Development of a human in-vitro pathophysiological model of FUS-ALS based on the induced pluripotent stem-cell technique and translation to patient phenotypes

Background: The submitted cumulative dissertation is based on two intertwined main studies with biomolecular foundation and clinical perspective on FUS-ALS complemented by two follow-up projects. This subtype of Amyotrophic lateral sclerosis is caused by heterozygous mutations mainly in the NLS of the FUS gene, which interferes with the proper nuclear import of the gene product. To date, there is no sufficient therapy available for this devastating neurodegenerative disease due to an incomplete pathophysiological understanding. Furthermore, not much is known about the specific clinical phenotype of FUS-ALS patients, including the influence of distinct FUS mutations due to the rarity of the disease. FUS is a DNA/RNA-binding protein that is mainly located in the nucleus and has essential functions in splicing, mRNA transport, transcription, and DNA damage repair. Hypothesis:1. It was hypothesized that the human-induced pluripotent stem-cell technique enables to create a sufficient motor neuron in-vitro cell model, which should provide new insights into unknown pathophysiological processes compared to previous cell models of FUS-ALS due to its patient-specific and human character. Thus, screening for potential therapeutic substances should be feasible using this model system. 2. Judging from the previously demonstrated, essential function of FUS in the DNA damage repair, FUS mutations are expected to increase the risk of malignant diseases in affected patients. Moreover, specific correlations between the nature of the mutation and the clinical, neurological phenotype appear plausible.Material & methods: First, an in-vitro cell culture model of FUS-ALS was established. For this purpose, a patient-specific, induced pluripotent stem cell-derived sMN culture was generated, which contained spinal motor neurons with mutations in the gene FUS or WT control cells. The Microfluidic Chamber system was used for the selective analysis of axons, which enabled the live-cell imaging of lysosomes and mitochondria using TIRF microscopy. For the analysis of DNA damage and its repair, gamma-H2A.X immunofluorescence staining was used on the one hand and live-cell laser ablation microscopy on the other, which allowed the precise induction of DNA damage and the monitoring of the repair response. For this purpose, isogenic FUS-GFP cell lines generated via CRISPR-Cas9n were used. A multicentre, retrospective cross-sectional study was conducted to determine genotype-phenotype correlations and the prevalence of malignant neoplasms in FUS-ALS. Previously published FUS-ALS cases have been added to perform a meta-analysis of clinical features.Results: Primarily, correct neuronal differentiation was observed prior to neurodegenerative phenotypes, perfectly mimicking a neurodegenerative disease in the dish. The typical cellular pathology of cytoplasmatic FUS deposition could be reproduced, making it a suitable model for more in-depth pathophysiological studies. Furthermore, the use of Microfluidic Chambers enabled the guided cultivation of neurons with somato-axonal direction of neurite outgrow along tiny microchannels in silico, resulting in a pure motoneuronal, axonal model. Within the distal axonal compartment of these channels, a loss of motility of both lysosomes and mitochondria was observed in parallel with a loss of the mitochondrial membrane potential, followed by the secondary degeneration of the distal axons of the sMNs with FUS mutation. A pathological increase in nuclear DNA damage has been identified as the cause of the distal-axonal phenotypes. This was due to a reduced nuclear FUS abundance as a result of the FUS-NLS mutation, which impaired proper nuclear import. There was evidence of a vicious cycle in this setting because the loss of the nuclear function of FUS disrupted the proper PAR-dependent DNA damage response, resulting in sustained DNA damage. Moreover, the remaining nuclear FUS was transferred into the cytoplasm upon phosphorylation by DNA-PK in a DNA damage response dependent manner, which is to date a process of unclear biological relevance. However, pharmacological inhibition of either the degradation of the PAR biopolymer or DNA-PK improved the nuclear presence of mutant FUS, restored its function in the DNA damage response, and finally prevented the distal axonal phenotype. Furthermore, the multicentric cohort study included 36 newly diagnosed patients. Only one in 40 patients was diagnosed with a malignant disease. By combining the newly diagnosed patients with previously published cases (186 cases in total), the so far most comprehensive database of FUS-ALS patients has been created. This allowed a thorough genotype-phenotype analysis, which showed a clear correlation between individual FUS mutations and the clinical phenotype. Conclusion: The experimental results indicated a primary nuclear insufficiency of mutated FUS, which is due to an impaired nuclear import and leads to a secondary axonal degeneration and finally to neuronal demise (“Dying-Back”). Potential therapeutic options have been identified, but their applicability and safety must be determined in prospective studies. The hypothesis of a generally increased risk of malignant diseases in the analysed FUS-ALS patient group was rejected. However, the clinical data of the meta-analysis are helpful in the counselling of newly diagnosed FUS-ALS patients, including the decision making of the therapeutic management and clearly add FUS-ALS to the family of diseases characterised by deficient DNA damage repair with purely neurological phenotypes such as AOA1, AOA2, and SCAN1

Antibody-coating of bacteria in the urine in relation to various immunologic indexes

Antibody-coating of bacteria in the urine in relation to various immunologic indexes. Little is known about the immunologic aspects of antibody coating, though the test for determining antibody-coated bacteria in urine has been examined for its diagnostic uses by many authors after its inauguration in 1974. In adults with chronic pyelonephritis with and without antibody-coated bacteria in the urine, we tested whether bacterial coating is correlated with the homologous O-antibody titre in the urine. We also determined Ig levels in urine and serum, as well as homologous O-antibody titres in serum. By means of indirect immunofluorescence technique, we were able to show homologous O-antibodies in the urine of all patients with and without antibody-coated bacteria. IgG and IgA levels in urine were mostly raised, as were often the O-antibody titres in the serum. There were no significant differences in the immunologic parameters within the patient groups with or without antibody-coating. The presence of homologous O-antibodies in urine does not therefore necessarily lead to coating of the bacteria.Recouvrement des bactéries par des anticorps en relation avec divers index immunologiques. Les connaissances concernant l'aspect immunologique du recouvrement par des anticorps sont peu nombreuses bien que le test de détection dans l'urine de bactéries recouvertes d'anticorps ait été évalué pour son utilité diagnostique par de nombreux auteurs depuis sa description en 1974. Chez des adultes atteints de pyélonéphrite chronique avec et sans présence dans l'urine de bactéries recouvertes d'anticorps nous avons recherché une corrélation entre ce recouvrement et le titre d'anticorps homologue O dans l'urine. Nous avons aussi déterminé les concentrations d'Ig dans l'urine et le sérum, de même que le titre de l'anticorps homologue O dans le sérum. Au moyen de la technique d'immunofluorescence indirecte nous avons pu mettre en évidence des anticorps homologues O dans les urines de tous les malades, qu'elles contiennent ou non des bactéries recouvertes d'anticorps. Les concentrations d'IgG et d'IgA dans l'urine étaient le plus souvent élevées de même que les titres d'anticorps O dans le sérum. Il n'a pas été observé de différence significative dans les index immunologiques dans les groupes de malades avec ou sans recouvrement des bactéries par des anticorps. La présence d'anticorps homologues O dans l'urine n'implique pas nécessairement le recouvrement des bactéries

Beiträge zur Geschichte des Landkreises Regensburg 44

Altbayerisches Weinland an der Donau - Der Baierwein in der Umgebung Regensburgs; darin: Heimrath, Margarete: Das Biethaus in Bach, ein historisches Denkmal zum Weinbau (S. 5-13); Heimrath, Margarete: Das Arbeitsjahr des Winzers (S. 14-16); Naumann, Günter: Das Biethaus in Bach an der Donau - Baugeschichte und bauliches Konzept (S. 17-21); Häußler, Theodor, Die Baumpresse (S. 22-27); Häußler, Theodor: Der Baierwein einst und jetzt (S. 28-44

CXCR7 Functions as a Scavenger for CXCL12 and CXCL11

CXCR7 (RDC1), the recently discovered second receptor for CXCL12, is phylogenetically closely related to chemokine receptors, but fails to couple to G-proteins and to induce typical chemokine receptor mediated cellular responses. The function of CXCR7 is controversial. Some studies suggest a signaling activity in mammalian cells and zebrafish embryos, while others indicate a decoy activity in fish. Here we investigated the two propositions in human tissues. We provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation. Consistently, CXCR7 continuously cycles between the plasma membrane and intracellular compartments in the absence and presence of ligand, both in mammalian cells and in zebrafish. In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations. Active CXCL12 sequestration by CXCR7 is demonstrated in adult mouse heart valves and human umbilical vein endothelium. The finding that CXCR7 specifically scavenges CXCL12 suggests a critical function of the receptor in modulating the activity of the ubiquitously expressed CXCR4 in development and tumor formation. Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs

CXCR7 functions as a scavenger for CXCL12 and CXCL11

Background: CXCR7 (RDC1), the recently discovered second receptor for CXCL12, is phylogenetically closely related to chemokine receptors, but fails to couple to G-proteins and to induce typical chemokine receptor mediated cellular responses. The function of CXCR7 is controversial. Some studies suggest a signaling activity in mammalian cells and zebrafish embryos, while others indicate a decoy activity in fish. Here we investigated the two propositions in human tissues. Methodology/Principal Findings: We provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation. Consistently, CXCR7 continuously cycles between the plasma membrane and intracellular compartments in the absence and presence of ligand, both in mammalian cells and in zebrafish. In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations. Active CXCL12 sequestration by CXCR7 is demonstrated in adult mouse heart valves and human umbilical vein endothelium. Conclusions/Significance: The finding that CXCR7 specifically scavenges CXCL12 suggests a critical function of the receptor in modulating the activity of the ubiquitously expressed CXCR4 in development and tumor formation. Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs

Citrullination Licenses Calpain to Decondense Nuclei in Neutrophil Extracellular Trap Formation

Neutrophils respond to various stimuli by decondensing and releasing nuclear chromatin characterized by citrullinated histones as neutrophil extracellular traps (NETs). This achieves pathogen immobilization or initiation of thrombosis, yet the molecular mechanisms of NET formation remain elusive. Peptidyl arginine deiminase-4 (PAD4) achieves protein citrullination and has been intricately linked to NET formation. Here we show that citrullination represents a major regulator of proteolysis in the course of NET formation. Elevated cytosolic calcium levels trigger both peptidylarginine deiminase-4 (PAD4) and calpain activity in neutrophils resulting in nuclear decondensation typical of NETs. Interestingly, PAD4 relies on proteolysis by calpain to achieve efficient nuclear lamina breakdown and chromatin decondensation. Pharmacological or genetic inhibition of PAD4 and calpain strongly inhibit chromatin decondensation of human and murine neutrophils in response to calcium ionophores as well as the proteolysis of nuclear proteins like lamin B1 and high mobility group box protein 1 (HMGB1). Taken together, the concerted action of PAD4 and calpain induces nuclear decondensation in the course of calcium-mediated NET formation

Revisionen des Porträts. Jenseits von Mimesis und Repräsentation

Noch immer wird das Phänomen ›Porträt‹ im kunsthistorischen Diskurs zumeist unter Begrifflichkeiten wie Identität, Individualität, Repräsentation oder Ähnlichkeit diskutiert. Zeitgenössische amimetische, konzeptuelle und performative Porträtformen werden mit solchen Konzepten jedoch nicht mehr vollständig eingeholt. Der Band befragt deshalb einerseits kritisch diese traditionellen, mimetischen Begriffe anhand von Fallstudien. Andererseits werden ihnen dynamische und offene Konzepte (teils aus Nachbardisziplinen) wie Spur, Berührung, Fraktalität, Defazialisierung oder Dividualität an die Seite gestellt, um den kunsthistorischen Porträt-Begriff in einem fachübergreifenden Diskurs aufzufächern, der auch die Digitalisierung umfasst. ›Porträt‹ wird somit explizit als Konstruktion, self-fashioning und konzeptuelle Praxis des Performativen betrachtet

Polarised Quark Distributions in the Nucleon from Semi-Inclusive Spin Asymmetries

We present a measurement of semi-inclusive spin asymmetries for positively and negatively charged hadrons from deep inelastic scattering of polarised muons on polarised protons and deuterons in the range $0.003$1 GeV$^2$. Compared to our previous publication on this subject, with the new data the statistical errors have been reduced by nearly a factor of two. From these asymmetries and our inclusive spin asymmetries we determine the polarised quark distributions of valence quarks and non-strange sea quarks at $Q^2$=10 GeV$^2$. The polarised $u$ valence quark distribution, $\Delta u_v(x)$, is positive and the polarisation increases with $x$. The polarised $d$ valence quark distribution, $\Delta d_v(x)$, is negative and the non-strange sea distribution, $\Delta \bar q(x)$, is consistent with zero over the measured range of $x$. We find for the first moments $\int_0^1 \Delta u_v(x) dx = 0.77 \pm 0.10 \pm 0.08$, $\int_0^1 \Delta d_v(x) dx = -0.52 \pm 0.14 \pm 0.09$ and $\int_0^1 \Delta \bar q(x) dx= 0.01 \pm 0.04 \pm 0.03$, where we assumed $\Delta \bar u(x) = \Delta \bar d(x)$. We also determine for the first time the second moments of the valence distributions $\int_0^1 x \Delta q_v(x) dx$.Comment: 17 page

Searches at HERA for Squarks in R-Parity Violating Supersymmetry

A search for squarks in R-parity violating supersymmetry is performed in e^+p collisions at HERA at a centre of mass energy of 300 GeV, using H1 data corresponding to an integrated luminosity of 37 pb^(-1). The direct production of single squarks of any generation in positron-quark fusion via a Yukawa coupling lambda' is considered, taking into account R-parity violating and conserving decays of the squarks. No significant deviation from the Standard Model expectation is found. The results are interpreted in terms of constraints within the Minimal Supersymmetric Standard Model (MSSM), the constrained MSSM and the minimal Supergravity model, and their sensitivity to the model parameters is studied in detail. For a Yukawa coupling of electromagnetic strength, squark masses below 260 GeV are excluded at 95% confidence level in a large part of the parameter space. For a 100 times smaller coupling strength masses up to 182 GeV are excluded.Comment: 32 pages, 14 figures, 3 table

Measurements of Transverse Energy Flow in Deep-Inelastic Scattering at HERA

Measurements of transverse energy flow are presented for neutral current deep-inelastic scattering events produced in positron-proton collisions at HERA. The kinematic range covers squared momentum transfers Q^2 from 3.2 to 2,200 GeV^2, the Bjorken scaling variable x from 8.10^{-5} to 0.11 and the hadronic mass W from 66 to 233 GeV. The transverse energy flow is measured in the hadronic centre of mass frame and is studied as a function of Q^2, x, W and pseudorapidity. A comparison is made with QCD based models. The behaviour of the mean transverse energy in the central pseudorapidity region and an interval corresponding to the photon fragmentation region are analysed as a function of Q^2 and W.Comment: 26 pages, 8 figures, submitted to Eur. Phys.