570 Biowissenschaften; Biologie
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Genome-scale metabolic models are mathematical representations of all known reactions occurring in a cell. Combined with constraints based on physiological measurements, these models have been used to accurately predict metabolic fluxes and effects of perturbations (e.g. knock-outs) and to inform metabolic engineering strategies. Recently, protein-constrained models have been shown to increase predictive potential (especially in overflow metabolism), while alleviating the need for measurement of nutrient uptake rates. The resulting modelling frameworks quantify the upkeep cost of a certain metabolic flux as the minimum amount of enzyme required for catalysis. These improvements are based on the use of in vitro turnover numbers or in vivo apparent catalytic rates of enzymes for model parameterization. In this thesis several tools for the estimation and refinement of these parameters based on in vivo proteomics data of Escherichia coli, Saccharomyces cerevisiae, and Chlamydomonas reinhardtii have been developed and applied. The difference between in vitro and in vivo catalytic rate measures for the three microorganisms was systematically analyzed. The results for the facultatively heterotrophic microalga C. reinhardtii considerably expanded the apparent catalytic rate estimates for photosynthetic organisms. Our general finding pointed at a global reduction of enzyme efficiency in heterotrophy compared to other growth scenarios. Independent of the modelled organism, in vivo estimates were shown to improve accuracy of predictions of protein abundances compared to in vitro values for turnover numbers. To further improve the protein abundance predictions, machine learning models were trained that integrate features derived from protein-constrained modelling and codon usage. Combining the two types of features outperformed single feature models and yielded good prediction results without relying on experimental transcriptomic data. The presented work reports valuable advances in the prediction of enzyme allocation in unseen scenarios using protein constrained metabolic models. It marks the first successful application of this modelling framework in the biotechnological important taxon of green microalgae, substantially increasing our knowledge of the enzyme catalytic landscape of phototrophic microorganisms.
Aging is associated with bone loss, which can lead to osteoporosis and high fracture risk. This coincides with the enhanced formation of bone marrow adipose tissue (BMAT), suggesting a negative effect of bone marrow adipocytes on skeletal health. Increased BMAT formation is also observed in pathologies such as obesity, type 2 diabetes and osteoporosis. However, a subset of bone marrow adipocytes forming the constitutive BMAT (cBMAT), arise early in life in the distal skeleton, contain high levels of unsaturated fatty acids and are thought to provide a physiological function. Regulated BMAT (rBMAT) forms during aging and obesity in proximal regions of the bone and contain a large proportion of saturated fatty acids. Paradoxically, BMAT accumulation is also enhanced during caloric restriction (CR), a life-span extending dietary intervention. This indicates, that different types of BMAT can form in response to opposing nutritional stimuli with potentially different functions.
To this end, two types of nutritional interventions, CR and high fat diet (HFD), that are both described to induce BMAT accumulation were carried out. CR markedly increased BMAT formation in the proximal tibia and led to a higher proportion of unsaturated fatty acids, making it similar to the physiological cBMAT. Additionally, proximal and diaphyseal tibia regions displayed higher adiponectin expression. In aged mice, CR was associated with an improved trabecular bone structure. Taken together, these findings demonstrate, that the type of BMAT that forms during CR might provide beneficial effects for local bone stem/progenitor cells and metabolic health. The HFD intervention performed in this thesis showed no effect on BMAT accumulation and bone microstructure. RNA Seq analysis revealed alterations in the composition of the collagen-containing extracellular matrix (ECM).
In order to investigate the effects of glucose homeostasis on osteogenesis, differentiation capacity of immortalized multipotent mesenchymal stromal cells (MSCs) and osteochondrogenic progenitor cells (OPCs) was analyzed. Insulin improved differentiation in both cell types, however, combination of with a high glucose concentration led to an impaired mineralization of the ECM. In the MSCs, this was accompanied by the formation of adipocytes, indicating negative effects of the adipocytes formed during hyperglycemic conditions on mineralization processes. However, the altered mineralization pattern and structure of the ECM was also observed in OPCs, which did not form any adipocytes, suggesting further negative effects of a hyperglycemic environment on osteogenic differentiation.
In summary, the work provided in this thesis demonstrated that differentiation commitment of bone-resident stem cells can be altered through nutrient availability, specifically glucose. Surprisingly, both high nutrient supply, e.g. the hyperglycemic cell culture conditions, and low nutrient supply, e.g. CR, can induce adipogenic differentiation. However, while CR-induced adipocyte formation was associated with improved trabecular bone structure, adipocyte formation in a hyperglycemic cell-culture environment hampered mineralization. This thesis provides further evidence for the existence of different types of BMAT with specific functions.
Moss-microbe associations are often characterised by syntrophic interactions between the microorganisms and their hosts, but the structure of the microbial consortia and their role in peatland development remain unknown.
In order to study microbial communities of dominant peatland mosses, Sphagnum and brown mosses, and the respective environmental drivers, four study sites representing different successional stages of natural northern peatlands were chosen on a large geographical scale: two brown moss-dominated, circumneutral peatlands from the Arctic and two Sphagnum-dominated, acidic peat bogs from subarctic and temperate zones.
The family Acetobacteraceae represented the dominant bacterial taxon of Sphagnum mosses from various geographical origins and displayed an integral part of the moss core community. This core community was shared among all investigated bryophytes and consisted of few but highly abundant prokaryotes, of which many appear as endophytes of Sphagnum mosses. Moreover, brown mosses and Sphagnum mosses represent habitats for archaea which were not studied in association with peatland mosses so far. Euryarchaeota that are capable of methane production (methanogens) displayed the majority of the moss-associated archaeal communities. Moss-associated methanogenesis was detected for the first time, but it was mostly negligible under laboratory conditions. Contrarily, substantial moss-associated methane oxidation was measured on both, brown mosses and Sphagnum mosses, supporting that methanotrophic bacteria as part of the moss microbiome may contribute to the reduction of methane emissions from pristine and rewetted peatlands of the northern hemisphere.
Among the investigated abiotic and biotic environmental parameters, the peatland type and the host moss taxon were identified to have a major impact on the structure of moss-associated bacterial communities, contrarily to archaeal communities whose structures were similar among the investigated bryophytes. For the first time it was shown that different bog development stages harbour distinct bacterial communities, while at the same time a small core community is shared among all investigated bryophytes independent of geography and peatland type.
The present thesis displays the first large-scale, systematic assessment of bacterial and archaeal communities associated both with brown mosses and Sphagnum mosses. It suggests that some host-specific moss taxa have the potential to play a key role in host moss establishment and peatland development.
Arachidonsäurelipoxygenasen (ALOX-Isoformen) sind Lipid-peroxidierenden Enzyme, die bei der Zelldifferenzierung und bei der Pathogenese verschiedener Erkrankungen bedeutsam sind. Im menschlichen Genom gibt es sechs funktionelle ALOX-Gene, die als Einzelkopiegene vorliegen. Für jedes humane ALOX-Gen gibt es ein orthologes Mausgen. Obwohl sich die sechs humanen ALOX-Isoformen strukturell sehr ähnlich sind, unterscheiden sich ihre funktionellen Eigenschaften deutlich voneinander. In der vorliegenden Arbeit wurden vier unterschiedliche Fragestellungen zum Vorkommen, zur biologischen Rolle und zur Evolutionsabhängigkeit der enzymatischen Eigenschaften von Säugetier-ALOX-Isoformen untersucht:
1) Spitzhörnchen (Tupaiidae) sind evolutionär näher mit dem Menschen verwandt als Nagetiere und wurden deshalb als Alternativmodelle für die Untersuchung menschlicher Erkrankungen vorgeschlagen. In dieser Arbeit wurde erstmals der Arachidonsäurestoffwechsel von Spitzhörnchen untersucht. Dabei wurde festgestellt, dass im Genom von Tupaia belangeri vier unterschiedliche ALOX15-Gene vorkommen und die Enzyme sich hinsichtlich ihrer katalytischen Eigenschaften ähneln. Diese genomische Vielfalt, die weder beim Menschen noch bei Mäusen vorhanden ist, erschwert die funktionellen Untersuchungen zur biologischen Rolle des ALOX15-Weges. Damit scheint Tupaia belangeri kein geeigneteres Tiermodel für die Untersuchung des ALOX15-Weges des Menschen zu sein.
2) Entsprechend der Evolutionshypothese können Säugetier-ALOX15-Orthologe in Arachidonsäure-12-lipoxygenierende- und Arachidonsäure-15-lipoxygenierende Enzyme eingeteilt werden. Dabei exprimieren Säugetierspezies, die einen höheren Evolutionsgrad als Gibbons aufweisen, Arachidonsäure-15-lipoxygenierende ALOX15-Orthologe, während evolutionär weniger weit entwickelte Säugetiere Arachidonsäure-12 lipoxygenierende Enzyme besitzen. In dieser Arbeit wurden elf neue ALOX15-Orthologe als rekombinante Proteine exprimiert und funktionell charakterisiert. Die erhaltenen Ergebnisse fügen sich widerspruchsfrei in die Evolutionshypothese ein und verbreitern deren experimentelle Basis. Die experimentellen Daten bestätigen auch das Triadenkonzept.
3) Da humane und murine ALOX15B-Orthologe unterschiedliche funktionelle Eigenschaften aufweisen, können Ergebnisse aus murinen Krankheitsmodellen zur biologischen Rolle der ALOX15B nicht direkt auf den Menschen übertragen werden. Um die ALOX15B-Orthologen von Maus und Mensch funktionell einander anzugleichen, wurden im Rahmen der vorliegenden Arbeit Knock-in Mäuse durch die In vivo Mutagenese mittels CRISPR/Cas9-Technik hergestellt. Diese exprimieren eine humanisierte Mutante (Doppelmutation von Tyrosin603Asparaginsäure+Histidin604Valin) der murinen Alox15b. Diese Mäuse waren lebens- und fortpflanzungsfähig, zeigten aber geschlechtsspezifische Unterschiede zu ausgekreuzten Wildtyp-Kontrolltieren im Rahmen ihre Individualentwicklung.
4) In vorhergehenden Untersuchungen zur Rolle der ALOX15B in Rahmen der Entzündungsreaktion wurde eine antiinflammatorische Wirkung des Enzyms postuliert. In der vorliegenden Arbeit wurde untersucht, ob eine Humanisierung der murinen Alox15b die Entzündungsreaktion in zwei verschiedenen murinen Entzündungsmodellen beeinflusst. Eine Humanisierung der murinen Alox15b führte zu einer verstärkten Ausbildung von Entzündungssymptomen im induzierten Dextran-Natrium-Sulfat-Kolitismodell. Im Gegensatz dazu bewirkte die Humanisierung der Alox15b eine Abschwächung der Entzündungssymptome im Freund‘schen Adjuvans Pfotenödemmodell. Diese Daten deuten darauf hin, dass sich die Rolle der ALOX15B in verschiedenen Entzündungsmodellen unterscheidet.
In the present thesis, AC electrokinetic forces, like dielectrophoresis and AC electroosmosis, were demonstrated as a simple and fast method to functionalize the surface of nanoelectrodes with submicrometer sized biological objects. These nanoelectrodes have a cylindrical shape with a diameter of 500 nm arranged in an array of 6256 electrodes. Due to its medical relevance influenza virus as well as anti-influenza antibodies were chosen as a model organism. Common methods to bring antibodies or proteins to biosensor surfaces are complex and time-consuming. In the present work, it was demonstrated that by applying AC electric fields influenza viruses and antibodies can be immobilized onto the nanoelectrodes within seconds without any prior chemical modification of neither the surface nor the immobilized biological object. The distribution of these immobilized objects is not uniform over the entire array, it exhibits a decreasing gradient from the outer row to the inner ones. Different causes for this gradient have been discussed, such as the vortex-shaped fluid motion above the nanoelectrodes generated by, among others, electrothermal fluid flow. It was demonstrated that parts of the accumulated material are permanently immobilized to the electrodes. This is a unique characteristic of the presented system since in the literature the AC electrokinetic immobilization is almost entirely presented as a method just for temporary immobilization. The spatial distribution of the immobilized viral material or the anti-influenza antibodies at the electrodes was observed by either the combination of fluorescence microscopy and deconvolution or by super-resolution microscopy (STED). On-chip immunoassays were performed to examine the suitability of the functionalized electrodes as a potential affinity-based biosensor. Two approaches were pursued: A) the influenza virus as the bio-receptor or B) the influenza virus as the analyte. Different sources of error were eliminated by ELISA and passivation experiments. Hence, the activity of the immobilized object was inspected by incubation with the analyte. This resulted in the successful detection of anti-influenza antibodies by the immobilized viral material. On the other hand, a detection of influenza virus particles by the immobilized anti-influenza antibodies was not possible. The latter might be due to lost activity or wrong orientation of the antibodies. Thus, further examinations on the activity of by AC electric fields immobilized antibodies should follow. When combined with microfluidics and an electrical read-out system, the functionalized chips possess the potential to serve as a rapid, portable, and cost-effective point-of-care (POC) device. This device can be utilized as a basis for diverse applications in diagnosing and treating influenza, as well as various other pathogens.
Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.5 (hNaV1.5; SCN5A) in 3 h. Subsequently, CB-nMVs were isolated from fractions of nitrogen-cavitated CHO cells overexpressing the hNaV1.5. In an integrative approach, nMVs were micro-transplanted into Xenopus laevis oocytes. CB-nMVs expressed native lidocaine-sensitive hNaV1.5 currents within 24 h; CF-nMVs did not elicit any response. Both the CB- and CF-nMV preparations evoked single-channel activity on the planar lipid bilayer while retaining sensitivity to lidocaine application. Our findings suggest a high usability of the quick-synthesis CF-nMVs and maintenance-free CB-nMVs as ready-to-use tools for in-vitro analysis of electrogenic membrane proteins and large, voltage-gated ion channels.
No evidence of growth impairment after forced migration in Polish school children after World War II
(2023)
Background: Migration is omnipresent. It can come hand in hand with emotional stress which is known to influence the growth of children.
Objective: The aim of this study was to analyse whether type of migration (forced or voluntary) and the geographic direction had influenced the growth of Polish children after World War II.
Sample and Methods: A sub dataset of 2,208 individuals between the ages of 2-20, created from data of the 2nd Polish Anthropological Survey carried out in 1966–1969, including anthropometrical data and social and demographic information based on questionnaire, was used to analyse migration effects.
Results: No association could be found between the direction of migration and the height of the children. The confidence intervals of the means of all classified migration categories overlap significantly and the effect size of the influence of migration category on height is ds=.140, which is too low to see any effects, even if there were one.
Conclusion: Neither forced nor voluntary migration in Poland after World War II led to a change in height in children of migrating families.
Background: Assessing short-term growth in humans is still fraught with difficulties. Especially when looking for small variations and increments, such as mini growth spurts, high precision instruments or frequent measurements are necessary. Daily measurements however require a lot of effort, both for anthropologists and for the subjects. Therefore, new sophisticated approaches are needed that reduce fluctuations and reveal underlying patterns.
Objectives: Changepoints are abrupt variations in the properties of time series data. In the context of growth, such variations could be variation in mean height. By adjusting the variance and using different growth models, we assessed the ability of changepoint analysis to analyse short-term growth and detect mini growth spurts.
Sample and Methods: We performed Bayesian changepoint analysis on simulated growth data using the bcp package in R. Simulated growth patterns included stasis, linear growth, catch-up growth, and mini growth spurts. Specificity and a normalised variant of the Matthews correlation coefficient (MCC) were used to assess the algorithm’s performance. Welch’s t-test was used to compare differences of the mean.
Results: First results show that changepoint analysis can detect mini growth spurts. However, the ability to detect mini growth spurts is highly dependent on measurement error. Data preparation, such as ranking and rotating time series data, showed negligible improvements. Missing data was an issue and may affect the prediction quality of the classification metrics.
Conclusion: Changepoint analysis is a promising tool to analyse short-term growth. However, further optimisation and analysis of real growth data is needed to make broader generalisations.
Human growth data analysis and statistics – the 5th Gülpe International Student Summer School
(2023)
The Summer School in Gülpe (Ecological Station of the University of Potsdam) offers an exceptional learning opportunity for students to apply their knowledge and skills to real-world problems. With the guidance of experienced human biologists, statisticians, and programmers, students have the unique chance to analyze their own data and gain valuable insights. This interdisciplinary setting not only bridges different research areas but also leads to highly valuable outputs. The progress of students within just a few days is truly remarkable, especially when they are motivated and receive immediate feedback on their questions, problems, and results. The Summer School covers a wide range of topics, with this year’s focus mainly on two areas: understanding the impact of socioeconomic and physiological factors on human development and mastering statistical techniques for analyzing data such as changepoint analysis and the St. Nicolas House Analysis (SNHA) to visualize interacting variables. The latter technique, born out of the Summer School’s emphasis on gaining comprehensive data insights and understanding major relationships, has proven to be a valuable tool for researchers in the field. The articles in this special issue demonstrate that the Summer School in Gülpe stands as a testament to the power of practical learning and collaboration. Students who attend not only gain hands-on experience but also benefit from the expertise of professionals and the opportunity to engage with peers from diverse disciplines.
Twenty-four scientists met for the annual Auxological conference held at Krobielowice castle, Poland, to discuss the diverse influences of the environment and of social behavior on growth following last year’s focus on growth and public health concerns (Hermanussen et al., 2022b). Growth and final body size exhibit marked plastic responses to ecological conditions. Among the shortest are the pygmoid people of Rampasasa, Flores, Indonesia, who still live under most secluded insular conditions. Genetics and nutrition are usually considered responsible for the poor growth in many parts of this world, but evidence is accumulating on the prominent impact of social embedding on child growth. Secular trends not only in the growth of height, but also in body proportions, accompany the secular changes in the social, economic and political conditions, with major influences on the emotional and educational circumstances under which the children grow up (Bogin, 2021). Aspects of developmental tempo and aspects of sports were discussed, and the impact of migration by the example of women from Bangladesh who grew up in the UK. Child growth was considered in particular from the point of view of strategic adjustments of individual size within the network of its social group. Theoretical considerations on network characteristics were presented and related to the evolutionary conservation of growth regulating hypothalamic neuropeptides that have been shown to link behavior and physical growth in the vertebrate species. New statistical approaches were presented for the evaluation of short term growth measurements that permit monitoring child growth at intervals of a few days and weeks.