@phdthesis{Schjeide2021,
  author    = {Schjeide, Brit-Maren},
  title     = {Development and characterization of the MoN-Light BoNT assay to determine the toxicity of botulinum neurotoxin in motor neurons differentiated from CRISPR-modified induced pluripotent stem cells},
  doi       = {10.25932/publishup-51627},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-516278},
  school      = {Universit{\"a}t Potsdam},
  pages     = {e, xviii, 265},
  year      = {2021},
  abstract  = {Botulinum neurotoxin (BoNT) is produced by the anaerobic bacterium Clostridium botulinum. It is one of the most potent toxins found in nature and can enter motor neurons (MN) to cleave proteins necessary for neurotransmission, resulting in flaccid paralysis. The toxin has applications in both traditional and esthetic medicine. Since BoNT activity varies between batches despite identical protein concentrations, the activity of each lot must be assessed. The gold standard method is the mouse lethality assay, in which mice are injected with a BoNT dilution series to determine the dose at which half of the animals suffer death from peripheral asphyxia. Ethical concerns surrounding the use of animals in toxicity testing necessitate the creation of alternative model systems to measure the potency of BoNT. Prerequisites of a successful model are that it is human specific; it monitors the complete toxic pathway of BoNT; and it is highly sensitive, at least in the range of the mouse lethality assay. One model system was developed by our group, in which human SIMA neuroblastoma cells were genetically modified to express a reporter protein (GLuc), which is packaged into neurosecretory vesicles, and which, upon cellular depolarization, can be released - or inhibited by BoNT - simultaneously with neurotransmitters. This assay has great potential, but includes the inherent disadvantages that the GLuc sequence was randomly inserted into the genome and the tumor cells only have limited sensitivity and specificity to BoNT. This project aims to improve these deficits, whereby induced pluripotent stem cells (iPSCs) were genetically modified by the CRISPR/Cas9 method to insert the GLuc sequence into the AAVS1 genomic safe harbor locus, precluding genetic disruption through non-specific integrations. Furthermore, GLuc was modified to associate with signal peptides that direct to the lumen of both large dense core vesicles (LDCV), which transport neuropeptides, and synaptic vesicles (SV), which package neurotransmitters. Finally, the modified iPSCs were differentiated into motor neurons (MNs), the true physiological target of BoNT, and hypothetically the most sensitive and specific cells available for the MoN-Light BoNT assay. iPSCs were transfected to incorporate one of three constructs to direct GLuc into LDCVs, one construct to direct GLuc into SVs, and one "no tag" GLuc control construct. The LDCV constructs fused GLuc with the signal peptides for proopiomelanocortin (hPOMC-GLuc), chromogranin-A (CgA-GLuc), and secretogranin II (SgII-GLuc), which are all proteins found in the LDCV lumen. The SV construct comprises a VAMP2-GLuc fusion sequence, exploiting the SV membrane-associated protein synaptobrevin (VAMP2). The no tag GLuc expresses GLuc non-specifically throughout the cell and was created to compare the localization of vesicle-directed GLuc. The clones were characterized to ensure that the GLuc sequence was only incorporated into the AAVS1 safe harbor locus and that the signal peptides directed GLuc to the correct vesicles. The accurate insertion of GLuc was confirmed by PCR with primers flanking the AAVS1 safe harbor locus, capable of simultaneously amplifying wildtype and modified alleles. The PCR amplicons, along with an insert-specific amplicon from candidate clones were Sanger sequenced to confirm the correct genomic region and sequence of the inserted DNA. Off-target integrations were analyzed with the newly developed dc-qcnPCR method, whereby the insert DNA was quantified by qPCR against autosomal and sex-chromosome encoded genes. While the majority of clones had off-target inserts, at least one on-target clone was identified for each construct. Finally, immunofluorescence was utilized to localize GLuc in the selected clones. In iPSCs, the vesicle-directed GLuc should travel through the Golgi apparatus along the neurosecretory pathway, while the no tag GLuc should not follow this pathway. Initial analyses excluded the CgA-GLuc and SgII-GLuc clones due to poor quality protein visualization. The colocalization of GLuc with the Golgi was analyzed by confocal microscopy and quantified. GLuc was strongly colocalized with the Golgi in the hPOMC-GLuc clone (r = 0.85±0.09), moderately in the VAMP2-GLuc clone (r = 0.65±0.01), and, as expected, only weakly in the no tag GLuc clone (r = 0.44±0.10). Confocal microscopy of differentiated MNs was used to analyze the colocalization of GLuc with proteins associated with LDCVs and SVs, SgII in the hPOMC-GLuc clone (r = 0.85±0.08) and synaptophysin in the VAMP2-GLuc clone (r = 0.65±0.07). GLuc was also expressed in the same cells as the MN-associated protein, Islet1. A significant portion of GLuc was found in the correct cell type and compartment. However, in the MoN-Light BoNT assay, the hPOMC-GLuc clone could not be provoked to reliably release GLuc upon cellular depolarization. The depolarization protocol for hPOMC-GLuc must be further optimized to produce reliable and specific release of GLuc upon exposure to a stimulus. On the other hand, the VAMP2-GLuc clone could be provoked to release GLuc upon exposure to the muscarinic and nicotinic agonist carbachol. Furthermore, upon simultaneous exposure to the calcium chelator EGTA, the carbachol-provoked release of GLuc could be significantly repressed, indicating the detection of GLuc was likely associated with vesicular fusion at the presynaptic terminal. The application of the VAMP2-GLuc clone in the MoN-Light BoNT assay must still be verified, but the results thus far indicate that this clone could be appropriate for the application of BoNT toxicity assessment.},
  language  = {en}
}
@phdthesis{Burkhardt2021,
  author    = {Burkhardt, Wiebke},
  title     = {Role of dietary sulfonates in the stimulation of gut bacteria promoting intestinal inflammation},
  doi       = {10.25932/publishup-51368},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-513685},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XX, 79, XXXIX},
  year      = {2021},
  abstract  = {The interplay between intestinal microbiota and host has increasingly been recognized as a major factor impacting health. Studies indicate that diet is the most influential determinant affecting the gut microbiota. A diet rich in saturated fat was shown to stimulate the growth of the colitogenic bacterium Bilophila wadsworthia by enhancing the secretion of the bile acid taurocholate (TC). The sulfonated taurine moiety of TC is utilized as a substrate by B. wadsworthia. The resulting overgrowth of B. wadsworthia was accompanied by an increased incidence and severity of colitis in interleukin (IL)-10-deficient mice, which are genetically prone to develop inflammation. Based on these findings, the question arose whether the intake of dietary sulfonates also stimulates the growth of B. wadsworthia and thereby promotes intestinal inflammation in genetically susceptible mice. Dietary sources of sulfonates include green vegetables and cyanobacteria, which contain the sulfolipids sulfoquinovosyl diacylglycerols (SQDG) in considerable amounts. Based on literature reports, the gut commensal Escherichia coli is able to release sulfoquinovose (SQ) from SQDG and in further steps, convert SQ to 2,3-dihydroxypropane-1-sulfonate (DHPS) and dihydroxyacetone phosphate. DHPS may then be utilized as a growth substrate by B. wadsworthia, which results in the formation of sulfide. Both, sulfide formation and a high abundance of B. wadsworthia have been associated with intestinal inflammation. In the present study, conventional IL-10-deficient mice were fed either a diet supplemented with the SQDG-rich cyanobacterium Spirulina (20\%, SD) or a control diet. In addition SQ, TC, or water were orally applied to conventional or gnotobiotic IL-10-deficient mice. The gnotobiotic mice harbored a simplified human intestinal microbiota (SIHUMI) either with or without B. wadsworthia. During the intervention period, the body weight of the mice was monitored, the colon permeability was assessed and fecal samples were collected. After the three-week intervention, the animals were examined with regard to inflammatory parameters, microbiota composition and sulfonate concentrations in different intestinal sites. None of the mice treated with the above-mentioned sulfonates showed weight loss or intestinal inflammation. Solely mice fed SD or gavaged with TC displayed a slight immune response. These mice also displayed an altered microbiota composition, which was not observed in mice gavaged with SQ. The abundance of B. wadsworthia was strongly reduced in mice fed SD, while that of mice treated with SQ or TC was in part slightly increased. The intestinal SQ-concentration was elevated in mice orally treated with SD or SQ, whereas neither TC nor taurine concentrations were consistently elevated in mice gavaged with TC. Additional colonization of SIHUMI mice with B. wadsworthia resulted in a mild inflammatory response, but only in mice treated with TC. In general, TC-mediated effects on the immune system and abundance of B. wadsworthia were not as strong as described in the literature. In summary, neither the tested dietary sulfonates nor TC led to bacteria-induced intestinal inflammation in the IL-10-deficient mouse model, which was consistently observed in both conventional and gnotobiotic mice. For humans, this means that foods containing SQDG, such as spinach or Spirulina, do not increase the risk of intestinal inflammation.},
  language  = {en}
}
@phdthesis{Baeseler2021,
  author    = {Baeseler, Jessica},
  title     = {Trace element effects on longevity and neurodegeneration with focus on C. elegans},
  school      = {Universit{\"a}t Potsdam},
  pages     = {X,114,VIII},
  year      = {2021},
  abstract  = {The trace elements zinc and manganese are essential for human health, especially due to their enzymatic and protein stabilizing functions. If these elements are ingested in amounts exceeding the requirements, regulatory processes for maintaining their physiological concentrations (homeostasis) can be disturbed. Those homeostatic dysregulations can cause severe health effects including the emergence of neurodegenerative disorders such as Parkinson's disease (PD). The concentrations of essential trace elements also change during the aging process. However, the relations of cause and consequence between increased manganese and zinc uptake and its influence on the aging process and the emergence of the aging-associated PD are still rarely understood. This doctoral thesis therefore aimed to investigate the influence of a nutritive zinc and/or manganese oversupply on the metal homeostasis during the aging process. For that, the model organism Caenorhabditis elegans (C. elegans) was applied. This nematode suits well as an aging and PD model due to properties such as its short life cycle and its completely sequenced, genetically amenable genome. Different protocols for the propagation of zinc- and/or manganese-supplemented young, middle-aged and aged C. elegans were established. Therefore, wildtypes, as well as genetically modified worm strains modeling inheritable forms of parkinsonism were applied. To identify homeostatic and neurological alterations, the nematodes were investigated with different methods including the analysis of total metal contents via inductively-coupled plasma tandem mass spectrometry, a specific probe-based method for quantifying labile zinc, survival assays, gene expression analysis as well as fluorescence microscopy for the identification and quantification of dopaminergic neurodegeneration.. During aging, the levels of iron, as well as zinc and manganese increased.. Furthermore, the simultaneous oversupply with zinc and manganese increased the total zinc and manganese contents to a higher extend than the single metal supplementation. In this relation the C. elegans metallothionein 1 (MTL-1) was identified as an important regulator of metal homeostasis. The total zinc content and the concentration of labile zinc were age-dependently, but differently regulated. This elucidates the importance of distinguishing these parameters as two independent biomarkers for the zinc status. Not the metal oversupply, but aging increased the levels of dopaminergic neurodegeneration. Additionally, nearly all these results yielded differences in the aging-dependent regulation of trace element homeostasis between wildtypes and PD models. This confirms that an increased zinc and manganese intake can influence the aging process as well as parkinsonism by altering homeostasis although the underlying mechanisms need to be clarified in further studies.},
  language  = {en}
}
@phdthesis{Mancini2021,
  author    = {Mancini, Carola},
  title     = {Analysis of the effects of age-related changes of metabolic flux on brown adipocyte formation and function},
  doi       = {10.25932/publishup-51266},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xvii, 134},
  year      = {2021},
  abstract  = {Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis, thereby allowing mammals to maintain a constant body temperature in a cold environment. Thermogenic capacity of this tissue is due to a high mitochondrial density and expression of uncoupling protein 1 (UCP1), a unique brown adipocyte marker which dissipates the mitochondrial proton gradient to produce heat instead of ATP. BAT is actively involved in whole-body metabolic homeostasis and during aging there is a loss of classical brown adipose tissue with concomitantly reduced browning capacity of white adipose tissue. Therefore, an age-dependent decrease of BAT-related energy expenditure capacity may exacerbate the development of metabolic diseases, including obesity and type 2 diabetes mellitus. Given that direct effects of age-related changes of BAT-metabolic flux have yet to be unraveled, the aim of the current thesis is to investigate potential metabolic mechanisms involved in BAT-dysfunction during aging and to identify suitable metabolic candidates as functional biomarkers of BAT-aging. To this aim, integration of transcriptomic, metabolomic and proteomic data analyses of BAT from young and aged mice was performed, and a group of candidates with age-related changes was revealed. Metabolomic analysis showed age-dependent alterations of metabolic intermediates involved in energy, nucleotide and vitamin metabolism, with major alterations regarding the purine nucleotide pool. These data suggest a potential role of nucleotide intermediates in age-related BAT defects. In addition, the screening of transcriptomic and proteomic data sets from BAT of young and aged mice allowed identification of a 60-kDa lysophospholipase, also known as L-asparaginase (Aspg), whose expression declines during BAT-aging. Involvement of Aspg in brown adipocyte thermogenic function was subsequently analyzed at the molecular level using in vitro approaches and animal models. The findings revealed sensitivity of Aspg expression to β3-adrenergic activation via different metabolic cues, including cold exposure and treatment with β3-adrenergic agonist CL. To further examine ASPG function in BAT, an over-expression model of Aspg in a brown adipocyte cell line was established and showed that these cells were metabolically more active compared to controls, revealing increased expression of the main brown-adipocyte specific marker UCP1, as well as higher lipolysis rates. An in vitro loss-of-function model of Aspg was also functionally analyzed, revealing reduced brown adipogenic characteristics and an impaired lipolysis, thus confirming physiological relevance of Aspg in brown adipocyte function. Characterization of a transgenic mouse model with whole-body inactivation of the Aspg gene (Aspg-KO) allowed investigation of the role of ASPG under in vivo conditions, indicating a mild obesogenic phenotype, hypertrophic white adipocytes, impairment of the early thermogenic response upon cold-stimulation and dysfunctional insulin sensitivity. Taken together, these data show that ASPG may represent a new functional biomarker of BAT-aging that regulates thermogenesis and therefore a potential target for the treatment of age-related metabolic disease.},
  language  = {en}
}
@phdthesis{Rausch2021,
  author    = {Rausch, Ann-Kristin},
  title     = {Development of LC-MS/MS Multi-Methods for the Analysis of Contaminants and Residues},
  school      = {Universit{\"a}t Potsdam},
  pages     = {IX, 234, v},
  year      = {2021},
  abstract  = {Mycotoxins are secondary metabolites produced by several filamentous fungal species, thus occurring ubiquitously in the environment and food. While the heterogeneous group shows differences in their bioavailability and toxicity, the low-molecular-weight xenobiotics are capable of impacting human and animal health acutely and chronically. Therefore, maximum levels for the major mycotoxins in food and feed are regulated in the current European legislation. Besides free mycotoxins, naturally occurring modified mycotoxins are gaining more attention in recent years. Modified mycotoxins constitute toxins altered by plants, microorganisms, and living organisms in different metabolic pathways or food processing steps. The toxicological relevant compounds often co-occur with their free forms in infested food and feed. Thus, the toxins may contribute to the overall toxicity of mycotoxins, wherefore their presence and toxicity should be considered in risk assessment. Until now, however, there are no regulated limits for modified mycotoxins within the European Union. In this thesis, rapid, sensitive, and robust methods for the analysis of mycotoxins and their modified forms were developed and validated using state-of-the-art high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) systems. Firstly, two analytical methods for determining 38 mycotoxins in cereals and 41 mycotoxins in beer were established since agricultural products count as the primary source of mycotoxin contamination. For the analysis of cereal samples, a QuEChERS- based extraction approach was pursued, while analytes from beer samples were extracted using an acetonitrile precipitation scheme. Validation in cereals, namely wheat, corn, rice, and barley, as well as in beer, demonstrated satisfactory results. To obtain information regarding the natural occurrence of mycotoxins in food products, the developed methods were applied to the analysis of several commercial samples partly produced worldwide. The Fusarium toxins deoxynivalenol and its conjugated metabolite deoxynivalenol-3-glucoside turned out to be the most abundant toxins. None of the other modified mycotoxins were quantified in the samples. However, one cereal sample showed traces of zearalenone- 14-sulfate below the limit of quantification. Moreover, pesticides, plant growth regulators, and tropane alkaloids were investigated in this thesis. Pesticides present biologically highly effective compounds applied in the environment to protect humans from the hazardous effects of pests. While plant growth regulators show similar functions, mainly improving agricultural production, tropane alkaloids are naturally occurring secondary metabolites mainly in the species of Solanaceae that may pose unintended poisoning of humans. The third part of the present thesis aimed to analyze cereal-relevant compounds simultaneously, wherefore a multi-method for the analysis of (modified) mycotoxins, pesticides, plant growth regulators, and tropane alkaloids was established. After processing the samples, this should be done in a single extraction step with subsequent one-time measurements. Various sample preparation procedures were compared, whereby an approach based on an acidified acetonitrile/water extraction, followed by an online clean-up, was finally chosen. The simultaneous determination of more than 350 analytes required an analytical tool that offered an increased resolving power, represented as an enhanced peak capacity, and the possibility of analyzing a broad polarity range. Thus, a two-dimensional LC-MS/MS system based on two different separation mechanisms that performed orthogonal to one another was used for the analysis. Validation of the developed method revealed good performance characteristics for most analytes, while subsequent application showed that 86\% of the samples were contaminated with at least one compound. In summary, this thesis provides novel insights into the analysis of food-relevant (modified) mycotoxins. Different sample preparation and LC-MS/MS approaches were introduced, resulting in the development of three new analytical methods. For the first time, such a high number of modified mycotoxins was included in multi-mycotoxin methods and a multi-method ranging both contaminants and residues. Although first steps towards the analysis of modified mycotoxins have been made, further research is needed to elucidate their (co-) occurrence and toxicological behavior in order to understand their relevance to human health in the future.},
  language  = {en}
}
@phdthesis{Hauffe2021,
  author    = {Hauffe, Robert},
  title     = {Investigating metabolic consequences of an HSP60 reduction during diet-induced obesity},
  doi       = {10.25932/publishup-50929},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-509294},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xxi, 116},
  year      = {2021},
  abstract  = {The mitochondrial chaperone complex HSP60/HSP10 facilitates mitochondrial protein homeostasis by folding more than 300 mitochondrial matrix proteins. It has been shown previously that HSP60 is downregulated in brains of type 2 diabetic (T2D) mice and patients, causing mitochondrial dysfunction and insulin resistance. As HSP60 is also decreased in peripheral tissues in T2D animals, this thesis investigated the effect of overall reduced HSP60 in the development of obesity and associated co-morbidities. To this end, both female and male C57Bl/6N control (i.e. without further alterations in their genome, Ctrl) and heterozygous whole-body Hsp60 knock-out (Hsp60+/-) mice, which exhibit a 50 \% reduction of HSP60 in all tissues, were fed a normal chow diet (NCD) or a highfat diet (HFD, 60 \% calories from fat) for 16 weeks and were subjected to extensive metabolic phenotyping including indirect calorimetry, NMR spectroscopy, insulin, glucose and pyruvate tolerance tests, vena cava insulin injections, as well as histological and molecular analysis. Interestingly, NCD feeding did not result in any striking phenotype, only a mild increase in energy expenditure in Hsp60+/- mice. Exposing mice to a HFD however revealed an increased body weight due to higher muscle mass in female Hsp60+/- mice, with a simultaneous decrease in energy expenditure. Additionally, these mice displayed decreased fasting glycemia. Opposingly, male Hsp60+/- compared to control mice showed lower body weight gain due to decreased fat mass and an increased energy expenditure, strikingly independent of lean mass. Further, only male Hsp60+/- mice display improved HOMA-IR and Matsuda insulin sensitivity indices. Despite the opposite phenotype in regards to body weight development, Hsp60+/- mice of both sexes show a significantly higher cell number, as well as a reduction in adipocyte size in the subcutaneous and gonadal white adipose tissue (sc/gWAT). Curiously, this adipocyte hyperplasia - usually associated with positive aspects of WAT function - is disconnected from metabolic improvements, as the gWAT of male Hsp60+/- mice shows mitochondrial dysfunction, oxidative stress, and insulin resistance. Transcriptomic analysis of gWAT shows an up regulation of genes involved in macroautophagy. Confirmatory, expression of microtubuleassociated protein 1A/1B light chain 3B (LC3), as a protein marker of autophagy, and direct measurement of lysosomal activity is increased in the gWAT of male Hsp60+/- mice. In summary, this thesis revealed a novel gene-nutrient interaction. The reduction of the crucial chaperone HSP60 did not have large effects in mice fed a NCD, but impacted metabolism during DIO in a sex-specific manner, where, despite opposing body weight and body composition phenotypes, both female and male Hsp60+/- mice show signs of protection from high fat diet-induced systemic insulin resistance.},
  language  = {en}
}
@phdthesis{RodriguezSillke2021,
  author    = {Rodriguez-Sillke, Yasmina},
  title     = {Der Einfluss von Nahrungsmittelantigenen auf die mukosale sowie periphere Hom{\"o}ostase und Entz{\"u}ndung bei chronisch entz{\"u}ndlichen Darmerkrankungen},
  school      = {Universit{\"a}t Potsdam},
  pages     = {134},
  year      = {2021},
  language  = {de}
}
@phdthesis{Nieschalke2021,
  author    = {Nieschalke, Kai},
  title     = {Proteinaddukte und Urinmetaboliten des Nagetierkanzerogens Methyleugenol als Biomarker der Exposition},
  school      = {Universit{\"a}t Potsdam},
  pages     = {142, XLIV},
  year      = {2021},
  language  = {de}
}
@phdthesis{HaferkornStarke2021,
  author    = {Haferkorn-Starke, Robert Christian},
  title     = {Entwicklung eines Lebensmitteluntersuchungssystems f{\"u}r mikrobielle Erreger mit Hilfe molekularbiologischer Methoden},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XVII, 239, vi},
  year      = {2021},
  language  = {de}
}
@phdthesis{LenihanGeels2020,
  author    = {Lenihan-Geels, Georgia Ngawai},
  title     = {The regulation of metabolic flexibility by p53 in skeletal muscle and brown adipose tissue},
  school      = {Universit{\"a}t Potsdam},
  year      = {2020},
  language  = {en}
}