@phdthesis{Akimzhanov2005,
  author    = {Akimzhanov, Askar M.},
  title     = {Epigenetic repression of the NFATc1 transcription factor in human lymphomas},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-12921},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2005},
  abstract  = {We examined the regulation of NFATc1 in different lymphomas and observed an inversed correlation between the methylation status and expression of NFATc1. Our data demonstrate that aberrant DNA methylation associated with chromatin remodeling within nfatc1 locus is a major mechanism for the repression of NFATc1 expression, suggesting that the DNA methylation-mediated transcriptional silencing of NFATc1 may be a critical event in the tumorogenesis of ALCLs and cHLs. Furthermore, the DNA methylation of human nfatc1 promoter region could be used as a novel biomarker of tumor progression. Our results indicate a close link between the loss of immunoreceptor signaling and NFATc1 expression in human lymphomas. For both ALCLs and cHLs, defects in immunoreceptor signaling have been described which result in a loss of receptor-mediated gene expression programs (Schwering et al., 2003; Bonzheim et al., 2004; Marafioti et al., 2004). In T cells, one indicator gene of these programs appears to be the nfatc1 gene whose expression is controlled by TCR signals (Chuvpilo et al., 2002a). In contrast, in T cells NFATc1 expression is unaffected by TCR signals, and NFATc2 was found to be expressed at normal levels in ALCLs and cHLs (L.K., unpubl. data). Moreover, the activity of NF-kappaB factors which can bind to certain NFAT binding sites and share a distantly-related DNA binding domain with NFATs is strongly elevated in cHL cells (Bargou et al., 1997; Hinz et al., 2001; Hinz et al., 2002) suggesting that NFATs and NF-kappaBs exert very different effects on generation and maintenance of Hodgkin's lymhomas. However, it should be mentioned that in Burkitt's and further B cell lymphomas in which NFATc1 proteins are strongly expressed and controlled by receptor signals (Kondo et al., 2003), they could exert a promoting function in tumor development. The genes of p53 family members p63 and p73 are prominent examples for mammalian genes whose products can act both as oncoproteins and tumor suppressor genes (Hibi et al., 2000; Stiewe and Putzer, 2002), and it is likely that more genes exist which encode both tumor suppressors and oncoproteins. It remains to be shown whether the nfatc1 gene is one of them.},
  subject      = {Lymphom},
  language  = {en}
}
@phdthesis{Hofstetter2014,
  author    = {Hofstetter, Christine},
  title     = {Inhibition of H3K27me-Specific Demethylase Activity During Murine ES cell Differentiation Induces DNA Damage Response},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-107023},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Stem cells are defined by their capacity to self-renew and their potential to differentiate into multiple cell lineages. Pluripotent embryonic stem (ES) cells can renew indefinitely while keeping the potential to differentiate into any of the three germ layers (ectoderm, endoderm or mesoderm). For decades, ES cells are in the focus of research because of these unique features. When ES cells differentiate they form spheroid aggregates termed "embryoid bodies" (EBs). These EBs mimic post- implantation embryonic development and therefore facilitate the understanding of developmented mechanisms. During ES cell differentiation, de-repression or repression of genes accompanies the changes in chromatin structure. In ES cells, several mechanisms are involved in the regulation of the chromatin architecture, including post-translational modifications of histones. Post-translational histone methylation marks became one of the best- investigated epigenetic modifications, and they are essential for maintaining pluripotency. Until the first histone demethylase KDM1A was discovered in 2004 histone modifications were considered to be irreversible. Since then, a great number of histone demethylases have been identified. Their activity is linked to gene regulation as well as to stem cell self-renewal and differentiation. KDM6A and KDM6B are H3K27me3/2-specific histone demethylases, which are known to play a central role in the regulation of posterior development by regulating HOX gene expression. So far less is known about the molecular function of KDM6A or KDM6B in undifferentiated and differentiating ES cells. In order to completely abrogate KDM6A and KDM6B demethylase activity in undifferentiated and differentiating ES cells, a specific inhibitor (GSK-J4) was employed. Treatment with GSK-J4 had no effect on the viability or proliferation on ES cells. However, in the presence of GSK-J4 ES cell differentiation was completely abrogated with cells arrested in G1-phase and an increased rate of apoptosis. Global transcriptome analyses in early-differentiating ES cells revealed that only a limited set of genes were differentially regulated in response to GSK-J4 treatment with more genes up- regulated than down-regulated. Many of the up-regulated genes are linked to DNA damage response (DDR). In agreement with this, DNA damage was found in EBs incubated with GSK-J4. A co-localization of H3K27me3 or KDM6B with γH2AX foci, marking DNA breaks, could be excluded. However, differentiating Eed knockout (KO) ES cells, which are devoid of the H3K27me3 mark, showed an attenuated GSK-J4- induced DDR. Finally, hematopoietic differentiation in the presence of GSK-J4 resulted in a reduced colony-forming potential. This leads to the conclusion that differentiation in the presence of GSK-J4 is also restricted to hematopoietic differentiation. In conclusion, my results show that the enzymatic activity of KDM6A and KDM6B is not essential for maintaining the pluripotent state of ES cells. In contrast, the enzymatic activity of both proteins is indispensable for ES cell and hematopoietic differentiation. Additionally KDM6A and KDM6B enzymatic inhibition in differentiating ES cells leads to increased DNA damage with an activated DDR. Therefore, KDM6A and KDM6B are associated with DNA damage and in DDR in differentiating ES cells.},
  subject      = {Embryonale Stammzelle},
  language  = {en}
}
@phdthesis{Jakob2012,
  author    = {Jakob, Sissi},
  title     = {Molecular mechanisms of early-life stress in 5-Htt deficient mice: Gene x environment interactions and epigenetic programming},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74150},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Early-life stress has been shown to influence the development of the brain and to increase the risk for psychiatric disorders later in life. Furthermore, variation in the human serotonin transporter (5-HTT, SLC6A4) gene is suggested to exert a modulating effect on the association between early-life stress and the risk for depression. At the basis of these gene x environment (G x E) interactions, epigenetic mechanisms, such as DNA-methylation, seem to represent the primary biological processes mediating early-life programming for stress susceptibility or resilience, respectively. The exact molecular mechanisms however remain to be elucidated, though. In the present study, we used two different stress paradigms to assess the molecular mechanisms mediating the relationship between early-life stress and disorders of emotion regulation later in life. First, a 5-Htt x prenatal stress (PS) paradigm was applied to investigate whether the effects of PS are dependent on the 5-Htt genotype. For this purpose, the effects of PS on cognition and anxiety- / depression-related behavior were examined using a maternal restraint stress paradigm of PS in C57BL/6 wild-type (WT) and heterozygous 5-Htt deficient (5-Htt+/-) mice. Additionally, in female offspring, a genome-wide hippocampal gene expression and DNA methylation profiling was performed using the Affymetrix GeneChip® Mouse Genome 430 2.0 Array and the AffymetrixGeneChip® Mouse Promoter 1.0R Array. Some of the resulting candidate genes were validated by quantitative real-time PCR. Further, the gene expression of these genes was measured in other brain regions of the PS animals as well as in the hippocampus of offspring of another, 5-Htt x perinatal stress (PeS) paradigm, in which pregnant and lactating females were stressed by an olfactory cue indicating infanticide. To assess resilience to PS and PeS, correlation studies between gene expression and behaviour were performed based on an initial performance-based LIMMA analysis of the gene expression microarray. 5-Htt+/- offspring of the PS paradigm showed enhanced memory performance and signs of reduced anxiety as compared to WT offspring. In contrast, exposure of 5-Htt+/- mice to PS was associated with increased depression-like behavior, an effect that tended to be more pronounced in female offspring. Further, 5-Htt genotype, PS and their interaction differentially affected the expression and DNA methylation of numerous genes and related pathways within the female hippocampus. Specifically, MAPK and neurotrophin signaling were regulated by both the 5-Htt+/- genotype and PS exposure, whereas cytokine and Wnt signaling were affected in a 5-Htt genotype x PS manner, indicating a gene x environment interaction at the molecular level. The candidate genes of the expression array could be validated and their expression patterns were partly consistent in the prefrontal cortex and striatum. Furthermore, the genotype effect of XIAP associated factor 1 (Xaf1) was also detected in the mice of the PeS paradigm. Concerning resilience, we found that the expression of growth hormone (Gh), prolactin (Prl) and fos-induced growth factor (Figf) were downregulated in WTPS mice that performed well in the forced swim test (FST). At the same time, the results indicated that Gh and Prl expression correlated positively with adrenal weight, whereas Figf expression correlated positively with basal corticosteron concentration, indicating an intricate relationship between depression-like behavior, hippocampal gene expression and the hypothalamo-pituitary-adrenal (HPA) axis activity. Correlation studies in the PeS animals revealed a link between Gh / Prl expression and anxiety-like behavior. In conclusion, our data suggest that although the 5-Htt+/- genotype shows clear adaptive capacity, 5-Htt+/- mice, particularly females, appear to be more vulnerable to developmental stress exposure when compared to WT offspring. Moreover, hippocampal gene expression and DNA methylation profiles suggest that distinct epigenetic mechanisms at the molecular level mediate the behavioral effects of the 5-Htt genotype, PS exposure, and their interaction. Further, resilience to early-life stress might be conferred by genes whose expression is linked to HPA axis function.},
  subject      = {Stressreaktion},
  language  = {en}
}
@phdthesis{Kollert2021,
  author    = {Kollert, Leonie},
  title     = {Epigenetics of anxiety and depression - a differential role of TGFB-Inducible Early Growth Response Protein 2 gene promoter methylation},
  doi       = {10.25972/OPUS-21126},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-211268},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Among mental disorders, panic disorder (PD) is one of the most common anxiety disorders characterized by recurring and unexpected episodes of extreme fear i.e. panic attacks. PD displays lifetime prevalence rates in the general population between 2.1-4.7 \% and in about 30 to 40 \% occurs comorbid with major depressive disorder (MDD). Differential methylation levels of the monoamine oxidase A (MAOA) gene have previously been associated with the etiology of both PD and MDD. The TGFB-Inducible Early Growth Response Protein 2 (TIEG2; alias KLF11), an activating transcription factor of the MAOA gene, has been reported to be increased in MDD, but has not yet been investigated in PD on any level. Therefore, in an attempt to further define the role of an impaired TIEG2-MAOA pathway in anxiety and affective disorders, in the present thesis TIEG2 promoter DNA methylation was analyzed in two independent samples of I) PD patients with or without comorbid MDD in a case/control design and II) MDD patients with and without anxious depression. Additionally, in PD patients of sample I), TIEG2 methylation was correlated with Beck Depression Inventory (BDI-II) scores. Finally, in a third independent healthy control sample, correlation of TIEG2 promoter methylation levels with Anxiety Sensitivity Index (ASI) scores as a PD-related measure was analyzed. No overall association of TIEG2 promoter methylation with PD was detected. However, PD patients with comorbid MDD showed significant TIEG2 hypomethylation compared to PD patients without comorbid MDD (p=.008) as well as to healthy controls (p=.010). In addition, MDD patients without anxious features displayed a statistical trend in decreased TIEG2 methylation in comparison to MDD patients with anxious depression (p=.052). Furthermore, TIEG2 methylation was negatively correlated with BDI-II scores in PD patients (p=.013) and positively correlated with ASI scores in the healthy control sample (p=.043). In sum, the current study suggests TIEG2 promoter hypomethylation as a potential epigenetic marker of MDD comorbidity in PD or of non-anxious depression, respectively. If replicated and verified in future studies, altered TIEG2 methylation might therefore represent a differential pathomechanism of anxiety and mood disorders.},
  subject      = {Epigenetik},
  language  = {en}
}
@phdthesis{Li2013,
  author    = {Li, Xiaoli},
  title     = {Functional analyses of ES cell pluripotency by inducible knockdown of the Polycomb group protein Pcgf6},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-84015},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Polycomb group (PcG) proteins are chromatin modifiers involved in heritable gene repression. Two main PcG complexes have been characterized: Polycomb repressive complex (PRC) 2 is involved in the initiation of gene silencing, whereas PRC1 participates in the stable maintenance of gene repression. Pcgf4 (Polycomb group protein, Bmi1) is one of the most studied PRC1 members with essential functions for embryonic development and adult stem cell self renewal. In embryonic stem cells (ES cells), Pcgf4 is poorly expressed while its paralogs (Pcgf1, Pcgf2, Pcgf3, Pcgf5 and Pcgf6) are expressed at higher levels. The relevance of the Pcgf paralog Pcgf6 for the maintenance of ESC pluripotency has not been addressed so far. My analyses revealed that Pcgf6 was the most expressed Pcgf paralog in undifferentiated ES cells. When ES cells differentiated, gene expression of Pcgf6 strongly declined. To investigate the functions of Pcgf6 in ES cells, we established a doxycycline (dox) inducible shRNA-targeted knockdown system according to publications by Seibler et al. (Seibler et al. 2005; Seibler et al. 2007). Following dox-induced knockdown (KD) of Pcgf6, we observed decreased ES cell colony formation. In parallel, gene expression of pluripotency markers Oct4, Nanog and Sox2 was reduced upon dox-treatment, wheras the expression of mesoderm genes such as T (Brachyury) were up-regulated. Further, microarray analysis revealed de-repression of several spermatogenesis-specic genes upon Pcgf6-KD, suggesting that Pcgf6 may play a role during spermatogenesis. Upon in vitro differentiation, Pcgf6-KD ES cells showed increased hemangioblast formation, paralleled by increased hematopoietic development. In summary, results of this study suggest that Pcgf6 is involved in maintaining ES cell identity by repressing lineage-specific gene expression in undifferentiated ES cells.},
  subject      = {Embryonale Stammzelle},
  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{Pennington2018,
  author    = {Pennington, Laura Sophie},
  title     = {The role of Cadherin-13 in serotonergic neurons during different murine developmental stages},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161331},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Abstract Background: Attention-deficit/ hyperactivity disorder (ADHD) ranges among the most common neurodevelopmental disorders worldwide with a prevalence of 3-12\% in childhood and 1-5\% for adults. Over the last decade extensive genetic research has been conducted in order to determine its causative genetic factors. None of the so far identified susceptibility genes, however, could explain the estimated ADHD heritability of 76\%. In this thesis one of the most promising candidates -Cadherin 13 (Cdh13) - was examined in terms of its influence on the central serotonergic (5-HT) system. In addition to that, the Cdh13 protein distribution pattern was analysed over time. Methods: The developing serotonergic system was compared over three embryonic and postnatal stages (E13.5, E17.5 and P7) in different Cdh13 genotypes (WT, HZ and KO) using immunohistochemistry and various double staining protocols. Results: The raphe nuclei of the 5-HT system develop in spite of Cdh13 absence and show a comparable mature constellation. The cells in the KO, however, are slightly more scattered than in the WT. Furthermore the dynamics of their formation is altered, with a transient delay in migration at E13.5. In early developmental stages the total amount of serotonergic cells is reduced in KO and HZ, though their proportional distribution to the raphe nuclei stays constant. Strikingly, at P7 the absolute numbers are comparable again. Concerning the Cdh13 protein, it shows high concentrations on fibres running through hindbrain and midbrain areas at E13.5. This, however, changes over time, and it becomes more evenly spread until P7. Furthermore, its presence in serotonergic cells could be visualised using confocal microscopy. Since the described pattern is only in parts congruent to the localisation of serotonergic neurons, it is most likely that Cdh13 is present in other developing neurotransmitter systems, such as the dopaminergic one, as well. Conclusion: It could be proven that Cdh13 is expressed in serotonergic cells and that its knockout does affect the developing serotonergic system to some degree. Its absence, however, only slightly and transiently affects the measured parameters of serotonergic system development, indicating a possible compensation of CDH13 function by other molecules in the case of Cdh13 deficiency. In addition further indicators could be found for an influence of Cdh13 on outgrowth and path finding of neuronal processes.},
  subject      = {Cadherine},
  language  = {en}
}
@phdthesis{Prell2024,
  author    = {Prell, Andreas},
  title     = {The effects of paternal age on DNA methylation of developmentally important genes in human and bovine sperm},
  doi       = {10.25972/OPUS-34786},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-347866},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Western societies are steadily becoming older undergoing a clear trend of delayed parenthood. Children of older fathers have an undeniably higher risk for certain neurodevelopmental disorders and other medical conditions. Changes in the epigenetic landscape and especially in DNA methylation patterns are likely to account for a portion of this inherited disease susceptibility. DNA methylation changes during the ageing process are a well-known epigenetic feature. These so-called age-DMRs exist in developmentally important genes in the methylome of several mammalian species. However, there is only a minor overlap between the age-DMR datasets of different studies. We therefore replicated age-DMRs (which were obtained from a genome wide technique) by applying a different technical approach in a larger sample number. Here, this study confirmed 10 age-DMRs in the human and 4 in the bovine sperm epigenome from a preliminary candidate list based on RRBS. For this purpose, we used bisulphite Pyrosequencing in 94 human and 36 bovine sperm samples. These Pyrosequencing results confirm RRBS as an effective and reliable method to screen for age-DMRs in the vertebrate genome. To decipher whether paternal age effects are an evolutionary conserved feature of mammalian development, we compared methylation patterns between human and bovine sperm in orthologous regulatory regions. We discovered that the level of methylation and the age effect are both species-specific and speculate that these methylation marks reflect the lineage-specific development of each species to hit evolutionary requirements and adaptation processes. Different methylation levels between species in developmentally important genes also imply a differing mutational burden, representing a potential driver for point mutations and consequently deviations in the underlying DNA sequence of different species. Using the example of different haplotypes, this study showed the great effect of single base variations on the methylation of adjacent CpGs. Nonetheless, this study could not provide further evidence or a mechanism for the transfer of epigenetic marks to future generations. Therefore, further research in tissues from the progeny of old and young fathers is required to determine if the observed methylation changes are transmitted to the next generation and if they are associated with altered transcriptional activity of the respective genes. This could provide a direct link between the methylome of sperm from elderly fathers and the development potential of the next generation.},
  subject      = {Epigenetik},
  language  = {en}
}
@phdthesis{Reichenbach2020,
  author    = {Reichenbach, Juliane Renate},
  title     = {Paternal age effects on sperm DNA methylation and its impact on the next generation},
  doi       = {10.25972/OPUS-19980},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199805},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The effect of late parenthood on the offspring´s physical and mental health status has recently become an increasingly important topic of discussion. Studies on neurodevelopmental disorders in children of older parents (Naserbakht et al., 2011) outline the negative consequences of aging fathers as unpredictable compared to the better-understood unfavorable maternal influences (Cedars et al. 2015). This may be due to the fact that lifelong production of male gametes becomes more susceptible to error, not only for somatic mutations. Non-genomic mechanisms such as epigenetic methylation also alter DNA dynamically throughout life (Jones et al., 2015) and influence the aging human sperm DNA (Jenkins et al., 2014). These methylation changes may be transmitted to the next generation via epigenetic inheritance mechanisms (Milekic et al., 2015), which may negatively impact the sensitive epigenetic regulation of cell differentiation in the embryonic period (Curley et al., 2011; Spiers et al., 2015). Accordingly, Nardone et al. (2014) reported several hypomethylated regions in autistic patients, illustrating potential epigenetic influences on the multifactorial pathogenesis of neuropsychiatric disorders. In the present study, the methylation status of five gene regions in the sperm DNA of males of different ages was analyzed by two techniques - pyrosequencing and deep bisulfite sequencing. Two gene regions, FOXK1 and DMPK, showed a highly significant age-related methylation loss and FOXK1 a reduced methylation variation at the level of single alleles. In addition, the examined gene region of FOXK1 showed significant methylation changes in the fetal cord blood DNA of the respective offspring of the sperm donor. This fact suggests a transfer of age-related methylation loss to the next generation. Interestingly, a methylation analysis at the level of single alleles showed that the methylation loss was inherited exclusively by the father. FOXK1 is a transcription factor that plays an important role in the epigenetic regulation of the cell cycle during embryonic neuronal development (Huang et al., 2004; Wijchers et al., 2006). For this reason, the methylation status of FOXK1 in the blood of autistic patients and an age- and sex-matched control group was investigated. While both groups showed age-associated FOXK1 methylation loss, a faster dynamics of methylation change was observed in the autistic group. Although further studies are needed to uncover inheritance mechanisms of epigenetic information, the present results show an evident influence of age-related methylation changes on offspring. When advising future fathers, it is important to consider how the paternal epigenome is altered by aging and can have a negative impact on the developing embryo.},
  subject      = {Epigenetik},
  language  = {en}
}
@phdthesis{Riemens2023,
  author    = {Riemens, Renzo J. M.},
  title     = {Neuroepigenomics in Alzheimer's disease: The single cell ADds},
  isbn      = {978-94-6423-524-1},
  doi       = {10.25972/OPUS-25457},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254574},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Die Forschung, die in dieser Arbeit zusammengestellt wird, kann in zwei Teile geteilt werden. Der erste Teil, bestehend aus vier Kapiteln, konzentriert sich auf die Rolle der epigenetischen Dysregulation in der {\"A}tiopathophysiologie der sporadischen Alzheimer-Krankheit (sAD). Neben Einblicken in die neuesten Entwicklungen in neuroepigenomischen Studien zu dieser Krankheit geht der erste Teil der Arbeit auch auf verbleibende Herausforderungen ein und gibt einen Ausblick auf m{\"o}gliche Entwicklungen auf diesem Gebiet. Der zweite Teil, der drei weitere Kapitel umfasst, konzentriert sich auf die Anwendung von auf induzierten pluripotenten Stammzellen (iPSC) basierenden Krankheitsmodellen f{\"u}r das Studium der AD, einschließlich, aber nicht beschr{\"a}nkt auf mechanistische Studien zur epigenetischen Dysregulation unter Verwendung dieser Plattform. Neben der Skizzierung der bisherigen Forschung mit iPSC-basierten Modellen f{\"u}r sAD gibt der zweite Teil der Arbeit auch Einblicke in die Gewinnung krankheitsrelevanter Nervenkulturen auf Basis der gezielten Differenzierung von iPSCs und beinhaltet dar{\"u}ber hinaus einen experimentellen Ansatz f{\"u}r den Aufbau eines solchen Modellsystems.},
  subject      = {Epigenetik},
  language  = {en}
}
@phdthesis{Schraut2015,
  author    = {Schraut, Karla-Gerlinde},
  title     = {Epigenetic programming by prenatal stress in female serotonin transporter deficient mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-120270},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Early life stress, including exposure to prenatal stress (PS), has been shown to affect the developing brain and induce severe effects on emotional health in later life, concomitant with an increased risk for psychopathology. However, some individuals are more vulnerable to early-life stress, while others adapt successfully, i.e. they are resilient and do not succumb to adversity. The molecular substrates promoting resilience in some individuals and vulnerability in other individuals are as yet poorly investigated. A polymorphism in the serotonin transporter gene (5­HTT/SLC6A4) has been suggested to play a modulatory role in mediating the effects of early-life adversity on psychopathology, thereby rendering carriers of the lower-expressing short (s)-allele more vulnerable to developmental adversity, while long (l)-allele carriers are relatively resilient. The molecular mechanisms underlying this gene x environment interaction (GxE) are not well understood, however, epigenetic mechanisms such as DNA methylation and histone modifications have been discussed to contribute as they are at the interface of environment and the genome. Moreover, developmental epigenetic programming has also been postulated to underlie differential vulnerability/resilience independent of genetic variation. The present work comprises two projects investigating the effects of prenatal maternal restraint stress in 5-HTT deficient mice. In the first study, we examined to which extent previously observed changes in behavior and hippocampal gene expression of female 5-Htt+/- prenatally stressed (PS) offspring were associated with changes in DNA methylation patterns. Additionally, we investigated the expression of genes involved in myelination in hippocampus and amygdala of those animals using RT-qPCR. The genome-wide hippocampal DNA methylation screening was performed using methylated-DNA immunoprecipitation (MeDIP) on Affymetrix GeneChip® Mouse Promoter 1.0R arrays. In order to correlate individual gene-specific DNA methylation, mRNA expression and behavior, we used hippocampal DNA from the same mice as assessed before. 5-Htt genotype, PS and their interaction differentially affected the DNA methylation signature of numerous genes, a part of which were also differentially expressed. More specifically, we identified a differentially methylated region in the Myelin basic protein (Mbp) gene, which was associated with Mbp expression in a 5-Htt-, PS- and 5-Htt x PS-dependent manner. Subsequent fine-mapping linked the methylation status of two specific CpG sites in this region to Mbp expression and anxiety-related behavior. We furthermore found that not only the expression of Mbp but of large gene set associated with myelination was affected by a 5-Htt x PS interaction in a brain-region specific manner. In conclusion, hippocampal DNA methylation patterns and expression profiles of female PS 5-Htt+/- mice suggest that distinct molecular mechanisms, some of which are associated with changes in gene promoter methylation, and processes associated with myelination contribute to the behavioral effects of the 5-Htt genotype, PS exposure, and their interaction. In the second study, we aimed at investing the molecular substrates underlying resilience to PS. For this purpose, we exposed 5-Htt+/+ dams to the same restraint stress paradigm and investigated the effects of PS on depression- and anxiety-like behavior and corticosterone (CORT) secretion at baseline and after acute restraint stress in female 5-Htt+/+ and 5-Htt+/- offspring. We found that PS affected the offspring's social behavior in a negative manner. When specifically examining those PS animals, we grouped the PS offspring of each genotype into a social, resilient and an unsocial, vulnerable group. While anxiety-like behavior in the EPM was reduced in unsocial, but not social, PS 5-Htt+/+ animals when compared to controls, this pattern could not be found in animals of the other genotype, indicating that social anxiety and state anxiety in the EPM were independent of each other. We then assessed genome-wide hippocampal gene expression profiles using mRNA sequencing in order to identify pathways and gene ontology (GO) terms enriched due to 5-Htt genotype (G), PS exposure (E) and their interaction (GxE) as well as enriched in social, but not unsocial, PS offspring, and vice versa. Numerous genes were affected by 5-Htt genotype, PS and most of all a GxE-interaction. Enrichment analysis using enrichr identified that the genotype affected mitochondrial respiration, while GxE-interaction-affected processes associated primarily with myelination and chromatin remodeling. We furthermore found that 5-Htt+/- mice showed profound expression changes of numerous genes in a genomic region located 10 mio kb upstream of the 5 Htt locus on the same chromosome. When looking at social vs. unsocial mice, we found that a much higher number of genes was regulated in 5 Htt+/- animals than in 5-Htt+/+ animals, reflecting the impact of GxE-interaction. Double the number of genes was regulated in social PS vs. control mice when compared to unsocial PS vs. control in both genotypes, suggesting that the successful adaption to PS might have required more active processes from the social group than the reaction to PS from the unsocial group. This notion is supported by the up-regulation of mitochondrial respiration in social, but not in unsocial, PS 5-Htt+/- mice when compared to controls, as those animals might have been able to raise energy resources the unsocial group was not. Next to this, processes associated with myelination seemed to be down-regulated in social 5-Htt+/- mice, but not in unsocial animals, when compared to controls. Taken together, PS exposure affected sociability and anxiety-like behavior dependent on the 5-Htt genotype in female offspring. Processes associated with myelination and epigenetic mechanisms involved in chromatin remodeling seemed be affected in a GxE-dependent manner in the hippocampus of these offspring. Our transcriptome data furthermore suggest that mitochondrial respiration and, with this, energy metabolism might be altered in 5-Htt+/- offspring when compared to 5-Htt+/+ offspring. Moreover, myelination and mitochondrial respiration might contribute to resilience towards PS exposure in 5-Htt+/- offspring, possibly by affecting brain connectivity and energy capabilities.},
  subject      = {Stress},
  language  = {en}
}
@phdthesis{Varagnolo2014,
  author    = {Varagnolo, Linda},
  title     = {PRC2 inhibition counteracts the culture-associated loss of engraftment potential of human cord blood-derived hematopoietic stem/progenitor cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108073},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Cord blood hematopoietic stem cells (CB-HSCs) are an outstanding source for the treatment of a variety of malignant and non-malignant disorders. However, the low amount of cells collected per donor is often insufficient for treatment of adult patients. In order to make sufficient numbers of CB-HSCs available for adults, expansion is required. Different approaches were described for HSC expansion, however these approaches are impeded by the loss of engrafting potential during ex vivo culture. Little is known about the underlying molecular mechanisms. Epigenetic mechanisms play essential roles in controlling stem cell potential and fate decisions and epigenetic strategies are considered for HSC expansion. Therefore, this study aimed to characterize global and local epigenotypes during the expansion of human CB-CD34+, a well established CB progenitor cell type, to better understand the molecular mechanisms leading to the culture-associated loss of engrafting potential. Human CB-CD34+ cells were cultured using 2 different cytokine cocktails: the STF cocktail containing SCF, TPO, FGF-1 and the STFIA cocktail, which combines STF with Angiopoietin-like 5 (Angptl5) and Insulin-like growth factor-binding protein 2 (IGFBP2). The latter expands CB-HSCs ex vivo. Subsequently, the NOD-scid gamma (NSG) mouse model was used to study the engraftment potential of expanded cells. Engraftment potential achieved by fresh CB-CD34+ cells was maintained when CB-CD34+ cells were expanded under STFIA but not under STF conditions. To explore global chromatin changes in freshly isolated and expanded CB-CD34+ cells, levels of the activating H3K4me3 and the repressive H3K27me3 histone marks were determined by chromatin flow cytometry and Western blot analyses. For analysis of genome-wide chromatin changes following ex vivo expansion, transcriptome profiling by microarray and chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) were performed. Additionally, local chromatin transitions were monitored by ChIP analyses on promoter regions of developmental and self-renewal factors. On a global level, freshly isolated CD34+ and CD34- cells differed in H3K4me3 and H3K27me3 levels. After 7 days of expansion, CD34+ and CD34- cells adopted similar levels of active and repressive marks. Expanding the cells without IGFBP2 and Angptl5 led to a higher global H3K27me3 level. ChIP-seq analyses revealed a cytokine cocktail-dependent redistribution of H3K27me3 profiles. Chemical inhibition of the H3K27 methyltransferase EZH2 counteracted the culture-associated loss of NSG engraftment potential. Collectively, the data presented in this study revealed that by adding epigeneticly active compounds in the culture media we observed changes on a chromatin level which counteracted the loss of engraftment potential. H3K27me3 rather than H3K4me3 may be critical to establish a specific engraftment supporting transcriptional program. Furthermore, I identified a critical function for the Polycomb repressive complex 2-component EZH2 in the loss of engraftment potential during the in vitro expansion of HPSCs. Taken together this thesis provides a better molecular understanding of chromatin changes upon expansion of CB-HSPCs and opens up new perspectives for epigenetic ex vivo expansion strategies.},
  subject      = {Epigenetik},
  language  = {en}
}
@phdthesis{Wedel2018,
  author    = {Wedel, Carolin},
  title     = {The impact of DNA sequence and chromatin on transcription in \(Trypanosoma\) \(brucei\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173438},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {For cellular viability, transcription is a fundamental process. Hereby, the DNA plays the most elemental and highly versatile role. It has long been known that promoters contain conserved and often well-defined motifs, which dictate the site of transcription initiation by providing binding sites for regulatory proteins. However, research within the last decade revealed that it is promoters lacking conserved promoter motifs and transcribing constitutively expressed genes that constitute the majority of promoters in eukaryotes. While the process of transcription initiation is well studied, whether defined DNA sequence motifs are required for the transcription of constitutively expressed genes in eukaryotes remains unknown. In the highly divergent protozoan parasite Trypanosoma brucei, most of the proteincoding genes are organized in large polycistronic transcription units. The genes within one polycistronic transcription unit are generally unrelated and transcribed by a common transcription start site for which no RNA polymerase II promoter motifs have been identified so far. Thus, it is assumed that transcription initiation is not regulated but how transcription is initiated in T. brucei is not known. This study aimed to investigate the requirement of DNA sequence motifs and chromatin structures for transcription initiation in an organism lacking transcriptional regulation. To this end, I performed a systematic analysis to investigate the dependence of transcription initiation on the DNA sequence. I was able to identify GT-rich promoter elements required for directional transcription initiation and targeted deposition of the histone variant H2A.Z, a conserved component during transcription initiation. Furthermore, nucleosome positioning data in this work provide evidence that sites of transcription initiation are rather characterized by broad regions of open and more accessible chromatin than narrow nucleosome depleted regions as it is the case in other eukaryotes. These findings highlight the importance of chromatin during transcription initiation. Polycistronic RNA in T. brucei is separated by adding an independently transcribed miniexon during trans-splicing. The data in this work suggest that nucleosome occupancy plays an important role during RNA maturation by slowing down the progressing polymerase and thereby facilitating the choice of the proper splice site during trans-splicing. Overall, this work investigated the role of the DNA sequence during transcription initiation and nucleosome positioning in a highly divergent eukaryote. Furthermore, the findings shed light on the conservation of the requirement of DNA motifs during transcription initiation and the regulatory potential of chromatin during RNA maturation. The findings improve the understanding of gene expression regulation in T. brucei, a eukaryotic parasite lacking transcriptional Regulation.},
  subject      = {Transkription},
  language  = {en}
}
@phdthesis{Ziegler2016,
  author    = {Ziegler, Christiane},
  title     = {Epigenetic Mechanisms in the Pathogenesis and Therapy of Anxiety Disorders},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146815},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Anxiety disorders (AD) are common, disabling mental disorders, which constitute the most prevalent mental health condition conveying a high individual and socioeconomic burden. Social anxiety disorder (SAD), i.e. fear in social situations particularly when subjectively scrutinized by others, is the second most common anxiety disorder with a life time prevalence of 10\%. Panic disorder (PD) has a life time prevalence of 2-5\% and is characterized by recurrent and abrupt surges of intense fear and anticipatory anxiety, i.e. panic attacks, occurring suddenly and unexpected without an apparent cue. In recent years, psychiatric research increasingly focused on epigenetic mechanisms such as DNA methylation as a possible solution for the problem of the so-called "hidden heritability", which conceptualizes the fact that the genetic risk variants identified so far only explain a small part of the estimated heritability of mental disorders. In the first part of this thesis, oxytocin receptor (OXTR) gene methylation was investigated regarding its role in the pathogenesis of social anxiety disorder. In summary, OXTR methylation patterns were implicated in different phenotypes of social anxiety disorder on a categorical, neuropsychological, neuroendocrinological as well as on a neural network level. The results point towards a multilevel role of OXTR gene hypomethylation particularly at one CpG site (CpG3, Chr3: 8 809 437) within the protein coding region of the gene in SAD. The second part of the thesis investigated monoamine oxidase A (MAOA) gene methylation regarding its role in the pathogenesis of panic disorder as well as - applying a psychotherapy-epigenetic approach - its dynamic regulation during the course of cognitive behavioural therapy (CBT) in PD patients. First, MAOA hypomethylation was shown to be associated with panic disorder as well as with panic disorder severity. Second, in patients responding to treatment MAOA hypomethylation was shown to be reversible up to the level of methylation in healthy controls after the course of CBT. This increase in MAOA methylation along with successful psychotherapeutic treatment was furthermore shown to be associated with symptom improvement regarding agoraphobic avoidance in an independent replication sample of non-medicated patients with PD. Taken together, in the future the presently identified epigenetic patterns might contribute to establishing targeted preventive interventions and personalized treatment options for social anxiety disorder or panic disorder, respectively.},
  subject      = {Angst},
  language  = {en}
}