Es wird gezeigt, wie das Zusammentreffen hervorragender Wissenschaftler mit breitem Wissen und Fähigkeiten zur interdisziplinären Kooperation im letzten Jahrzehnt vor der Machtergreifung der Nationalsozialisten zu einer Glanzzeit naturwissenschaftlicher Forschung an der Freiburger Universität führte. / It is demonstrated, how the coincidence of excellent scientists with broad knowledge und ability for interdisciplinary cooperation led to a height of scientific research at the Freiburg University.

Orientador: Antonio Herbert Lancha Junior / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T15:31:34Z (GMT). No. of bitstreams: 1 Campos_PatriciaLopesde_D.pdf: 998065 bytes, checksum: 6a4a069b0d9422cc32f9686a61e72b14 (MD5) Previous issue date: 2005 / Resumo: Durante o exercício sub-máximo, a utilização de estoques de glicogênio muscular é de suma importância para possibilitar a continuidade do mesmo. A suplementação de Aminoácidos de Cadeia Ramificada (AACR ou BCAA, do inglês Branched Chain Amino Acids) tem sido experimentada como forma de gerar energia para o músculo nessas condições. Através da utilização dos seus esqueletos de carbono, podem ser gerados intermediários do ciclo dos ácidos tricarboxílicos (CAT ou TCA, do inglês Tricarboxilic Acids Cycle); no entanto, há controvérsias quanto à eficiência dos AACR em aumentar a atividade do ciclo, uma vez que autores sugerem a diminuição da eficiência do mesmo após tal estratégia de suplementação. Por outro lado, há toda uma discussão na literatura acerca da importância do aumento dos intermediários do TCA nos primeiros momentos do exercício para apoiar o metabolismo oxidativo, assim como o impacto que as reações anapleróticas, que promovem esse fenômeno, apresentam, considerando-se o fluxo de energia total do TCA. O objetivo desse trabalho foi avaliar se a suplementação dos aminoácidos em questão seria capaz de alterar o desempenho de ratos treinados em exercício sub-máximo, e também a formação de alguns intermediários do TCA ou mesmo a atividade de enzimas importantes para esse fenômeno. Para isso foram utilizados ratos Wistar machos adultos, que foram treinados por 8 semanas em sistema de natação, e receberam na última semana, a suplementação de isoleucina, leucina ou valina, exclusivamente, ou então os três aminoácidos em conjunto, ou placebo, por 7 dias. No sexto dia de suplementação, eles fizeram a última sessão de treinamento de 1 hora de natação, foram mantidos em jejum por 24 horas e, no dia seguinte, foram submetidos a um teste sub-máximo de natação, até a exaustão. O grupo AACR teve aumento na síntese de malato e o grupo isoleucina apresentou maior atividade das transaminases de alanina e aspartato (AAT e AST), quando comparados ao placebo. Não houve diferença no desempenho dos animais em relação à resistência à fadiga, apesar do grupo leucina apresentar menores concentrações de glicogênio muscular que o grupo placebo. Concluiu-se que os AACR não foram capazes de alterar atividade do ciclo de Krebs, nem o desempenho nessas condições experimentais / Abstract: During submaximal exercise, muscle glycogen utilization is important. Branched-chain AminoAcids supplementation has been utilized during the past years to provide energy for muscles in this condition, through their carbon skeletons, which might generate tricarboxilic acids cycle's intermediates (TCAI). However, this hypothesis remains controversial. On the other hand, there is a recent discussion on the literature about the real importance of the rapid muscle TCAI raise and its role for de TCA cycle. This study aimed to evaluate if BCAA supplementation in trained rats submitted to sub-maximal exercise were capable of changing TCAI activity or transaminases (AAT and AST) concentration, or even performance in muscle depletion. Wistar Male Adult rats were trained for 8 weeks in a proper swimming system. In the last week they received BCAA supplementation. Before the final experiment, they trained for 1 hour, and then wento through a 24 h fasting. The final test consisted on swimming until exhaustion, then they were euthanized and tissues were kept in liquid Nitrogen until further analysis. BCAA group had higher muscle concentrations of malate. Isoleucine group had higher muscle transaminases¿ concentration, although rats supplemented with leucine alone had lower muscle glycogen levels than placebo group. We concluded that none of the BCAA supplementation was able to affect performance or TCA activity in our experimental conditions / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Proteins and their interactions are fundamental to cellular life. Disruption of protein-protein, protein-RNA, or protein-DNA interactions can lead to disease, by affecting the function of protein complexes or by affecting gene regulation. A better understanding of these interactions on the molecular level gives rise to new methods to predict protein interaction, and is critical for the rational design of new therapeutic agents that disrupt disease-causing interactions. This thesis consists of three parts that focus on various aspects of protein interactions and their prediction in the context of disease. In the first part of this thesis, we classify interfaces of protein-protein interactions. We do so by systematically computing all binding sites between protein domains in protein complex structures solved by X-ray crystallography. The result is SCOPPI, the Structural Classification of Protein Protein Interfaces. Clustering and classification of geometrically similar interfaces reveals interesting examples comprising viral mimicry of human interface binding sites, gene fusion events, conservation of interface residues, and diversity of interface localisations. We then develop a novel method to predict protein interactions which is based on these structural interface templates from SCOPPI. The method is applied in three use cases covering osteoclast differentiation, which is relevant for osteoporosis, the microtubule-associated network in meiosis, and proteins found deregulated in pancreatic cancer. As a result, we are able to reconstruct many interactions known to the expert molecular biologist, and predict novel high confidence interactions backed up by structural or experimental evidence. These predictions can facilitate the generation of hypotheses, and provide knowledge on binding sites of promising disease-relevant candidates for targeted drug development. In the second part, we present a novel algorithm to search for protein binding sites in RNA sequences. The algorithm combines RNA structure prediction with sequence motif scanning and evolutionary conservation to identify binding sites on candidate messenger RNAs. It is used to search for binding sites of the PTBP1 protein, an important regulator of glucose secretion in the pancreatic beta cell. First, applied to a benchmark set of mRNAs known to be regulated by PTBP1, the algorithm successfully finds significant binding sites in all benchmark mRNAs. Second, collaborators carried out a screen to identify changes in the proteome of beta cells upon glucose stimulation while inhibiting gene expression. Analysing this set of post-transcriptionally controlled candidate mRNAs for PTBP1 binding, the algorithm produced a ranked list of 11 high confident potential PTBP1 binding sites. Experimental validation of predicted targets is ongoing. Overall, identifying targets of PTBP1 and hence regulators of insulin secretion may contribute to the treatment of diabetes by providing novel protein drug targets or by aiding in the design of novel RNA-binding therapeutics. The third part of this thesis deals with gene regulation in disease. One of the great challenges in medicine is to correlate genotypic data, such as gene expression measurements, and other covariates, such as age or gender, to a variety of phenotypic data from the patient. Here, we address the problem of survival prediction based on microarray data in cancer patients. To this end, a computational approach was devised to find genes in human cancer tissue samples whose expression is predictive for the survival outcome of the patient. The central idea of the approach is the incorporation of background knowledge information in form of a network, and the use of an algorithm similar to Google s PageRank. Applied to pancreas cancer, it identifies a set of eight genes that allows to predict whether a patient has a poor or good prognosis. The approach shows an accuracy comparable to studies that were performed in breast cancer or lymphatic malignancies. Yet, no such study was done for pancreatic cancer. Regulatory networks contain information of transcription factors that bind to DNA in order to regulate genes. We find that including background knowledge in form of such regulatory networks gives highest improvement on prediction accuracy compared to including protein interaction or co-expression networks. Currently, our collaborators test the eight identified genes for their predictive power for survival in an independent group of 150 patients. Under a therapeutic perspective, reliable survival prediction greatly improves the correct choice of therapy. Whereas the live expectancy of some patients might benefit from extensive therapy such as surgery and chemotherapy, for other patients this may only be a burden. Instead, for this group, a less aggressive or different treatment could result in better quality of the remaining lifetime. Conclusively, this thesis contributes novel analytical tools that provide insight into disease-relevant interactions of proteins. Furthermore, this thesis work contributes a novel algorithm to deal with noisy microarray measurements, which allows to considerably improve prediction of survival of cancer patients from gene expression data.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Recent data from the literature have suggested that some Krebs cycle intermediates could act as potent antioxidant agents, both in vitro and in vivo, against a variety of pro-oxidant agents. However, the mechanism(s) involved in the antioxidant effect of Krebs cycle intermediates are not fully understood. Additionally, there are scarce data in the literature taking into account the in vitro effect of Krebs cycle intermediates during oxidative stress conditions. Thus, the aim of this study was to determine the effect of some Krebs cycle intermediates on lipid peroxidation induced in vitro by different pro-oxidant agents, and the mechanism(s) by which they act. Firstly, we investigated the effect and the mechanism(s) by which malonate and quinolinic acid modulate the thiobarbituric acid- reactive species (TBARS) production in vitro, using rat brain S1 preparations (Article 1). The present results showed that the malonate-induced TBARS production was not changed by potassium cyanide or MK-801. However, the pro-oxidant effect of quinolinic acid was significantly prevented by MK-801. In addition we found that malonate was able to form complexes with iron ions (Fe2+), but these complexes were not able to interfere with in vitro deoxyribose degradation assays. Based on the results presented, we conclude that malonate pro-oxidant activity in vitro seems to be independent of the NMDA receptors activity. Additionally, we suggest that the malonate effect, in these conditions, is due to its ability to form complexes with iron ions, thus modulating an adequate ratio Fe2+/Fe3+ that could cause an increase in free radicals generation. In contrast, the quinolinic acid effect seems to be dependent of the NMDA receptors activation. However, we can not rule out the involvement of iron ions in quinolinic acid toxicity under our assay conditions. An other objective of this study was to investigate the effect of some Krebs cycle intermediates on quinolinic acid- or iron (Fe2+)-induced TBARS production in the rat brain S1 preparations, and the mechanism(s) by which they act (Article 2). The results showed that oxaloacetate, citrate, succinate, and malate were able to significantly prevent both basal and quinolinic acid- or iron-induced TBARS production. However, α-ketoglutarate induced per se a significant increase in basal TBARS production. The addition of potassium cyanide or the heat-treatment of S1 at 100ºC during 10 min completely abolished the antioxidant succinate activity, without change the effect of other Krebs cycle intermediates studied. Except for succinate, all intermediates used in this study were able to form complexes with iron (Fe2+) ions, however only oxaloacetate and α-ketoglutarate significantly prevented deoxyribose degradation induced by hydrogen peroxide. Based on the results presented, we concluded that oxaloacetate, malate, succinate, and citrate could act as antioxidants under basal, and under quinolinic acid- or iron- induced TBARS production, whereas α-ketoglutarate act as a pro-oxidant agent per se. The mechanism(s) by which citrate, malate, and oxaloacetate acts seems to be related to their ability to form complexes with iron (Fe2+) ions, thus modulating the iron redox cycle. In contrast, the succinate antioxidant effect seems to be dependent of the succinate dehydrogenase (SDH) activity. / Dados recentes na literatura têm relatado que alguns intermediários do ciclo de Krebs podem agir como potentes antioxidantes, tanto in vitro, quanto in vivo, em diversos sistemas pró-oxidantes. Porém, o(s) mecanismo(s) através dos qual(is) os intermediários do ciclo de Krebs exercem suas atividades antioxidantes não são completamente entendidas. Considerando a escassez de dados in vitro na literatura a respeito do efeito desses intermediários durante situações de estresse oxidativo, o presente trabalho tem como objetivo determinar o efeito de intermediários do ciclo de Krebs sob a peroxidação lipídica induzida por diferentes agentes pró-oxidantes in vitro, bem como investigar o(s) mecanismo(s) de ação dos mesmos. Primeiramente investigamos o efeito e o(s) mecanismo(s) pelo(s) qual(is) o malonato e o ácido quinolínico modulam a produção de espécies reativas ao ácido tiobarbitúrico (TBARS) em S1 de cérebro de ratos, in vitro (artigo 1). Os resultados obtidos mostraram um aumento na produção de TBARS induzido pelo malonato, o qual não foi modificado pela adição de cianeto de potássio, nem pelo MK-801. Por outro lado, o efeito pró-oxidante do ácido quinolínico foi significativamente prevenido pelo MK-801. Observamos ainda que o malonato foi capaz de formar complexos com íons ferrosos e que esses complexos não foram capazes de interferir nos ensaios da degradação da desoxirribose in vitro. Portanto, com base nos resultados encontrados, concluímos que o efeito pró-oxidante do malonato in vitro parece ser independente da atividade dos receptores NMDA. Os resultados sugerem que o efeito do malonato nessas condições deve-se principalmente a sua capacidade de interagir com íons ferro, modulando uma razão Fe2+/Fe3+ que favorece a geração de radicais livres. Por outro lado, o efeito do ácido quinolínico parece ser devido à ativação dos receptores NMDA. Porém, não podemos excluir a participação dos íons ferro para a toxicidade do mesmo nessas condições. Outro foco deste estudo foi investigar o efeito de alguns intermediários do ciclo de Krebs na produção de TBARS induzida por ácido quinolínico ou ferro em S1 de cérebro de ratos in vitro, bem como investigar o(s) mecanismo(s) de ação dos mesmos (artigo 2). Os resultados mostraram que o oxaloacetato, o citrato, o sucinato e o malato foram capazes de reduzir significativamente a produção de TBARS basal, bem como a induzida por ácido quinolínico ou ferro. Por outro lado, o α-cetoglutarato foi capaz de induzir per se um significativo aumento na produção de TBARS. A adição de cianeto de potássio, bem como o pré-tratamento do S1 por 10 min a 100ºC aboliram completamente o efeito antioxidante do sucinato, sem interferir significativamente no efeito dos demais intermediários estudados. Todos os intermediários estudados, exceto o sucinato, foram capazes de quelar íons ferro, porém somente o oxaloacetato e o α-cetoglutarato foram capazes de prevenir a degradação da desoxirribose induzida por peróxido de hidrogênio. Com base nos resultados obtidos, podemos concluir que o oxaloacetato, o malato o sucinato e o citrato agem como antioxidantes sob condições basais ou em presença do ácido quinolínico ou ferro, enquanto que o α-cetoglutarato age como um agente pró-oxidante per se. O mecanismo pelo qual o citrato, o malato e o oxaloacetato exercem seus efeitos antioxidantes parece ser devido à capacidade desses em interagir com íons ferro modulando o ciclo redox desse. Por outro lado, o efeito do sucinato parece ser devido à atividade da enzima succinato desidrogenase (SDH).

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Previous data from the literature have shown that some Krebs cycle intermediates could act as antioxidant in several models, both in vitro and in vivo. However, the mechanism(s) involved in the antioxidant effect of Krebs cycle intermediates are not fully understood. Additionally, there are scarce data in the literature taking into account the in vitro effect of Krebs cycle intermediates during oxidative stress conditions. Thus, the aim of this study was to determine the effect of some Krebs cycle intermediates on lipid peroxidation induced in vitro by different pro-oxidant agents, and the mechanism(s) by which they act. Furthermore, it was necessary elucidate the mechanisms by which the different pro-oxidants acts under in vitro conditions. The present results showed that the malonate-induced TBARS production was not changed by potassium cyanide or MK-801. However, the pro-oxidant effect of quinolinic acid was significantly prevented by MK-801. In addition we found that both malonate and oxalate were able to form complexes with iron ions (Fe2+). Based on the presented results, we conclude that malonate pro-oxidant activity in vitro seems to be independent of the secondary excitotoxicity via indirect NMDA receptors activation. Additionally, we suggest that both the malonate and oxalate effect, in these experimental conditions, is due to its ability to form complexes with iron ions, thus modulating an adequate ratio Fe2+/Fe3+ that could cause an increase in free radicals generation. In contrast, the quinolinic acid effect seems to be dependent of the NMDA receptors activation. However, we can not rule out the involvement of iron ions in quinolinic acid toxicity under our assay conditions. Another objective of this study was to investigate the effect of some Krebs cycle intermediates against either basal or induced TBARS production, using rat brain S1 preparations and the mechanism(s) by which they act. The results showed that oxaloacetate, citrate, succinate, and malate were able to significantly prevent both basal and quinolinic acid-, iron- or malonate-induced TBARS production. On the other hand, fumarate prevented only malonate-induced TBARS production, without effect under basal conditions. However, α-ketoglutarate induced per se a significant increase in basal TBARS production. The antioxidant activity of fumarate and succinate were completely abolished when S1 was submitted to heat-treatment at 100ºC during 10 min. Likewise, potassium cyanide completely abolished the antioxidant effect of succinate. The effect of other Krebs cycle intermediates studied was unchanged with respect to heat-treatment, or cyanide. Except for succinate and fumarate, all intermediates used in this study were able to form complexes with iron (Fe2+) ions, however only oxaloacetate and α-ketoglutarate significantly prevented deoxyribose degradation induced by hydrogen peroxide. Based on the results presented, we concluded that oxaloacetate, malate, succinate, fumarate and citrate could act as antioxidants under such conditions, whereas α-ketoglutarate acts as a pro-oxidant agent per se. The mechanism(s) by which citrate, malate, and oxaloacetate acts seems to be related to their ability to form complexes with iron (Fe2+) ions, thus modulating the iron redox cycle. In contrast, the succinate and fumarate antioxidant effect seems to be dependent of the some enzymatic system. / Dados prévios da literatura têm mostrado que alguns intermediários do ciclo de Krebs podem agir como antioxidantes em diversos modelos, tanto in vitro, quanto in vivo. Porém, o(s) mecanismo(s) através dos qual(is) esses intermediários exercem suas atividades antioxidantes não são completamente entendidas. Considerando a escassez de dados na literatura a respeito do efeito dos intermediários do ciclo de Krebs durante situações de estresse oxidativo, o presente trabalho teve por objetivo determinar o efeito desses sob a peroxidação lipídica induzida por diferentes agentes pró-oxidantes in vitro, bem como investigar o(s) mecanismo(s) de ação dos mesmos. Além disso, faz-se necessário caracterizar o(s) mecanismos(s) pelo(s) qual(is) os diferentes pró-oxidantes agem nos sistemas in vitro. Os resultados dessa tese mostraram que a atividade pró-oxidante in vitro do malonato não foi modificada pela adição de cianeto de potássio, nem pelo MK-801. Por outro lado, o efeito pró-oxidante do ácido quinolínico foi significativamente prevenido pelo MK-801. Observamos ainda que o malonato, e também o oxalato foram capazes de formar complexos com íons ferrosos. Portanto, com base nos resultados encontrados, concluímos que o efeito pró-oxidante do malonato in vitro parece ser independente da excitotoxicidade secundária, conseqüência da ativação indireta dos receptores NMDA. Os resultados sugerem que o efeito do malonato e do oxalato nessas condições experimentais deve-se principalmente a sua capacidade de interagir com íons ferro, modulando uma razão Fe2+/Fe3+ que favorece a geração de radicais livres. Por outro lado, o efeito do ácido quinolínico parece ser devido à ativação dos receptores NMDA. Porém, não podemos excluir a participação dos íons ferro para a toxicidade do mesmo nessas condições. Outro foco deste estudo foi investigar o efeito de alguns intermediários do ciclo de Krebs na produção de TBARS basal ou induzida por diferentes pró-oxidantes em S1 de cérebro de ratos in vitro, bem como investigar o(s) mecanismo(s) de ação dos mesmos. Os resultados mostraram que o oxaloacetato, o citrato, o sucinato e o malato foram capazes de reduzir significativamente a produção de TBARS basal, bem como a induzida por ácido quinolínico, ferro ou malonato. O fumarato, por sua vez, teve efeito antioxidante somente sobre a produção de TBARS induzida. Por outro lado, o α-cetoglutarato foi capaz de induzir per se um significativo aumento na produção de TBARS. O efeito antioxidante do fumarato e do sucinato foi completamente abolido quando o S1 foi submetido a um prétratamento por 10 min a 100ºC, enquanto que o efeito dos demais intermediários permaneceu inalterado. Da mesma forma, a adição de cianeto de potássio aboliu completamente o efeito antioxidante do sucinato sem interferir significativamente no efeito dos demais intermediários estudados. Todos os intermediários estudados, exceto o sucinato e o fumarato, foram capazes de quelar íons ferro, porém somente o oxaloacetato e o α- cetoglutarato foram capazes de prevenir a degradação da desoxirribose induzida por peróxido de hidrogênio. Com base nos resultados obtidos, podemos concluir que o oxaloacetato, o malato, o sucinato, o fumarato e o citrato agem como antioxidantes sob determinadas condições, enquanto que o α-cetoglutarato age como um agente pró-oxidante per se. O mecanismo pelo qual o citrato, o malato e o oxaloacetato exercem seus efeitos antioxidantes parece ser devido à capacidade desses em interagir com íons ferro modulando o ciclo redox desse. Por outro lado, o efeito do sucinato e do fumarato parece ser devido a alguma atividade enzimática.

Es wird gezeigt, wie das Zusammentreffen hervorragender Wissenschaftler mit breitem Wissen und Fähigkeiten zur interdisziplinären Kooperation im letzten Jahrzehnt vor der Machtergreifung der Nationalsozialisten zu einer Glanzzeit naturwissenschaftlicher Forschung an der Freiburger Universität führte. / It is demonstrated, how the coincidence of excellent scientists with broad knowledge und ability for interdisciplinary cooperation led to a height of scientific research at the Freiburg University.

Cell cycle (CC) and TP53 regulatory networks are frequently deregulated in cancer. While numerous genome-wide studies of TP53 and CC-regulated genes have been performed, significant variation between studies has made it difficult to assess regulation of any given gene of interest. To overcome the limitation of individual studies, we developed a meta-analysis approach to identify high confidence target genes that reflect their frequency of identification in independent datasets. Gene regulatory networks were generated by comparing differential expression of TP53 and CC-regulated genes with chromatin immunoprecipitation studies for TP53, RB1, E2F, DREAM, B-MYB, FOXM1 and MuvB. RNA-seq data from p21-null cells revealed that gene downregulation by TP53 generally requires p21 (CDKN1A). Genes downregulated by TP53 were also identified as CC genes bound by the DREAM complex. The transcription factors RB, E2F1 and E2F7 bind to a subset of DREAM target genes that function in G1/S of the CC while B-MYB, FOXM1 and MuvB control G2/M gene expression. Our approach yields high confidence ranked target gene maps for TP53, DREAM, MMB-FOXM1 and RB-E2F and enables prediction and distinction of CC regulation. A web-based atlas at www.targetgenereg.org enables assessing the regulation of any human gene of interest.

Background: The Patient Health Questionnaire PHQ-9 is a widely used instrument to screen for depression in clinical research. The first aim of this study was to psychometrically test the PHQ-9 in a large sample of cancer patients. The second aim was to calculate unbiased estimates of the depression burden for several cancer groups taking into account age and gender distributions. Methods: A sample of 2,059 cancer patients with varying diagnoses were examined in this study six months after discharge from a rehabilitation clinic. A representative sample of 2,693 people from the general population served as controls. Expected PHQ-9 mean scores of the general population sample, regressed on age and gender, were calculated to enable a fair comparison of different groups of cancer patients. Results: While the reliability (Cronbach’s alpha) for the PHQ-9 scale was good (alpha ≥ 0.84), the CFA fit indices of the one-dimensional solution were unsatisfactory in the patients’ sample. The factorial analysis confirmed two factors. PHQ-9 mean scores for 15 types of cancer are given, ranging from 4.0 (prostate) to 8.2 (thyroid gland). Differences between expected mean scores (derived from the general population) and raw mean scores of the cancer subsamples are reported that provide a better estimate of the depression burden. Conclusions: The results confirmed that the PHQ-9 performs well in testing depression in cancer patients. Regression coefficients can be used for performing unbiased comparisons among cancer groups, not only for this study. The burden of patients with testis cancer and Hodgkin lymphoma is underestimated when age and gender are not taken into account.

The fundamental subject of this thesis is the development of tools for the analysis of DNA methylation data as well as their application on bisulfite sequencing data comprising a large number of samples. DNA methylation is one of the major epigenetic modifications. It affects the cytosines of the DNA and is essential for the normal development of cells and tissues. Unusual alterations are associated with a variety of diseases and, specially, in cancergeneous tissues global changes in the DNA methylation level have been detected. To sequence DNA methylation on single nucleotide resolution, the sequences are treated with sodium bisulfite before sequencing, whereby unmethylated cytosines are represented as thymines. Thus, specialized techniques are required to process and analyze these kind of data. Here, the bisulfite analysis toolkit BAT is introduced, that is designed to facilitate an quick analysis of bisulfite treated DNA methylation sequencing data. It covers all steps of processing raw sequencing data up to calling of differential DNA methylation. At the begin of analysis, sodium bisulfite treated sequence data are aligned and DNA methylation rates for each covered cytosine in the reference genome are called. Subsequently, BAT integrates annotation data and performs basic analysis, i. e., methylation rate distribution plots and hierarchical clustering of the samples. In addition, calling of differentially methylated regions is performed and statistics of called regions are automatically created. Finally, DNA methylation and gene expression data integration is covered by the calculation of correlating regions. Secondly, a novel algorithm, metilene, for the calculation of differentially methylated regions (DMRs) between two groups of samples is introduced. Existing methods are limited in terms of detection sensitivity as well as time and memory consumption. Our approach is based on a circular binary segmentation, using a scoring function to detect sub-regions that show a stronger difference between the mean methylation levels of two groups than the surrounding background. These sub-regions are tested using a two-dimensional Kolmogorov Smirnov test (2D-KS test) [Fasano 1987] for significant differences taking all samples of each group into account. The use of the non-parametric 2D-KS test allows to avoid assumptions about a background distribution. Furthermore, the two dimensions of the problem, i. e., (i) the detection of a region, such that (ii) the methylation rates of the samples in the groups are significantly different, are taken into account in a single test. The algorithm calls DMRs in sufficiently short time on single sample comparisons as well as on about 50 samples per group. Furthermore, it works on whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) data and is able so estimate missing data points from the methylation rates of other samples in the group. Benchmarks on simulated and real data sets show that metilene outperforms other existing methods and is especially suitable for noisy datasets often found for example in cancer analysis. In the framework of this thesis, the previously introduced methods and algorithms are used to analyze a WGBS dataset of two different subtypes of germinal-center derived B-cell lymphomas and healthy controls. In both lymphoma subgroups genome-wide hypomethylation was found, with an exception for a specific type of promoter regions, i. e., poised promoters, that were frequently found to be hypermethylated. Using the previously presented algorithm, DMRs were called between the three entities. A strong enrichment of DMRs immediately downstream of the transcription start site was observed, indicating the regulatory relevance of this regions. The integration of gene expression data of the same samples, revealed that a considerable amount of the DMRs showed significant correlation between gene expression and DNA methylation. Finally, transcription factor binding sites and mutation data were combined with the methylation and expression data analysis. This identified strongly altered signaling pathways and cancer subtype specific genes. Furthermore, the data integration indicates that mutations and DNA methylation changes may act complementary to another. Finally, findings from the lymphoma study regarding the hypermethylation of poised promoters in cancer were extended to a huge data set comprising a variety of cancers. We could show that the relation of DNA methylation at a small set of frequently poised regions with respect to the background methylation level is sufficient to classify almost all samples based on DNA methylation data from 450k BeadChips into cancer or non-cancer probes. In addition, we found that the increase in methylation co-occurs with upregulated gene expression of several poised promoter regulated genes in almost all fresh cancer samples, implying a de-poising of poised regions. This upregulated gene expression is in contrast to the silencing of those genes in cancer cell lines, indicating that the upregulated gene expression might be a temporary status and possibly contributes to cancerogenesis.

Ethyl pyruvate, a known ROS scavenger and anti-inflammatory drug was found to combat leukemia cells. Tumor cell killing was achieved by concerted action of necrosis/apoptosis induction, ATP depletion, and inhibition of glycolytic and para-glycolytic enzymes. Ethyl lactate was less harmful to leukemia cells but was found to arrest cell cycle in the G0/G1 phase. Both, ethyl pyruvate and ethyl lactate were identified as new inhibitors of GSK-3β. Despite the strong effect of ethyl pyruvate on leukemia cells, human cognate blood cells were only marginally affected. The data were compiled by immune blotting, flow cytometry, enzyme activity assay and gene array analysis. Our results inform new mechanisms of ethyl pyruvate-induced cell death, offering thereby a new treatment regime with a high therapeutic window for leukemic tumors.