@phdthesis{Kiss2024,
  author    = {Kiss, Andrea},
  title     = {Moss-associated bacterial and archaeal communities of northern peatlands: key taxa, environmental drivers and potential functions},
  doi       = {10.25932/publishup-63064},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-630641},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XX, 139, liv},
  year      = {2024},
  abstract  = {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.},
  language  = {en}
}
@phdthesis{Schauer2006,
  author    = {Schauer, Nicolas},
  title     = {Quantitative trait loci (QTL) for metabolite accumulation and metabolic regulation : metabolite profiling of interspecific crosses of tomato},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-7643},
  school      = {Universit{\"a}t Potsdam},
  year      = {2006},
  abstract  = {The advent of large-scale and high-throughput technologies has recently caused a shift in focus in contemporary biology from decades of reductionism towards a more systemic view. Alongside the availability of genome sequences the exploration of organisms utilizing such approach should give rise to a more comprehensive understanding of complex systems. Domestication and intensive breeding of crop plants has led to a parallel narrowing of their genetic basis. The potential to improve crops by conventional breeding using elite cultivars is therefore rather limited and molecular technologies, such as marker assisted selection (MAS) are currently being exploited to re-introduce allelic variance from wild species. Molecular breeding strategies have mostly focused on the introduction of yield or resistance related traits to date. However given that medical research has highlighted the importance of crop compositional quality in the human diet this research field is rapidly becoming more important. Chemical composition of biological tissues can be efficiently assessed by metabolite profiling techniques, which allow the multivariate detection of metabolites of a given biological sample. Here, a GC/MS metabolite profiling approach has been applied to investigate natural variation of tomatoes with respect to the chemical composition of their fruits. The establishment of a mass spectral and retention index (MSRI) library was a prerequisite for this work in order to establish a framework for the identification of metabolites from a complex mixture. As mass spectral and retention index information is highly important for the metabolomics community this library was made publicly available. Metabolite profiling of tomato wild species revealed large differences in the chemical composition, especially of amino and organic acids, as well as on the sugar composition and secondary metabolites. Intriguingly, the analysis of a set of S. pennellii introgression lines (IL) identified 889 quantitative trait loci of compositional quality and 326 yield-associated traits. These traits are characterized by increases/decreases not only of single metabolites but also of entire metabolic pathways, thus highlighting the potential of this approach in uncovering novel aspects of metabolic regulation. Finally the biosynthetic pathway of the phenylalanine-derived fruit volatiles phenylethanol and phenylacetaldehyde was elucidated via a combination of metabolic profiling of natural variation, stable isotope tracer experiments and reverse genetic experimentation.},
  subject      = {Tomate},
  language  = {en}
}
@phdthesis{Mubeen2018,
  author    = {Mubeen, Umarah},
  title     = {Regulation of central carbon and nitrogen metabolism by Target of Rapamycin (TOR) kinase in Chlamydomonas reinhardtii},
  school      = {Universit{\"a}t Potsdam},
  pages     = {vii, 153},
  year      = {2018},
  abstract  = {The highly conserved protein complex containing the Target of Rapamycin (TOR) kinase is known to integrate intra- and extra-cellular stimuli controlling nutrient allocation and cellular growth. This thesis describes three studies aimed to understand how TOR signaling pathway influences carbon and nitrogen metabolism in Chlamydomonas reinhardtii. The first study presents a time-resolved analysis of the molecular and physiological features across the diurnal cycle. The inhibition of TOR leads to 50\% reduction in growth followed by nonlinear delays in the cell cycle progression. The metabolomics analysis showed that the growth repression is mainly driven by differential carbon partitioning between anabolic and catabolic processes. Furthermore, the high accumulation of nitrogen-containing compounds indicated that TOR kinase controls the carbon to nitrogen balance of the cell, which is responsible for biomass accumulation, growth and cell cycle progression. In the second study the cause of the high accumulation of amino acids is explained. For this purpose, the effect of TOR inhibition on Chlamydomonas was examined under different growth regimes using stable 13C- and 15N-isotope labeling. The data clearly showed that an increased nitrogen uptake is induced within minutes after the inhibition of TOR. Interestingly, this increased N-influx is accompanied by increased activities of nitrogen assimilating enzymes. Accordingly, it was concluded that TOR inhibition induces de-novo amino acid synthesis in Chlamydomonas. The recognition of this novel process opened an array of questions regarding potential links between central metabolism and TOR signaling. Therefore a detailed phosphoproteomics study was conducted to identify the potential substrates of TOR pathway regulating central metabolism. Interestingly, some of the key enzymes involved in carbon metabolism as well as amino acid synthesis exhibited significant changes in the phosphosite intensities immediately after TOR inhibition. Altogether, these studies provide a) detailed insights to metabolic response of Chlamydomonas to TOR inhibition, b) identification of a novel process causing rapid upshifts in amino acid levels upon TOR inhibition and c) finally highlight potential targets of TOR signaling regulating changes in central metabolism. Further biochemical and molecular investigations could confirm these observations and advance the understanding of growth signaling in microalgae.},
  language  = {en}
}
@phdthesis{Folikumah2022,
  author    = {Folikumah, Makafui Yao},
  title     = {Stimuli-promoted in situ formation of hydrogels with thiol/thioester containing peptide precursors},
  doi       = {10.25932/publishup-56971},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-569713},
  school      = {Universit{\"a}t Potsdam},
  pages     = {159},
  year      = {2022},
  abstract  = {Hydrogels are potential synthetic ECM-like substitutes since they provide functional and structural similarities compared to soft tissues. They can be prepared by crosslinking of macromolecules or by polymerizing suitable precursors. The crosslinks are not necessarily covalent bonds, but could also be formed by physical interactions such as π-π interactions, hydrophobic interactions, or H-bonding. On demand in situ forming hydrogels have garnered increased interest especially for biomedical applications over preformed gels due to the relative ease of in vivo delivery and filling of cavities. The thiol-Michael addition reaction provides a straightforward and robust strategy for in situ gel formation with its fast reaction kinetics and ability to proceed under physiological conditions. The incorporation of a trigger function into a crosslinking system becomes even more interesting since gelling can be controlled with stimulus of choice. The use of small molar mass crosslinker precursors with active groups orthogonal to thiol-Michael reaction type electrophile provides the opportunity to implement an on-demand in situ crosslinking without compromising the fast reaction kinetics. It was postulated that short peptide sequences due to the broad range structural-function relations available with the different constituent amino acids, can be exploited for the realisation of stimuli-promoted in situ covalent crosslinking and gelation applications. The advantages of this system over conventional polymer-polymer hydrogel systems are the ability tune and predict material property at the molecular level. The main aim of this work was to develop a simplified and biologically-friendly stimuli-promoted in situ crosslinking and hydrogelation system using peptide mimetics as latent crosslinkers. The approach aims at using a single thiodepsipeptide sequence to achieve separate pH- and enzyme-promoted gelation systems with little modification to the thiodepsipeptide sequence. The realization of this aim required the completion of three milestones. In the first place, after deciding on the thiol-Michael reaction as an effective in situ crosslinking strategy, a thiodepsipeptide, Ac-Pro-Leu-Gly-SLeu-Leu-Gly-NEtSH (TDP) with expected propensity towards pH-dependent thiol-thioester exchange (TTE) activation, was proposed as a suitable crosslinker precursor for pH-promoted gelation system. Prior to the synthesis of the proposed peptide-mimetic, knowledge of the thiol-Michael reactivity of the would-be activated thiol moiety SH-Leu, which is internally embedded in the thiodepsipeptide was required. In line with pKa requirements for a successful TTE, the reactivity of a more acidic thiol, SH-Phe was also investigated to aid the selection of the best thiol to be incorporated in the thioester bearing peptide based crosslinker precursor. Using 'pseudo' 2D-NMR investigations, it was found that only reactions involving SH-Leu yielded the expected thiol-Michael product, an observation that was attributed to the steric hindrance of the bulkier nature of SH-Phe. The fast reaction rates and complete acrylate/maleimide conversion obtained with SH-Leu at pH 7.2 and higher aided the direct elimination of SH-Phe as a potential thiol for the synthesis of the peptide mimetic. Based on the initial studies, for the pH-promoted gelation system, the proposed Ac-Pro-Leu-Gly-SLeu-Leu-Gly-NEtSH was kept unmodified. The subtle difference in pKa values between SH-Leu (thioester thiol) and the terminal cysteamine thiol from theoretical conditions should be enough to effect a 'pseudo' intramolecular TTE. In polar protic solvents and under basic aqueous conditions, TDP successfully undergoes a 'pseudo' intramolecular TTE reaction to yield an α,ω-dithiol tripeptide, HSLeu-Leu-Gly-NEtSH. The pH dependence of thiolate ion generation by the cysteamine thiol aided the incorporation of the needed stimulus (pH) for the overall success of TTE (activation step) - thiol-Michael addition (crosslinking) strategy. Secondly, with potential biomedical applications in focus, the susceptibility of TDP, like other thioesters, to intermolecular TTE reaction was probed with a group of thiols of varying thiol pKa values, since biological milieu characteristically contain peptide/protein thiols. L-cysteine, which is a biologically relevant thiol, and a small molecular weight thiol, methylthioglycolate both with relatively similar thiol pKa, values, led to an increase concentration of the dithiol crosslinker when reacted with TDP. In the presence of acidic thiols (p-NTP and 4MBA), a decrease in the dithiol concentration was observed, an observation that can be attributed to the inability of the TTE tetrahedral intermediate to dissociate into exchange products and is in line with pKa requirements for successful TTE reaction. These results additionally makes TDP more attractive and the potentially the first crosslinker precursor for applications in biologically relevant media. Finally, the ability of TDP to promote pH-sensitive in situ gel formation was probed with maleimide functionalized 4-arm polyethylene glycol polymers in tris-buffered media of varying pHs. When a 1:1 thiol: maleimide molar ratio was used, TDP-PEG4MAL hydrogels formed within 3, 12 and 24 hours at pH values of 8.5, 8.0 and 7.5 respectively. However, gelation times of 3, 5 and 30 mins were observed for the same pH trend when the thiol: maleimide molar was increased to 2:1. A direct correlation of thiol content with G' of the gels at each pH could also be drawn by comparing gels with thiol: maleimide ratios of 1:1 to those with 2:1 thiol: maleimide mole ratios. This is supported by the fact that the storage modulus (G') is linearly dependent on the crosslinking density of the polymer. The values of initial G′ for all gels ranged between (200 - 5000 Pa), which falls in the range of elasticities of certain tissue microenvironments for example brain tissue 200 - 1000 Pa and adipose tissue (2500 - 3500 Pa). Knowledge so far gained from the study on the ability to design and tune the exchange reaction of thioester containing peptide mimetic will give those working in the field further insight into the development of new sequences tailored towards specific applications. TTE substrate design using peptide mimetic as presented in this work has revealed interesting new insights considering the state-of-the-art. Using the results obtained as reference, the strategy provides a possibility to extend the concept to the controlled delivery of active molecules needed for other robust and high yielding crosslinking reactions for biomedical applications. Application for this sequentially coupled functional system could be seen e.g. in the treatment of inflamed tissues associated with urinary tract like bladder infections for which pH levels above 7 were reported. By the inclusion of cell adhesion peptide motifs, the hydrogel network formed at this pH could act as a new support layer for the healing of damage epithelium as shown in interfacial gel formation experiments using TDP and PEG4MAL droplets. The versatility of the thiodepsipeptide sequence, Ac-Pro-Leu-Gly-SLeu-Leu-Gly-(TDPo) was extended for the design and synthesis of a MMP-sensitive 4-arm PEG-TDPo conjugate. The purported cleavage of TDPo at the Gly-SLeu bond yields active thiol units for subsequent reaction of orthogonal Michael acceptor moieties. One of the advantages of stimuli-promoted in situ crosslinking systems using short peptides should be the ease of design of required peptide molecules due to the predictability of peptide functions their sequence structure. Consequently the functionalisation of a 4-arm PEG core with the collagenase active TDPo sequence yielded an MMP-sensitive 4-arm thiodepsipeptide-PEG conjugate (PEG4TDPo) substrate. Cleavage studies using thiol flourometric assay in the presence of MMPs -2 and -9 confirmed the susceptibility of PEG4TDPo towards these enzymes. The resulting time-dependent increase in fluorescence intensity in the presence of thiol assay signifies the successful cleavage of TDPo at the Gly-SLeu bond as expected. It was observed that the cleavage studies with thiol flourometric assay introduces a sigmoid non-Michaelis-Menten type kinetic profile, hence making it difficult to accurately determine the enzyme cycling parameters, kcat and KM . Gelation studies with PEG4MAL at 10 \% wt. concentrations revealed faster gelation with MMP-2 than MMP-9 with 28 and 40 min gelation times respectively. Possible contributions by hydrolytic cleavage of PEG4TDPo has resulted in the gelation of PEG4MAL blank samples but only after 60 minutes of reaction. From theoretical considerations, the simultaneous gelation reaction would be expected to more negatively impact the enzymatic than hydrolytic cleavage. The exact contributions from hydrolytic cleavage of PEG4TDPo would however require additional studies. In summary this new and simplified in situ crosslinking system using peptide-based crosslinker precursors with tuneable properties exhibited in situ crosslinking gelation kinetics on similar levels with already active dithiols reported. The advantageous on-demand functionality associated with its pH-sensitivity and physiological compatibility makes it a strong candidate worth further research as biomedical applications in general and on-demand material synthesis is concerned. Results from MMP-promoted gelation system unveils a simple but unexplored approach for in situ synthesis of covalently crosslinked soft materials, that could lead to the development of an alternative pathway in addressing cancer metastasis by making use of MMP overexpression as a trigger. This goal has so far not being reach with MMP inhibitors despite the extensive work this regard.},
  language  = {en}
}
@phdthesis{Rinne2024,
  author    = {Rinne, Theresa Charlotte},
  title     = {The effects of nutrients on bone stem cell function and regeneration},
  school      = {Universit{\"a}t Potsdam},
  pages     = {V, 134},
  year      = {2024},
  abstract  = {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.},
  language  = {en}
}
@phdthesis{Drobyshev2023,
  author    = {Drobyshev, Evgenii},
  title     = {Toxic or beneficial? What is the role of food-relevant selenium species selenoneine?},
  doi       = {10.25932/publishup-57379},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-573794},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xiv, 100},
  year      = {2023},
  abstract  = {Selenium (Se) is an essential trace element that is ubiquitously present in the environment in small concentrations. Essential functions of Se in the human body are manifested through the wide range of proteins, containing selenocysteine as their active center. Such proteins are called selenoproteins which are found in multiple physiological processes like antioxidative defense and the regulation of thyroid hormone functions. Therefore, Se deficiency is known to cause a broad spectrum of physiological impairments, especially in endemic regions with low Se content. Nevertheless, being an essential trace element, Se could exhibit toxic effects, if its intake exceeds tolerable levels. Accordingly, this range between deficiency and overexposure represents optimal Se supply. However, this range was found to be narrower than for any other essential trace element. Together with significantly varying Se concentrations in soil and the presence of specific bioaccumulation factors, this represents a noticeable difficulty in the assessment of Se epidemiological status. While Se is acting in the body through multiple selenoproteins, its intake occurs mainly in form of small organic or inorganic molecular mass species. Thus, Se exposure not only depends on daily intake but also on the respective chemical form, in which it is present. The essential functions of selenium have been known for a long time and its primary forms in different food sources have been described. Nevertheless, analytical capabilities for a comprehensive investigation of Se species and their derivatives have been introduced only in the last decades. A new Se compound was identified in 2010 in the blood and tissues of bluefin tuna. It was called selenoneine (SeN) since it is an isologue of naturally occurring antioxidant ergothioneine (ET), where Se replaces sulfur. In the following years, SeN was identified in a number of edible fish species and attracted attention as a new dietary Se source and potentially strong antioxidant. Studies in populations whose diet largely relies on fish revealed that SeN represents the main non-protein bound Se pool in their blood. First studies, conducted with enriched fish extracts, already demonstrated the high antioxidative potential of SeN and its possible function in the detoxification of methylmercury in fish. Cell culture studies demonstrated, that SeN can utilize the same transporter as ergothioneine, and SeN metabolite was found in human urine. Until recently, studies on SeN properties were severely limited due to the lack of ways to obtain the pure compound. As a predisposition to this work was firstly a successful approach to SeN synthesis in the University of Graz, utilizing genetically modified yeasts. In the current study, by use of HepG2 liver carcinoma cells, it was demonstrated, that SeN does not cause toxic effectsup to 100 μM concentration in hepatocytes. Uptake experiments showed that SeN is not bioavailable to the used liver cells. In the next part a blood-brain barrier (BBB) model, based on capillary endothelial cells from the porcine brain, was used to describe the possible transfer of SeN into the central nervous system (CNS). The assessment of toxicity markers in these endothelial cells and monitoring of barrier conditions during transfer experiments demonstrated the absence of toxic effects from SeN on the BBB endothelium up to 100 μM concentration. Transfer data for SeN showed slow but substantial transfer. A statistically significant increase was observed after 48 hours following SeN incubation from the blood-facing side of the barrier. However, an increase in Se content was clearly visible already after 6 hours of incubation with 1 μM of SeN. While the transfer rate of SeN after application of 0.1 μM dose was very close to that for 1 μM, incubation with 10 μM of SeN resulted in a significantly decreased transfer rate. Double-sided application of SeN caused no side-specific transfer of SeN, thus suggesting a passive diffusion mechanism of SeN across the BBB. This data is in accordance with animal studies, where ET accumulation was observed in the rat brain, even though rat BBB does not have the primary ET transporter - OCTN1. Investigation of capillary endothelial cell monolayers after incubation with SeN and reference selenium compounds showed no significant increase of intracellular selenium concentration. Speciesspecific Se measurements in medium samples from apical and basolateral compartments, as good as in cell lysates, showed no SeN metabolization. Therefore, it can be concluded that SeN may reach the brain without significant transformation. As the third part of this work, the assessment of SeN antioxidant properties was performed in Caco-2 human colorectal adenocarcinoma cells. Previous studies demonstrated that the intestinal epithelium is able to actively transport SeN from the intestinal lumen to the blood side and accumulate SeN. Further investigation within current work showed a much higher antioxidant potential of SeN compared to ET. The radical scavenging activity after incubation with SeN was close to the one observed for selenite and selenomethionine. However, the SeN effect on the viability of intestinal cells under oxidative conditions was close to the one caused by ET. To answer the question if SeN is able to be used as a dietary Se source and induce the activity of selenoproteins, the activity of glutathione peroxidase (GPx) and the secretion of selenoprotein P (SelenoP) were measured in Caco-2 cells, additionally. As expected, reference selenium compounds selenite and selenomethionine caused efficient induction of GPx activity. In contrast to those SeN had no effect on GPx activity. To examine the possibility of SeN being embedded into the selenoproteome, SelenoP was measured in a culture medium. Even though Caco-2 cells effectively take up SeN in quantities much higher than selenite or selenomethionine, no secretion of SelenoP was observed after SeN incubation. Summarizing, we can conclude that SeN can hardly serve as a Se source for selenoprotein synthesis. However, SeN exhibit strong antioxidative properties, which appear when sulfur in ET is exchanged by Se. Therefore, SeN is of particular interest for research not as part of Se metabolism, but important endemic dietary antioxidant.},
  language  = {en}
}