Genomic imprinting is the epigenetic modification of loci, primarily by DNA methylation, which results in parent-of-origin-specific monoallelic expression of a small subset of genes. In plants, imprinting occurs during endosperm development and a balance of maternally- and paternally-expressed imprinted genes is essential for normal seed development. Dependence on DNA methylation for imprinting highlights the potential to manipulate seed development, and consequently seed size, by altering DNA methyltransferase activity. DNA METHYLTRANSFERASE 1 (MET1) is the primary plant maintenance DNA methyltransferase and plays a significant role in imprinting. However, no evaluation of the potential role for other MET1 family members in genomic imprinting has been reported. The current model for the control of imprinting in plants suggests that maintenance DNA methyltransferases are required throughout development, yet the tissue-specific requirement of these enzymes is unconfirmed as analysis has relied solely on constitutive DNA methyltransferase mutants. To address these problems and to evaluate the potential to alter seed size, the work reported in this thesis investigated the potential involvement of putative maintenance DNA methyltransferases MET2a, MET2b and MET3 and the tissue-specific role of MET1 in imprinting. Imprinting was not significantly altered in met2a-1, met2b-1 and met3-1 mutants, indicating that MET1 is the sole DNA methyltransferase required for imprinting. Transcriptional analysis suggested MET1 is expressed throughout floral organ development and in the male and female gametophyte generation indicating that MET1 is potentially available to maintain imprinting-dependent methylation in these tissues. Tools to suppress MET1 tissuespecifically were developed to investigate the tissue-specific requirement of MET1 for imprinting. Analysis indicates that such tools could also be used to alter seed size by manipulating imprinting in commercially important species. Further work is needed to validate this approach.

Germ cell tumours (GCTs) are clinically and pathologically heterogeneous neoplasms that arise at gonadal (testicular/ovarian) and extra-gonadal sites. The chemotherapy burden for patients with malignant germ cell tumours (mGCTs) that require treatment results in substantial longterm side-effects, and, furthermore, poor-risk patients have < 50% survival. Consequently, identifying common molecular changes and novel therapeutic targets in mGCTs is of major clinical importance. MicroRNAs are short, non-protein coding RNAs that regulate gene expression. We previously showed that miR-99a-5p/-100-5p and miR-125b-5p are among the most frequently underexpressed microRNAs in mGCTs, regardless of anatomical site, histological type or patient age. The present study investigates the upstream causes and downstream consequences of such under-expression. The mature form of miR-125b-5p is the product of two genomic loci, which form a cluster with either miR-99a-5p (on chromosome 21q) or miR-100-5p (on chromosome 11q). MiR-99a-5p/- 100-5p share identical 'seed' regions (at nucleotide positions 2-7), which determine their mRNA targets. Cross-reactivity experiment revealed that both miR-99a-5p and miR-100-5p probes were highly cross-reactive to each other's target (from 91% to 95%), indicating functional overlap. Linear regression analysis of qRT-PCR data reveals a strong positive correlation between miR-99a-5p/-100-5p and miR-125b-5p levels (R2 =0.989) in mGCTs, strongly suggesting co-regulation. Primary microRNA transcripts (pri-miR-99a/-100 and pri-miR-125b), and other genes that colocalise to these miRNA clusters (e.g. BLID on chromosome 11), were quantified by RT-qPCR in four representative cell lines - TCam2, 1411H, 2102Ep, and GCT44 - which were derived from a range of common histological types of mGCTs. A significant down-regulation (p < 0.0001) of all primary transcripts was observed, suggesting transcriptional repression of the entire cluster regions. Treatment of the cell lines with 5'-azacytidine resulted in significant upregulation of all three miRNAs (p < 0.002), as well as BLID (p < 0.02). The methylation status of potential CpG islands at the region of interest on chromosome 11 and chromosome 21 was therefore investigated by Pyrosequencing. Significant hyper methylation was found in 2102Ep, 1411H and GCT44 cell lines, suggesting that the miR-99a-5p/-100-5p and miR-125b-5p clusters are likely transcriptionally silenced by DNA methylation. To assess the functional relevance of these microRNAs in GCT progression, co-transfection of microRNA mimics (8.3 nM miR-99a-5p/-100-5p + 8.3 nM miR-125b-5p) was performed. A significant decrease in cell growth was seen in 1411H (p < 0.01) and TCam2 (p < 0.03) cells. To identify the mimics' downstream mRNA targets, HumanHT-12 v4 Expression Bead Chip (Illumina) mRNA arrays were used and data analysed using Sylamer. This analysis showed that mimic-treated cells were enriched in downregulated genes involved in pro-proliferative mechanisms. Among those, further functional characterisation focussed in particular on TRIM71, FGFR3, E2F7 and LIN28A. Moreover, restoring miR-99a-5p/-100-5p and miR-125b-5p in TCam2 cells also resulted in G0-G1 accumulation, consistent with a cell cycle effect. These data support a functionally important role for miR99a-5p/-100-5p and miR-125b-5p in GCT progression. They also raise the possibility of a therapeutic replenishment approach for treating these, and potentially other, tumours.

Pendant la méiose, la division cellulaire qui forme les cellules haploïdes, les chromosomes homologues hérités de chacun des deux parents sont appariés et échangent des segments réciproques appelés crossing-overs (CO). Les CO ne sont pas distribués au hasard dans le génome et leur taux varie le long des chromosomes. Certains des mécanismes responsable ont été décrits chez les mammifères et la levure mais ne sont pas conservés chez les plantes. Les CO sont fortement inhibés dans l'hétérochromatine qui est riche en éléments répétés. Le degré élevé de méthylation d l'ADN qui caractérise les séquences répétées pourrait être un inhibiteur des CO. Cela a été clairement démontré chez le champignon Ascobolus immersus et des études récentes ont montré que la perte de méthylation modifiait la distribution des CO chez Arabidopsis thaliana. Le but de ma thèse a été de décrire plus précisément le rôle de la méthylation de l'ADN dans le contrôle des CO en l'absence de polymorphisme de séquence qui affecte aussi la recombinaison.Pour cela, j'ai mesuré la recombinaison dans différentes plantes dans lesquelles la méthylation de l'ADN a été partiellement ou totalement enlevée grâce à la mutation du gène ddm1. Pour tester l'effet opposé d'un gain de méthylation, j'ai aussi essayé de cibler la methylation de l'ADN à un point chaud de recombinaison connu. Mes résultats montrent que la parte de la méthylation de l'ADN entraîne une augmentation globale de la recombinaison. Paradoxalement, l'heterochromatine qui est normalement très méthylée est moins affectée par la perte de méthylation que le reste du chromosome, probablement car la méthylation de l'ADN a des effets à distance. L'augmentation de CO est accentuée dans les générations successives du mutant ddm1. Cependant, l'effet le plus important est observé dans les hétérozygotes où la moitié du génome seulement est hypométhylée, ce qui suggère un rôle complexe de la méthylation. Finalement, j'ai pu montrer que le polymorphisme affecte la recombinaison surtout dans l'hétérochromatine mais pas dans le sens attendu puisque les plantes homozygotes recombinent moins que les plantes hétérozygotes. / During meiosis, the cellular division that gives rise to haploid cells, homologous chromosomes inherited from each parent are paired and are subjected to reciprocal exchanges of chromosome segments called crossing-overs (COs). COs are not randomly distributed in the genome. Some of the involved mechanisms have recently been described in mammals and yeast bu they are not conserved in plants. Repeat-rich heterochromatin is suppressed for COs. The high level of DNA methylation associated with repeats could be an inhibitor of COs. This was clearly demonstrated in the fungus Ascobolus immersus and recent studies have shown that the loss of DNA methylation also affects COs in Arabidopsis thaliana. The aim of my thesis was to describe more precisely the role of DNA methylation in the control of CO distribution in the absence of any DNA sequence polymorphism which are known to affect recombination. For this purpose, I measured recombination in different plants where DNA methylation has been partially or completely removed thanks to the mutation of the DDM1 gene. To test the opposed effect of a gain of DNA methylation,.I also tried to target DNA methylation at a known recombination hotspot. My results show that the loss of DNA methylation induces a global increase of recombination. Paradoxically, the normally highly methylated heterochromatin is less affected by this loss than the rest of the chromosome, probably because DNA methylation has distal effects. The increased recombination is exacerbated in successive generations of the hypomethylated ddm1 mutants. However, the strongest effect is seen in the heterozygotes where only half of the genome is hypomethylated, suggesting a complex role in the control of CO distribution. Finally, I show that DNA sequence polymorphism affects mainly recombination in the heterochromatin but not in the expected sense, since homozygous plants recombine less than heterozygous.

Background: Over 200 million individuals worldwide are chronically exposed to arsenic (As) in drinking water at concentrations above the World Health Organization (WHO) guideline of 10 µg/L. Arsenic exposure is of particular concern in Bangladesh, where it is estimated that 35-77 million people are exposed to As in well water at concentrations above the WHO guideline. Chronic As exposure is associated with neurological impairments, respiratory disease, cardiovascular disease, skin lesions, and cancers of the skin, liver, lung and bladder. The mechanisms of As toxicity in humans are not well-characterized: there are considerable interspecies differences in As toxicokinetics, and until recently, there were no animal models to study As carcinogenesis. However, two of several proposed pathways of As toxicity in humans involve DNA methylation and oxidative stress. Arsenic metabolism, DNA methylation, and glutathione (GSH) are metabolically connected through the one-carbon metabolism and transsulfuration pathways, and their interactions are remarkably complex. The epidemiologic studies in this dissertation are designed to address the overarching hypothesis that one-carbon metabolism and the transsulfuration pathway interact to influence susceptibility to As toxicity. Introduction: Arsenic is methylated in the liver to monomethyl (MMA) and dimethyl (DMA) arsenical species by arsenic(III)-methyltransferase (AS3MT), which requires a methyl group from S-adenosylmethionine (SAM) and the presence of a reductant, such as glutathione (GSH). SAM is the universal methyl donor for transmethylation reactions, including DNA methylation, and is a product of folate-dependent one-carbon metabolism. GSH is the primary endogenous antioxidant and determinant of the intracellular redox state, and the rate-limiting precursor for GSH synthesis, cysteine (Cys), is a product of the transsulfuration pathway. One-carbon metabolism and the transsulfuration pathway are connected through homocysteine (Hcys). In humans, aberrant DNA methylation, oxidative stress, hyperhomocysteinemia (HHcys), and impaired As methylation capacity have been identified as risk factors for As-related conditions, including As-induced skin lesions. However, there are knowledge gaps regarding the relationships among these risk factors in humans, namely (1) the dose-response relationship between chronic As exposure and global DNA methylation over a wide range of As concentrations, as well as the influence of As exposure on the newly-discovered epigenetic modification, 5-hydroxymethylcytosine (5hmC); (2) whether an oxidized GSH redox state impairs the capacity to methylate As and DNA; and (3) whether variants in one-carbon metabolism genes are associated with HHcys and susceptibility to As-induced skin lesions. Methods: We addressed these questions in five self-contained epidemiological studies of As-exposed Bangladeshi adults, which employed cross-sectional (Chapters 3-6) and nested case-control (Chapter 7) designs. First, we examined the dose-response relationship between As exposure and global methylation of peripheral blood mononuclear cell (PBMC) DNA (Chapter 3). Second, we optimized a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure global 5-methylcytosine (5mC) and 5hmC content in human DNA samples, and we examined the associations of As exposure with global %mC and %hmC in two independent samples of As-exposed adults (Chapter 4). Third, we measured GSH and its "oxidized" form, glutathione disulfide (GSSG) in plasma, and we examined the interaction of plasma GSH redox state and folate nutritional status on As methylation capacity (Chapter 5). Fourth, we examined the relationships between blood GSH redox, blood SAM, and global methylation of PBMC DNA (Chapter 6). Fifth, we conducted a nested case-control study (Chapter 7) to determine whether nonsynonymous single nucleotide polymorphisms (SNPs) in methylene-tetrahydrofolate reductase (MTHFR) and other one-carbon metabolism genes were associated with HHcys and risk for As-induced precancerous skin lesions, and we conducted an exploratory genome-wide association study (GWAS) of Hcys in a subset of participants. Results: Chronic As exposure was associated with increased global DNA methylation over a wide range of well water As concentrations (Chapter 3), but the relationship between As exposure and global %hmC was gender-specific, with a positive association in males and negative association in females (Chapter 4). We found that an oxidized GSH redox state was associated with both decreased As methylation capacity (Chapter 5) and global DNA hypomethylation (Chapter 6). Finally, in the nested-case control study, we confirmed previous findings that serum HHcys was a risk factor for As-induced skin lesions, and gene variants in MTHFR were found to explain a substantial proportion of the variance in serum Hcys concentrations (Chapter 7). However, we did not find that one-carbon metabolism gene variants were risk factors for As-induced skin lesions. The GWAS of serum Hcys identified one genome-wide significant SNP in the pregnane X receptor (PXR) gene, along with other SNPs in genes involved in cell signaling and the establishment of epithelial cell polarity. Taken together, our findings suggest that indices of one-carbon metabolism and the transsulfuration pathway--DNA methylation, GSH redox, and As methylation--interact with one another to influence susceptibility to As toxicity in humans. In addition, to our knowledge, this is the first report of an association between As exposure and global 5hmC.

The first half of this dissertation demonstrates development of a novel method for DNA methylation profiling based on site specific conversion of cytosine in CpG sites catalyzed by DNA methyltransferases. DNA methylation, a chemical process by which DNA bases are modified by methyl groups, is one of the key epigenetic mechanisms used by cells to regulate gene expression. It predominantly occurs at the 5-position of cytosines in CpG sites and is essential in normal development. Aberrant methylation is associated with many diseases including cancer. Bisulfite Genomic Sequencing (BGS), the gold standard in DNA methylation profiling, works on the principle of converting unmethylated cytosines to uracils using sodium bisulfite under strong basic conditions that cause extensive DNA damage limiting its applications. This dissertation focuses on the research and development of a new method for single cell whole-genome DNA methylation profiling that will convert the unmethylated cytosines in CpG sites to thymine analogs with the aid of DNA methyltransferase and photo-irradiation. Previously we synthesized a model deoxycytidine containing an optimized allyl chemical group at the 5-position and demonstrated that this molecule undergoes photo-conversion to its deoxythymidine analog (C to T conversion) with irradiation at 300 nm. The C to T conversion also proved feasible using synthetic DNA molecules. In this thesis, we demonstrate the conversion of a novel modified deoxycytidine molecule (PhAll-dC) using 350 nm photo-irradiation and a triplet photosensitizer (thioxanthone, TX) to avoid potential DNA damage. The new photoproduct was identified as the deoxythymidine analog of the starting molecule as assessed by IR, MS and NMR. An AdoMet analog containing the optimized chemical group was also synthesized and tested for enzymatic transfer to the C5-position of CpG cytosines using DNA methyltransferases. DNA methyltansferase M.SssI was engineered for more efficient enzymatic transfer. In the future, we will incorporate a triplet photosensitizer into the photoreactive moiety on AdoMet to increase energy transfer efficiency for photo-conversion of C to the T analog. Incorporating this into an overall method followed by amplification and sequencing should allow us to assess the methylation status of all CpGs in the genome in an efficient manner. The second half of this dissertation demonstrates development of a DNA sequencing by synthesis (SBS) method, The Sequence Walking Approach, using novel nucleotide reversible terminators (NRTs) together with natural nucleotides. Following the completion of The Human Genome Project, next generation DNA sequencing technologies emerged to overcome the limitations of Sanger Sequencing, the prominent DNA sequencing technology of the time. These technologies led to significant improvements in throughput, accuracy and economics of DNA sequencing. Today, fluorescence-based sequencing by synthesis methods dominate the high-throughput sequencing market. One of the major challenges facing fluorescence-based SBS methods is their read length limitation which constitutes a big barrier for applications such as de novo genome assembly and resolving structurally complex regions of the genome. In this regard, we have developed a novel SBS method called ‘The Sequence Walking Approach’ to overcome current challenges in increasing the single pass read length of DNA sequencing. Our method utilizes three dNTPs together with one nucleotide reversible terminator in reactions called ‘walks’ that terminate at predetermined bases instead of after each incorporation. In this method, the primer extended via 4-color SBS is stripped off and replaced by the original primer for walking reactions. By reducing the accumulation of cleavage artifacts of incorporated NRTs in a single run, our method aims to reach longer read lengths. In this thesis, we have demonstrated a variation of The Sequence Walking Approach in which 4-color sequencing steps are interspersed with walking steps over a continuous length of DNA without stripping off extended primers and reannealing the original primer. The improvements introduced with this method will enable the use of fluorescence-based SBS in many applications such as detection of genomic variants and de novo genome assemblies while preserving low costs and high accuracy.

Os tumores extra-axias do sistema nervoso são de localização extra-cerebral e na maioria das vezes benignos; meningiomas, schwanomas e metástases fazem parte deste grupo. O aparecimento de um tumor ocorre a partir do acúmulo de alterações genéticas e epigenéticas nas células. Para entender o mecanismo molecular da progressão tumoral e a formação de metástases é indispensável identificar os genes que acumulam essas alterações. Sendo assim, este trabalho teve como objetivo analisar o perfil de metilação dos genes TP16, TP53, DAL-1, GSTP-1, MEN-1, NDRG2 e das DNA metiltransferases 3A, 3B e 3L e sua associação com os tumores extra-axiais e ainda, avaliar, através de um estudo caso-controle, a influência dos SNPs TP53 Pro47Ser e Arg72Pro, EGF + 61, GSTP-1 Ile105Val e WRN Cys1367Arg no desenvolvimento e prognóstico desses tumores. A técnica utilizada para a análise de hipermetilação foi a MSP, e através dela observamos que a atividade das DNMTs não está associada à metilação dos tumores extra-axiais e ainda, os perfis de metilação das DNMTs de novo não estão associados com alterações no padrão de metilação dos genes TP16, TP53, DAL-1, GSTP-1, MEN- 1 e NDRG2. Observamos que a metilação do gene TP53 está associada principalmente aos tumores de maior grau de malignidade, a uma deficiência na resposta a tratamentos e, conseqüentemente, a um maior número de óbitos. A metilação do gene TP16 está envolvida mais freqüentemente na formação de schwanomas e a de NDRG2 na progressão dos meningiomas. A análise de polimorfismos foi realizada através da técnica de PCR-RFLP e observamos diferenças nas distribuições genotípicas entre pacientes e controles nos SNPs TP53 Pro47Ser e Arg72Pro, EGF + 61 e GSTP-1 Ile105Val, onde as variantes Ser47, Pro72, EGF G61 e Val105 foram observadas com maior freqüência entre os portadores de tumores extra-axiais. Dessa forma, estas variantes podem ser fatores de susceptibilidade para o desenvolvimento dos tumores. / The extra-axial brain tumors have extra-brain localization and in most of the time they are benign, meningiomas, schwannomas and metastasis are included in this group. The appearance of a tumor occurs because of the accumulation of genetic and epigenetic alterations in the cells. In order to understand the molecular mechanism of the tumor progression and the metastasis formation it is important to identify the genes that accumulate the alterations. Thereby, the objective of this study was to analyze the methylation profile of the genes TP16, TP53, DAL-1, GSTP-1, MEN-1, NDRG2 and the DNA methyltransferases 3A, 3B and 3L and their association with the extra-axial brain tumors. Another purpose was to determine, in a case-control study, the roles of the TP53 Pro47Ser and Arg72Pro, EGF + 61, GSTP-1 Ile105Val and WRN Cys1367Arg SNPs in the development and prognosis of these tumors. We used the MSP to screen the hypermethylation profile and we observed no association between the DNMTs activity and the hypermethylation of the tumors. We also did not find association between the methylation of the DNMTs de novo and alterations in the methylation profile of the genes TP16, TP53, DAL-1, GSTP-1, MEN-1 and NDRG2. We observed that TP53 hypermethylation was associated with the high grade tumors, a poor response to the treatments and, consequently, the high number of obits. The TP16 methylation was involved with the shwannomas formation and the NDRG2 gene was involved in the meningiomas progression. For the polymorphism analysis, we used the PCR-RFLP technique and we observed differences in the genotype distributions between cases and controls of TP53 Pro47Ser and Arg72Pro, EGF + 61 and GSTP-1 Ile105Val SNPs, where the variants Ser47, Pro72, EGF G61 and Val105 were more frequent in patients than in controls. Thus, these variants can be important factors of susceptibility to the tumor development.

O comportamento materno é essencial para a sobrevivência e desenvolvimento do filhote mamífero. Durante a prenhez, as fêmeas recebem estímulos sensoriais e hormonais capazes de modificar e preparar o cérebro da mãe para o início dos padrões de comportamento materno (por exemplo, aumentando o número neurônios produtores de oxitocina no hipotálamo). Estudos têm identificado o hipotálamo como o principal responsável por estas mudanças, porém outras áreas do cérebro também estão envolvidas no processo do comportamento materno. Peg3, um gene marcado paternalmente expresso, é conhecido por controlar o comportamento materno em camundongos. Fêmeas nocautes para o gene Peg3 falham em aumentar a ingestão de alimentos, na ejeção de leite e em algumas atividades maternais, como placentofagia e construção do ninho. Este estudo teve como objetivo determinar os padrões de metilação da região diferencialmente metilada de Peg3 (Peg3DMR) de animais da raça Nelore de bovinos em diversas áreas do cérebro. Amostras foram coletadas das seguintes áreas: córtex frontal, occipital, temporal e parietal, hipocampo e hipotálamo, num total de 8 animais (4 machos e 4 fêmeas). O padrão de metilação destas amostras foi analisado pelo protocolo COBRA (do inglês, Combined Bisulfite-Restriction Analysis), que combina a modificação do DNA por bissulfito de sódio, amplificação por PCR e digestão por enzima de restrição. Foram encontrados diferentes padrões de metilação entre as amostras, ocorrendo uma predominância de hipometilação entre as amostras do sexo masculino, e padrões mais variados nas amostras do sexo feminino. As variações nos padrões de metilação ocorreram de maneira mais marcante entre as amostras de uma mesma região cerebral de diferentes animais, do que entre as amostras de várias regiões de um mesmo animal. Os resultados indicam que pode haver uma variação no status de imprinting em nível populacional, porém estudos com um número maior de amostras são necessários para a verificação da significância estatística destas variações. / The maternal behavior is essential to survival and development of mammalian offspring. Throughout pregnancy, females receive sensory and hormonal stimuli which promote modifications and prepare the mothers brain to the onset of maternal behavior patterns (for example, by increasing numbers of neurons producing oxytocin in the hypothalamus). Studies have identified the hypothalamus as the main responsible for these changes, but other areas of the brain are also involved in the maternal behavior process. Peg3, an imprinted paternally expressed gene, is known to control maternal behavior in mice. Peg3 knockout females failed in increasing food intake, milk ejection and some maternal activities as placentofagia and nest building. This study aimed to determine the methylation patterns of the differently methylated region of Peg3 (DMR-Peg3) of animals from Nellore cattle breed in several areas of the brain. Samples were collected from the following areas of cattle brain: the frontal, occipital, temporal and parietal cortices, hippocampus and hypothalamus, in a total of 8 animals (4 males and 4 females). The methylation pattern of these samples was analyzed by the protocol COBRA (Combined Bisulfite-Restriction Analysis), which combines DNA modification by sodium bisulfite, PCR amplification and digestion by restriction enzymes. It was found different methylation patterns among the samples. There was a predominance of hypomethylation among male samples, while different patterns were found among the female samples. Variation in the methylation patterns was more markedly observed among samples of the same cerebral region among different animals, then among samples of several regions within an animal. The results suggest that there may be a variation in the imprinting status at a population level, but further assays, with an increased number of samples are needed to verify the statistical significance of this variation.

All mC5-MTases are monomeric enzymes, except M. EcoHK31I and M. AquI which are MTases composed of two poly peptides. M.EcoHK31I is a mC5-MTase which recognizes the sequence 5-YGGCCR-3' and consists of polypeptide alpha and beta, with the latter gene encoded in an alternative reading frame of the former. All of the conserved motifs in mC5-MTases can be found in polypeptide alpha, except motif IX, which is located in polypeptide beta. Both polypeptides are required for in vitro methylation. / Methylation of cytosine residues in DNA occurs in diverse organisms from bacteria to humans. In higher eukaryotic organisms cytosine-C5 methyltransferase (mC5-MTase) is the only type of DNA MTase and it plays an important role in controlling a number of cellular processes including transcription genomic imprinting and DNA repair. In bacteria, there are three types of MTases, mC4-, mC5- and mAb-, classified according to the methylation site of the DNA. MTase and its cognate restriction endonuclease (ENase) form restriction-modification system. The role of MTase is to protect the host from its own ENase digestion while the ENase acts to degrade the invasion of foreign DNA. Sequence comparison of nearly 50 bacterial mC5-MTases has shown that these enzymes share an overall common protein architecture. Ten conserved motifs (I to X), each 10 to 20 amino acids in length, have been identified, five of which are highly conserved (I, IV, VI, VIII and X). In addition, all of these enzymes have a hypervariable region lying between motifs VIII and IX. It is called the target recognition domain (TRD), and is responsible for the specificity of DNA recognition and the choice of base to be methylated. / Since both of the polypeptides alpha and beta of M.EcoHK31I are sequenced and cloned into the expression vector separately, the role of DNA recognition and subunits interaction of individual polypeptides can be studied. By electromobility shift assay, we found that polypeptides alpha and beta complex recognize specific double strand oligos substrate. Polypeptide alpha-DNA formed aggregates and polypeptide beta alone did not bind DNA. Therefore, polypeptide beta assists the proper binding of polypeptide alpha to DNA substrate. Complex of polypeptide alpha and a polypeptide beta variant with N-terminal deletion of 41 amino acids showed a 16-fold reduction in methylation activity. Further deletion resulted in an inactive MTase. By surface plasmon resonance assay, the dissociation equilibrium constant (KD) of polypeptides alpha and beta complex was found to be 56.2nM and the KD for polypeptide alpha and DeltaN46-polypeptide beta complex was increased by about 95 folds, contributing by a drastic decrease in dissociate rate constant (kd) and an increase in association rate constant (ka). This indicated that the N-terminal region of polypeptide beta takes part in subunit interaction. / To pinpoint which amino acid residues located at the variable region of polypeptide alpha are important for DNA binding and subunits interaction, "charge-to-alanine scanning mutagenesis" were performed on 16 charge residues between Asp213 and Glu271 in the small domain. It was found that the five charge residues upstream of motif X are not required for activity. For other residues except K225, E240 and D245, the protein is active when the same charge is maintained. / Fung Wai To. / "March 2006." / Adviser: P. C. Shaw. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6376. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 180-201). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Os tumores odontogênicos são um grupo heterogéneo de lesões formadas a partir de tecidos que dão origem ao dente. A metilação do ADN, uma adição covalente de um grupo metilo na posição 5 de carbono de um nucleótideo de citosina, é considerado um importante regulador da expressão génica. A adição do radical metil é catalisada por ADN metiltransferases (DNMTs). Embora alguns estudos epigenéticos tenham sido realizados em tumores odontogênicos, um estudo com os três tipos de DNMTs em vários membros desse grupo está em falta. Este estudo analisa a expressão de DNMTs em tumores odontogênicos. Amostras de vinte ameloblastomas, dez Calcificante tumores odontogênicos císticos, dez calcificados tumores epiteliais, dez tumor odontogênico adenomatóide, dez tumores odontogênicos queratocísticos, quatro fibromas ameloblásticos, dois fibro-odontoma ameloblástico, quatro fibroma centrais odontogênicos, sete tecidos de fibromas odontogênicos periféricos e dez mixomas odontogênicos foram incluídos. DNMT1, 3A e 3B foram expressas no núcleo e / ou citoplasma de todos os tumores odontogênicos. A alta expressão de DNMTs em células de tumor odontogênico sugere metilação como um mecanismo importante para este grupo de tumores. / Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. DNA methylation, a covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalyzed by DNA methyltransferases (DNMTs). Although some epigenetic studies have been conducted in odontogenic tumours, a study with the three types of DNMTs in several different members of this group is missing. This study analyzes the expression of DNMTs in odontogenic tumours. Formalin-fixed and paraffin-embedded tissue samples of twenty ameloblastomas, ten calcifying cystic odontogenic tumors, ten calcifying epithelial tumors, ten adenomatoid odontogenic tumors, ten keratocystic odontogenic tumors, five ameloblastic fibromas, two ameloblastic fibro-odontoma, four central odontogenic fibroma, seven peripheral odontogenic fibroma and ten odontogenic mixoma were included. DNMT1, 3A and 3B were expressed in the nucleus and/or cytoplasm of all odontogenic tumours. The high expression of DNMTs in odontogenic tumour cells suggests methylation as an important mechanism for this group of tumours.

A hipermatilação aberrante de regiões gênicas promotoras foi recentemente sugerida como meio de detecção do carcinoma epidermóide de cabeça e pescoço. Neste estudo nós avaliamos o status de metilação de um painel de 7 genes já relatados na literatura e sua correlação com lesões orais malignas e cancerizáveis de boca. Inicialmente, nós utilizamos amostras de enxágues salivares de pacientes com lesões benignas, displásicas e malignas para determinar a hipermetilação em regiões gênicas promotoras em pacientes de alto risco. Uma avaliação clínica de risco foi realizada e correlacionada com o diagnóstico histológico e status dos biomarcadores. A partir dos resultados analisados nas lesões intraorais, o gene DCC, que obteve a melhor performance entre os 7 genes, foi testado em lesões de queilite actínica e carcinoma epidermoide de lábio. Foram realizadas reações de PCR específica para metilação, quantitativa (Q-MSP) para os 7 genes (CCNA1, MGMT, MINT31, TIMP3, P16, DAPK, DCC) em enxágues salivares de 191 pacientes com lesões intraorais, e do gene DCC em 39 lesões de lábio. Análises de regressão logística e curva ROC foram utilizadas para avaliar a associação do status de metilação com o diagnóstico histológico e para estimar a acurácia da classificação respectivamente, nas amostras de enxágue salivar. Na análise multivariada, o diagnóstico displasia/ câncer foi associado com a idade (OR=1.3, 95% CI= (1.01-1.6, p=0.014) e a metilação do painel de 7 genes (OR=2.2, 95% CI=(1.34.0), p=0.006); a metilação do DCC também foi fortemente associada (OR=3.3, 95% CI=(1.7-6.6), p=0.004). Na análise multivariada, o diagnóstico histológico foi independentemente associado com a metilação do painel de 7 genes (OR=2.0, 95% CI=(1.1-3.6), p=0.027) ou do DCC (OR=2.8, 95% CI=(1.4-5.7), p=0.004). Uma nova análise, excluindo pacientes com diagnóstico prévio de câncer (n=30), e levando em conta uma classificação clínica de risco, foi realizada. Na análise univariada, DCC (OR=2.6, 95% CI=(1.1-6.1), p=0.026) e a classificação clínica de risco (OR=2.5, 95% CI=(1.3-5.1), p=0.008) foram associados com o diagnóstico de displasia/ câncer, e permaneceram significante na análise multivariada (DCC: OR=2.5, 95% CI= (1.1- 6.0), p=0.037, classificação de risco: OR=2.5, 95% CI=(1.2-5.0), p=0.012). A classificação clínica de risco identificou displasia/ câncer com a sensibilidade de (95% CI=4171%) e especificidade de 66% (95% CI= 5775%). A sensibilidade da classificação clínica de risco combinada com a metilação do painel de 7 genes melhorou, chegando a 71% (95% CI=5683%) e com a metilação do DCC chegou a 69% (95% CI=5481%). O status de metilação do painel de 7 genes, como também o DCC como um marcador individual, foi independentemente associado com o diagnóstico histológico em enxágues salivares. Nas lesões labiais não foi possível observar a mesma correlação entre o status de metilação do gene DCC e o diagnóstico histológico, provavelmente, devido a diferente etiologia das lesões intraorais e labiais. Os resultados mostram a potencial habilidade destes biomarcadores em prever o risco de presença de lesões intraorais cancerizáveis, usando uma abordagem não invasiva, além do potencial de melhorar a eficiência da classificação clínica de risco. Mas reforça a diferente etiologia das lesões intraorais e labiais e a necessidade de diferentes marcadores para essas lesões. / Aberrant promoter hypermethylation has been recently proposed as a means for detection of HNSCC in salivary rinses. Here we evaluate the ability of a previously reported 7-gene methylation panel status to correlate with premalignant and malignant oral lesions. We used a large prospective cohort of salivary rinses obtained from patients with benign, dysplastic, and cancer diagnoses to determine promoter hypermethylation in high-risk patients. Clinical risk assessment was performed and correlated with histological diagnosis and biomarker status. Also, a cohort of lip lesions was selected and methylation status of DCC gene was correlated with histology. Quantitative methylation-specific PCR (Q-MSP) was performed analyzing methylation status of 7 genes (CCNA1, MGMT, MINT31, TIMP3, P16, DAPK, DCC) in salivary rinses of 191 patients with oral lesion and 39 lip lesions. Logistic regression and receiver operating characteristic (ROC) analyses were used to examine the association of methylation status with histologic diagnosis and to estimate classification accuracy, respectively. On univariate analysis, diagnosis of dysplasia/cancer was associated with age (OR=1.3, 95% CI= (1.01-1.6, p=0.014) and 7-gene panel methylation (OR=2.2, 95% CI=(1.34.0), p=0.006); DCC methylation was also strongly associated (OR=3.3, 95% CI=(1.7-6.6), p=0.004). On multivariable modeling, histologic diagnosis was independently associated with 7 gene panel (OR=2.0, 95% CI=(1.1-3.6), p=0.027) or DCC (OR=2.8, 95% CI=(1.4- 5.7), p=0.004) methylation. A subset analyzed (n=161) without prior biopsy proven malignancy received clinical risk classification based on lesion examination. On univariate analysis, DCC (OR=2.6, 95% CI=(1.1-6.1), p=0.026) and clinical risk classification (OR=2.5, 95% CI=(1.3-5.1), p=0.008) were associated with diagnosis of dysplasia/cancer, and remained significant on multivariate analysis (DCC: OR=2.5, 95% CI= (1.1-6.0), p=0.037, risk classification: OR=2.5, 95% CI=(1.2-5.0), p=0.012). Clinical risk classification identified dysplasia/cancer with a sensitivity of 56% (95% CI=4171%) and specificity of 66% (95% CI= 5775%). The sensitivity of clinical risk classification combined with 7-gene panel methylation improved to 71% (95% CI=56 83%) and with DCC methylation improved to 69% (95% CI=5481%). The 7-gene panel methylation, as well DCC as a single marker, was independently associated with histologic diagnosis in salivary rinses. No correlation was found between DCC methylation status and histologic diagnosis of lip lesions, probably due different etiology of oral and lip lesions. The results show the potential ability of these biomarkers to predict risk for presence of oral premalignancy and malignancy using a non-invasive approach using salivary rinse and can improve the efficiency of clinical risk classification. Also, reinforces the different etiologic origins of intraoral and lip lesions and the need of different markers for these lesions.