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

Understanding Smart Product-Service System Value Offerings: A Comparative Case Analysis

Although smart product-service systems (SPSS) have attracted increasing interest from manufacturers in recent years, their commercialization can pose major challenges. This study aims to advance the SPSS literature by examining the pillars of manufacturers’ SPSS value offering strategies. Using a sociotechnical perspective and a configurational approach, this study examines how manufacturers configure SPSS value offerings. A fuzzy-set qualitative comparative analysis based on data from a qualitative study reveals three different configurations of sociotechnical factors that are consistently sufficient to achieve an attractive SPSS value offering. From a theoretical perspective, insight into these configurations advances the understanding of complementarities among sociotechnical factors for SPSS. From a managerial perspective, the configurations provide templates for evaluating existing organizational work systems as well as design options for developing new ones

Variation in fruit chemical and mineral composition of Kenyan guava (Psidium guajava L.): Inferences from climatic conditions, and fruit morphological traits

There is limited knowledge about the impact of climatic conditions and fruit morphological traits on the nutritional composition of the guava fruit. Fruits were gathered from 128 guava trees across four geographically diverse regions of Kenya. The fruits were morphologically characterized and analysed for their chemical and mineral composition. The ascorbic acid content correlated positively only with total annual precipitation, while total soluble solids (TSS) correlated positively with mean annual temperature. TSS correlated negatively with pulp weight and was higher in white-fleshed fruits than in the red-fleshed types. The mineral content of the fruits correlated negatively with most of the fruit weight- and size-based morphologicaltraits, as well as with the total annual precipitation, but positively with fruit seed proportion. This information could act as a guide in the selection of specific regions for upscaling guava production and aid in the selection of accessions for improvement programmes that enhance guava fruit nutritional composition

Assessment of sensory profile and instrumental analyzed attributes influenced by different potassium fertilization levels in three tomato cultivars

Sensory properties are an essential quality aspect when the consumption of fresh tomato is under consideration. The flavor of tomato is defined as a combination of taste sensations (sweetness, sourness), aroma (volatile compounds), and texture (firmness, mealiness), some of which are proven to be affected by insufficient nutrient supply − especially potassium (K). This study intends to undertake a holistic assessment of the K fertilization effect on the flavor of tomato by connecting the use of sensorial and instrumental methods. An optimal K supply significantly increased the sensory descriptors sweetness, sourness, and aroma as well as the instrumental estimated color, firmness, total soluble solids (TSS), titratable acids (TA), and dry matter (DM) in a cultivar-specific manner. The volatile organic compounds (VOCs) were not significantly affected by K fertilization. The evaluation by the panelists confirmed the results of the instru-mental analyses, by which an increment in the fruit quality with rising K supply could be detected. An optimal K supply of 3.66 g/plant could be suggested to increase tomato flavor in the cocktail cultivars studied: Primavera and Yellow Submarine. Cultivar effects should, therefore, be considered for defining the optimal K fertilizer dose that favors high tomato fruit quality and, hence, better flavor

Grinding Media Motion and Collisions in Different Zones of Stirred Media Mills

Product fineness during grinding in stirred media mills is mainly influenced by the specific energy input, the stress energy transferred by the colliding grinding media and the stress frequency. The stress energy from grinding media collisions is heterogeneously distributed in stirred media mills. Herein, in order to characterize the stress energy distribution and the local grinding media collision frequencies, the grinding media motion was calculated using discrete element method (DEM) simulations coupled with computational fluid dynamics (CFD). The local grinding media concentration, velocity profiles, grinding media collisions and stress energies were compared for varied total grinding media fillings and stirrer speeds. It was confirmed that the normalized grinding media velocity profile can be used to divide the grinding chamber into four types of zones that allow the modeling of the stress energy distribution. However, the collision frequency showed very different distributions for varied stirrer velocities and grinding media fillings