@phdthesis{Schubert2015,
  author    = {Schubert, Andreas},
  title     = {Protein kinases as targets for the development of novel drugs against alveolar echinococcosis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-113694},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {The metacestode larval stage of the fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), one of the most lethal zoonosis of the northern hemisphere. The development of metacestode vesicles by asexual multiplication and the almost unrestricted infiltrative growth within the host organs is ensured from a population of undifferentiated, proliferative cells, so-called germinative cells. AE treatment options include surgery, if possible, as well as Benzimidazole-based chemotherapy (BZ). Given that the cellular targets of BZs, the -tubulins, are highly conserved between cestodes and humans, the chemotherapy is associated with considerable side-effects. Therefore, BZ can only be applied in parasitostatic doses and has to be given lifelong. Furthermore, the current anti-AE chemotherapy is ineffective in eliminating the germinative cell population of the parasite, which leads to remission of parasite growth as soon as therapy is discontinued. This work focuses on protein kinases involved in the proliferation and development of the parasite with the intention of developing novel anti-AE therapies. Polo-like kinases (Plks) are important regulators of the eukaryotic cell cycle and are involved in the regulation and formation of the mitotic spindles during the M-phase of the cell cycle. Plks have already been shown to be associated with deregulated cellular growth in human cancers and have been investigated as novel drug targets in the flatworm parasite Schistosoma mansoni. In the first part of this work, the characterisation of a novel and druggable parasite enzyme, EmPlk1, which is homologous to the polo-like kinase 1 (Plk1) of humans and S. mansoni (SmPlk1), is presented. Through in situ hybridisation, it could be demonstrated that emplk1 is specifically expressed in the Echinococcus germinative cells. Upon heterologous expression in the Xenopus oocyte system, EmPlk1 induced germinal vesicle breakdown, thus indicating that it is an active kinase. Furthermore, BI 2536, a compound originally designed to inhibit the human ortholog of EmPlk1, inhibited the EmPlk1 activity at a concentration of 25 nM. In vitro treatment of parasite vesicles with similar concentrations of BI 2536 led to the elimination of the germinative cells from Echinococcus larvae, thus preventing the growth and further development of the parasite. In in vitro cultivation systems for parasite primary cells, BI 2536 effectively inhibited the formation of new metacestode vesicles from germinative cells. Thus, BI 2536 has profound anti-parasitic activities in vitro at concentrations well within the range of plasma levels measured after the administration of safe dosages to patients (50 nM after 24 h). This implies that EmPlk1 is a promising new drug target for the development of novel anti-AE drugs that would specifically affect the parasite's stem cell population, namely the only parasite cells capable of proliferation. In addition to the chemotherapeutic aspects of this work, the inhibitor BI 2536 could be further used to study the function of stem cells in this model organism, utilising a method of injection of parasite stem cells into metacestode vesicles, for instance, as has been developed in this work. In the second part of this work, a novel receptor tyrosine kinase, the Venus flytrap kinase receptor (EmVKR) of E. multilocularis has been characterised. Members of this class of single-pass transmembrane receptors have recently been discovered in the related trematode S. mansoni and are associated with the growth and differentiation of sporocyst germinal cells and ovocytes. The ortholog receptor in EmVKR is characterised by an unusual domain composition of an extracellular Venus flytrap module (VFT), which shows significant similarity to GABA receptors, such as the GABAB receptor (γ-amino butyric acid type B) and is linked through a single transmembrane domain to an intracellular tyrosine kinase domain with similarities to the kinase domains of human insulin receptors. Based upon the size (5112bp) of emvkr and nucleotide sequence specificities, efforts have been made to isolate the gene from cell culture samples to study the ligand for the activation of this receptor type in Xenopus oocytes. To date, this type of receptor has only been described in invertebrates, thus making it an attractive target for drug screening. In a first trial, the ATP competitive inhibitor AG 1024 was tested in our in vitro cell culture. In conclusion, the EmVKR represents a novel receptor tyrosine kinase in E. multilocularis. Further efforts have to be made to identify the activating ligand of the receptor and its cellular function, which might strengthen the case for EmVKR as a potential drug target. The successful depletion of stem cells in the metacestode vesicle by the Plk1 inhibitor BI 2536 gives rise to optimising the chemical component for EmPlk1 as a new potential drug target. Furthermore, this inhibitor opens a new cell culture technique with high potential to study the cellular behaviour and influencing factors of stem cells in vitro.},
  subject      = {Chemotherapie},
  language  = {en}
}
@phdthesis{Obier2010,
  author    = {Obier, Nadine},
  title     = {Defining the end of pluripotency in mouse embryonic stem cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-53722},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Stammzellen mit ihrer besonderen F{\"a}higkeit sich selbst zu erneuern und zu differenzieren stellen einen faszinierenden Zelltyp f{\"u}r Grundlagenforschung und angewandte Wissenschaften dar. Pluripotente embryonale Stammzellen (ES Zellen), die aus Zellen der inneren Zellmasse von Pr{\"a}implantationsembryonen etabliert werden, k{\"o}nnen ekto-, meso- und endodermale Zelltypen sowie Keimzellen hervorbringen. Im Gegensatz dazu sind multipotente adulte Stammzellen in ihrem Entwicklungspotential eingeschr{\"a}nkt, sie differenzieren sich zu allen Zelltypen ihres Gewebes. Zum Beispiel h{\"a}matopoetische Stammzellen (HSZs), die sich in Blut-bildenden Geweben wie dem Knochenmark befinden, verm{\"o}gen sich in alle Blutzellen zu differenzieren. W{\"a}hrend der Differenzierung von Stammzellen {\"a}ndert sich nicht deren Genom, sondern ihre epigenetische Regulation. Durch epigenetische Mechanismen werden Zelltypen mit verschiedensten Ph{\"a}notypen und Funktionen generiert. F{\"u}r Stammzelltherapien ist ein tieferes Verst{\"a}ndnis des Zusammenhangs von Epigenom und zellul{\"a}rer Funktion wichtig. Im Rahmen dieser Dissertation war es mein Ziel, differenzierende Stammzellkulturen auf ihre Genexpression, ihre Chromatinregulation und ihr Differenzierungspotiential hin zu analysieren. Um Histonmodifikationen, die einen m{\"o}glichen Mechanismus epigenetischer Regulation darstellen, global untersuchen zu k{\"o}nnen, sind zun{\"a}chst, durchusszytometrische Protokolle etabliert worden, die die Analyse einzelner Zellen erm{\"o}glichen sollten. Mit dieser Methode konnten reduzierte Levels von Histonazetylierung in differenzierten ES Zellen gezeigt werden. Im Gegensatz dazu beobachtete ich vergleichbare Levels von Histonazetylierung in unreifen und reifen Knochenmarkzellen. Zus{\"a}tzlich untersuchte ich die Wirkung des Histondeazetylase-Inhibitors (HDI) Trichostatin A (TSA) auf Knochenmarkzellkulturen, in denen auch HSZs enhalten sind. Nach Behandlung mit TSA erh{\"o}hte sich der Anteil von Zellen mit in vitro und in vivo h{\"a}matopoetischer Aktivit{\"a}t, w{\"a}hrend vor allem differenzierte Zellen in Apoptose gingen. Außerdem wurde der Verlust der Pluripotenz in differenzierenden ES Zellkulturen untersucht. Marker-basierte Analysen und funktionelle Tests wurden mit ES Zellen durchgef{\"u}hrt, die kurzfristig in vitro differenziert wurden. Es stellte sich heraus, dass nach funktionellen Gesichtspunkten die Pluripotenz bereits nach 2 Tagen Differenzierung deutlich reduziert war, beurteilt anhand der F{\"a}higkeit Kolonien zu bilden, embryoide K{\"o}rperchen (EK) zu formieren und zu kontrahierenden Herzmuskelzelltypen zu differenzieren. Im Gegensatz dazu verringerte sich die Expression von Pluripotenzmarkern erst zu sp{\"a}teren Zeitpunkten. Ich habe weiterhin beobachten k{\"o}nnen, dass die Wahl des Differenzierungssystems (Aggregations-EK, klonale EKs oder als adh{\"a}rente Einzelzellschicht) einen Einfluss auf den Fortschritt und die Homogenit{\"a}t der Differenzierung hatte. Um das Ende der Pluripotenz genauer zu untersuchen, wurden differenzierte ES Zellen zur{\"u}ck in ES Zellkulturbedingungen gebracht. Die Ergebnisse deuten an, dass 3 Tage differenzierte ES Zellen einen Punkt {\"u}berschritten haben, an dem eine R{\"u}ckkehr zur Pluripotenz allein durch Kulturbedingungen noch m{\"o}glich ist. Durch die Behandlung mit HDIs starben selektiv differenzierte ES Zellen. Des Weiteren war es Ziel dieser Arbeit, den Einuss von EED - einer essentiellen Untereinheit des Histon-methylierenden Polycomb repressive complex 2 (PRC2) - auf das Chromatin und die Funktion von ES Zellen hin zu analysieren. ES Zellen ohne EED wiesen neben dem bereits bekannten Verlust der Trimethylierung von Histon 3 an Lysin 27 (H3K27me3), global reduzierte H3K9me3 Levels sowie erh{\"o}hte Histonazetylierung auf. Trotz typischer ES Zell-Morphologie und normaler Expression von Pluripotenzgenen, besaßen EED knockout (KO)ES Zellen eine ver{\"a}nderte Organisation der Heterochromatinstruktur im Zellkern, eine verlangsamte Chromatinmobilit{\"a}t und Probleme bei der Differenzierung. Zusammenfassend gew{\"a}hren meine Daten Einblick in die epigenetische Regulation von Stammzellen. Im Besonderen konnte ich zeigen, dass die Behandlung mit HDIs f{\"u}r differenzierende Knochenmarkzellen und differenzierende ES Zellen nachteilig war und zu deren selektivem Zelltod f{\"u}hrte. Die hier durchgef{\"u}hrten Analysen ergaben, dass ES Zellen nach 3 Tagen Differenzierung das Ende der Pluripotenz erreicht hatten. Schließlich zeigten die Versuche mit EED KO ES Zellen, dass sie sich zwar selbst erneuerten und morphologisch identisch mit wildtypischen ES Zellen waren, jedoch Defekte bei der Differenzierung besaßen. Dies deutet darauf hin, dass EED nicht nur f{\"u}r undifferenzierte ES Zellen wichtig ist, sondern auch w{\"a}hrend der Differenzierung von Bedeutung ist.},
  subject      = {Stammzelle},
  language  = {en}
}
@phdthesis{Muehlemann2018,
  author    = {M{\"u}hlemann, Markus},
  title     = {Intestinal stem cells and the Na\(^+\)-D-Glucose Transporter SGLT1: potential targets regarding future therapeutic strategies for diabetes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169266},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The pancreas and the small intestine are pivotal organs acting in close synergism to regulate glucose metabolism. After absorption and processing of dietary glucose within the small intestine, insulin and glucagon are released from pancreatic islet cells to maintain blood glucose homeostasis. Malfunctions affecting either individual, organ-specific functions or the sophisticated interplay of both organs can result in massive complications and pathologic conditions. One of the most serious metabolic diseases of our society is diabetes mellitus (DM) that is hallmarked by a disturbance of blood glucose homeostasis. Type 1 (T1DM) and type 2 (T2DM) are the main forms of the disease and both are characterized by chronic hyperglycemia, a condition that evokes severe comorbidities in the long-term. In the past, several standard treatment options allowed a more or less adequate therapy for diabetic patients. Albeit there is much effort to develop new therapeutic interventions to treat diabetic patients in a more efficient way, no cure is available so far. In view of the urgent need for alternative treatment options, a more systemic look on whole organ systems, their biological relation and complex interplay is needed when developing new therapeutic strategies for DM. T1DM is hallmarked by an autoimmune-mediated destruction of the pancreatic β-cell mass resulting in a complete lack of insulin that is in most patients restored by applying a life-long recombinant insulin therapy. Therefore, novel regenerative medicine-based concepts focus on the derivation of bioartificial β-like cells from diverse stem cell sources in vitro that survive and sustain to secrete insulin after implantation in vivo. In this context, the first part of this thesis analyzed multipotent intestinal stem cells (ISCs) as alternative cell source to derive bioartificial, pancreatic β-like cells in vitro. From a translational perspective, intestinal stem cells pose a particularly attractive cell source since intestinal donor tissues could be obtained via minimal invasive endoscopy in an autologous way. Furthermore, intestinal and pancreatic cells both derive from the same developmental origin, the endodermal gut tube, favoring the differentiation process towards functional β-like cells. In this study, pancreas-specific differentiation of ISCs was induced by the ectopic expression of the pancreatic transcription factor 1 alpha (Ptf1a), a pioneer transcriptional regulator of pancreatic fate. Furthermore, pancreatic lineage-specific culture media were applied to support the differentiation process. In general, ISCs grow in vitro in a 3D Matrigel®-based environment. Therefore, a 2D culture platform for ISCs was established to allow delivery and ectopic expression of Ptf1a with high efficiency. Next, several molecular tools were applied and compared with each other to identify the most suitable technology for Ptf1a delivery and expression within ISCs as well as their survival under the new established 2D conditions. Success of differentiation was investigated by monitoring changes in cellular morphology and induction of pancreatic differentiation-specific gene expression profiles. In summary, the data of this project part suggest that Ptf1a harbors the potential to induce pancreatic differentiation of ISCs when applying an adequate differentiation media. However, gene expression analysis indicated rather an acinar lineage-determination than a pancreatic β-cell-like specification. Nevertheless, this study proved ISCs not only as interesting stem cell source for the generation of pancreatic cell types with a potential use in the treatment of T1DM but alsoPtf1a as pioneer factor for pancreatic differentiation of ISCs in general. Compared to T1DM, T2DM patients suffer from hyperglycemia due to insulin resistance. In T2DM management, the maintenance of blood glucose homeostasis has highest priority and can be achieved by drugs affecting the stabilization of blood glucose levels. Recent therapeutic concepts are aiming at the inhibition of the intestinal glucose transporter Na+-D-Glucose cotransporter 1 (SGLT1). Pharmacological inhibition of SGLT1 results in reduced postprandial blood glucose levels combined with a sustained and increased Glucagon-like peptide 1 (GLP-1) secretion. So far, systemic side effects of this medication have not been addressed in detail. Of note, besides intestinal localization, SGLT1 is also expressed in various other tissues including the pancreas. In context of having a closer look also on the interplay of organs when developing new therapeutic approaches for DM, the second part of this thesis addressed the effects on pancreatic islet integrity after loss of SGLT1. The analyses comprised the investigation of pancreatic islet size, cytomorphology and function by the use of a global SGLT1 knockout (SGLT1-/-) mouse model. As SGLT1-/- mice develop the glucose-galactose malabsorption syndrome when fed a standard laboratory chow, these animals derived a glucose-deficient, fat-enriched (GDFE) diet. Wildtype mice on either standard chow (WTSC) or GDFE (WTDC) allowed the discrimination between diet- and knockout-dependent effects. Notably, GDFE fed mice showed decreased expression and function of intestinal SGLT1, while pancreatic SGLT1 mRNA levels were unaffected. Further, the findings revealed increased isled sizes, reduced proliferation- and apoptosis rates as well as an increased α-cell and reduced β-cell proportion accompanied by a disturbed cytomorphology in islets when SGLT1 function is lost or impaired. In addition, pancreatic islets were dysfunctional in terms of insulin- and glucagon-secretion. Moreover, the release of intestinal GLP-1, an incretin hormone that stimulates insulin-secretion in the islet, was abnormal after glucose stimulatory conditions. In summary, these data show that intestinal SGLT1 expression and function is nutrient dependent. The data obtained from the islet studies revealed an additional and new role of SGLT1 for maintaining pancreatic islet integrity in the context of structural, cytomorphological and functional aspects. With special emphasis on SGLT1 inhibition in diabetic patients, the data of this project indicate an urgent need for analyzing systemic side effects in other relevant organs to prove pharmacological SGLT1 inhibition as beneficial and safe. Altogether, the findings of both project parts of this thesis demonstrate that focusing on the molecular and cellular relationship and interplay of the small intestine and the pancreas could be of high importance in context of developing new therapeutic strategies for future applications in DM patients.},
  subject      = {Stammzelle},
  language  = {en}
}
@phdthesis{Koziol2014,
  author    = {Koziol, Uriel},
  title     = {Molecular and developmental characterization of the Echinococcus multilocularis stem cell system},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-105040},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {The metacestode larva of Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), one of the most dangerous zoonotic diseases in the Northern Hemisphere. Unlike "typical" metacestode larvae from other tapeworms, it grows as a mass of interconnected vesicles which infiltrates the liver of the intermediate host, continuously forming new vesicles in the periphery. From these vesicles, protoscoleces (the infective form for the definitive host) are generated by asexual budding. It is thought that in E. multilocularis, as in other flatworms, undifferentiated stem cells (so-called germinative cells in cestodes and neoblasts in free-living flatworms) are the sole source of new cells for growth and development. Therefore, this cell population should be of central importance for the progression of AE. In this work, I characterized the germinative cells of E. multilocularis, and demonstrate that they are indeed the only proliferating cells in metacestode vesicles. The germinative cells are a population of undifferentiated cells with similar morphology, and express high levels of transcripts of a novel non-autonomous retrotransposon family (ta-TRIMs). Experiments of recovery after hydroxyurea treatment suggest that individual germinative cells have extensive self-renewal capabilities. However, germinative cells also display heterogeneity at the molecular level, since only some of them express conserved homologs of fgfr, nanos and argonaute genes, suggesting the existence of several distinct sub-populations. Unlike free-living flatworms, cestode germinative cells lack chromatoid bodies. Furthermore, piwi and vasa orthologs are absent from the genomes of cestodes, and there is widespread expression of some conserved neoblast markers in E. multilocularis metacestode vesicles. All of these results suggest important differences between the stem cell systems of free-living flatworms and cestodes. Furthermore, I describe molecular markers for differentiated cell types, including the nervous system, which allow for the tracing of germinative cell differentiation. Using these molecular markers, a previously undescribed nerve net was discovered in metacestode vesicles. Because the metacestode vesicles are non-motile, and the nerve net of the vesicle is independent of the nervous system of the protoscolex, we propose that it could serve as a neuroendocrine system. By means of bioinformatic analyses, 22 neuropeptide genes were discovered in the E. multilocularis genome. Many of these genes are expressed in metacestode vesicles, as well as in primary cell preparations undergoing complete metacestode regeneration. This suggests a possible role for these genes in metacestode development. In line with this hypothesis, one putative neuropeptide (RGFI-amide) was able to stimulate the proliferation of primary cells at a concentration of 10-7 M, and the corresponding gene was upregulated during metacestode regeneration.},
  subject      = {Fuchsbandwurm},
  language  = {en}
}
@phdthesis{Jansch2021,
  author    = {Jansch, Charline},
  title     = {Effects of SLC2A3 copy number variants on neurodevelopment and glucose metabolism in ADHD patient-specific neurons},
  doi       = {10.25972/OPUS-21620},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216201},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Neuropsychiatric disorders, such as attention-deficit/hyperactivity disorder (ADHD), represent a burden which deeply impair the patient's life. Neurobiological research has therefore increasingly focused on the examination of brain neurotransmitter systems, such as the serotonin (5-HT) system, since a dysfunction has been repeatedly implicated in the pathology of these diseases. However, investigation of functional human neurons in vitro has been restricted by technical limitations for a long time until the discovery of human induced pluripotent stem cells (iPSCs) revolutionized the field of experimental disease models. Since the pathogenesis of neuropsychiatric disorders involves a complex genetic component, genome-wide association studies (GWAS) revealed numerous risk genes that are associated with an increased risk for ADHD. For instance, the novel ADHD candidate gene SLC2A3 which encodes the glucose transporter-3 (GLUT3), facilitates the transport of glucose across plasma membranes and is essential for the high energy demand of several cell types, such as stem cells and neurons. Specifically, copy number variants (CNVs) of SLC2A3 might therefore impact cerebral glucose metabolism as well as the assembly of synaptic proteins in human neurons which might contribute to the pathogenesis of ADHD. We hypothesized that an altered SLC2A3 gene dosage in human neurons can exert diverse protective or detrimental effects on neurodevelopmental processes as well as the coping of glucometabolic stress events, such as hypo- and hyperglycaemic conditions. The generation of specific iPSC lines from ADHD patients and healthy probands served as basis to efficiently differentiate stem cells into 5-HT specific neurons. Using this neuronal culture, we were able to examine effects of SLC2A3 CNVs on the basal expression of SCL2A3 and GLUT3 in human neurons. Furthermore, the focus was on potentially altered coping of the cells with glucose deprivation and the treatment with specific high- and low glycaemic media. High-resolution fluorescence imaging in combination with electrophysiological and molecular biological techniques showed that: 1) The generated human iPSCs are fully reprogrammed human stem cells showing typical characteristics of embryonic stem cell-like morphology, growth behaviour, the ability to differentiate into different cell types of the human body and the expression of pluripotency-specific markers. 2) The neuronal subtype derived from our stem cells display typical characteristics of 5-HT specific median and dorsal neurons and forms synapses reflected by the expression of pre- and postsynaptic proteins. 3) Even if SLC2A3 CNVs influence SLC2A3 and GLUT3 basal expression, no significant alterations in gene and protein expression caused by hyper- and hypoglycaemic conditions, nor in the assembly of proteins associated with synapse formation could be observed in human iPSC-derived neurons.},
  subject      = {Stammzelle},
  language  = {en}
}
@phdthesis{Herz2021,
  author    = {Herz, Michaela},
  title     = {Genome wide expression profiling of Echinococcus multilocularis},
  doi       = {10.25972/OPUS-20380},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-203802},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Alveolar echinococcosis, which is caused by the metacestode stage of the small fox tapeworm Echinococcus multilocularis, is a severe zoonotic disease with limited treatment options. For a better understanding of cestode biology the genome of E. multilocularis, together with other cestode genomes, was sequenced previously. While a few studies were undertaken to explore the E. multilocularis transcriptome, a comprehensive exploration of global transcription profiles throughout life cycle stages is lacking. This work represents the so far most comprehensive analysis of the E. multilocularis transcriptome. Using RNA-Seq information from different life cycle stages and experimental conditions in three biological replicates, transcriptional differences were qualitatively and quantitatively explored. The analyzed datasets are based on samples of metacestodes cultivated under aerobic and anaerobic conditions as well as metacestodes obtained directly from infected jirds. Other samples are stem cell cultures at three different time points of development as well as non-activated and activated protoscoleces, the larval stage that can develop into adult worms. In addition, two datasets of metacestodes under experimental conditions suitable for the detection of genes that are expressed in stem cells, the so-called germinative cells, and one dataset from a siRNA experiment were analyzed. Analysis of these datasets led to expression profiles for all annotated genes, including genes that are expressed in the tegument of metacestodes and play a role in host-parasite interactions and modulation of the host's immune response. Gene expression profiles provide also further information about genes that might be responsible for the infiltrative growth of the parasite in the liver. Furthermore, germinative cell-specific genes were identified. Germinative cells are the only proliferating cells in E. multilocularis and therefore of utmost importance for the development and growth of the parasite. Using a combination of germinative cell depletion and enrichment methods, genes with specific expression in germinative cells were identified. As expected, many of these genes are involved in translation, cell cycle regulation or DNA replication and repair. Also identified were transcription factors, many of which are involved in cell fate commitment. As an example, the gene encoding the telomerase reverse transcriptase (TERT) was studied further. Expression of E. multilocularis tert in germinative cells was confirmed experimentally. Cell culture experiments indicate that TERT is required for proliferation and development of the parasite, which makes TERT a potentially interesting drug target for chemotherapy of alveolar echinococcosis. Germinative cell specific genes in E. multilocularis also include genes of densoviral origin. More than 20 individual densovirus loci with information for non-structural and structural densovirus proteins were identified in the E. multilocularis genome. Densoviral elements were also detected in many other cestode genomes. Genomic integration of these elements suggests that densovirus-based vectors might be suitable tools for genetic manipulation of tapeworms. Interestingly, only three of more than 20 densovirus loci in the E. multilocularis genome are expressed. Since the canonical piRNA pathway is lacking in cestodes, this raises the question about potential silencing mechanisms. Exploration of RNA-Seq information indicated natural antisense transcripts as a potential gene regulation mechanism in E. multilocularis. Preliminary experiments further suggest DNA-methylation, which was previously shown to occur in platyhelminthes, as an interesting avenue to explore in future. The transcriptome datasets also contain information about genes that are expressed in differentiated cells, for example the serotonin transporter gene that is expressed in nerve cells. Cell culture experiments indicate that serotonin and serotonin transport play an important role in E. multilocularis proliferation, development and survival. Overall, this work provides a comprehensive transcription data atlas throughout the E. multilocularis life cycle. Identification of germinative cell-specific genes and genes important for host-parasite interactions will greatly facilitate future research. A global overview of gene expression profiles will also aide in the detection of suitable drug targets and the development of new chemotherapeutics against alveolar echinococcosis.},
  subject      = {Fuchsbandwurm},
  language  = {en}
}
@phdthesis{Herrmann2023,
  author    = {Herrmann, Ruth Magdalena},
  title     = {Molekular- und zellbiologische Untersuchung zur Rolle des kanonischen Wnt-Signalwegs bei der Entwicklung von \(Echinococcus\) \(multilocularis\)},
  doi       = {10.25972/OPUS-27193},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-271937},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Die alveol{\"a}re Echinokokkose (AE) ist eine lebensbedrohliche Erkrankung des Menschen, welche durch das infiltrative Wachstum des Metazestoden-Larvenstadiums des Fuchsbandwurms (Echinococcus multilocularis) in der Leber verursacht wird. Das tumorartige Wachstum des Metazestoden beruht auf einer Echinococcus-spezifischen Modifikation der anterior-posterioren-K{\"o}rperachse (AP Achse). Es wird vermutet, dass dabei der anteriore Pol der invadierenden Oncosp{\"a}ren-Larve zun{\"a}chst abgeschaltet wird und sich der Metazestode anschließend asexuell als vesikul{\"a}res, posteriorisiertes Gewebes im Wirt vermehrt. Nach massiver Proliferation wird der anteriore Pol reetabliert und f{\"u}hrt zur Bildung zahlreicher Bandwurm-Kopfanlagen (Protoskolizes). Da die Ausbildung der AP K{\"o}rperachse evolutionsgeschichtlich konserviert {\"u}ber den wingless-related (Wnt)-Signalweg gesteuert wird, wurde in dieser Arbeit die Rolle von Wnt-Signaling bei der Musterbildung von E. multilocularis {\"u}ber molekular- und zellbiologische Studien n{\"a}her beleuchtet. Zentraler methodischer Ansatz der vorliegenden Arbeit war ein E. multilocularis Stammzell-Kultursystem, das Prim{\"a}rzellsystem, welches die in vitro-Generierung von Metazestoden-Vesikeln durch Proliferation und Differenzierung von germinativen Zellen (Stammzellen) erlaubt. {\"U}ber RNA-Sequenzierung wurde zun{\"a}chst gezeigt, dass in Prim{\"a}rzellkulturen sowohl Markergene f{\"u}r posteriore Entwicklung in Richtung Metazestode wie auch f{\"u}r Anterior-und Protoskolexmarker exprimiert werden. Unter Verwendung von RNA-Interferenz (RNAi) wurde anschließend ein erfolgreicher Knockdown des vermuteten Hauptregulators des kanonischen Wnt-Signalwegs, β Catenin (em-bcat1), erreicht und f{\"u}hrte zu einem charakteristischen, sogenannten ‚red dot' Ph{\"a}notyp, dem ersten jemals beschriebenen RNAi Ph{\"a}notyp f{\"u}r E. multilocularis-Prim{\"a}rzellen. Prim{\"a}rzellkulturen nach em-bcat1 RNAi zeigten eine stark verminderte F{\"a}higkeit, Metazestoden-Vesikel zu bilden sowie eine {\"U}berproliferation von germinativen Zellen. Zus{\"a}tzliche RNA-Seq-Analysen des Transkriptoms von RNAi(em-bcat1)-Kulturen zeigten eine signifikant verringerte Expression von Posterior- und Metazestodenmarkern, w{\"a}hrend Anterior- und Protoskolexmarker deutlich {\"u}berexprimiert wurden. Durch umfangreiche Whole-mount-in-situ-Hybridisierung (WMISH)-Experimente wurden diese Daten f{\"u}r eine Reihe ausgew{\"a}hlter Markergene f{\"u}r posteriore (Metazestode; em-wnt1, em-wnt11b, em-muc1) und f{\"u}r anteriore Entwicklung (Protoskolex; em sfrp, em-nou-darake, em npp36, em-frizzled10) verifiziert. In allen genannten F{\"a}llen zeigte sich durch {\"A}nderung der Polarit{\"a}t eine verminderte Genexpression von Posteriormarkern, w{\"a}hrend Anteriormarker deutlich erh{\"o}ht exprimiert wurden. {\"A}hnlich wie bei den verwandten, freilebenden Planarien, f{\"u}hrt demnach ein Knockdown des zentralen Wnt-Regulators β-Catenin bei E. multilocularis zu einer anteriorisierten, Anterior- und Protoskolexmarker dominierte Genexpression, welche der posteriorisierten Entwicklung zum Metazestoden entgegenwirkt. Neben Markergenen f{\"u}r die Ausbildung der AP-Achse wurden in dieser Arbeit auch solche f{\"u}r die medio-laterale (ML)-K{\"o}rperachse bei Zestoden erstmals beschrieben. So zeigte sich, dass ein Slit-Ortholog (em slit) im E. multilocularis Protoskolex im Bereich der K{\"o}rper-Mittellinie exprimiert wird und lieferte Hinweise darauf, dass, {\"a}hnlich zur Situation bei Planarien, die ML Achse von E. multilocularis durch Morphogengradienten aus slit (Mittellinie) und wnt5 (lateral) definiert wird. Im Metazestoden wird hingegen nur em-slit exprimiert. Der Metazestode besitzt damit als posterior-medianisiertes Gewebe Anlagen zur Polarit{\"a}t zur AP- und ML-Achse, welche erst mit Bildung von Protoskolizes vollst{\"a}ndig etabliert werden. Schließlich deuten die Ergebnisse dieser Arbeit darauf hin, dass bei der Wiederherstellung der K{\"o}rperachsen w{\"a}hrend der Entwicklung von Protoskolizes Hedgehog (Hh)-Signale entscheidend mitwirken. Zusammenfassend wurde in dieser Arbeit der zentrale Faktor des kanonischen Wnt Signalwegs, β-Catenin, als Hauptregulator der Entwicklung des tumorartig wachsenden E. multilocularis-Metazestoden identifiziert. Zudem wurde gezeigt, dass zur Metazestodenbildung neben einer Echinococcus-spezifischen Modifikation der AP K{\"o}rperachse auch eine solche der ML Achse beitr{\"a}gt. In humanen malignen Tumoren sind der Wnt-, Slit-Robo- und Hh-Signalweg gut erforschte Wirkstofftargets und k{\"o}nnten in Zukunft in {\"a}hnlicher Weise f{\"u}r eine zielgerichtete Therapie von AE dienen.},
  subject      = {Fuchsbandwurm},
  language  = {de}
}
@phdthesis{Fey2023,
  author    = {Fey, Philipp},
  title     = {KI-gest{\"u}tzte MR-Klassifizierung von Zellen und zellul{\"a}rer Differenzierung},
  doi       = {10.25972/OPUS-34516},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-345164},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {F{\"u}r die Verwendung von zellbasierten Therapeutika ist vor allem die korrekt Identifikation sowohl vom Ausgangsmaterial wie auch dem produziertem Material von zentraler Wichtigkeit. In dieser Arbeit wurde eine Methodik entwickelt, welche eine nicht-invasive Klassifizierung von Zellen und zellul{\"a}rer Entwicklung aufgrund ihrer zweidimensionalen Magnetresonanz-Korrelationsspektren erm{\"o}glichte. Hierzu wurde ein mobiler MR-Scanner mit einer Feldst{\"a}rke von 0.5T und einem Isozentrum von 1 cm3 verwendet. Aufgrund der kompakten und leichten Bauweise war es m{\"o}glich, das System in normalen Zellkulturlaboren zu verwenden. Von den Proben wurde ein zweidimensionales T1/T2 -Korrelationsspektrum aufgenommen, anhand dessen die Zellen klassifiziert werden sollten. Mithilfe von Agarose-Dotagraf® -Zell- Phantomen konnte die Stabilit{\"a}t und Reproduzierbarkeit des Messsystems und der verwendeten Sequenz validiert werden. Aufgrund der unter Umst{\"a}nden recht langen Messzeiten der MR-Technologie war auch die Handhabung und Kultur der Zellproben w{\"a}hrend des Messprozesses von großer Bedeutung. Um hierf{\"u}r den Durchsatz an Proben zu erh{\"o}hen, wurde eine kosteng{\"u}nstige und ebenfalls mobile Robotikanlage entwickelt. Diese basierte auf dem kommerziell erh{\"a}ltlichen Roboterarm Braccio, welcher durch einen Arduino Mega Mikrocontroller gesteuert wurde. Mit bis zu 24 Proben pro Tag konnte durch die Automatisierung der Durchsatz an Proben um den Faktor 3 - 4 gesteigert werden. Durch den entwickelten Prozess war es m{\"o}glich, eine umfangreiche Datenbank - bestehend aus 362 unabh{\"a}ngigen Messungen (biologische Replikate) - aufzubauen. Die Datenbank enthielt Messungen von zehn unterschiedlichen Zelllinien. Zus{\"a}tzlich wurden T1/T2 -Korrelationsspektren von mesenchymalen Stromazellen (MSCs) vor und nach deren Differenzierung zu Adipocyten aufgenommen, um ihre zellul{\"a}re Entwicklung nicht-invasiv charakterisieren zu k{\"o}nnen. Die aufgenommenen Daten wurden mithilfe einer geeigneten Support Vector Machine wie auch angepassten k{\"u}nstlichen neuronalen Netzwerken klassifiziert. Mithilfe dieser Methoden konnten die Zelllinien und MSCs anhand ihrer aufgenommenen Korrelationsspektren mit einer Genauigkeit von bis zu 98\% klassifiziert werden. Diese hohe Treffsicherheit legte den Schluss nahe, dass die Kombination aus nichtinvasiver, zweidimensionaler T1/T2 -MR-Relaxometrie und der Verwendung von geeigneten Methoden des machine learning und der k{\"u}nstlichen Intelligenz eine effiziente Methodik f{\"u}r die nicht-invasive Klassifizierung von Zellen sowie zellul{\"a}rer Entwicklung darstellt.},
  subject      = {Kernspintomografie},
  language  = {de}
}
@phdthesis{Ahmad2012,
  author    = {Ahmad, Ruhel},
  title     = {Neurogenesis from parthenogenetic human embryonic stem cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75935},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Imprinted genes play important roles in brain development. As the neural developmental capabilities of human parthenogenetic embryonic stem cells (hpESCs) with only a maternal genome were not assessed in great detail, hence here the potential of hpESCs to differentiate into various neural subtypes was determined. In addition DNA methylation and expression of imprinted genes upon neural differentiation was also investigated. The results demonstrated that hpESC-derived neural stem cells (hpNSCs) showed expression of NSC markers Sox1, Nestin, Pax6, and Musashi1 (MS1), the silencing of pluripotency genes (Oct4, Nanog) and the absence of activation of neural crest (Snai2, FoxD3) and mesodermal (Acta1) markers. Moreover, confocal images of hpNSC cultures exhibited ubiquitous expression of NSC markers Nestin, Sox1, Sox2 and Vimentin. Differentiating hpNSCs for 28 days generated neural subtypes with neural cell type-specific morphology and expression of neuronal and glial markers, including Tuj1, NeuN, Map2, GFAP, O4, Tau, Synapsin1 and GABA. hpNSCs also responded to region-specific differentiation signals and differentiated into regional phenotypes such as midbrain dopaminergic- and motoneuron-type cells. hpESC-derived neurons showed typical neuronal Na+/K+ currents in voltage clamp mode, elicited multiple action potentials with a maximum frequency of 30 Hz. Cell depicted a typical neuron-like current pattern that responded to selective pharmacological blockers of sodium (tetrodotoxin) and potassium (tetraethylammonium) channels. Furthermore, in hpESCs and hpNSCs the majority of CpGs of the differentially methylated regions (DMRs) KvDMR1 were methylated whereas DMR1 (H19/Igf2 locus) showed partial or complete absence of CpG methylation, which is consistent with a parthenogenetic (PG) origin. Upon differentiation parent-of-origin-specific gene expression was maintained in hpESCs and hpNSCs as demonstrated by imprinted gene expression analyses. Together this shows that despite the lack of a paternal genome, hpNSCs are proficient in differentiating into glial- and neuron-type cells, which exhibit electrical activity similar to newly formed neurons. Moreover, maternal-specific gene expression and imprinting-specific DNA-methylation are largely maintained upon neural differentiation. hpESCs are a means to generate histocompatible and disease allele-free ESCs. Additionally, hpESCs are a unique model to study the influence of imprinting on neurogenesis.},
  subject      = {Embryonale Stammzelle},
  language  = {en}
}