER - TY - THES A1 - Banaschewski, Nora Malaika Marcia Cathérine T1 - Erleichterungslernen bei Jugendlichen mit nicht-suizidalem selbstverletzendem Verhalten T1 - Pain relief learning in adolescents with non-suicidal self-injury N2 - Die Erleichterung von einem körperlichen Schmerzreiz besitzt appetitiven Charakter (Leknes et al., 2008; 2011; Seymour et al., 2005), aktiviert belohnungsassoziierte Hirnstrukturen (Leknes et al., 2011; Leknes & Brock, 2014; Leknes & Tracey, 2008; Navratilova & Porreca, 2014) und fördert durch ihre Konditionierbarkeit als Erleichterungslernen bezeichnete appetitive Lern- und Konditionierungsprozesse (Andreatta et al., 2010, 2012; 2013; 2017; Gerber et al., 2014; Tanimoto et al., 2004; Yarali et al., 2008). Die vorliegende Arbeit bestätigt das angewandte Versuchsparadigma als valides Modell für Erleichterungslernen im Menschen und zeigt erstmals, dass der appetitive Charakter von Schmerzerleichterung auch in Jugendlichen konditionierbar ist. Erfolgreiches Erleichterungslernen zeigte sich dabei in der untersuchten Stichprobe lediglich auf impliziter, nicht aber auf expliziter, kognitiver Ebene. Dies stützt Thesen und vorherige Forschungsbefunde einer Dualität assoziativen Lernens in ein implizites Lernen, welches vornehmlich subkortikale Strukturen erfordert und ein explizites Lernen, das vorrangig kortikale Strukturen wie den präfrontalen Cortex involviert (Andreatta et al., 2010; Strack & Deutsch, 2004; Williams et al., 2001). Die Beobachtungen einer differenten Furcht- versus Erleichterungs-Extinktion bestärken die Thesen eines diversen neuronalen Hintergrunds dieser beiden Lernformen (Diegelmann et al., 2013; Gerber et al., 2014; Yarali et al., 2009; Yarali & Gerber, 2010). Gleichzeitig werfen die Studienergebnisse die Frage auf, ob und inwiefern im Erleichterungslernen von Jugendlichen Unterschiede zu jenem in Erwachsenen bestehen. Die Hypothese einer verstärkten Akquisition von Erleichterungslernen bei Jugendlichen mit NSSV im Vergleich zu gesunden Jugendlichen ließ sich in der vorliegenden Studie nicht bestätigen. Somit liefern die Ergebnisse keinen direkten Hinweis darauf, dass ein verstärktes Lernen durch Schmerzerleichterung an der Ätiopathogenese von NSSV beteiligt sein könnte. Die vorliegende Arbeit zeigte vielmehr die Tendenz eines abgeschwächten impliziten Erleichterungslernens bei den Jugendlichen mit NSSV. Die tendenziellen Gruppenunterschiede ließen sich nicht hinreichend durch eine differente aktuelle Stimmungslage oder durch eine unterschiedlich starke Ausprägung aversiver emotionaler Anspannungen oder momentaner Angstaffekte erklären. Innerhalb der Gruppe Jugendlicher mit NSSV zeigte sich auch kein Hinweis darauf, dass der Erfolg von Erleichterungslernen vom Schweregrad des NSSV oder von der aktuellen Einnahme von Antidepressiva abhängig sein könnte. Explorative Analysen ergaben, dass Gruppeneffekte in der vorliegenden Studie womöglich aufgrund einer statistischen Unterschätzung, bedingt durch einen zu geringen Stichprobenumfang, nicht das Signifikanzniveau erreichten und dass Unterschiede im Erleichterungslernen von Jugendlichen mit und ohne NSSV tatsächlich sogar noch stärker ausgeprägt sein könnten. Somit sollte die vorliegende Arbeit als Pilotstudie für zukünftige größer angelegte Studien zu Erleichterungslernen bei NSSV betrachtet werden. Zukünftige Studien erscheinen insbesondere sinnvoll mit Blick auf die hohe klinische sowie gesellschaftliche Relevanz von NSSV für welches, trotz der hohen Prävalenzen und des deutlich erhöhten Morbiditäts- und Mortalitätsrisikos, zum aktuellen Zeitpunkt noch keine hinreichenden Erklärungsmodelle bestehen. Die Studie bestätigte das Vorliegen eines erhöhten Grades aversiver emotionaler Anspannung in Jugendlichen mit NSSV, welcher zuvor nur an Erwachsenen mit einer BPD untersucht und festgestellt worden war (Niedtfeld et al., 2010; Stiglmayr et al., 2005). Die Abnahme negativer Affekte bei den Jugendlichen mit NSSV im Studienverlauf repliziert die Ergebnisse vorheriger Studien, in denen eine Reduktion selbst-berichteter negativer Affekte durch die Beendigung eines Schmerzreizes beobachtet wurde (Bresin et al., 2010; Bresin & Gordon, 2013). Damit bestärken die Studienergebnisse bestehende Erklärungsmodelle für NSSV, welche eine entscheidende Beteiligung der körperlichen Schmerzen und der Schmerzerleichterung bei der Selbstverletzung an der Affektregulation vermuten. Weiterhin wirft die vorliegende Arbeit die Frage auf, welche Rolle eine veränderte Wahrnehmung von Schmerz und Schmerzerleichterung in der Ätiopathogenese von NSSV einnimmt und wie diese sich auf Lernprozesse auswirkt. Insgesamt erbrächten weitere Erkenntnisse über den potenziellen Zusammenhang von NSSV und abweichendem Erleichterungslernen ein besseres Verständnis für Mechanismen der Entstehung und Aufrechterhaltung von NSSV und böten zudem möglicherweise Ansätze für neue Therapiemöglichkeiten des Störungsbildes. N2 - Relief from a physical pain stimulus has an appetitive character (Leknes et al., 2008; 2011; Seymour et al., 2005), activates reward-associated brain structures (Leknes et al., 2011; Leknes & Brock, 2014; Leknes & Tracey, 2008; Navratilova & Porreca, 2014) and, due to its conditionability, promotes learning and conditioning processes called relief learning (Andreatta et al., 2010, 2012; 2013; 2017; Gerber et al., 2014; Tanimoto et al., 2004; Yarali et al., 2008). The present work confirms the applied experimental paradigm as a valid model for relief learning in humans and shows for the first time that the appetitive nature of pain relief is also conditionable in adolescents. Successful relief learning was shown in the investigated sample only on an implicit, but not on an explicit, cognitive level. This supports theses and prior research findings of a duality of associative learning into implicit learning, which primarily requires subcortical structures, and explicit learning, which primarily involves cortical structures such as the prefrontal cortex (Andreatta et al., 2010; Strack & Deutsch, 2004; Williams et al., 2001). The observations of differential fear versus relief extinction reinforce the hypotheses of a diverse neural background of these two forms of learning (Diegelmann et al., 2013; Gerber et al., 2014; Yarali et al., 2009; Yarali & Gerber, 2010). At the same time, the study results raise the question of whether and to what extent differences exist in the relief learning of adolescents compared to that in adults. The hypothesis of increased acquisition of relief learning in adolescents with non-suicidal self-injury (NSSI) compared with healthy adolescents could not be confirmed in the present study. Thus, the results do not provide direct evidence that enhanced relief learning may be involved in the etiopathogenesis of NSSI. Rather, the present work demonstrated a tendency for attenuated implicit relief learning among adolescents with NSSI. The tendential group differences could not be adequately explained by a differential current mood state or by different degrees of aversive emotional tension or momentary anxiety effects. Within the group of adolescents with NSSI, there was also no evidence that the success of relief learning might depend on the severity of NSSI or on the current use of antidepressants. Exploratory analyses revealed that group effects in the present study did not reach the significance level possibly because of statistical underestimation due to an insufficient sample size and that differences in relief learning between adolescents with and without NSSI might actually be even bigger. Thus, the present work should be considered as a pilot study for future larger-scale studies on relief learning in NSSI. Future studies seem particularly useful in view of the high clinical as well as societal relevance of NSSI for which, despite the high prevalences and the significantly increased risk of morbidity and mortality, no adequate explanatory models exist at the present time. The study confirmed the presence of increased levels of aversive emotional tension in adolescents with NSSI, which had previously been studied and found only in adults with a borderline personality disorder (Niedtfeld et al., 2010; Stiglmayr et al., 2005). The decrease in negative affect in adolescents with NSSI over the course of the study replicates the findings of previous studies in which a reduction in self-reported negative affect was observed as a result of the cessation of a pain stimulus (Bresin et al., 2010; Bresin & Gordon, 2013). Thus, the study results reinforce existing explanatory models for NSSI that suggest a crucial involvement of physical pain and pain relief during self-injury in affect regulation. Furthermore, the present work raises the question of the role of altered perception of pain and pain relief in the etiopathogenesis of NSSI and how this affects learning processes. Overall, further insights into the potential link between NSSI and deviant relief learning would provide a better understanding of mechanisms involved in the development and maintenance of NSSI, and, on top of that, might offer approaches for new treatment options for the disorder. KW - Selbstbeschädigung KW - Erleichterungslernen KW - Nicht-suizidales selbstverletzendes Verhalten KW - NSSV Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323673 ER - TY - JOUR A1 - Gronwald, Thomas A1 - Hoos, Olaf A1 - Hottenrott, Kuno T1 - Effects of Acute Normobaric Hypoxia on Non-linear Dynamics of Cardiac Autonomic Activity During Constant Workload Cycling Exercise JF - Frontiers in Physiology N2 - Aim: Measurements of Non-linear dynamics of heart rate variability (HRV) provide new possibilities to monitor cardiac autonomic activity during exercise under different environmental conditions. Using detrended fluctuation analysis (DFA) technique to assess correlation properties of heart rate (HR) dynamics, the present study examines the influence of normobaric hypoxic conditions (HC) in comparison to normoxic conditions (NC) during a constant workload exercise. Materials and Methods: Nine well trained cyclists performed a continuous workload exercise on a cycle ergometer with an intensity corresponding to the individual anaerobic threshold until voluntary exhaustion under both NC and HC (15% O2). The individual exercise duration was normalized to 10% sections (10–100%). During exercise HR and RR-intervals were continuously-recorded. Besides HRV time-domain measurements (meanRR, SDNN), fractal correlation properties using short-term scaling exponent alpha1 of DFA were calculated. Additionally, blood lactate (La), oxygen saturation of the blood (SpO2), and rating of perceived exertion (RPE) were recorded in regular time intervals. Results: We observed significant changes under NC and HC for all parameters from the beginning to the end of the exercise (10% vs. 100%) except for SpO2 and SDNN during NC: increases for HR, La, and RPE in both conditions; decreases for SpO2 and SDNN during HC, meanRR and DFA-alpha1 during both conditions. Under HC HR (40–70%), La (10–90%), and RPE (50–90%) were significantly-higher, SpO2 (10–100%), meanRR (40–70%), and DFA-alpha1 (20–60%) were significantly-lower than under NC. Conclusion: Under both conditions, prolonged exercise until voluntary exhaustion provokes a lower total variability combined with a reduction in the amplitude and correlation properties of RR fluctuations which may be attributed to increased organismic demands. Additionally, HC provoked higher demands and loss of correlation properties at an earlier stage during the exercise regime, implying an accelerated alteration of cardiac autonomic regulation. KW - autonomic nervous system KW - heart rate variability KW - detrended fluctuation analysis KW - endurance exercise KW - voluntary exhaustion KW - hypoxia Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369199 VL - 10 ER - TY - JOUR A1 - Fahmy-Garcia, Shorouk A1 - Farrell, Eric A1 - Witte-Bouma, Janneke A1 - Robbesom-van den Berge, Iris A1 - Suarez, Melva A1 - Mumcuoglu, Didem A1 - Walles, Heike A1 - Kluijtmans, Sebastiaan G. J. M. A1 - van der Eerden, Bram C. J. A1 - van Osch, Gerjo J. V. M. A1 - van Leeuwen, Johannes P. T. M. A1 - van Driel, Marjolein T1 - Follistatin Effects in Migration, Vascularization, and Osteogenesis in vitro and Bone Repair in vivo JF - Frontiers in Bioengineering and Biotechnology N2 - The use of biomaterials and signaling molecules to induce bone formation is a promising approach in the field of bone tissue engineering. Follistatin (FST) is a glycoprotein able to bind irreversibly to activin A, a protein that has been reported to inhibit bone formation. We investigated the effect of FST in critical processes for bone repair, such as cell recruitment, osteogenesis and vascularization, and ultimately its use for bone tissue engineering. In vitro, FST promoted mesenchymal stem cell (MSC) and endothelial cell (EC) migration as well as essential steps in the formation and expansion of the vasculature such as EC tube-formation and sprouting. FST did not enhance osteogenic differentiation of MSCs, but increased committed osteoblast mineralization. In vivo, FST was loaded in an in situ gelling formulation made by alginate and recombinant collagen-based peptide microspheres and implanted in a rat calvarial defect model. Two FST variants (FST288 and FST315) with major differences in their affinity to cell-surface proteoglycans, which may influence their effect upon in vivo bone repair, were tested. In vitro, most of the loaded FST315 was released over 4 weeks, contrary to FST288, which was mostly retained in the biomaterial. However, none of the FST variants improved in vivo bone healing compared to control. These results demonstrate that FST enhances crucial processes needed for bone repair. Further studies need to investigate the optimal FST carrier for bone regeneration. KW - follistatin 315 (FST315) KW - follistatin 288 (FST288) KW - migration KW - vascularization KW - osteogenesis KW - injectable in situ gelling slow release system KW - bone tissue engineering KW - regenerative medicine Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227617 VL - 7 ER - TY - JOUR A1 - Gál, Bernadett I. A1 - Kilencz, Tünde A1 - Albert, Anita A1 - Demeter, Ildikó A1 - Hegedűs, Klára Mária A1 - Janka, Zoltán A1 - Csifcsák, Gábor A1 - Álmos, Péter Z. T1 - Mild Effect of Nalmefene on Alcoholic Cue-Induced Response Invigoration in Alcohol Use Disorder Without Accompanying Changes in Electrophysiological Signatures of Early Visual Processing and Executive Control JF - Frontiers in Pharmacology N2 - Nalmefene is approved for as-needed pharmacological treatment in alcohol use disorder (AUD) by the European Medicines Agency. While the cellular effects of nalmefene have been thoroughly investigated, data are very limited on how this agent influences neural signals associated with inhibitory control and the visual analysis of environmental cues. This double-blind crossover study assessed the behavioral and neural effects of acute nalmefene administration in patients diagnosed with AUD. In experiment 1, we validated our experimental paradigm (electroencephalography combined with a modified Go/NoGo task using images of alcoholic and nonalcoholic drinks as prime stimuli) in 20 healthy adults to ensure that our protocol is suitable for assessing the behavioral and neural aspects of executive control. In experiment 2, we recruited 19 patients with AUD, and in a double-blind crossover design, we investigated the effects of nalmefene versus placebo on task performance (response accuracy, the sensitivity index, and reaction times), visual responses to appetitive cues (occipital P1, N1, and P2 components), and electrophysiological markers of conflict detection and response inhibition (frontal N2 and P3 waveforms). Under placebo, patients produced faster reaction times to alcohol-primed Go stimuli, an effect that was weak despite being statistically significant. However, the effect of alcoholic cues on the speed of response initiation disappeared after receiving nalmefene. We found no placebo versus nalmefene difference regarding our patients’ ability to accurately inhibit responses to NoGo stimuli or for occipital and frontal event-related potentials. Our results suggest that nalmefene might be potent in reducing the vigor to act upon alcoholic cues in AUD patients, but this effect is most probably mediated via subcortical (rather than cortical) neural circuits. KW - nalmefene KW - alcohol use disorder KW - response inhibition KW - incentive salience KW - event-related potentials KW - Go/NoGo task Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369182 VL - 10 ER - TY - JOUR A1 - Franke, Maximilian A1 - Conzelmann, Annette A1 - Grünblatt, Edna A1 - Werling, Anna M. A1 - Spieles, Helen A1 - Wewetzer, Christoph A1 - Warnke, Andreas A1 - Romanos, Marcel A1 - Walitza, Susanne A1 - Renner, Tobias J. T1 - No Association of Variants of the NPY-System With Obsessive-Compulsive Disorder in Children and Adolescents JF - Frontiers in Molecular Neuroscience N2 - Obsessive-compulsive disorder (OCD) causes severe distress and is therefore counted by the World Health Organisation (WHO) as one of the 10 most impairing illnesses. There is evidence for a strong genetic underpinning especially in early onset OCD (eoOCD). Though several genes involved in neurotransmission have been reported as candidates, there is still a need to identify new pathways. In this study, we focussed on genetic variants of the Neuropeptide Y (NPY) system. NPY is one of the most abundant neuropeptides in the human brain with emerging evidence of capacity to modulate stress response, which is of high relevance in OCD. We focussed on tag-SNPs of NPY and its receptor gene NPY1R in a family-based approach. The sample comprised 86 patients (children and adolescents) with eoOCD with both their biological parents. However, this first study on genetic variants of the NPY-system could not confirm the association between the investigated SNPs and eoOCD. Based on the small sample size results have to be interpreted as preliminary and should be replicated in larger samples. However, also in an additional GWAS analysis in a large sample, we could not observe an associations between NPY and OCD. Overall, these preliminary results point to a minor role of NPY on the stress response of OCD. KW - NPY KW - obsessive-compulsive KW - children KW - anxiety KW - neuropeptide Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229051 VL - 12 ER - TY - JOUR A1 - Geran, Rohat A1 - Uecker, Florian C. A1 - Prüss, Harald A1 - Haeusler, Karl Georg A1 - Paul, Friedemann A1 - Ruprecht, Klemens A1 - Harms, Lutz A1 - Schmidt, Felix A. T1 - Olfactory and Gustatory Dysfunction in Patients With Autoimmune Encephalitis JF - Frontiers in Neurology N2 - Objective: To test the hypothesis that olfactory (OF) and gustatory function (GF) is disturbed in patients with autoimmune encephalitides (AE). Methods: The orthonasal OF was tested in 32 patients with AE and 32 age- and sex-matched healthy controls (HC) with the standardized Threshold Discrimination Identification (TDI) score. This validated olfactory testing method yields individual scores for olfactory threshold (T), odor discrimination (D), and identification (I), along with a composite TDI score. The GF was determined by the Taste Strip Test (TST). Results: Overall, 24/32 (75%) of patients with AE, but none of 32 HC (p < 0.001) had olfactory dysfunction in TDI testing. The results of the threshold, discrimination and identification subtests were significantly reduced in patients with AE compared to HC (all p < 0.001). Assessed by TST, 5/19 (26.3%) of patients with AE, but none of 19 HC presented a significant limitation in GF (p < 0.001). The TDI score was correlated with the subjective estimation of the olfactory capacity on a visual analog scale (VAS; rs = 0.475, p = 0.008). Neither age, sex, modified Rankin Scale nor disease duration were associated with the composite TDI score. Conclusions: This is the first study investigating OF and GF in AE patients. According to unblinded assessment, patients with AE have a reduced olfactory and gustatory capacity compared to HC, suggesting that olfactory and gustatory dysfunction are hitherto unrecognized symptoms in AE. Further studies with larger number of AE patients would be of interest to verify our results. KW - autoimmune encephalitis KW - olfactory dysfunction KW - gustatory dysfunction KW - olfactory testing KW - threshold discrimination identification test Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232921 VL - 10 ER - TY - JOUR A1 - Jarick, Katja J. A1 - Mokhtari, Zeinab A1 - Scheller, Lukas A1 - Hartweg, Julia A1 - Thusek, Sina A1 - Le, Duc-Dung A1 - Ranecky, Maria A1 - Shaikh, Haroon A1 - Qureischi, Musga A1 - Heinze, Katrin G. A1 - Beilhack, Andreas T1 - Photoconversion of Alloreactive T Cells in Murine Peyer’s Patches During Acute Graft-Versus-Host Disease: Tracking the Homing Route of Highly Proliferative Cells In Vivo JF - Frontiers in Immunology N2 - The regulation of immune cell migration throughout the body is essential to warrant immunosurveillance and to maintain immune homeostasis. Marking and tracking of these cells has proven important to study mechanisms of immune cell trafficking and cell interaction in vivo. Photoconversion is a well-suited technique for intravital application because it enables contactless time- and location-specific marking of cells in the tissue without surgically manipulating the microenvironment of the cells in question. However, in dividing cells the converted fluorescent protein may decline quickly. Here, we provide a detailed description of the photoconversion technique and its applicability to tracking highly proliferating T cells from the priming site of T cell activation to peripheral target organs of effector function in a preclinical model. Dendra2+ T cells were photoconverted in the Peyer’s patches during the initiation phase of acute graft-versus-host disease (GvHD) and tracked through the mesenteric lymph nodes and the peripheral blood to the small intestine with flow cytometry and intravital two-photon microscopy. Photoconverted alloreactive T cells preserved the full proliferative capacity, homing, and migration of alloreactive T cells in the intestinal lamina propria. We conclusively proved that photoconversion of highly proliferative alloreactive T cells in the Peyer’s patches is an effective tool to study trafficking of alloreactive T cells under physiologic conditions and to GvHD target tissues. This technique can also be applied to the study of immune cell tracking under inflammatory and non-inflammatory conditions. KW - T cell migration KW - acute graft-versus-host disease KW - mouse models KW - photoconversion KW - Dendra2 KW - Peyer's patch KW - in vivo cell tracking KW - lymphocyte homing Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323309 VL - 9 ER - TY - JOUR A1 - Herster, Franziska A1 - Bittner, Zsofia A1 - Codrea, Marius Cosmin A1 - Archer, Nathan K. A1 - Heister, Martin A1 - Löffler, Markus W. A1 - Heumos, Simon A1 - Wegner, Joanna A1 - Businger, Ramona A1 - Schindler, Michael A1 - Stegner, David A1 - Schäkel, Knut A1 - Grabbe, Stephan A1 - Ghoreschi, Kamran A1 - Miller, Lloyd S. A1 - Weber, Alexander N. R. T1 - Platelets Aggregate With Neutrophils and Promote Skin Pathology in Psoriasis JF - Frontiers in Immunology N2 - Psoriasis is a frequent systemic inflammatory autoimmune disease characterized primarily by skin lesions with massive infiltration of leukocytes, but frequently also presents with cardiovascular comorbidities. Especially polymorphonuclear neutrophils (PMNs) abundantly infiltrate psoriatic skin but the cues that prompt PMNs to home to the skin are not well-defined. To identify PMN surface receptors that may explain PMN skin homing in psoriasis patients, we screened 332 surface antigens on primary human blood PMNs from healthy donors and psoriasis patients. We identified platelet surface antigens as a defining feature of psoriasis PMNs, due to a significantly increased aggregation of neutrophils and platelets in the blood of psoriasis patients. Similarly, in the imiquimod-induced experimental in vivo mouse model of psoriasis, disease induction promoted PMN-platelet aggregate formation. In psoriasis patients, disease incidence directly correlated with blood platelet counts and platelets were detected in direct contact with PMNs in psoriatic but not healthy skin. Importantly, depletion of circulating platelets in mice in vivo ameliorated disease severity significantly, indicating that both PMNs and platelets may be relevant for psoriasis pathology and disease severity. KW - psoriasis KW - neutrophil KW - platelet KW - platelet-neutrophil complexes (PNCs) KW - imiquimod Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-320175 VL - 10 ER - TY - JOUR A1 - Kasaragod, Vikram B. A1 - Schindelin, Hermann T1 - Structure–Function Relationships of Glycine and GABAA Receptors and Their Interplay With the Scaffolding Protein Gephyrin JF - Frontiers in Molecular Neuroscience N2 - Glycine and γ-aminobutyric acid (GABA) are the major determinants of inhibition in the central nervous system (CNS). These neurotransmitters target glycine and GABAA receptors, respectively, which both belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGICs). Interactions of the neurotransmitters with the cognate receptors result in receptor opening and a subsequent influx of chloride ions, which, in turn, leads to hyperpolarization of the membrane potential, thus counteracting excitatory stimuli. The majority of glycine receptors and a significant fraction of GABAA receptors (GABAARs) are recruited and anchored to the post-synaptic membrane by the central scaffolding protein gephyrin. This ∼93 kDa moonlighting protein is structurally organized into an N-terminal G-domain (GephG) connected to a C-terminal E-domain (GephE) via a long unstructured linker. Both inhibitory neurotransmitter receptors interact via a short peptide motif located in the large cytoplasmic loop located in between transmembrane helices 3 and 4 (TM3-TM4) of the receptors with a universal receptor-binding epitope residing in GephE. Gephyrin engages in nearly identical interactions with the receptors at the N-terminal end of the peptide motif, and receptor-specific interaction toward the C-terminal region of the peptide. In addition to its receptor-anchoring function, gephyrin also interacts with a rather large collection of macromolecules including different cytoskeletal elements, thus acting as central scaffold at inhibitory post-synaptic specializations. Dysfunctions in receptor-mediated or gephyrin-mediated neurotransmission have been identified in various severe neurodevelopmental disorders. Although biochemical, cellular and electrophysiological studies have helped to understand the physiological and pharmacological roles of the receptors, recent high resolution structures of the receptors have strengthened our understanding of the receptors and their gating mechanisms. Besides that, multiple crystal structures of GephE in complex with receptor-derived peptides have shed light into receptor clustering by gephyrin at inhibitory post-synapses. This review will highlight recent biochemical and structural insights into gephyrin and the GlyRs as well as GABAA receptors, which provide a deeper understanding of the molecular machinery mediating inhibitory neurotransmission. KW - glycine receptors KW - GABAA receptors KW - gephyrin KW - moonlighting protein KW - inhibitory post-synaptic specialization KW - cytoskeletal proteins Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325607 VL - 11 ER - TY - JOUR A1 - Kervarrec, Thibault A1 - Samimi, Mahtab A1 - Guyétant, Serge A1 - Sarma, Bhavishya A1 - Chéret, Jérémy A1 - Blanchard, Emmanuelle A1 - Berthon, Patricia A1 - Schrama, David A1 - Houben, Roland A1 - Touzé, Antoine T1 - Histogenesis of Merkel Cell Carcinoma: A Comprehensive Review JF - Frontiers in Oncology N2 - Merkel cell carcinoma (MCC) is a primary neuroendocrine carcinoma of the skin. This neoplasia features aggressive behavior, resulting in a 5-year overall survival rate of 40%. In 2008, Feng et al. identified Merkel cell polyomavirus (MCPyV) integration into the host genome as the main event leading to MCC oncogenesis. However, despite identification of this crucial viral oncogenic trigger, the nature of the cell in which MCC oncogenesis occurs is actually unknown. In fact, several hypotheses have been proposed. Despite the large similarity in phenotype features between MCC tumor cells and physiological Merkel cells (MCs), a specialized subpopulation of the epidermis acting as mechanoreceptor of the skin, several points argue against the hypothesis that MCC derives directly from MCs. Alternatively, MCPyV integration could occur in another cell type and induce acquisition of an MC-like phenotype. Accordingly, an epithelial as well as a fibroblastic or B-cell origin of MCC has been proposed mainly based on phenotype similarities shared by MCC and these potential ancestries. The aim of this present review is to provide a comprehensive review of the current knowledge of the histogenesis of MCC. KW - merkel cell polyomavirus (MCPyV) KW - epithelial KW - fibroblast KW - B cell KW - Merkel cell carcinoma (MCC) KW - histogenesis KW - origin Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325733 VL - 9 ER - TY - JOUR A1 - Nagy, Magdolna A1 - van Geffen, Johanna P. A1 - Stegner, David A1 - Adams, David J. A1 - Braun, Attila A1 - de Witt, Susanne M. A1 - Elvers, Margitta A1 - Geer, Mitchell J. A1 - Kuijpers, Marijke J. E. A1 - Kunzelmann, Karl A1 - Mori, Jun A1 - Oury, Cécile A1 - Pircher, Joachim A1 - Pleines, Irina A1 - Poole, Alastair W. A1 - Senis, Yotis A. A1 - Verdoold, Remco A1 - Weber, Christian A1 - Nieswandt, Bernhard A1 - Heemskerk, Johan W. M. A1 - Baaten, Constance C. F. M. J. T1 - Comparative Analysis of Microfluidics Thrombus Formation in Multiple Genetically Modified Mice: Link to Thrombosis and Hemostasis JF - Frontiers in Cardiovascular Medicine N2 - Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin α6β1 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis. KW - arterial thrombus formation KW - bleeding KW - collagen KW - glycoprotein VI KW - platelets KW - microfluidics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232194 VL - 6 ER - TY - JOUR A1 - Vural, Atay A1 - Doppler, Kathrin A1 - Meinl, Edgar T1 - Autoantibodies Against the Node of Ranvier in Seropositive Chronic Inflammatory Demyelinating Polyneuropathy: Diagnostic, Pathogenic, and Therapeutic Relevance JF - Frontiers in Immunology N2 - Discovery of disease-associated autoantibodies has transformed the clinical management of a variety of neurological disorders. Detection of autoantibodies aids diagnosis and allows patient stratification resulting in treatment optimization. In the last years, a set of autoantibodies against proteins located at the node of Ranvier has been identified in patients with chronic inflammatory demyelinating polyneuropathy (CIDP). These antibodies target neurofascin, contactin1, or contactin-associated protein 1, and we propose to name CIDP patients with these antibodies collectively as seropositive. They have unique clinical characteristics that differ from seronegative CIDP. Moreover, there is compelling evidence that autoantibodies are relevant for the pathogenesis. In this article, we review the current knowledge on the characteristics of autoantibodies against the node of Ranvier proteins and their clinical relevance in CIDP. We start with a description of the structure of the node of Ranvier followed by a summary of assays used to identify seropositive patients; and then, we describe clinical features and characteristics linked to seropositivity. We review knowledge on the role of these autoantibodies for the pathogenesis with relevance for the emerging concept of nodopathy/paranodopathy and summarize the treatment implications. KW - autoantibody KW - seropositive KW - chronic inflammatory demyelinating polyneuropathy KW - node of Ranvier KW - paranode KW - neurofascin KW - contactin KW - contactin-associated protein 1 Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233279 VL - 9 ER - TY - JOUR A1 - Siebert, Claudia A1 - Ciato, Denis A1 - Murakami, Masanori A1 - Frei-Stuber, Ludwig A1 - Perez-Rivas, Luis Gustavo A1 - Monteserin-Garcia, José Luis A1 - Nölting, Svenja A1 - Maurer, Julian A1 - Feuchtinger, Annette A1 - Walch, Axel K. A1 - Haak, Harm R. A1 - Bertherat, Jérôme A1 - Mannelli, Massimo A1 - Fassnacht, Martin A1 - Korpershoek, Esther A1 - Reincke, Martin A1 - Stalla, Günter K. A1 - Hantel, Constanze A1 - Beuschlein, Felix T1 - Heat Shock Protein 90 as a Prognostic Marker and Therapeutic Target for Adrenocortical Carcinoma JF - Frontiers in Endocrinology N2 - Background: Adrenocortical carcinoma (ACC) is a rare tumor entity with restricted therapeutic opportunities. HSP90 (Heat Shock Protein 90) chaperone activity is fundamental for cell survival and contributes to different oncogenic signaling pathways. Indeed, agents targeting HSP90 function have shown therapeutic efficacy in several cancer types. We have examined the expression of HSP90 in different adrenal tumors and evaluated the use of HSP90 inhibitors in vitro as possible therapy for ACC. Methods: Immunohistochemical expression of HSP90 isoforms was investigated in different adrenocortical tumors and associated with clinical features. Additionally, a panel of N-terminal (17-allylamino-17-demethoxygeldanamycin (17-AAG), luminespib, and ganetespib) and C-terminal (novobiocin and silibinin) HSP90 inhibitors were tested on various ACC cell lines. Results: Within adrenocortical tumors, ACC samples exhibited the highest expression of HSP90β. Within a cohort of ACC patients, HSP90β expression levels were inversely correlated with recurrence-free and overall survival. In functional assays, among five different compounds tested luminespib and ganetespib induced a significant decrease in cell viability in single as well as in combined treatments with compounds of the clinically used EDP-M scheme (etoposide, doxorubicin, cisplatin, mitotane). Inhibition of cell viability correlated furthermore with a decrease in proliferation, in cell migration and an increase in apoptosis. Moreover, analysis of cancer pathways indicated a modulation of the ERK1/2—and AKT—pathways by luminespib and ganetespib treatment. Conclusions: Our findings emphasize HSP90 as a marker with prognostic impact and promising target with N-terminal HSP90 inhibitors as drugs with potential therapeutic efficacy toward ACC. KW - adrenal gland KW - cortisol KW - N-terminal HSP90 inhibitors KW - C-terminal HSP90 inhibitors KW - prognostic marker Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-238029 VL - 10 ER - TY - JOUR A1 - Nguyen, Minh-Thu A1 - Saising, Jongkon A1 - Tribelli, Paula Maria A1 - Nega, Mulugeta A1 - Diene, Seydina M. A1 - François, Patrice A1 - Schrenzel, Jacques A1 - Spröer, Cathrin A1 - Bunk, Boyke A1 - Ebner, Patrick A1 - Hertlein, Tobias A1 - Kumari, Nimerta A1 - Härtner, Thomas A1 - Wistuba, Dorothee A1 - Voravuthikunchai, Supayang P. A1 - Mäder, Ulrike A1 - Ohlsen, Knut A1 - Götz, Friedrich T1 - Inactivation of farR Causes High Rhodomyrtone Resistance and Increased Pathogenicity in Staphylococcus aureus JF - Frontiers in Microbiology N2 - Rhodomyrtone (Rom) is an acylphloroglucinol antibiotic originally isolated from leaves of Rhodomyrtus tomentosa. Rom targets the bacterial membrane and is active against a wide range of Gram-positive bacteria but the exact mode of action remains obscure. Here we isolated and characterized a spontaneous Rom-resistant mutant from the model strain Staphylococcus aureus HG001 (RomR) to learn more about the resistance mechanism. We showed that Rom-resistance is based on a single point mutation in the coding region of farR [regulator of fatty acid (FA) resistance] that causes an amino acid change from Cys to Arg at position 116 in FarR, that affects FarR activity. Comparative transcriptome analysis revealed that mutated farR affects transcription of many genes in distinct pathways. FarR represses for example the expression of its own gene (farR), its flanking gene farE (effector of FA resistance), and other global regulators such as agr and sarA. All these genes were consequently upregulated in the RomR clone. Particularly the upregulation of agr and sarA leads to increased expression of virulence genes rendering the RomR clone more cytotoxic and more pathogenic in a mouse infection model. The Rom-resistance is largely due to the de-repression of farE. FarE is described as an efflux pump for linoleic and arachidonic acids. We observed an increased release of lipids in the RomR clone compared to its parental strain HG001. If farE is deleted in the RomR clone, or, if native farR is expressed in the RomR strain, the corresponding strains become hypersensitive to Rom. Overall, we show here that the high Rom-resistance is mediated by overexpression of farE in the RomR clone, that FarR is an important regulator, and that the point mutation in farR (RomR clone) makes the clone hyper-virulent. KW - antibiotic KW - Gram-positive bacteria KW - rhodomyrtone KW - Staphylococcus KW - membrane active Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224117 VL - 10 ER - TY - JOUR A1 - John, Cathy N. A1 - Abrantes, Pedro M. D. S. A1 - Prusty, Bhupesh K. A1 - Ablashi, Dharam V. A1 - Africa, Charlene W. J. T1 - K21 Compound, a Potent Antifungal Agent: Implications for the Treatment of Fluconazole-Resistant HIV-Associated Candida Species JF - Frontiers in Microbiology N2 - Background/Objectives: With mucocutaneous candidiasis being highly prevalent in HIV patients, the emergence of fluconazole-resistant Candida species forms a major challenge in treating and eradicating these infections. The objective of this study was to establish the antifungal activity of K21, a membrane-rupturing antimicrobial compound derived from a silica quaternary ammonium compound (SiQAC) with tetraethoxysilane (TEOS). Methods: The study sample included 81 Candida species of which 9 were type strains and 72 were clinical isolates. Minimum inhibitory concentrations, synergy, fractional inhibitory concentration index (FICI), and time kill assays were determined by broth microdilution. Electron microscopy (EM) was used to determine the qualitative changes brought about after treatment with K21. Results: K21 inhibited the growth of all fluconazole-resistant and susceptible Candida strains with only 2 h of exposure required to effectively kill 99.9% of the inoculum, and a definite synergistic effect was observed with a combination of K21 and fluconazole. EM demonstrated the presence of two forms of extracellular vesicles indicative of biofilm formation and cell lysis. Conclusion: The study established the efficacy of K21 as an antifungal agent and with fluconazole-resistant candidiasis on the increase, the development of K21 can provide a promising alternative to combat acquired drug resistance. KW - HIV-associated candidiasis KW - antimicrobial compounds KW - Candida KW - fluconazole KW - antifungal susceptibility KW - broth microdilution Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323505 VL - 10 ER - TY - JOUR A1 - Prusty, Bhupesh K. A1 - Gulve, Nitish A1 - Govind, Sheila A1 - Krueger, Gerhard R. F. A1 - Feichtinger, Julia A1 - Larcombe, Lee A1 - Aspinall, Richard A1 - Ablashi, Dharam V. A1 - Toro, Carla T. T1 - Active HHV-6 Infection of Cerebellar Purkinje Cells in Mood Disorders JF - Frontiers in Microbiology N2 - Early-life infections and associated neuroinflammation is incriminated in the pathogenesis of various mood disorders. Infection with human roseoloviruses, HHV-6A and HHV-6B, allows viral latency in the central nervous system and other tissues, which can later be activated causing cognitive and behavioral disturbances. Hence, this study was designed to evaluate possible association of HHV-6A and HHV-6B activation with three different groups of psychiatric patients. DNA qPCR, immunofluorescence and FISH studies were carried out in post-mortem posterior cerebellum from 50 cases each of bipolar disorder (BPD), schizophrenia, 15 major depressive disorder (MDD) and 50 appropriate control samples obtained from two well-known brain collections (Stanley Medical Research Institute). HHV-6A and HHV-6B late proteins (indicating active infection) and viral DNA were detected more frequently (p < 0.001 for each virus) in human cerebellum in MDD and BPD relative to controls. These roseolovirus proteins and DNA were found less frequently in schizophrenia cases. Active HHV-6A and HHV-6B infection in cerebellar Purkinje cells were detected frequently in BPD and MDD cases. Furthermore, we found a significant association of HHV-6A infection with reduced Purkinje cell size, suggesting virus-mediated abnormal Purkinje cell function in these disorders. Finally, gene expression analysis of cerebellar tissue revealed changes in pathways reflecting an inflammatory response possibly to HHV-6A infection. Our results provide molecular evidence to support a role for active HHV-6A and HHV-6B infection in BPD and MDD. KW - HHV-6 KW - bipolar disorder KW - schizophrenia KW - major depressive disorder KW - Purkinje cells Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-369222 VL - 9 ER - TY - JOUR A1 - Ticha, Olga A1 - Moos, Lukas A1 - Wajant, Harald A1 - Bekeredjian-Ding, Isabelle T1 - Expression of Tumor Necrosis Factor Receptor 2 Characterizes TLR9-Driven Formation of Interleukin-10-Producing B Cells JF - Frontiers in Immunology N2 - B cell-derived interleukin-10 (IL-10) production has been described as a hallmark for regulatory function in B lymphocytes. However, there is an ongoing debate on the origin of IL-10-secreting B cells and lack of specific surface markers has turned into an important obstacle for studying human B regulatory cells. In this study, we propose that tumor necrosis factor receptor 2 (TNFR2) expression can be used for enrichment of IL-10-secreting B cells. Our data confirm that IL-10 production can be induced by TLR9 stimulation with CpG ODN and that IL-10 secretion accompanies differentiation of peripheral blood B cells into plasma blasts. We further show that CpG ODN stimulation induces TNFR2 expression, which correlates with IL-10 secretion and terminal differentiation. Indeed, flow cytometric sorting of TNFR2+ B cells revealed that TNFR2+ and TNFR2− fractions correspond to IL-10+ and IL-10− fractions, respectively. Furthermore, CpG-induced TNFR2+ B cells were predominantly found in the IgM+ CD27+ B cell subset and spontaneously released immunoglobulin. Finally, our data corroborate the functional impact of TNFR2 by demonstrating that stimulation with a TNFR2 agonist significantly augments IL-10 and IL-6 production in B cells. Altogether, our data highlight a new role for TNFR2 in IL-10-secreting human B lymphocytes along with the potential to exploit this finding for sorting and isolation of this currently ill-defined B cell subset. KW - human KW - B cells KW - interleukin-10 KW - tumor necrosis factor receptor 2 KW - TLR 9 KW - Breg Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-241323 VL - 8 ER - TY - JOUR A1 - Klotz, Peter A1 - Higgins, Paul G. A1 - Schaubmar, Andreas R. A1 - Failing, Klaus A1 - Leidner, Ursula A1 - Seifert, Harald A1 - Scheufen, Sandra A1 - Semmler, Torsten A1 - Ewers, Christa T1 - Seasonal Occurrence and Carbapenem Susceptibility of Bovine Acinetobacter baumannii in Germany JF - Frontiers in Microbiology N2 - Acinetobacter baumannii is one of the leading causes of nosocomial infections in humans. To investigate its prevalence, distribution of sequence types (STs), and antimicrobial resistance in cattle, we sampled 422 cattle, including 280 dairy cows, 59 beef cattle, and 83 calves over a 14-month period. Metadata, such as the previous use of antimicrobial agents and feeding, were collected to identify putative determining factors. Bacterial isolates were identified via MALDI-TOF/MS and PCR, antimicrobial susceptibility was evaluated via VITEK2 and antibiotic gradient tests, resistance genes were identified by PCR. Overall, 15.6% of the cattle harbored A. baumannii, predominantly in the nose (60.3% of the A. baumannii isolates). It was more frequent in dairy cows (21.1%) than in beef cattle (6.8%) and calves (2.4%). A seasonal occurrence was shown with a peak between May and August. The rate of occurrence of A. baumannii was correlated with a history of use of 3rd generation cephalosporins in the last 6 months prior to sampling Multilocus sequence typing (Pasteur scheme) revealed 83 STs among 126 unique isolates. Nine of the bovine STs have previously been implicated in human infections. Besides known intrinsic resistance of the species, the isolates did not show additional resistance to the antimicrobial substances tested, including carbapenems. Our data suggest that cattle are not a reservoir for nosocomial A. baumannii but carry a highly diverse population of this species. Nevertheless, some STs seem to be able to colonize both cattle and humans. KW - ESKAPE KW - Acinetobacter baumannii KW - antimicrobial susceptibility KW - MLST KW - cattle KW - epidemiology Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325927 VL - 10 ER - TY - JOUR A1 - Schroeter, Matthias L. A1 - Pawelke, Sarah A1 - Bisenius, Sandrine A1 - Kynast, Jana A1 - Schuemberg, Katharina A1 - Polyakova, Maryna A1 - Anderl-Straub, Sarah A1 - Danek, Adrian A1 - Fassbender, Klaus A1 - Jahn, Holger A1 - Jessen, Frank A1 - Kornhuber, Johannes A1 - Lauer, Martin A1 - Prudlo, Johannes A1 - Schneider, Anja A1 - Uttner, Ingo A1 - Thöne-Otto, Angelika A1 - Otto, Markus A1 - Diehl-Schmid, Janine T1 - A Modified Reading the Mind in the Eyes Test Predicts Behavioral Variant Frontotemporal Dementia Better Than Executive Function Tests JF - Frontiers in Aging Neuroscience N2 - Behavioral variant frontotemporal dementia (bvFTD) is characterized by deep alterations in behavior and personality. Although revised diagnostic criteria agree for executive dysfunction as most characteristic, impairments in social cognition are also suggested. The study aimed at identifying those neuropsychological and behavioral parameters best discriminating between bvFTD and healthy controls. Eighty six patients were diagnosed with possible or probable bvFTD according to Rascovsky et al. (2011) and compared with 43 healthy age-matched controls. Neuropsychological performance was assessed with a modified Reading the Mind in the Eyes Test (RMET), Stroop task, Trail Making Test (TMT), Hamasch-Five-Point Test (H5PT), and semantic and phonemic verbal fluency tasks. Behavior was assessed with the Apathy Evaluation Scale, Frontal Systems Behavioral Scale, and Bayer Activities of Daily Living Scale. Each test’s discriminatory power was investigated by Receiver Operating Characteristic curves calculating the area under the curve (AUC). bvFTD patients performed significantly worse than healthy controls in all neuropsychological tests. Discriminatory power (AUC) was highest in behavioral questionnaires, high in verbal fluency tasks and the RMET, and lower in executive function tests such as the Stroop task, TMT and H5PT. As fluency tasks depend on several cognitive functions, not only executive functions, results suggest that the RMET discriminated better between bvFTD and control subjects than other executive tests. Social cognition should be incorporated into diagnostic criteria for bvFTD in the future, such as in the International Classification of Diseases (ICD)-11, as already suggested in the Diagnostic and Statistical Manual for Mental Disorders (DSM)-5. KW - behavioral variant frontotemporal dementia KW - diagnostic criteria KW - executive function KW - social cognition KW - theory of mind Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234254 VL - 10 ER - TY - JOUR A1 - Lang, Isabell A1 - Füllsack, Simone A1 - Wajant, Harald T1 - Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies JF - Frontiers in Immunology N2 - Progranulin (PGRN) is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1) and TNFR2 and inhibition of tumor necrosis factor (TNF)-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources. However, even under consideration of these speculations, there is still a striking discrepancy in the literature between the concentrations of PGRN needed to inhibit TNF signaling and the concentrations required to block TNF binding to TNFR1 and TNFR2. While signaling events induced by 0.2–2 nM of TNF have been efficiently inhibited by low, near to equimolar concentrations (0.5–2.5 nM) of PGRN in various studies, the reported inhibitory effects of PGRN on TNF-binding to TNFR1/2 required a huge excess of PGRN (100–1,000-fold). Therefore, we investigated the effect of PGRN on TNF binding to TNFR1 and TNFR2 in highly sensitive cellular binding studies. Unlabeled TNF inhibited >95% of the specific binding of a Gaussia princeps luciferase (GpL) fusion protein of TNF to TNFR1 and TNFR2 and blocked binding of soluble GpL fusion proteins of TNFR1 and TNFR2 to membrane TNF expressing cells to >95%, too. Purified PGRN, however, showed in both assays no effect on TNF–TNFR1/2 interaction even when applied in huge excess. To rule out that tags and purification- or storage-related effects compromise the potential ability of PGRN to bind TNF receptors, we directly co-expressed PGRN, and as control TNF, in TNFR1- and TNFR2-expressing cells and looked for binding of GpL-TNF. While expression of TNF strongly inhibited binding of GpL-TNF to TNFR1/2, co-expression of PGRN had not effect on the ability of the TNFR1/2-expressing cells to bind TNF. KW - binding studies KW - Gaussia princeps luciferase fusion protein KW - progranulin KW - tumor necrosis factor KW - tumor necrosis factor receptor-1 KW - tumor necrosis factor receptor-2 Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236373 VL - 9 ER - TY - JOUR A1 - Weiss, Esther A1 - Ziegler, Sabrina A1 - Fliesser, Mirjam A1 - Schmitt, Anna-Lena A1 - Hünniger, Kerstin A1 - Kurzai, Oliver A1 - Morton, Charles-Oliver A1 - Einsele, Hermann A1 - Loeffler, Juergen T1 - First Insights in NK—DC Cross-Talk and the Importance of Soluble Factors During Infection With Aspergillus fumigatus JF - Frontiers in Cellular and Infection Microbiology N2 - Invasive aspergillosis (IA) is an infectious disease caused by the fungal pathogen Aspergillus fumigatus that mainly affects immunocompromised hosts. To investigate immune cell cross-talk during infection with A. fumigatus, we co-cultured natural killer (NK) cells and dendritic cells (DC) after stimulation with whole fungal structures, components of the fungal cell wall, fungal lysate or ligands for distinct fungal receptors. Both cell types showed activation after stimulation with fungal components and were able to transfer activation signals to the counterpart not stimulated cell type. Interestingly, DCs recognized a broader spectrum of fungal components and thereby initiated NK cell activation when those did not recognize fungal structures. These experiments highlighted the supportive function of DCs in NK cell activation. Furthermore, we focused on soluble DC mediated NK cell activation and showed that DCs stimulated with the TLR2/Dectin-1 ligand zymosan could maximally stimulate the expression of CD69 on NK cells. Thus, we investigated the influence of both receptors for zymosan, Dectin-1 and TLR2, which are highly expressed on DCs but show only minimal expression on NK cells. Specific focus was laid on the question whether Dectin-1 or TLR2 signaling in DCs is important for the secretion of soluble factors leading to NK cell activation. Our results show that Dectin-1 and TLR2 are negligible for NK cell activation. We conclude that besides Dectin-1 and TLR2 other receptors on DCs are able to compensate for the missing signal. KW - natural killer cells KW - dendritic cells KW - NK-DC cross-talk KW - Aspergillus fumigatus KW - soluble factors KW - innate immunity Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233565 VL - 8 ER - TY - JOUR A1 - Schurig, Johannes A1 - Haeusler, Karl Georg A1 - Grittner, Ulrike A1 - Nolte, Christian H. A1 - Fiebach, Jochen B. A1 - Audebert, Heinrich J. A1 - Endres, Matthias A1 - Rocco, Andrea T1 - Frequency of Hemorrhage on Follow Up Imaging in Stroke Patients Treated With rt-PA Depending on Clinical Course JF - Frontiers in Neurology N2 - Background: According to current guidelines, stroke patients treated with rt-PA should undergo brain imaging to exclude intracerebral bleeding 24 h after thrombolysis, before the start of medical secondary prevention. However, the usefulness of routine follow-up imaging with regard to changes in therapeutic management in patients without neurological deterioration is unclear. We hypothesized that follow up brain imaging solely to exclude bleeding in patients who clinically improved after rt-PA application may not be necessary. Methods: Retrospective single-center analysis including stroke patients treated with rt-PA. Records were reviewed for hemorrhagic transformation one day after systemic thrombolysis and brain imaging-based changes in therapeutic management. Twenty-four hour after thrombolysis patients were divided into four groups: (1) increased NIHSS score; (2) unchanged NIHSS score; (3) improved NIHSS score and; (4) NIHSS score = 0. Results: Out of 188 patients (mean age 73 years, 100 female) receiving rt-PA, 32 (17%) had imaging-proven hemorrhagic transformation including 11 (6%) patients with parenchymal hemorrhage. Patients in group (1, 2) more often had hypertension (p = 0.015) and more often had parenchymal hemorrhage (9 vs. 4%; p < 0.206) compared to group (3, 4) and imaging-based changes in therapeutic management were more frequent (19% vs. 6%; p = 0.007). Patients of group (3, 4) had no changes in therapeutic management in 94% of the cases. Patients in group (4) had no hemorrhagic transformation in routine follow-up brain imaging. Conclusions: Frequency of hemorrhagic transformation in Routine follow-up brain imaging and consecutive changes in therapeutic management were different depending on clinical course measured by NHISS score. KW - thrombolysis KW - stroke KW - stroke management KW - magnetic resonance imaging KW - computerized tomography KW - intracerebral hemorrhage Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234947 VL - 10 ER -