Introduction: Body Mass Index (BMI) is a relative measure of whether an individual’s weight is at a healthy level for their height. A higher BMI is associated with an increased risk of cancer and cardiovascular disease (CVD). However, the biologic mechanisms are not well understood. One proposed mechanism is through changes in global DNA methylation levels, particularly global DNA hypomethylation. Global DNA hypomethylation refers to lower levels of DNA methylation across the entire genome and hypermethylation refers to higher levels of DNA methylation across the entire genome. Changes in methylation levels can affect gene expression, genomic stability, and chromosomal structure. The methylation status of repetitive sequences in the DNA, such as LINE-1, is commonly used to represent a surrogate measure of global DNA methylation levels. Objectives: 1. Quantify and describe LINE-1 DNA methylation in leukocytes in a large sample of healthy volunteers. 2. Examine the relationship between BMI and LINE-1 DNA methylation levels. 3. Assess if sex is an effect modifier of the relationship between BMI and LINE-1 DNA methylation levels. Methods: A nested cross-sectional study was composed of 502 healthy volunteers between the ages of 20 and 50. Subjects completed a study questionnaire and provided blood samples for laboratory analyses. For each subject, DNA was isolated, underwent bisulfite conversion, and LINE-1 DNA methylation levels were measured by Polymerase Chain Reaction (PCR) High-Resolution Melting Curve analysis. For the main analysis, a multivariate linear regression model was used to examine the relationship between BMI and LINE-1 DNA methylation levels, while controlling for confounders. Results: LINE-1 DNA methylation was normally distributed with a mean of 84.52% and a standard deviation of 3.19%. BMI (normal, overweight, and obese categories) was not significantly associated with LINE-1 DNA methylation levels in the adjusted linear regression model (p=0.41) and the interaction term between BMI and sex was not significant (p=0.50). Conclusions: LINE-1 DNA methylation was measured with a high degree of reliability in a sample of healthy volunteers. This research provided a description of LINE-1 DNA methylation levels in a large healthy population and showed that BMI was not associated with global DNA methylation. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2012-04-03 17:23:59.843

In eukaryotes, the SET1 family of methyltransferases carry out the methylation of Lysine 4 on Histone H3. Alone, these enzymes exhibit low enzymatic activity and require the presence of additional regulatory proteins, which include RbBP5, Ash2L, WDR5 and DPY-30, to stimulate their catalytic activity. While previous structural studies established the structural basis underlying the interaction between RbBP5, Ash2L and WDR5, the formation of the Ash2L/DPY-30 complex remains elusive. Here we report the crystal structure of the Ash2L/DPY-30 complex solved at 2.2Å. Our results show that a Cterminal amphipathic α-helix on Ash2L makes several hydrophobic interactions with the DPY-30 homodimer. Moreover, the structure reveals that a tryptophan residue on Ash2L, which directly precedes its C-terminal amphipathic α-helix, makes key interactions with one of DPY-30 α-helix. Finally, biochemical studies of Ash2L revealed a hitherto unknown ability of this protein to bind anionic lipids.

Epigenetic regulators are commonly mutated in cancer. Activating mutations and overexpression of EZH2 occur in lymphoma and other malignancies, while loss-of-function mutations are found in myeloid malignancies. This thesis is a study of this apparent contradiction, examining the importance of cellular context for Ezh2 loss during the evolution of a single malignancy, Acute Myeloid Leukaemia (AML). This work demonstrates diametrically opposite functions for Ezh2 at early and late stages during the evolution of leukemias generated by disparate AML-associated fusion-oncogenes. Ezh2 functions as an oncogene that may be therapeutically targeted during disease maintenance. In contrast, Ezh2 behaves as a tumour suppressor gene during AML induction. Integrated genomic analysis demonstrates that largely different expression programmes are de-repressed during disease induction and maintenance following Ezh2 loss. Studying disease induction, Ezh2 represses a subset of bivalent promoters that resolve towards gene activation upon Ezh2 loss, inducing a feto-oncogene programme including genes like Plag1, whose overexpression phenocopies Ezh2 loss to accelerate AML induction in mouse models. This data highlights the importance of cellular context and phase of disease evolution for Ezh2 function. Moreover, the work herein identifies EZH2 as a potential target in AML, whilst providing reassurance of the safety of this therapeutic strategy.

Made available in DSpace on 2014-12-02T11:16:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-26Bitstream added on 2014-12-02T11:21:03Z : No. of bitstreams: 1 000774986_20150601.pdf: 1296692 bytes, checksum: 21e36e9a6a948cafbd5e35952816c786 (MD5) Bitstreams deleted on 2015-06-03T11:42:46Z: 000774986_20150601.pdf,. Added 1 bitstream(s) on 2015-06-03T11:44:11Z : No. of bitstreams: 1 000774986.pdf: 9422731 bytes, checksum: e40055dff4f7f745ade353914c6e7948 (MD5) / Os produtos naturais têm desempenhado um papel importante pela humanidade, no uso da medicina popular e na geração da descoberta de novos fármacos. Fungos endofíticos são micro-organismos que vivem em associação mutualística com a espécie hospedeira trazendo benefícios a ambos, e são uma promissora fonte de produtos naturais. A espécie vegetal Eugenia jambolana tem sido intensamente explorada química e biologicamente devido a seus usos populares e por apresentar diversas atividades biológicas. No intuito de explorar a potencionalidade dessa espécie vegetal, E. jambolana foi submetida ao isolamento dos fungos endofíticos presentes nas folhas, caule e frutos, dos quais foram isolados treze fungos endofíticos. Estes endófitos foram cultivados em escala reduzida em Czapek (400 mL) para obtenção dos extratos brutos AcOEt. Os extratos brutos AcOEt foram submetidos a análises químicas (cromatografia em camada delgada comparativa, cromatografia líquida de alta eficiência e ressonância magnética nuclear de 1H) e biológicas (ensaios antioxidante, antifúngico, anticolinesterásico e citotóxico). Todos os extratos brutos apresentaram pelo menos uma atividade biológica positiva, o que adicionado as análises químicas permitiram selecionar dois fungos endofíticos para crescimento em escala ampliada, e isolamento dos metabólitos secundários. Destes, o fungo endofítico isolado do caule de E. jambolana codificado como Ej-c3 foi identificado como Saccharicola sp., foi possível isolar 7 substâncias das quais 4 são inéditas (4-7), os compostos 4 e 5 foram inativo nos ensaios realizados. Do estudo químico do fungo endofítico Botryosphaeria parva (Ej-f1), isolado das folhas, foram isolados 4 substâncias da classe das isocumarinas [meleina (3), 4-hidroximeleina (11), 5-hidroximeleina (13), 7-hidroximeleina (12)], as substâncias 3 e 13 foram ativas no ensaio antifúngico contra C. sphaerospermum. No intuito de aumentar a... / The natural products have occuped an important role for humanity, in the use of folk medicine and in the generation of drug discovery. Endophytic fungi are microorganism that living in a mutualist association with host species, refleting benefits to both, and they are a promising source for natural products. The vegetal species Eugenia jambolana has been intensively explored chemically and biologically, because of its popular uses and it has several biological activities. In order to explore the potencionality of this plant species, E. jambolana was submitted to isolation of endophytic fungi in the leaves stems and fruits, of which thirteen endophytic fungi were isolated. The endophytes were cultived in small scale in Czapek (400 mL) to give the EtOAc crude extract. The EtOAc crude extract were submited to chemistry (thin layer chromatography, high performace liquid chromatography and 1H nuclear magnetic ressonance) and biological (antioxidant, antifungal, anticholinesterase and cytotoxic activities) analysis. All of crude extract showed at least one positive biological activity, which added the chemical analyzes allowed us to select two endophytic fungi to growing on a larger scale, and isolation of secondary metabolites. The endophytic fungus isolated from stems of E. jambolana encoded Ej-c3 was identified as Saccharicola sp. it was isolated 7 substances of which 4 are new (4-7). The substances 4 e 5 were inactive in the biological assays. Chemical study of Botryosphaeria parva (Ej-f1) isolated from leaves, was isolated 4 isocoumains [melein (3), 4-hidroxymelein (11), 5-hidroxymelein (13), 7-hidroxymelein (12)]. The substances 3 and 13 were active in antifungal assay agains the fungus C. sphaerospermum. In order to increase the metabolite production and activate the silent biossintetic pathway, the epigenetic appear as a useful and simple tool to gene expression. Lecythophora sp. an endolichenic fungus from Parmotrema tinctorum, was...

Orientador: Angela Regina Araujo / Banca: Isabele Rodrigues Nascimento / Banca: Edson Rodrigues Filho / Banca: Carmen Lúcia Cardoso / Banca: Luiz Alberto Beraldo de Moraes / Resumo: Os produtos naturais têm desempenhado um papel importante pela humanidade, no uso da medicina popular e na geração da descoberta de novos fármacos. Fungos endofíticos são micro-organismos que vivem em associação mutualística com a espécie hospedeira trazendo benefícios a ambos, e são uma promissora fonte de produtos naturais. A espécie vegetal Eugenia jambolana tem sido intensamente explorada química e biologicamente devido a seus usos populares e por apresentar diversas atividades biológicas. No intuito de explorar a potencionalidade dessa espécie vegetal, E. jambolana foi submetida ao isolamento dos fungos endofíticos presentes nas folhas, caule e frutos, dos quais foram isolados treze fungos endofíticos. Estes endófitos foram cultivados em escala reduzida em Czapek (400 mL) para obtenção dos extratos brutos AcOEt. Os extratos brutos AcOEt foram submetidos a análises químicas (cromatografia em camada delgada comparativa, cromatografia líquida de alta eficiência e ressonância magnética nuclear de 1H) e biológicas (ensaios antioxidante, antifúngico, anticolinesterásico e citotóxico). Todos os extratos brutos apresentaram pelo menos uma atividade biológica positiva, o que adicionado as análises químicas permitiram selecionar dois fungos endofíticos para crescimento em escala ampliada, e isolamento dos metabólitos secundários. Destes, o fungo endofítico isolado do caule de E. jambolana codificado como Ej-c3 foi identificado como Saccharicola sp., foi possível isolar 7 substâncias das quais 4 são inéditas (4-7), os compostos 4 e 5 foram inativo nos ensaios realizados. Do estudo químico do fungo endofítico Botryosphaeria parva (Ej-f1), isolado das folhas, foram isolados 4 substâncias da classe das isocumarinas [meleina (3), 4-hidroximeleina (11), 5-hidroximeleina (13), 7-hidroximeleina (12)], as substâncias 3 e 13 foram ativas no ensaio antifúngico contra C. sphaerospermum. No intuito de aumentar a... / Abstract: The natural products have occuped an important role for humanity, in the use of folk medicine and in the generation of drug discovery. Endophytic fungi are microorganism that living in a mutualist association with host species, refleting benefits to both, and they are a promising source for natural products. The vegetal species Eugenia jambolana has been intensively explored chemically and biologically, because of its popular uses and it has several biological activities. In order to explore the potencionality of this plant species, E. jambolana was submitted to isolation of endophytic fungi in the leaves stems and fruits, of which thirteen endophytic fungi were isolated. The endophytes were cultived in small scale in Czapek (400 mL) to give the EtOAc crude extract. The EtOAc crude extract were submited to chemistry (thin layer chromatography, high performace liquid chromatography and 1H nuclear magnetic ressonance) and biological (antioxidant, antifungal, anticholinesterase and cytotoxic activities) analysis. All of crude extract showed at least one positive biological activity, which added the chemical analyzes allowed us to select two endophytic fungi to growing on a larger scale, and isolation of secondary metabolites. The endophytic fungus isolated from stems of E. jambolana encoded Ej-c3 was identified as Saccharicola sp. it was isolated 7 substances of which 4 are new (4-7). The substances 4 e 5 were inactive in the biological assays. Chemical study of Botryosphaeria parva (Ej-f1) isolated from leaves, was isolated 4 isocoumains [melein (3), 4-hidroxymelein (11), 5-hidroxymelein (13), 7-hidroxymelein (12)]. The substances 3 and 13 were active in antifungal assay agains the fungus C. sphaerospermum. In order to increase the metabolite production and activate the silent biossintetic pathway, the epigenetic appear as a useful and simple tool to gene expression. Lecythophora sp. an endolichenic fungus from Parmotrema tinctorum, was... / Doutor

Deaf1 regulates 5-HT1A receptor gene (HTR1A) transcription by binding a 26-bp palindromic DNA sequence containing the rs6295 C(-1019) HTR1A polymorphism. However, Deaf1 cannot bind the G(-1019) HTR1A allele, which is associated with major depression. MeCP2 is impli-cated in serotonergic regulation and because the C(-1019) is a methylation site, I addressed whether MeCP2 alters Deaf1 regulation of HTR1A. MeCP2 and Deaf1 co-precipitated and bound the putative Deaf1 sites of the mouse HTR1A promoter in vivo. Binding of MeCP2 to the Deaf1 site was dependent on Deaf1 and MeCP2 increased Deaf1 recruitment to its site independ-ently of methylation. MeCP2, Deaf1 and their combination modulated transcription of human and mouse HTR1A luciferase reporter constructs, with Deaf1 having a dominant effect, Deaf1 actions were disrupted by mutation of the Deaf1 site(s). Deaf1 KO or MeCP2 cKO in mice in-creased functional 5-HT1A autoreceptor-mediated hypothermia response. Overall, MeCP2 and Deaf1 coordinately regulate the HTR1A promoter.

In the last years, the amount of available data in the field of computational biology steadily increased. In order to be able to analyze these data, various algorithms have been developed by bioinformaticians to process them efficiently. Moreover, computational models were developed to predict for instance biological relationships of species. Furthermore, the prediction of properties like the structure of certain biological molecules is modeled by complex algorithms. Despite these advances in handling such complicated tasks with automated workflows and a huge variety of freely available tools, the expert still needs to supervise the data analysis pipeline inspecting the quality of both the input data and the results. Additionally, choosing appropriate parameters of a model is quite involved. Visual support puts the expert into the data analysis loop by providing visual encodings of the data and the analysis results together with interaction facilities. In order to meet the requirements of the experts, the visualizations usually have to be adapted for the application purpose or completely new representations have to be developed. Furthermore, it is necessary to combine these visualizations with the algorithms of the experts to prepare the data. These in-situ visualizations are needed due to the amount of data handled within the analysis pipeline in this domain. In this thesis, algorithms and visualizations are presented that were developed in two different research areas of computational biology. On the one hand, the multi-replicate peak-caller Sierra Platinum was developed, which is capable of predicting significant regions of histone modifications occurring in genomes based on experimentally generated input data. This algorithm can use several input data sets simultaneously to calculate statistically meaningful results. Multiple quality measurements and visualizations were integrated into to the data analysis pipeline to support the analyst. Based on these in-situ visualizations, the analyst can modify the parameters of the algorithm to obtain the best results for a given input data set. Furthermore, Sierra Platinum and related algorithms were benchmarked against an artificial data set to evaluate the performance under specific conditions of the input data set, e.g., low read quality or undersequenced data. It turned out that Sierra Platinum achieved the best results in every test scenario. Additionally, the performance of Sierra Platinum was evaluated with experimental data confirming existing knowledge. It should be noticed that the results of the other algorithms seemed to contradict this knowledge. On the other hand, this thesis describes two new visualizations for RNA secondary structures. First, the interactive dot plot viewer iDotter is described that is able to visualize RNA secondary structure predictions as a web service. Several interaction techniques were implemented that support the analyst exploring RNA secondary structure dot plots. iDotter provides an API to share or archive annotated dot plots. Additionally, the API enables the embedding of iDotter in existing data analysis pipelines. Second, the algorithm RNApuzzler is presented that generates (outer-)planar graph drawings for all RNA secondary structure predictions. Previously presented algorithms failed in always producing crossing-free graphs. First, several drawing constraints were derived from the literature. Based on these, the algorithm RNAturtle was developed that did not always produced planar drawings. Therefore, some drawing constraints were relaxed and additional drawing constraints were established. Building on these modified constraints, RNApuzzler was developed. It takes the drawing generated by RNAturtle as an input and resolves the possible intersections of the graph. Due to the resolving mechanism, modified loops can become very large during the intersection resolving step. Therefore, an optimization was developed. During a post-processing step the radii of the heavily modified loops are reduced to a minimum. Based on the constraints and the intersection resolving mechanism, it can be shown that RNApuzzler is able to produce planar drawings for any RNA secondary structure. Finally, the results of RNApuzzler are compared to other algorithms.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20990号 / 医博第4336号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 山田 亮, 教授 藤渕 航 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A Dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2015 / The free radical theory of aging postulates that accumulation of oxidative damage in major cellular components is the predominant underlying cause of the aging phenotype. This damage is caused most commonly by reactive oxygen species (ROS) and antioxidant enzymes such as the superoxide dismutases (SOD) that neutralize ROS, are therefore vital. Manganese superoxide dismutase (MnSOD) is particularly critical as it is functional in the mitochondria, a major site for ROS generation. Numerous studies have demonstrated a tissue-specific decrease in the activity and mRNA levels of major antioxidants, including MnSOD, with aging, however the exact mechanism of this regulation is unclear. It was hypothesized that a general down-regulation of various antioxidant enzymes such as this may occur at the transcriptional level. In order to investigate SOD2 regulation, a comprehensively annotated rat SOD2 promoter region was established using the appropriate bioinformatics tools. Following this, SOD2 mRNA levels in tissues from young and old rat tissue were compared using quantitative PCR. The results showed increased and decreased SOD2 mRNA levels in old compared to young liver tissue and brain tissue, respectively, however these trends were not statistically significant. As MnSOD has been shown to be epigenetically downregulated in various age-related diseases it was hypothesized that the decrease in MnSOD mRNA levels seen in aging brain tissue may be a result of epigenetic regulation at the SOD2 (MnSOD gene) promoter, specifically, through DNA methylation. A methylation assay assessing the SOD2 gene promoter revealed no significant evidence of hypermethylation. Although this suggests that promoter methylation is an unlikely mechanism of SOD2 regulation in aging, further work would need to be implemented in order to prove this conclusively.

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