Protein arginine methylation is an important modification of proteins, involved in many cellular processes. Some examples are transcription, RNA editing, cellular communication, DNA repair, viral replication and chromatin remodeling. In recent years, the significance of protein arginine methyltransferases (PRMTs) in human diseases has been increasingly studied, especially in cardiovascular disease and cancer. Although the importance of these enzymes is recognized, the understanding of how exactly PRMTs function is still limited. Very little information is available to explain how or why any of the different PRMTs interact with other proteins or, what determines where in that protein to place their methyl marks. Adding to this complexity, placing one of the three different methylation marks (products) or the other (mono methyl arginine MMA, asymmetric dimethyl ADMA, or symmetric dimethyl SDMA) on a protein can cause a cell to respond differently. Therefore, if we really want to understand how this family of proteins functions and how to control them, it’s essential that we understand how they achieve their product specificity; this means, how they decide which methyl mark to place on an interacting protein. In order to better understand the product specificity of this family of enzymes, I have been using as a model two Protein arginine methyltransferases that are responsible different methylation marks: PRMT1, which can make both ADMA and MMA and TbPRMT7, which can only make MMA. Using the information that crystal structure of these enzymes provide and what we already know about how PRMT activity is regulated, my aim is to better understand the mechanisms by which these enzymes achieve their product specificity.

No description available.

The 5' end of eukaryotic and viral mRNAs contain a "cap" structure with the sequence m7G(5')pppN(5'). The methylation of the 7-position on the guanine cap is very important to proper mRNA processing and initiation of translation. The enzyme responsible for this methylation, RNA guanine-7-methyltransferase, has been cloned and studied from a number of different species, including human, X. laevis, yeast, and C. elegans. The sequences for mouse guanine-7-methyltransferase cDNA and protein have been deduced based upon identity of mouse ESTs to the cDNA of the human enzyme. The deduced mouse cDNA encodes an ORF of 465 amino acids and is 76.4% identical to the human enzyme, or 86.5% within the C-terminal domain. Active site characterization of mouse and human guanine-7-methyltransferase indicates a cysteine residue is important to proper enzyme activity. Enzyme activity was completely eliminated when N-ethylmaleimide (NEM) was added to the assay mixture. When the product of the reaction, S-adenosyl-L-homocysteine (SAH), was added at a concentration of 40uM the mouse enzyme retained 60% activity while enzyme isolated from Human Osteosarcoma (HOS) cells retained 100% of the original activity. SAH demonstrated no protective effects on the cloned human enzyme. Factors that affect binding of RNA to the active site were also investigated. UV-cross-linking of RNA to the active site of the mouse enzyme was inhibited 35% by NEM. Cap analog, GpppG, at a concentration of 1mM, inhibited cross-linking, but the similar nucleotide GMP, at a concentration of 1mM, did not inhibit cross-linking. These analyses have given a clearer understanding of this very important enzyme. / Master of Science

In eukaryotic genomes ranging from plants to mammals, DNA methylation is a major epigenetic modification of DNA by adding a methyl group exclusively to cytosine residuals. In mammalian genomes such as humans, these cytosine bases are usually followed by guanine. Although it does not change the primary DNA sequence, this covalent modification plays critical roles in several regulatory processes and can impact gene activity in a heritable fashion. What is more important, DNA methylation is essential for mammalian embryonic development and aberrant DNA methylation is implicated in several human diseases, in particular in neuro-developmental syndromes (such as the fragile X and Rett syndromes) and cancer. These biological significances disclose the importance of understanding genomic patterns and function role of DNA methylation in human, as a initial step to get to know the epigenotype and its manner in connecting the phenotype and genotype. Two key papers back in 1975 independently suggested that methylation of CpG dinucleotides in vertebrates could be established de novo and inherited through somatic cell divisions by protein machineries of DNA methyltransferases that recognizes hemi-methylated CpG palindromes. They also indicated that the methyl group could be recognized by DNA-binding proteins and that DNA methylation directly silences gene expression. After almost four decades, several key points in these foundation papers are proved to be true. Take the mammalian genome for example, there are several findings indicating the epigenetic repression of gene expression by DNA methylation. These include X-chromosome inactivation, gene imprinting and suppressing the proliferation of transposable elements and repeat elements of viral or retroviral origin. In addition to these, many novel roles of DNA methylation have also been revealed. For example, DNA methylation can regulate alternative splicing by preventing CTCF, an evolutionarily conserved zinc-finger protein, binding to DNA. By using the technique of fluorescence resonance energy transfer (FRET) and fluorescence polarization, DNA methylation has also been shown to increase nucleosome compaction through DNA-histone contacts. What is more important, DNA methylation is essential for mammalian embryonic development and aberrant change of DNA methylation has been related to disease such as cancer. However, it is also notable there are several lines of evidence contradicting the relationship between DNA methylation and gene silencing. For example, comparison of DNA methylation levels in human genome on the active and inactive X chromosomes showed reduced methylation specifically over gene bodies on inactive X chromosomes. Not only in human, DNA methylation is found to be usually targeted to the transcription units of actively transcribed genes in invertebrate species. These results prove that the function of DNA methylation is challenging to be unravel. Besides, due to the development of sequencing technique, whole genome DNA methylation profiles have been detected in diverse species. Comparing genomic patterns of DNA methylation shows considerable variation among taxa, especially between vertebrates and invertebrates. However, even though extensive studies reveal the patterns and functions of DNA methylation in different species, in the mean time, they also highlight the limits to our understanding of this complex epigenetic system. During my Ph.D., in order to perform in-depth studies of DNA methylation in diverse animals as a way to understand the complexity of DNA methylation and its functions, I dedicated my efforts in investigating and analyzing the DNA methylation profiles in diverse species, ranging from insects to primates, including both model and non-model organisms. This dissertation, which constitutes an important part of my research, mainly focuses on the DNA methylation profile in primates including human and chimpanzee. In general, I will use three chapters to elucidate my work in generating and interpreting the whole genome DNA methylation data. Firstly, we generated nucleotide-resolution whole-genome methylation maps of the prefrontal cortex of multiple humans and chimpanzees, then comprehensive comparative studies for these DNA methylation maps have been performed, by integrating data on gene expression as well. This work demonstrates that differential DNA methylation might be an important molecular mechanism driving gene-expression divergence between human and chimpanzee brains and also potentially contribute to the human-specific traits, such as evolution of disease vulnerabilities. Secondly , we performed global analyses of CpG islands (CGIs) methylation across multiple methylomes of distinctive cellular origins in human. The results from this work show that the human CpG islands can be distinctly classified into different clusters solely based upon the DNA methylation profiles, and these CpG islands clusters reflect their distinctive nature at many biological levels, including both genomic characteristics and evolutionary features. Moreover, these CpG islands clusters are non-randomly associated with several important biological phenomena and processes such as diseases, aging, and gene imprinting. These new findings shed lights in deciphering the regulatory mechanisms of CpG islands in human health and diseases. At last, by utilizing the DNA methylome from human sperm and genetic map generated from the International HapMap Consortium project, we investigated the hypothesis suggesting a potential role of germ line DNA methylation in affecting meiotic recombination, which is essential for successful meiosis and various evolutionary processes. Even thought the results imply that DNA methylation is a important factor affecting regional recombination rate, the strength of correlation between these two is not as strong as the previous report. Besides, high-throughput analyses indicate that other epigenetic modifications, tri-methylation of histone 3 lysine 4 and histone 3 lysine 27 are also global features at the recombination hotspots, and may interact with methylation to affect the recombination pattern simultaneously. This work suggests epigenetic mechanisms as additional factors affecting recombination, which cannot be fully explained by the DNA sequence itself. In summary, I hope the results from these work can expand our knowledge regarding the common and variable patterns of DNA methylation in different taxa, and shed light about the function role and its major change during animal evolution.

INTRODUÇÃO: A expansão e disseminação do consumo de crack e cocaína no Brasil vem se tornando um grave problema de saúde pública nos últimos vinte anos. Diferentes abordagens biológicas têm sido investigadas utilizando o fenótipo de abuso/dependência de crack/cocaína, cujos resultados têm demonstrado a participação importante do substrato genético, assim como a sua interação com os fatores ambientais no desenvolvimento desse transtorno. OBJETIVOS: Investigar o padrão de metilação do DNA do genoma de indivíduos que apresentam abuso/dependência de cocaína e de crack, comparando ao padrão de metilação de indivíduos controles. MÉTODOS: Foram selecionados 24 dependentes de cocaína e crack e 24 controles saudáveis, pareados por sexo e idade. Utilizando amostras de DNA extraídas de sangue periférico de cada um dos participantes, foi realizada a técnica de metilação global com o ensaio Illumina Infinium Human Methylation450 (450K) BeadChip. Os resultados iniciais foram normalizados considerando a heterogeneidade celular e analisados utilizando o pacote ChAMP (Chip Analysis Methylation Pipeline) para identificar genes e/ou regiões gênicas diferencialmente metiladas que possam representar fatores de vulnerabilidade para o comportamento de abuso/dependência do crack e da cocaína. Os processos biológicos e vias celulares com os quais os sítios diferencialmente metilados estão envolvidos foram explorados usando as ferramentas disponibilizadas pelo \"WebGestalt\" e pelo \"UCSC Genome Browser\". RESULTADOS: Foram observados 250 sítios diferencialmente metilados, associados a 246 genes na comparação dos perfis de metilação entre os casos e controles, sendo que 49% destes estavam localizados nas regiões promotoras dos genes, sugerindo que esses sítios podem estar relacionados com a expressão gênica. Alterações estatisticamente significantes no padrão de metilação entre casos x controles foram observadas em 23 sítios CpG (p-valor ajustado < 10-5 e |?beta| = 0,1). Observou-se também que três regiões diferencialmente metiladas foram associadas a genes hipometilados (BMP8A, GPR88 e RNF166) (p-valor ajustado < 0.05), cada uma com pelo menos três sítios. Alterações estatisticamente significantes também foram observadas em seis genes hiper-representados: CALCA, NCOA2, DRD2, EHMT1, EHMT2, MAP2K1, MAPK3 e MAPK1, envolvidos em processos biológicos e moleculares. CONCLUSÕES: Observou-se diferenças estatisticamente significativas no padrão de metilação genômico de usuários/dependentes de crack e cocaína quando comparados aos controles saudáveis em amostra de DNA extraídas de sangue periférico. Comparação de estudos de expressão em tecido cerebral correlacionaram-se parcialmente com os achados apresentados. Outros estudos utilizando amostras independentes são necessários para confirmar esses achados. A confirmação desses resultados poderá contribuir na identificação e compreensão dos mecanismos biológicos envolvidos na dependência do crack/cocaína / BACKGROUND: The expansion and dissemination of crack and cocaine in Brazil has become a progressive and serious public health problem during the last twenty years. Different biological approaches have been investigated using the crack/cocaine abuser/dependent phenotype, with results confirming the important role of the genetic component, as well as its interaction with environmental factors. OBJECTIVES: To investigate the DNA methylation pattern levels in the genome of individuals with crack and cocaine abuse/dependence and comparing it with the DNA methylation pattern of the genome of control subjects. METHODS: 24 crack and cocaine abusers/dependents and 24 healthy controls were selected and matched by sex and age. Using DNA samples from peripheral blood of each participant, a global methylation technique was performed using the Illumina Infinium Human Methylation450 (450K) Bead Chip assay. The initial results were normalized for cellular heterogeneity and re-analyzed using ChAMP package (Chip Methylation Analysis Pipeline) to identify differentially methylated genes or/and DNA regions that may represent biological/genetic risk factors for crack and cocaine abuse/dependence behavior. The biological processes and cellular pathways were explored using tools provided by \"WebGestalt\" and \"UCSC Genome Browser\". RESULTS: 250 differentially methylated sites associated with 246 genes in methylation comparison profiles between cases and controls were identified, of which almost half were located in the promoter regions of genes (49%), suggesting that these sites may be related to gene expression. Statistically significant changes in the methylation patterns between cases and controls were observed in 23 CpG sites (adjust p-value < 10-5 and |deltabeta| = 0.1). In addition, three differentially methylated regions were associated with hipomethylated genes (BMP8A, GPR88 e RNF166) (adjust p-value < 0.05), each one with at least three sites. Statistically significant changes were also observe with six hiper-represented genes: CALCA, NCOA2, DRD2, EHMT1, EHMT2, MAP2K1, MAPK3 e MAPK1, which are involved in biological and molecular processes. CONCLUSIONS: Crack and cocaine users/dependents presented significant statistical differences in the methylation pattern when compared to healthy controls in DNA samples extracted from peripheral blood. Results from gene expression studies using brain tissue can be correlated with our results. In order to confirm the present findings, future studies should be replicated using independent samples. The confirmation of these results will contribute to the understanding of the biological mechanisms involved in crack/cocaine dependence

INTRODUÇÃO: A expansão e disseminação do consumo de crack e cocaína no Brasil vem se tornando um grave problema de saúde pública nos últimos vinte anos. Diferentes abordagens biológicas têm sido investigadas utilizando o fenótipo de abuso/dependência de crack/cocaína, cujos resultados têm demonstrado a participação importante do substrato genético, assim como a sua interação com os fatores ambientais no desenvolvimento desse transtorno. OBJETIVOS: Investigar o padrão de metilação do DNA do genoma de indivíduos que apresentam abuso/dependência de cocaína e de crack, comparando ao padrão de metilação de indivíduos controles. MÉTODOS: Foram selecionados 24 dependentes de cocaína e crack e 24 controles saudáveis, pareados por sexo e idade. Utilizando amostras de DNA extraídas de sangue periférico de cada um dos participantes, foi realizada a técnica de metilação global com o ensaio Illumina Infinium Human Methylation450 (450K) BeadChip. Os resultados iniciais foram normalizados considerando a heterogeneidade celular e analisados utilizando o pacote ChAMP (Chip Analysis Methylation Pipeline) para identificar genes e/ou regiões gênicas diferencialmente metiladas que possam representar fatores de vulnerabilidade para o comportamento de abuso/dependência do crack e da cocaína. Os processos biológicos e vias celulares com os quais os sítios diferencialmente metilados estão envolvidos foram explorados usando as ferramentas disponibilizadas pelo \"WebGestalt\" e pelo \"UCSC Genome Browser\". RESULTADOS: Foram observados 250 sítios diferencialmente metilados, associados a 246 genes na comparação dos perfis de metilação entre os casos e controles, sendo que 49% destes estavam localizados nas regiões promotoras dos genes, sugerindo que esses sítios podem estar relacionados com a expressão gênica. Alterações estatisticamente significantes no padrão de metilação entre casos x controles foram observadas em 23 sítios CpG (p-valor ajustado < 10-5 e |?beta| = 0,1). Observou-se também que três regiões diferencialmente metiladas foram associadas a genes hipometilados (BMP8A, GPR88 e RNF166) (p-valor ajustado < 0.05), cada uma com pelo menos três sítios. Alterações estatisticamente significantes também foram observadas em seis genes hiper-representados: CALCA, NCOA2, DRD2, EHMT1, EHMT2, MAP2K1, MAPK3 e MAPK1, envolvidos em processos biológicos e moleculares. CONCLUSÕES: Observou-se diferenças estatisticamente significativas no padrão de metilação genômico de usuários/dependentes de crack e cocaína quando comparados aos controles saudáveis em amostra de DNA extraídas de sangue periférico. Comparação de estudos de expressão em tecido cerebral correlacionaram-se parcialmente com os achados apresentados. Outros estudos utilizando amostras independentes são necessários para confirmar esses achados. A confirmação desses resultados poderá contribuir na identificação e compreensão dos mecanismos biológicos envolvidos na dependência do crack/cocaína / BACKGROUND: The expansion and dissemination of crack and cocaine in Brazil has become a progressive and serious public health problem during the last twenty years. Different biological approaches have been investigated using the crack/cocaine abuser/dependent phenotype, with results confirming the important role of the genetic component, as well as its interaction with environmental factors. OBJECTIVES: To investigate the DNA methylation pattern levels in the genome of individuals with crack and cocaine abuse/dependence and comparing it with the DNA methylation pattern of the genome of control subjects. METHODS: 24 crack and cocaine abusers/dependents and 24 healthy controls were selected and matched by sex and age. Using DNA samples from peripheral blood of each participant, a global methylation technique was performed using the Illumina Infinium Human Methylation450 (450K) Bead Chip assay. The initial results were normalized for cellular heterogeneity and re-analyzed using ChAMP package (Chip Methylation Analysis Pipeline) to identify differentially methylated genes or/and DNA regions that may represent biological/genetic risk factors for crack and cocaine abuse/dependence behavior. The biological processes and cellular pathways were explored using tools provided by \"WebGestalt\" and \"UCSC Genome Browser\". RESULTS: 250 differentially methylated sites associated with 246 genes in methylation comparison profiles between cases and controls were identified, of which almost half were located in the promoter regions of genes (49%), suggesting that these sites may be related to gene expression. Statistically significant changes in the methylation patterns between cases and controls were observed in 23 CpG sites (adjust p-value < 10-5 and |deltabeta| = 0.1). In addition, three differentially methylated regions were associated with hipomethylated genes (BMP8A, GPR88 e RNF166) (adjust p-value < 0.05), each one with at least three sites. Statistically significant changes were also observe with six hiper-represented genes: CALCA, NCOA2, DRD2, EHMT1, EHMT2, MAP2K1, MAPK3 e MAPK1, which are involved in biological and molecular processes. CONCLUSIONS: Crack and cocaine users/dependents presented significant statistical differences in the methylation pattern when compared to healthy controls in DNA samples extracted from peripheral blood. Results from gene expression studies using brain tissue can be correlated with our results. In order to confirm the present findings, future studies should be replicated using independent samples. The confirmation of these results will contribute to the understanding of the biological mechanisms involved in crack/cocaine dependence

Glioblastoma (GBM) is the most common and malignant of the glial tumors. These tumors may develop from lower-grade astrocytomas (diffuse astrocytoma; grade II or anaplastic astrocytoma; grade III) through a progressive pathway, but, more frequently, they manifest de novo without any evidence of a pre-malignant lesion. The treatment of GBM includes surgery, radiotherapy, and chemotherapy with temozolomide. Despite improvements in treatment protocols, the median survival of GBM patients remains very low at 12-15 months. The cause of glioma (either development or progression) can be genetic and epigenetic modification driven changes. In contrast to genetic modifications, where DNA sequence is changed, epigenetic modifications are those gene expression regulatory mechanisms which do not involve the change in the DNA sequence. It includes DNA methylation, chromatin modifications and miRNA mediated changes in gene expression. Aberrant DNA methylation is one of the common molecular lesions occurring in the cancer cell. The 5th position of cytosine (CpG) is the most preferred site of DNA methylation in mammalian cells. The methylated cytosines are prone to undergo oxidative deamination, and get mutated to thymine in DNA. Consequently, this led to decrease in CpG abundance in the genome. In normal conditions, promoters of majority of the genes escape methylation, because of which CpG of these regions remain same. This phenomenon led to the restriction of CpGs in the promoter regions of most of the genes. These CpG rich regions of the promoters are known as CpG islands, and the methylation status of these islands have a major role in regulating gene expression. The cancer genome is shown to undergo genome-wide hypomethylation whereas CpG islands undergo hypermethylation compared to normal tissue, resulting in net loss of total methylation, as the CpGs from non-island areas far exceed in number compared to the CpGs from islands. The most studied change of DNA methylation in neoplasms is the silencing of the tumor suppressor genes by CpG island promoter hypermethylation. Apart from few studies, the role of DNA methylation in glioma development and progression is poorly known. With this background, we have focused our study on DNA methylation changes in GBM. To identify GBM specific DNA methylation alterations, we have performed the genome wide methylation profile of 44 GBM and 8 normal samples using Infinium methylation array. Beta value, which is a measure of methylation, was calculated for all the CpG probes. Beta value ranges between 0-1 (from no methylation to complete methylation). We sought to understand the clinical importance, with particular importance to patient survival, of the DNA methylation pattern observed. We also undertook steps to understand the contribution of the differential DNA methylation and the associated gene expression changes in GBM development. This work has been divided into three parts: Part I –Identification of GBM specific methylome and development of a DNA methylation prognostic signature for GBM To identify the differentially methylated genes in GBM, we compared the methylation levels of 27,578 CpGs between GBM and normal control samples using statistical methods. We then compared the list of differentially methylated genes with the expression data generated by The Caner Genome Atlas (TCGA) to find out genes whose expression oppositely correlates with the DNA methylation status. This resulted in the identification of 62 genes hypermethylated and down regulated, while 65 genes hypomethylated and up regulated. We believe that this set of differentially methylated genes may play important role in glioma development. Next, to identify GBM specific DNA methylation survival signature, we correlated the survival data of 44 GBM patients with beta values of all the 27,578 probes. Using Cox regression method, we identified a set of 9 genes, whose methylation predicted the survival in GBM patients. A risk score was then calculated using methylation values and regression co-efficient of each of the genes. The methylation risk score was found to be an independent predictor of survival in a multivariate analysis in TCGA data set and the Bent et al data set (independent validation sets). Using methylation risk score, we were able to divide the patients into low and high risk groups with significant difference in survival. To discover the biology behind the difference in the survival of low and high risk groups, we performed network analysis, using differentially expressed genes between low and high risk patients, which revealed an activated NFkB pathway association with poor prognosis. The inhibition of NFkB pathway sensitized the glioma cells for chemotherapeutic drugs only in NFkB activated cell lines, suggesting a pivotal role for NFkB pathway imparting chemoresistance in poor surviving group. Part II -NPTX2, a methylation silenced gene, inhibits NFkB through a p53-PTEN-PI3K-AKT signaling pathway To understand the mechanism behind the prediction of survival by methylation of 9 genes, we took NPTX2 as a candidate gene for further investigation. NPTX2, a risky methylated gene, is highly methylated in high risk group with poor survival, which suggests that it may have a growth inhibitory activity in GBM. Bisulphite sequencing confirmed the hypermethylation status of NPTX2 promoter in GBM samples and glioma cell lines compared to normal brain tissue. As expected, NPTX2 transcript level was significantly down regulated in GBMs and glioma cell lines compared to normal samples, and could be re-expressed upon methylation inhibitor treatment in glioma cells. Exogenous over expression of NPTX2 inhibited proliferation, colony formation and sensitized glioma cells to chemotherapeutic drugs. Moreover, NPTX2 also inhibited soft agar colony formation in vitro, which confirms its growth inhibitory function in GBM. As NPTX2 was methylated and silenced in the high risk group, which has high activation of NFkB pathway, we then checked if NPTX2 could inhibit NFkB activity. Indeed, we observed that NPTX2 overexpression inhibited expression from NFkB dependent luciferase reporter, sequence-specific DNA-binding of NFkB, nuclear translocation of NFkB sub unit (p65) and it also significantly repressed key NFkB target genes. We also show that NPTX2 mediated inhibition of NFkB could be abrogated by co-expression of constitutively active forms of PI3 kinase, AKT and IKKα, suggesting an involvement of PI3K-AKT-IKKα axis in NPTX2 mediated NFkB inhibition. Further, we found that NPTX2 repressed NFkB activity by inhibiting AKT through an ATM-p53-PTEN-PI3K dependent pathway. Thus, these results explain the need for hypermethylation and down regulation of NPTX2 in high risk GBM wherein the NFkB pathway is activated. Part III -Methylation silencing of ULK2, an autophagy gene, is important for astrocyte transformation and cell growth Among the differentially methylated genes (see part I), ULK2 was one of the most hypermethylated and down regulated genes. ULK2 is a known initiator protein in autophagy pathway, which is a type II cell death mechanism. There are many contradictory reports with respect to the role of autophagy in GBM development. For example, it has been shown that autophagy has a tumor suppressor activity and is essential for temozolomide mediated cell toxicity in GBM cells, whereas others studies implicate its involvement in tumor growth and progression. Hence, we carried out experiments to understand the role of ULK2 in GBM development. Using bisulphite sequencing, we validated ULK2 promoter hypermethylation status in GBM and glioma cell lines. In good correlation, ULK2 was found to be down regulated in GBMs and glioma cell lines, which was reexpressed by methylase inhibitor treatment in glioma cell lines. The over expression of ULK2 was found to inhibit colony formation, proliferation and soft agar colony formation of glioma cells. As expected, ULK2 overexpressing cells showed higher autophagy, compared to control cells. Interestingly, we also found increased apoptosis in ULK2 overexpressing cells. The cell death caused by ULK2 overexpression was compromised when cells were treated with 3-MA (an autophagy inhibitor) or Z-VAD-FMK (a pan caspase inhibitor). However, ULK2 failed to inhibit cell growth in autophagy deficient cells (ATG5-/-), thereby suggesting the importance of autophagy in ULK2 induced cell death. Further, ULK2 overexpression, increased catalase degradation and Reactive Oxygen Species (ROS) generation, which suggests that increase in ROS may play a role in ULK2 dependent cell death. In good correlation, N-Acetyl Cysteine, a ROS inhibitor, treatment rescued the cells from ULK2 mediated cell death, confirming the role of ROS in ULK2 induced cell death. Kinase deficient ULK2 overexpression failed to induce cell growth inhibition, autophagy and apoptosis, suggesting kinase activity of ULK2 is important for ULK2 function. Co-transfection of ULK2 inhibited Ras mediated transformation of immortalized normal human astrocytes. Taken together, we have identified and validated ULK2 as one of the DNA methylation silenced genes in GBM. ULK2 was found to be growth inhibitory in GBM cells by increasing autophagy dependent apoptosis. ULK2 inhibited Ras mediated transformation, suggesting essentiality of DNA methylation mediated ULK2 down regulation in GBM. In conclusion, the present work sheds light on the importance of methylation of genes in GBM progression. As observed, two of the genes, NPTX2 and ULK2 play as critical growth inhibitors in GBM. Also, we have identified a robust, independent and a highly sensitive 9 gene methylation signature, for GBM patient’s survival prediction.

Epigenetic mechanisms play tremendous roles in the development and management of neural processing. The important mechanisms include inactivation of transcription via methylation, histone modification via acetylation/deacetylation, and miRNA regulation. These modifications allow for expression or silencing of genes, without manipulation of nucleotide sequence. An individual's internal and external environments provide input for quotidian epigenetic regulation. Aberrations in the form of regulation have been increasingly linked to neurological disorders, in addition to the established correlation to tumorigenesis. In recent years, deviations from normal epigenetic patterns have been observed in cases of Alzheimer's disease (AD). The brains of patients with AD have been shown to display significantly less methylation overall, as compared to age-matched controls. Of particular concern, the methylation, which normally keeps the promoter of the APP gene silenced, occur far less frequently in AD patient allowing for the progression of amyloid deposition and subsequent tau pathology. In addition to the hypomethylation present in AD, many AD cases present with a concurrent hypoacetylation on histones in the hippocampus. There is strong evidence suggesting that the reduced levels of acetylation are due to over-activation of histone deacetylases. Post-mortem examinations of the brains of AD patients have shown that the brain-derived neurotrophic gene, which is crucial for neural processing associated with maturation and memory, has low levels of acetylation halting its transcription. While low levels of methylation and acetylation seem to contribute to the pathogenesis of AD, regulatory miRNA levels can have adverse effects whether they are aberrantly reduced or increased. Patients with AD tend to show abnormally augmented expression of miRNA-125b, miRNA-128, and miRNA-9 in the hippocampus, while a reduced expression of miRNA-107. Deregulation of these miRNAs have been linked to the progression of AD and include amyloid deposition, tau pathology, and oxidative stress through inflammatory processes. The latter quandary of oxidative stress has been shown to be crucial for the early progression of AD. Reactive oxygen species disallow the methylation of genes due to steric hindrance at the CpG islands of DNA where DNA methyltransferases act. Research shows that increases in oxidative stress are correlated to decreases in methylation, which allows for APP expression. While these alterations to normal epigenetic patterns occur internally, there is a breadth of changes that the external environment imposes to exacerbated AD pathogenesis. Most heavily studied of these external environmental factors is lead exposure. There is a strong correlation between lead exposure in individuals who carry the ApoE4 gene and increased mRNA transcription of the APP gene. Lead is thought to demethylate the promoter of the APP gene and allow for amyloid processes to occur. Inadequate nutrition, specifically deficits in choline and folate, has been linked to hypomethylated states due to an inefficient "methylation/remethylation cycle" leading to an accumulation of homocysteine characteristic of AD. With the emphasis epigenetic deregulation has in the progression of AD, epigenetic treatments need to be seriously considered as therapeutic avenues. Current drugs treat the symptoms and acute conditions of AD, but through epigenetic modifications, the pathology of the diseases can be directly addressed. Potential therapeutic avenues include the use of methyl donors, highly specific histone deacetylase inhibitors, and miRNA biomarkers. Methyl donors can help alleviate the hypomethylated state and prevent further APP expression and amyloid deposition. Currently, the histone deacetylase inhibitors are being used as global inhibitors, but have adverse effects including non-specific and premature cell death. By further researching these inhibitors and finding a mechanism to attack specific histone deacetylases (such as HDAC6 in AD), the efficacy of this aspect of treatment will be greatly increased. The current use of miRNAs as epigenetic regulators to turn off unwanted genetic expression is ineffective due to a major problem of effective delivery to target zones. By using the gene sequences of miRNAs as biomarkers, an AD patient's genomic sequence can be mapped, marking which areas require regulation. This process is necessary because of the inter-individuality of miRNA regulation between each case of AD. Also, the problem of some anti-miRNA molecules not being able to cross the blood brain barrier needs to be addressed using a novel transport mechanism, as direct brain injections are not feasible. The simplest, and highly effective, therapeutic avenue is a healthy lifestyle. Daily exercise and proper nutrition hinder inflammatory process and oxidative stress and can prevent progression of AD through allowing higher brain perfusion for cognitive functioning.

Call number: LD2668 .T4 1985 A28 / Master of Science

BACKGROUND: The study of genetic variants alone is not enough to explain a complex disease like cancer. Alterations in DNA methylation patterns have been associated with different types of tumor. In order to detect markers of susceptibility for the development of cutaneous melanoma and breast cancer in the Uruguayan population, we integrated genetic and epigenetic information of patients and controls. METHODS: We performed two case-control studies that included 49 individuals with sporadic cutaneous melanoma and 73 unaffected controls, and 179 women with sporadic breast cancer and 209 women controls. We determined the level of global leukocyte DNA methylation using relative quantification of 5mdC by HPLC, and we compared methylation levels between cases and controls with nonparametric statistical tests. Since the Uruguayan population is admixed and both melanoma and breast cancer have very high incidences in Uruguay compared to other populations, we examined whether individual ancestry influences global leucocyte DNA methylation status. We carried out a correlation analysis between the percentage of African, European and Native American individual ancestries, determined using 59 ancestry informative markers, and global DNA methylation in all participants. RESULTS: We detected global DNA hypomethylation in leukocytes of melanoma and breast cancer patients compared with healthy controls (p < 0.001). Additionally, we found a negative correlation between African ancestry and global DNA methylation in cancer patients (p <0.005). CONCLUSIONS: These results support the potential use of global DNA methylation as a biomarker for cancer risk. In addition, our findings suggest that the ancestral genome structure generated by the admixture process influences DNA methylation patterns, and underscore the importance of considering genetic ancestry as a modifying factor in epigenetic association studies in admixed populations such as Latino ones.