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For the differentiation of a embryonic stem cells (ESCs) to neuronal cells (NCs) a complex and coordinated gene regulation program is needed. One important control element for neuronal differentiation is the repressor element 1 silencing transcription factor (REST) complex, which represses neuronal gene expression in non-neuronal cells. Crucial effector proteins of the REST complex are small phosphatases such as the CTDSPs (C-terminal domain small phosphatases) that regulate polymerase II activity by dephosphorylating the C-terminal domain of the polymerase, thereby repressing target genes. The stepwise inactivation of REST, including the CTDSPs, leads to the induction of a neuron-specific gene program, which ultimately induces the formation of neurons. The spatio-temporal control of REST and its effector components is therefore a crucial step for neurogenesis.
In zebrafish it was shown that the REST-associated CTDSP2 is negatively regulated by the micro RNA (miR) -26b. Interestingly, the miR-26b is encoded in an intron of the primary transcript of CTDSP2. This gives the fundament of an intrinsic regulatory negative feedback loop, which is essential for the proceeding of neurogenesis. This feedback loop is active during neurogenesis, but inactive in non-neuronal cells. The reason for this is that the maturation of the precursor miR (pre-miR) to the mature miR-26 is arrested in non neuronal cells, but not in neurons. As only mature miRs are actively repressing genes, the regulation of miR-26 processing is an essential step in neurogenesis.
In this study, the molecular basis of miR-26 processing regulation in the context of neurogenesis was addressed. The mature miR is processed from two larger precursors: First the primary transcript is cleaved by the enzyme DROSHA in the nucleus to form the pre-miR. The pre-miR is exported from the nucleus and processed further through the enzyme DICER to yield the mature miR. The mature miR can regulate gene expression in association with the RNA-induced silencing complex (RISC).
Multiple different scenarios in which miR processing was regulated were proposed and experimentally tested. Microinjection studies using Xenopus leavis oocytes showed that slowdown or blockage of the nucleo-cytoplasmic transport are not the reason for delayed pre-miR-26 processing. Moreover, in vitro and in vivo miR-processing assays showed that maturation is most likely regulated through a in trans acting factor, which blocks processing in non neuronal cells.
Through RNA affinity chromatographic assays using zebrafish and murine lysates I was able to isolate and identify proteins that interact specifically with pre-miR-26 and could by this influence its biogenesis. Potential candidates are FMRP/FXR1/2, ZNF346 and Eral1, whose functional characterisation in the context of miR-biogenesis could now be addressed.
The second part of my thesis was executed in close colaboration with the laboratory of Prof. Albrecht Müller. The principal question was addressed how miR-26 influences neuronal gene expression and which genes are primarily affected. This research question could be addressed by using a cell culture model system, which mimics ex vivo the differentiation of ESCs to NCs via neuronal progenitor.
For the functional analysis of miR-26 knock out cell lines were generated by the CRISPR/Cas9 technology. miR-26 deficient ESC keep their pluripotent state and are able to develop NPC, but show major impairment in differentiating to NCs. Through RNA deep sequencing the miR-26 induced transcriptome differences could be analysed.
On the level of mRNAs it could be shown, that the expression of neuronal gene is downregulated in miR-26 deficient NCs. Interestingly, the deletion of miR-26 leads to selectively decreased levels of miRs, which on one hand regulate the REST complex and on the other hand are under transcriptional control by REST themself. This data and the discovery that induction of miR-26 leads to enrichment of other REST regulating miRs indicates that miR-26 initiates neurogenesis through stepwise inactivation of the REST complex.
Die Rolle von Chronophin bei Schlaganfall-induziertem Funktionsverlust der Blut-Hirn-Schranke
(2018)
Der ischämische Schlaganfall ist mit einer jährlichen Inzidenz von 200/100 000 Einwohnern die häufigste Gefäßerkrankung in Deutschland. Atherothrombose, arterielle Hypertonie und Embolien unterschiedlichen Ursprungs sind die wesentlichen Ursachen des ischämischen Schlaganfalls. Die neurologischen Defizite nach einem Schlaganfall resultieren aus einem gestörten zerebralen Blutfluss und somit einer insuffizienten Sauerstoffversorgung. Zusätzlich ist die Ödembildung, welche von einer gesteigerten Permeabilität der Blut-Hirn-Schranke verursacht wird, am neuronalen Zelltod beteiligt.
Chronophin ist eine Aktinzytoskelett-regulierende Serin-Phosphatase. In einem ischämischen Schlaganfall-Modell konnte im Rahmen dieser Arbeit gezeigt werden, dass der globale Verlust von Chronophin zu einer vermehrten Ödembildung und einem aggravierten neurologischen Zustand der Mäuse im Vergleich zu wildtypischen Kontrollen führte. Hirnlysate von wildtypischen Mäusen zeigten verringerte Chronophin-Level in der vom Schlaganfall betroffenen Hemisphäre. Jedoch konnten initiale immunhistochemische und zellbiologische Untersuchungen weder Chronophin-abhängige Veränderungen der Blut-Hirn-Schranke feststellen noch einen zerebralen Zelltyp identifizieren, der für den schützenden Effekt von Chronophin verantwortlich ist.
Diese Ergebnisse weisen auf einen komplexen, vielzelligen Mechanismus hin, dem die schützende Rolle von Chronophin im ischämischen Schlaganfall unterliegt. Die Entschlüsselung dieses Mechanismus ist Aufgabe künftiger Untersuchungen.
The work presented in this thesis covers the effects of early-life adversity in the context of altered serotonin (5-HT; 5-hydroxytryptamine) system functioning in mice. The main body is focussing on a screening approach identifying molecular processes, potentially involved in distinct behavioural manifestations that emerge from or are concomitant with early adversity and, with regard to some behavioural manifestations, dependent on the functioning of the 5-HT system.
For cellular viability, transcription is a fundamental process. Hereby, the DNA plays the most elemental and highly versatile role. It has long been known that promoters contain conserved and often well-defined motifs, which dictate the site of transcription initiation by providing binding sites for regulatory proteins. However, research within the last decade revealed that it is promoters lacking conserved promoter motifs and transcribing constitutively expressed genes that constitute the majority of promoters in eukaryotes. While the process of transcription initiation is well studied, whether defined DNA sequence motifs are required for the transcription of constitutively expressed genes in eukaryotes remains unknown. In the highly divergent protozoan parasite Trypanosoma brucei, most of the proteincoding genes are organized in large polycistronic transcription units. The genes within one polycistronic transcription unit are generally unrelated and transcribed by a common transcription start site for which no RNA polymerase II promoter motifs have been identified so far. Thus, it is assumed that transcription initiation is not regulated but how transcription is initiated in T. brucei is not known. This study aimed to investigate the requirement of DNA sequence motifs and chromatin structures for transcription initiation in an organism lacking transcriptional regulation. To this end, I performed a systematic analysis to investigate the dependence of transcription initiation on the DNA sequence. I was able to identify GT-rich promoter elements required for directional transcription initiation and targeted deposition of the histone variant H2A.Z, a conserved component during transcription initiation. Furthermore, nucleosome positioning data in this work provide evidence that sites of transcription initiation are rather characterized by broad regions of open and more accessible chromatin than narrow nucleosome depleted regions as it is the case in other eukaryotes. These findings highlight the importance of chromatin during transcription initiation. Polycistronic RNA in T. brucei is separated by adding an independently transcribed miniexon during trans-splicing. The data in this work suggest that nucleosome occupancy plays an important role during RNA maturation by slowing down the progressing polymerase and thereby facilitating the choice of the proper splice site during trans-splicing. Overall, this work investigated the role of the DNA sequence during transcription initiation and nucleosome positioning in a highly divergent eukaryote. Furthermore, the findings shed light on the conservation of the requirement of DNA motifs during transcription initiation and the regulatory potential of chromatin during RNA maturation. The findings improve the understanding of gene expression regulation in T. brucei, a eukaryotic parasite lacking transcriptional Regulation.
In mammals, anucleate blood platelets are constantly produced by their giant bone marrow (BM) progenitors, the megakaryocytes (MKs), which originate from hematopoietic stem cells. Megakaryopoiesis and thrombopoiesis have been studied intensively, but the exact mechanisms that control platelet generation from MKs remain poorly understood. Using multiphoton intravital microscopy (MP-IVM), thrombopoiesis and proplatelet formation were analyzed in the murine BM in real-time and in vivo, identifying an important role for several proteins, including Profilin1, TRPM7 and RhoA in thrombopoiesis. Currently, it is thought that blood cell precursors, such as MKs, migrate from the endosteal niche towards the vascular niche during maturation. In contrast to this paradigm, it was shown that MKs are homogeneously distributed within the dense BM blood vessel network, leaving no space for vessel-distant niches. By combining results from in vivo MP-IVM, in situ light-sheet fluorescence microscopy (LSFM) of the intact BM as well as computational simulations, surprisingly slow MK migration, limited intervascular space and a vessel-biased MK pool were revealed, contradicting the current concept of directed MK migration during thrombopoiesis.
Platelets play an essential role in hemostasis and thrombosis, but also in the pathogenesis of ischemic stroke. Ischemic stroke, which is mainly caused by thromboembolic occlusion of brain arteries, is among the leading causes of death and disability worldwide with limited treatment options. The platelet collagen receptor glycoprotein (GP) VI is a key player in arterial thrombosis and a critical determinant of stroke outcome, making its signaling pathway an attractive target for pharmacological intervention. The spleen tyrosine kinase (Syk) is an essential signaling mediator downstream of GPVI, but also of other platelet and immune cell receptors. In this thesis, it was demonstrated that mice lacking Syk specifically in platelets are protected from arterial thrombus formation and ischemic stroke, but display unaltered hemostasis. Furthermore, it was shown that mice treated with the novel, selective and orally bioavailable Syk inhibitor BI1002494 were protected in a model of arterial thrombosis and had smaller infarct sizes and a significantly better neurological outcome 24 h after transient middle cerebral artery occlusion (tMCAO), also when BI1002494 was administered therapeutically, i.e. after ischemia. These results provide direct evidence that pharmacological Syk inhibition might become a safe therapeutic strategy. The T cell receptor chain-associated protein kinase of 70 kDA (Zap-70) is also a spleen tyrosine kinase family member, but has a lower intrinsic activity compared to Syk and is expressed in T cells and natural killer (NK) cells, but not in platelets. Unexpectedly, arterial thrombus formation in vivo can occur independently of Syk kinase function as revealed by studies in Sykki mice, which express Zap-70 under the control of intrinsic Syk promoter elements.
Die Synthese der mRNA durch die RNA-Polymerase II ist der zentrale und kritische Prozess im Rahmen
der Transkriptionsregulation Protein-kodierender Gene. Viele Jahrzehnte der intensiven Erforschung brachten viele Details über diesen Mechanismus zu Tage, der von einer unglaublichen Komplexität und Dynamik geprägt ist. Dabei stellte sich heraus, dass der Mediatorkomplex eine zentrale Rolle bei der Regulation der Polymerase II-abhängigen Transkription spielt, im Besonderen der Initiation. In der Funktion einer Schnittstelle verknüpft er die allgemeine Transkriptionsmaschinerie mit den Gen- spezifischen Transkriptionsregulatoren. Durch die Interaktion des Schwanzmoduls mit diesen Regulatoren und der Interaktion des Kopfmoduls mit der Polymerase II verbindet er wie eine Brücke die oberhalb des Promotors liegenden Aktivatorsequenzen mit dem Kernpromotor und initiiert so die Ausbildung des Pre-Initiationskomplexes. Darüber hinaus mehren sich gerade in den letzten Jahren die Hinweise darauf, dass der Mediator auch noch an anderen Prozessen der Transkription beteiligt ist. Zu diesen gehören z.B. die Elongation, die Ausbildung von Genschlaufen oder auch der Umbau der Chromatinstruktur. In Anbetracht der Tatsachen, dass der Mediator (a) aus bis zu 25 Untereinheiten mit flexibler Zusammensetzung besteht, (b) eine flexible Struktur besitzt und (c) umfassend und dynamisch über posttranslationale Modifikationen modifiziert ist, erscheint es durchaus möglich, dass der Mediator all diese Funktionen ausfüllt und die Rolle einer allgemeinen Transkriptionsplattform einnimmt. Im Zusammenhang mit dieser Dissertationsschrift ist es gelungen, den Mediator innerhalb all dieser Funktionen „abzubilden“ und die bisher umfassendste Interaktomanalyse dieses Komplexes zu präsentieren. Durch die optimierten Bedingungen der Zelllyse und Co-Immunopräzipitation, gelang es auch transiente Interaktionspartner zu isolieren. Durch das metabolische Markieren der Wildtypkontrolle konnten außerdem unspezifische und spezifische Interaktionen eindeutig voneinander unterschieden werden. Über 400 Proteine wurden als signifikante Interaktionspartner des Mediators identifiziert. Viele dieser Proteine konnten als vollständige Komplexe zusammengefasst werden, z.B die RNA-Polymerase II, alle allgemeinen Transkriptionsfaktoren, der SAGA-Komplex, viele Komplexe des Chromatin Remodelings und stark acetylierte Histone. Viele weitere Interaktionspartner spielen zudem eine Rolle bei der co-transkriptionalen Prozessierung der mRNA, wie z.B dem Splicing, dem mRNA-decapping oder Abbau. Darüber hinaus gibt es starke Hinweise darauf, dass der Mediator auch mit der Polymerase I und III interagiert und an der ribosomalen Biogenese beteiligt ist. Weitere
Analysen zeigten, dass das Interaktom zudem hochdynamisch ist
Der Tumorsupressor APC ist in der Mehrzahl aller Fälle kolorektaler Karzinome bereits in
der initialen Phase der Karzinogenese mutiert. Diese Mutationen führen zu einer aberranten Aktivierung des Wnt-Signalweges sowie zu weiteren die Karzinogenese vorrantreibenden Aktivitäten, beispielsweise einem veränderten Migrationsverhalten. Dieser Dissertation
zu Grunde liegt die Idee, dass durch die Trunkierung des APC-Proteins aber auch Abhängigkeiten von Genaktivitäten entstehen, die zuvor entbehrlich waren. Solche synthetisch
letalen Gene sollten in einem high-content shRNA-Screen gefunden werden.
Für die Durchführung des Screens wurde ein von der SW480 Kolonkarzinomzelllinie
abgeleitetes, isogenes Zellsystem generiert, welches durch Induktion mit Doxyzyklin das
vollständige APC-Allel (FL-APC) exprimiert. Infolge dieser Expression zeigen die Zellen
einen weniger malignen Phänotyp. Dies spiegelt sich darin wider, dass die Zellen durch
FL-APC Expression in ihrer Wnt-Signalwegsaktivität eingeschränkt werden. Doxyzyklininduzierte Zellen sind schlechter in der Lage ohne Adhäsion zu proliferieren als nicht induzierte Zellen. Andererseits ist ihre Fähigkeit einem FKS-gradienten entlang zu migrieren
verbessert.
Der shRNA-Screen wurde mit der Decipher shRNA-Bibliothek durchgeführt. Diese
enthält 27.500 verschiedene shRNAs mit Interferenzaktivität gegen 5.000 mRNAs, die potentiell pharmakologisch inhibierbare Proteine kodieren. Die besten zwei Kandidaten für
eine synthetisch letale Interaktion mit trunkiertem APC, BCL2L1 und EIF2B5 wurden im
Verlauf einer Masterarbeit bzw. direkt in dieser Disseration validiert. EIF2B5 zeigte in vitro nach Depletion durch unterschiedliche shRNAs einen di erentiellen Proliferationse ekt
bei FL-APC induzierten im Vergleich zu kontrollbehandelten Zellen. Dieser di erentielle
E ekt konnte in einem weiteren Modellsystem, SW480 Zellen mit konstitutiver FL-APC
Expression, ebenfalls validiert werden.
Durch Expression einer shRNA mit Aktivität gegen EIF2B5 werden in beiden Zellsystem die unfolded protein response (UPR) Gene DDIT3 und splXBP1 aktiviert. Interessanterweise werden durch die Expression von FL-APC diese Gene reprimiert. Im Promotor
der EIF2B5-mRNA be ndet sich eine Bindestelle für MYC. Es ist denkbar, dass durch die
Expression von FL-APC eine globale Veränderung der Genexpression vorgenommen wird,
die einerseits eine Repression von EIF2B5 nach sich zieht aber andererseits eine hierdurch
ausgelöste ER-Stress Antwort verhindert. Eine Inhibition von EIF2B5 ohne diese Adaption
andererseits führt nach diesem Model zu einer UPR-aktivierten Apoptose.
In einem zweiten Projekt wurde das überraschende Verhalten von Kolonkarzinomzellen untersucht, die nach Zugabe von BEZ235, einem dualen PI3K/mTOR Inhibitor, trotz
gegenteiliger Erwartungen MYC-Proteinmengen erhöhen. Eine Repression wurde erwar-
tet, weil die Inhibition von PI3K einerseits zu einer proteasomalen Destabiliserung und
andererseits die mTOR Inhibition zu einer verringerten Synthese von MYC führen sollte.
Während bereits gezeigt werden konnte, dass durch einen FOXO-vermittelten Mechanismus MAPK-abhängig die MYC-Expression verstärkt wird, wurde in dieser Dissertation
die erwartete Translationsinhibition untersucht. BEZ235 inhibiert zwar CAP-abhängige
Translation, das MYC Protein wird jedoch aufgrund einer IRES-vermittelten Translation
weiterhin exprimiert. Silvestrol, ein Inhibitor der Helikase eIF4A andererseits interveniert
mit CAP- und IRES-abhängiger Translation und kann die MYC-Proteinkonzentrationen
verringern. Wir konnten zudem feststellen, dass die Applikation von Silvestrol auch in vivo
möglich und wirksam ist und zudem tolleriert wird. Dies gibt Anlass zur Ho nung, dass
eine Intervention der Translation auch im Menschen eine valide Strategie zur Behandlung
MYC-getriebener Tumore sein könnte.
The human-bacterial pathogen interaction is a complex process that results from
a prolonged evolutionary arms race in the struggle for survival. The pathogen employs
virulence strategies to achieve host colonization, and the latter counteracts using defense
programs. The encounter of both organisms results in drastic physiological changes
leading to stress, which is an ancient response accompanying infection. Recent evidence
suggests that the stress response in the host converges with the innate immune pathways
and influences the outcome of infection. However, the contribution of stress and the exact
mechanism(s) of its involvement in host defense remain to be elucidated. Using the model
bacterial pathogen Shigella flexneri, and comparing it with the closely related pathogen
Salmonella Typhimurium, this study investigated the role of host stress in the outcome of
infection.
Shigella infection is characterized by a pronounced pro-inflammatory response
that causes intense stress in host tissues, particularly the intestinal epithelium, which
constitutes the first barrier against Shigella colonization. In this study, inflammatory
stress was simulated in epithelial cells by inducing oxidative stress, hypoxia, and cytokine
stimulation. Shigella infection of epithelial cells exposed to such stresses was strongly
inhibited at the adhesion/binding stage. This resulted from the depletion of sphingolipidrafts
in the plasma membrane by the stress-activated sphingomyelinases. Interestingly,
Salmonella adhesion was not affected, by virtue of its flagellar motility, which allowed the
gathering of bacteria at remaining membrane rafts. Moreover, the intracellular replication
of Shigella lead to a similar sphingolipid-raft depletion in the membrane across adjacent
cells inhibiting extracellular bacterial invasion.
Additionally, this study shows that Shigella infection interferes with the host stress
granule-formation in response to stress. Interestingly, infected cells exhibited a nuclear
depletion of the global RNA-binding stress-granule associated proteins TIAR and TIA-1
and their accumulation in the cytoplasm.
Overall, this work investigated different aspects of the host stress-response in the
defense against bacterial infection. The findings shed light on the importance of the host
stress-pathways during infection, and improve the understanding of different strategies
in host-pathogen interaction.
This work summarizes the results of studies on three major aspects of platelet signaling and of the pathogenesis of immune thrombocytopenia. Therefore, this thesis is divided into three parts. i) Platelet activation and subsequent thrombus formation at sites of vascular injury is crucial for normal hemostasis, but it can also trigger myocardial infarction and stroke. The initial capture of flowing platelets to the injured vessel wall is mediated by the interaction of the glycoprotein (GP) Ib-V-IX complex with von Willebrand factor (vWF) immobilized on the exposed subendothelial extracellular matrix (ECM). The central importance of GPIb for platelet adhesion is well established, whereas GPV is generally considered to be of minor relevance for platelet physiology and thrombus formation. This study intended to clarify the relevance of this receptor during thrombus formation using Gp5-/- mice and mice with different double-deficiencies in GPV and in other platelet receptors. It was found that GPV and the collagen receptor integrin a2b1 have partially redundant functions in collagentriggered platelet aggregation. Further, it was revealed that GPV limits thrombus formation and impairs hemostasis in vivo. The data presented here demonstrate that the protective effect of GPVI-deficiency (another platelet collagen receptor) in arterial thrombosis and ischemic stroke depends on the expression of GPV. Moreover, it was demonstrated that lack of GPV restores the hemostatic function of mice lacking both GPVI and a2b1 or mice lacking GPVI and the C-type lectin receptor 2 (CLEC-2). Conclusively, GPV-depletion or blockade might have the potential to treat hemorrhagic disease states. ii) Platelets contain the two phospholipase (PL) D isoforms, PLD1 and PLD2, both of which presumably become activated upon platelet stimulation. However, the function of PLD in the process of platelet activation and aggregation has not been definitively explored. Thus, PLD-deficient mice were analyzed. Mice lacking PLD1 or PLD2 were viable, fertile and had normal platelet counts. PLD1 was found to be responsible for the inducible PLD-activity in platelets and to contribute to efficient integrin activation under static conditions. Moreover, flow adhesion experiments revealed that PLD1 is essential for efficient GPIb-mediated integrin activation. Consequently, Pld1-/- mice were protected from arterial thrombosis and ischemic brain infarction without affecting tail bleeding times. Hence, inhibition of PLD1 might be a novel approach for antithrombotic therapy. iii) Cellular activation of platelets or immune cells results in increased cytosolic calcium (Ca2+) levels. Store-operated calcium entry (SOCE) via the STIM1-Orai1 axis is the main route of Ca2+ entry downstream of immunoreceptor tyrosine-based activating motif (ITAM) receptor stimulation in mast cells and T cells. However, the requirement of Ca2+-mobilization in Fcg receptor (FcgR)-signaling and the relevance of STIM2 for T cell SOCE have been unclear. To address these questions, genetically modified mice lacking central molecules of the SOCE machinery were analyzed. Ca2+-measurements revealed that both STIM isoforms contribute to Ca2+-mobilization downstream of T cell receptor activation. Additionally, it was found that FcgR stimulation results in SOCE and is mediated by STIM1 and probably Orai1. Animal models of immune thrombocytopenia (ITP) revealed that SOCE is essential for platelet clearance and that both STIM isoforms contribute to the pathology of ITP. Moreover, in this work it was also demonstrated that STIM1 and Orai1 are essential in IgG-mediated systemic anaphylaxis. STIM2 contributes to IgG-mediated, but not to IgE-mediated anaphylaxis. The data indicate that interference with SOCE might become a new strategy to prevent or treat IgG-dependent autoimmune diseases.
This work considered the frequency-modulated balanced steady-state free precession (fm-bSSFP) sequence as a tool to provide banding free bSSFP MR images. The sequence was implemented and successfully applied to suppress bandings in various in vitro and in vivo examples. In combination with a radial trajectory it is a promising alternative for standard bSSFP applications. First, two specialized applications were shown to establish the benefits of the acquisition strategy in itself. In real time cardiac imaging, it was shown that the continuous shift in frequency causes a movement of the bandings across the FOV. Thus, no anatomical region is constantly impaired, and a suitable timeframe can be found to examine all important structures. Furthermore, a combination of images with different artifact positions, similar to phase-cycled acquisitions is possible. In this way, fast, banding-free imaging of the moving heart was realized. Second, acquisitions with long TR were shown. While standard bSSFP suffers from increasing incidence of bandings with higher TR values, the frequency-modulated approach provided banding free images, regardless of the TR.
A huge disadvantage of fm-bSSFP, in combination with the radial trajectory, is the decrease in signal intensity. In this work a specialized reconstruction method, the multifrequency reconstruction for frequency-modulated bSSFP (Muffm), was established, which successfully compensated that phenomena. The application of Muffm to several anatomical sites, such as inner ear, legs and cardiac acquisitions, proofed the advantageous SNR of the reconstruction.
Furthermore, fm-bSSFP was applied to the clinically highly relevant task of water-fat separation. Former approaches of a phase-sensitive separation procedure in combination with standard bSSFP showed promising results but failed in cases of high inhomogeneity or high field strengths where banding artifacts become a major issue. The novel approach of using the fm-bSSFP acquisition strategy with the separation approach provided robust, reliable images of high quality. Again, losses in signal intensity could be regained by Muffm, as both approaches are completely compatible.
Opposed to conventional banding suppression techniques, like frequency-scouts or phase-cycling, all reconstruction methods established in this work rely on a single radial acquisition, with scan times similar to standard bSSFP scans. No prolonged measurement times occur and patient time in the scanner is kept as short as possible, improving patient comfort, susceptibility to motion or physiological noise and cost of one scan.
All in all, the frequency-modulated acquisition in combination with specializes reconstruction methods, leads to a completely new quality of images with short acquisition times.