O produto comercial CI Disperse Blue 291 (CI DB291) é amplamente utilizado pela industria têxtil. Estudos mostram que corantes dinitrofenilazo são genotóxicos no ensaio de Ames/Salmonella, mas há poucos estudos sobre seus efeitos em células eucariontes. Este trabalho teve como objetivo investigar a genotoxicidade e citotoxicidade do corante DB291 em células humanas em cultura, bem como vias pelas quais o corante atua levando aos efeitos tóxicos. O corante comercial foi purificado por HPLC-DAD e utilizado para incubação com células HepG2 (5-100 µM por 3-72 h). A citotoxicidade foi avaliada pelos ensaios de XTT, corante cristal violeta, lactato desidrogenase extracelular e consumo de glicose. A geração de ROS intracelular foi verificada pela emissão de fluorescência da 2\',7\'-diclorofluoresceína. Análises de ciclo celular, fragmentação do DNA, potencial de membrana mitocondrial (Ψ) e cálcio intracelular foram realizadas por citometria de fluxo. A produção de ATP foi mensurada por quimiluminescência. Níveis de 8-oxodG e 5-mdC foram avaliados por HPLC-ESI-MS/MS e HPLC-UV, respectivamente. A expressão da enzima DNMT1 foi avaliada por western blot. Um produto de biotransformação foi identificado e caracterizado estruturalmente por MS/MS e 1H-RMN. A exposição ao corante DB291 diminuiu significativamente a sobrevivência das células em um modo tempo- e dose-dependente (IC50 = 74 µM), com a concomitante formação de um produto de biotransformação reduzido. A morte celular ocorreu sem lise da membrana plasmática. Nas células expostas, foi observado aumento da atividade enzimática mitocondrial, acompanhado por um aumento da taxa de consumo de glicose. Índices elevados de ATP, Ψ e cálcio foram verificados após a incubação das células com concentrações crescentes do corante. Alterações mitocondriais e o processo de biotransformação impeliram a aumento na produção de ROS intracelular, 8-oxodG e fragmentação do DNA. O dano ao DNA induziu a parada no ciclo celular e super-expressão de DNMT1, seguido por hipometilação global do DNA no ciclo celular subsequente. Os resultados obtidos apontam pela primeira vez para a toxicidade do corante DB291 via biotransformação, com alterações mitocondriais e indução de estresse oxidativo. / The commercial CI Disperse Blue 291 (CI DB291) is widely used by textile industry. It is mutagenic in the Ames/S. typhimurium assay, but there are few studies showing its effects in eukaryotic cells. We evaluated here the toxicity of CI DB291 in the human HepG2 cell line. The commercial dye was purified by HPLC-DAD and used for incubation with HepG2 cells (5-100 µM for 3-72 h). Cytotoxicity was assessed by the XTT, crystal violet dye, extracellular lactate dehydrogenase and glucose consumption assays. ROS formation was assessed by 2\',7\'-dichlorofluorescein fluorescence. Analyses of cell cycle, DNA fragmentation, mitochondrial membrane potential (Ψ) and intracellular calcium were analyzed by flow cytometry. ATP level was measured by chemiluminescence. Levels of 8-oxodG and 5-mdC were evaluated by HPLCESI-MS/MS and HPLC-UV, respectively. DNMT1 expression was assessed by western blot. A biotransformation product was identified and structurally characterized by MS/MS and 1H-NMR. DB291 significantly decreased cell survival in a time- and dose-dependent manner (IC50 = 74 µM), with concomitant formation of a reduced biotransformation product. Plasma membrane lysis did not occur. Increased mitochondrial enzymatic activity, accompanied by increase in glucose consumption rate, was observed in cells incubated with DB291. Elevated ATP, Ψ, and intracellular calcium was verified after cell incubation with increasing dye concentrations. Mitochondrial changes and the biotransformation process accounted for the observed raise in intracellular ROS, 8-oxodG, and DNA fragmentation. DNA damage induced cell cycle arrest and DNMT1 overexpression, followed by DNA hypomethylation in the subsequent cell cycle. Results point for the first time to toxicity of the dye through biotransformation, mitochondrial changes, and oxidative stress.

Liu, Xin. / "December 2009." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 119-126). / Abstracts in English and Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iv / Acknowledgements --- p.vi / List of abbreviations --- p.vii / List of Tables List of Figures --- p.X xii / List of Publications --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Gastric cancer epidemiology and etiology --- p.1 / Chapter 1.2 --- Molecular carcinogenesis --- p.4 / Chapter 1.3 --- Tumor suppressor gene and the modes of tumor suppressor gene inactivation --- p.4 / Chapter 1.4 --- DNA methylation and carcinogenesis --- p.8 / Chapter 1.5 --- Identification of tumor suppressor genes --- p.15 / Chapter 1.6 --- "Vitamins, vitamin B complex, thiamine transporters and diseases" --- p.18 / Chapter 1.7 --- "Glucose metabolism, glycolysis and carcinogenesis" --- p.22 / Chapter 1.8 --- Clinical implications of DNA methylation --- p.28 / Chapter Chapter 2 --- Research Aim and Procedure --- p.31 / Chapter Chapter 3 --- Materials and Methods --- p.35 / Chapter 3.1 --- Cell lines and human tissue samples --- p.35 / Chapter 3.2 --- Cell culture --- p.35 / Chapter 3.3 --- Total RNA extraction --- p.36 / Chapter 3.4 --- Genomic DNA extraction --- p.37 / Chapter 3.5 --- Reverse transcription PCR (RT-PCR) --- p.38 / Chapter 3.5.1 --- Reverse transcription (RT) --- p.38 / Chapter 3.5.2 --- Semi-quantitative RT-PCR --- p.40 / Chapter 3.5.3 --- Real time RT-PCR --- p.42 / Chapter 3.6 --- General techniques --- p.44 / Chapter 3.6.1 --- DNA and RNA quantification --- p.44 / Chapter 3.6.2 --- Gel electrophoresis --- p.44 / Chapter 3.6.3 --- LB medium and LB plate preparation --- p.44 / Chapter 3.6.4 --- Plasmid DNA extraction --- p.45 / Chapter 3.6.4a --- Plasmid DNA mini extraction --- p.45 / Chapter 3.6.4b --- Plasmid DNA midi extraction --- p.46 / Chapter 3.6.5 --- DNA sequencing --- p.46 / Chapter 3.7 --- Methylation status analysis --- p.49 / Chapter 3.7.1 --- CpG island analysis --- p.49 / Chapter 3.7.2 --- Sodium bisulfite modification of DNA --- p.49 / Chapter 3.7.3 --- Methylation-specific PCR (MSP) --- p.50 / Chapter 3.7.4 --- Bisulfite genomic sequencing (BGS) --- p.53 / Chapter 3.8 --- Construction of expression plasmid DNA --- p.55 / Chapter 3.8.1 --- Construction of the SLC19A3-expressing vector --- p.55 / Chapter 3.8.2 --- Construction of the FBP1-expressing vector --- p.57 / Chapter 3.9 --- Functional analyses --- p.58 / Chapter 3.9.1 --- Monolayer colony formation assay --- p.58 / Chapter 3.9.2 --- Cancer cell growth curve analysis --- p.59 / Chapter 3.9.3 --- Lactate assay --- p.60 / Chapter 3.10 --- Statistical analysis --- p.61 / Chapter Chapter 4 --- Results --- p.62 / Chapter 4.1 --- Identification of novel candidate tumor suppressor genes downregulated by DNA methylation --- p.62 / Chapter 4.2 --- Selection of genes for further study --- p.62 / Chapter 4.3 --- Identification of SLC19A3 as a novel candidate tumor suppressor gene in gastric cancer --- p.64 / Chapter 4.3.1 --- Pharmacological restoration of SLC 19A3 downregulation in gastric cancer --- p.64 / Chapter 4.3.2 --- Methylation analysis of SLC 19A3 promoter region --- p.66 / Chapter 4.3.3 --- Functional analysis of SLC 19A3 in gastric cancer --- p.72 / Chapter 4.3.4 --- Clinicopathologic characteristics of SLC 19A3 promoter methylation in gastric cancer --- p.75 / Chapter 4.3.5 --- Discussion --- p.78 / Chapter 4.4 --- Identification of FBP1 as a novel candidate tumor suppressor gene regulated by NF-kB in gastric cancer --- p.85 / Chapter 4.4.1 --- Pharmacological restoration of FBP1 downregulation in gastric cancer --- p.85 / Chapter 4.4.2 --- Methylation analysis of FBP 1 promoter region --- p.87 / Chapter 4.4.3 --- Functional analysis of FBP 1 in gastric cancer --- p.93 / Chapter 4.4.4 --- Reduction of lactate generation under FBP1 expression --- p.95 / Chapter 4.4.5 --- Clinicopathologic characteristics of FBP 1 promoter methylation in gastric cancer --- p.98 / Chapter 4.4.6 --- NF-kB mediated FBP1 promoter hypermethylation in gastric cancer --- p.104 / Chapter 4.4.7 --- Discussion --- p.106 / Chapter Chapter 5 --- General discussion --- p.112 / Chapter Chapter 6 --- Summary --- p.117 / Reference list --- p.119

Introdução: Mecanismos epigenéticos, como a metilação de DNA, desempenham um papel importante na neurobiologia da depressão. Enquanto o estresse aumenta a metilação de DNA e reduz a expressão de genes envolvidos na plasticidade neuronial, inibidores de DNA metiltransferases (DNMTi), enzimas que catalisam a metilação de DNA, aumentam rapidamente a expressão gênica e induzem efeitos tipo-antidepressivos em modelos animais. Considerando, ainda, que antidepressivos convencionais podem interferir com mecanismos epigenéticos, este trabalho testou a hipótese de que drogas DNMTi induzem efeito tipo-antidepressivo agudo e sustentado em modelos animais. Além disso, avaliamos se o efeito de antidepressivos convencionais e de DNMTis sobre os níveis de mRNA e de metilação de DNA em diferentes genes associados a depressão e regulados por mecanismos epigenéticos (BDNF, TrkB, 5-HT1A, NMDA e AMPA) em estruturas encefálicas (hipocampo dorsal, ventral e córtex pré-frontal) de animais submetidos a modelo animal de depressão. Métodos: Para tanto, ratos Wistar foram submetidos ao modelo do desamparo aprenddo [learned helplessness, LH, pré-teste (PT), 40 choques inescapáveis nas patas]. Os animais receberam injeções sistêmicas de DNMTi (5-AzaD ou RG108), antidepressivos (imipramina ou fluoxetina), ou veículo, por 1 ou 7 dias, e foram submetidos a sessão teste do desamparo aprendido (T, 30 choques escapáveis) no último dia. Adicionalmente, um grupo independente foi submetido ao mesmo protocolo experimental e sacrificados 1 h após a última injeção. As estruturas encefálicas foram dissecadas para posterior análise molecular [imunoprecipitação de DNA metilado (meDIP) e quantificação de RNAm por qRT-PCR). Resultados: O estresse dos choques nas patas aumentou o número de falhas no teste. O tratamento com DNMTi agudamente, assim como com antidepressivos (tratamento repetido), foi capaz de atenuar essas alterações comportamentais, efeito considerado tipo-antidepressivo nesse modelo. Ainda, o estresse aumentou a metilação de DNA e reduziu os níveis de RNAm para BDNF e TrkB, enquanto que o tratamento com RG108 atenuou essas alterações moleculares no córtex pré-frontal de ratos. Conclusão: Os presentes resultados indicam que DNMTi, diferente de antidepressivos convencionais, são capazes de induzir rápido e sustentado efeito tipo-antidepressivo. Além disso, BDNF e TrkB parecem ser importantes para a resposta comportamental induzida pela inibição de DNMTs no córtex pré-frontal de ratos submetidos ao LH. / Introduction: Epigenetic mechanisms, such as DNA methylation, are thought to play an important role in the neurobiology of depression. While stress increases DNA methylation and decreases the expression of genes involved in neuronal plasticity, DNA methyltransferases inhibitors (DNMTi) increases gene expression and induces antidepressant-like effects in animal models. Considering that conventional antidepressants could interfere with epigenetic mechanisms, this work tested the hypothesis that acute treatment with DNMTi would induce acute and long-lasting antidepressant-like effects. Furthermore, we evaluated whether the stress could induce changes in the mRNA and DNA methylation levels in different genes involved with depression and regulated by epigenetic mechanisms (BDNF, TrkB, 5-HT1A, NMDA and AMPA) in different brain structures [dorsal hippocampus, ventral hippocampus and prefrontal cortex (PFC)] and whether such changes would be attenuated by systemic treatment with DNMTi (acutely) and antidepressants (chronically). Methods: Male Wistar rats were submitted to the learned helplessness model (LH; pretest session, 40 inescapable foot shocks). The animals received systemic injection the DNMTi (5-AzaD or RG108), antidepressants (imipramine or fluoxetine) or vehicle for one or seven days and were submitted to the LH test (30 escapable foot shocks) in the last day. Additionally, one independent group were submitted to the same experimental protocol and sacrificed one hour after last injection for collection of brain samples to further molecular analyses (methylated DNA immunopreciptation and mRNA levels by qRT-PCR). Results: Exposure to inescapable footshocks increased the number of escape failures in the test. Treatment with DNMTi (acute), as well as with antidepressants (repeated treatment), attenuated stress-induced behavioral responses, an antidepressant-like effect in this model. Moroever, stress increased DNA methylation and decreased RNAm levels of BDNF and TrkB, while treatment with RG108 attenuated molecular changes induced by stress in rat PFC. Conclusion: The present results indicate that DNMTi, different from conventional antidepressants, are able to induce rapid and sustained antidepressant-like effects. In addition, BDNF and TrkB appear to be important for behavioral response induced by inhibition of DNMTs in the rat PFC submitted to the LH.

O principal objetivo deste trabalho é o de estabelecer uma estratégia confiável relacionada a genotoxicidade, proteção genômica e recombinação mitótica em Aspergillus nidulans. A genotoxicidade foi induzida por vapor de benzeno e, para testar a proteção genômica, a soja foi usada devido às suas propriedades anticarcinogênicas muito conhecidas. O principal resultado obtido foi que a soja transgênica mostrou maiores propriedades antigenotóxicas do que a soja tradicional. Isto foi duplamente confirmado, tanto através de estratégias de genética clássica, como molecular. Além disso, cada experimento foi repetido três vezes. Principais conclusões foram as seguintes: a) estabelecimento de um protocolo adequado para atender às complexidades dos eventos epigenéticos e, b) a aplicação desse tal protocolo e experimentos afins para testar outros agentes ambientais suspeitos de apresentarem propriedades antigenotóxicas, ou que precisem de sua identificação como tal. Evidentemente, levando-se em conta a grande preocupação relacionada aos alimentos transgênicos e aos muitos produtos de biotecnologia, que entram no mercado, o elenco de prováveis candidatos aos tipos de testes apresentados, será muito grande. Como os resultados obtidos sugerem íntima relação entre metilação de DNA eucariótico, a recombinação mitótica e outros eventos danosos às células, os eventos envolvidos serão epigeneticamente discutidos. / Main aim is to provide a reliable approach to deal with the aspects related to induced genotoxicity, genomic protection and eukaryotic mitotic recombination in Aspergillus nidulans. Genotoxicity was benzene fumes induced, and to test genomic protection soybean has been used on account of its putative anticarcinogenic properties. Main outcome is that transgenic soybean bears higher antigenotoxic properties than traditional soybean. This has been twofold confirmed through basic genetic approaches and molecular approaches, as well. In addition, each experimental approach has been three times repeated. Additional important outcomes are: a- establishment of a reliable protocol to deal with the complexities of the epigenetic events, and b- likely use of present protocol and experimental set-up to test other environmental agents of which antigenotoxic properties are either suspected, or need precise identification. Evidently, on account of present wide concern the transgenic foodstuffs, and many other biotechnological products, as well, are obvious candidates for similar approaches. Obtained results suggest close relationships among eukaryotic DNA methylation, mitotic recombination, and other cells damaging events reputedly leading to carcinogenesis.

La méthylation de l’ADN est une modification épigénétique sur les cytosines des dinucléotides CpG catalysée par les enzymes DNMT. Les cellules cancéreuses présentent des hyperméthylations aberrantes sur les promoteurs de gènes dits suppresseurs de tumeurs, ce qui contribue à leur répression transcriptionnelle et favorise la progression tumorale. De par sa nature réversible, la méthylation de l’ADN est une cible de choix pour des thérapies épigénétiques ; cependant, les inhibiteurs de DNMT ont une action de déméthylation globale du génome qui conduit à une forte toxicité. Mon travail a consisté à développer des stratégies de déméthylation ciblée sur des régions spécifiques du génome. Premièrement, j’ai validé une stratégie induisant une reprogrammation épigénétique spécifique et durable du gène suppresseur de tumeurs SERPINB5 dans des cellules de cancer du sein. Deuxièmement, j’ai optimisé des stratégies d'édition de l’épigénome comme outil en recherche fondamentale. / DNA methylation takes place on cytosines of CpG dinucleotides in mammals and is catalysed by DNMT enzymes. Cancer cells are characterised by frequent promoter hypermethylation leading to transcriptional repression of tumor suppressor genes and favouring tumor progression. Because of its reversible nature, DNA methylation is a target of choice in epigenetic therapies. However, current DNMT inhibitors act in a global and non-specific manner, leading to side effects and toxicity in normal cells. During my thesis I have developed strategies to perform targeted demethylation in specific regions of the genome without affecting global methylation. First, I have validated a strategy inducing the specific and durable epigenetic reprogramming of the tumor suppressor gene SERPINB5 in a breast cancer cell line, which can pave the way to further biomedical research. Second, I have optimised epigenome editing strategies as a regular tool in basic research.

Les notions selon lesquelles l’exposition environnementale peut être mémorisée et puisse dans des conditions défavorables favoriser l’apparition d’épimutations soulèvent la question d’une possible transmission transgénérationnelle chez l’Homme lorsque les gamètes sont atteints.Afin de répondre à la question du risque de transmission de dérégulation épigénétique (épimutation) chez l’Homme, nous l’avons abordé selon deux axes. Le premier nous a permis d’évaluer le risque de transmission intergénérationnelle chez un patient présentant un syndrome de Silver-Russell (SRS) et nous avons pu démontrer pour la première fois l’efficience de la reprogrammation épigénétique chez l’Homme pour des régions soumises à empreinte : disparition du défaut de méthylation du locus H19/IGF2 causal dans les gamètes du patient ainsi qu’une absence de transmission à sa descendance. Le second nous a conduit à dépister la présence d’épimutations spermatiques à partir de deux modèles : 1/ de pères d’enfants atteints de pathologies liées à l’empreinte parentale et 2/ de patients atteints de cancer testiculaire. Dans les 2 cas, l’analyse par pyroséquençage de leurs spermatozoïdes n’a pas mis en évidence de défaut d’empreinte. Néanmoins, nous avons retrouvé une association entre oligozoospermie et défaut d’empreinte spermatique.Le principal défi des études à venir est d’identifier les mécanismes intervenant dans l’apparition de ces épimutations. Les principales pistes convergent vers les petits ARNs non codants ou certaines régions de l’ADN dont les marques épigénétiques pourraient (au moins partiellement) échapper aux contrôles mis en place lors des phases successives de reprogrammation épigénétique. / The notion that environmental exposure can be memorized and promote epimutation (defined as defects on DNA methylation) raises the question of possible epigenetic transgenerational transmission in humans. To address whether an epimutation could be transmitted in humans, we pursued two axes. First, the evaluation of intergenerational transmission in the family of a Silver-Russell patient has shown, for the first time, the efficiency of epigenetic reprogramming in humans, specifically on imprinted regions. Indeed, no imprinted defect on causal H19/IGF2 locus was detected in the patient’s spermatozoa or in the DNA of his daughter. The second axis was to assess the presence of sperm epimutations 1/ from fathers of children diagnosed with imprinted syndromes and 2/ from men presenting testicular seminoma. Pyrosequencing analyses on imprinted genes did not reveal any alteration of sperm DNA methylation, though we confirmed an association between oligozoospermia and sperm imprinting defects.The next step will be to identify the mechanisms involved in the origin of the sperm epimutation. The main hypotheses converge to small non-coding RNAs or certain DNA regions which escape to controls setting up (at least partially) at the time of epigenetic reprogramming.

Most breast cancer patients undergo radiation diagnostics and are also treated with radiotherapy. In addition to being an important treatment modality, ionizing radiation (IR) is a potent tumour-causing agent that has been linked to breast cancer development. However, the exact molecular etiology of IR-induced mammary gland carcinogenesis remains unknown. We set out to analyze the role of DNA methylation in mammary gland responses to low dose IR using a well-established rat model. We also studied low dose IR effects on global gene expression and microRNAome. We found that exposure to low, mammography-like dose of IR led to a significant loss of global DNA methylation in rat mammary gland tissue. Furthermore, low dose IR significantly affected rat mammary gland transcriptome and microRNAome. The datasets generated within the scope of this thesis may be used to identify novel predictive biomarkers for assessment of the magnitude of IR effects on mammary gland tissue. / xi, 120 leaves ; 29 cm

The survival for high-grade glioma patients is poor and the treatment may cause severe side effects. A common obstacle in the treatment is chemoresistance. To improve the quality of life and prolong survival for these patients prognostic biomarkers and new approaches for chemotherapy are needed. To this end, a strategy to evade chemoresistance was evaluated by combining chemotherapeutic drugs with agents inhibiting resistance mechanisms identified by a bioinformatic analysis (paper I). The prognostic value of 13 different proteins was analyzed in this thesis (papers II-IV). Two of them, p38 mitogen-activated protein kinase (MAPK) and protein tyrosine phosphatase non-receptor type 6 (PTPN6, also known as SHP1) were analyzed for their potential as targets in combination chemotherapy (in paper III and IV, respectively).   We found that: PTPN6 expression and methylation status may be important for survival of anaplastic glioma patients, p38 MAPK phosphorylation may be a potential negative prognostic biomarker for high-grade glioma patients and FGF2 expression may be a potential negative prognostic biomarker for proneural glioma patients. PTPN6 may be a useful target for combination chemotherapy with cisplatin, melphalan or bortezomib in high-grade gliomas. The following drug combinations; camptothecin combined with an EGFR or RAC1 inhibitor, imatinib combined with a Notch or RAC1 inhibitor, temozolomide combined with an EGFR or FAK inhibitor and vandetanib combined with a p38 MAPK inhibitor may be useful combination chemotherapy for high-grade gliomas.

Biodiversity provides the raw material for evolution and adaptation of populations and species. In agricultural biodiversity, the within-population genetic diversity is of major importance. On one hand, it can provide a buffering effect against the year-to-year variation of climate or biotic pressures and on the other hand diversity serves as a resource for the population to respond to selective pressures due to specific local conditions, thus allowing for local adaptation, particularly in the case where a population is introduced into a new location. Due to its wide geographic distribution indicating a high adaptiveotential and its socio-economic importance, wheat was chosen as model crop in this study. Flowering time is a major adaptive trait which has allows wheat to grow over a wide range of ecological and climatic conditions. This PhD study was designed to gain insights about the influence of within population diversity on the short term response of populations to contrasting agro-climatic conditions by studying the genetic, epigenetic and phenotypic variation. But due to the lack of prior existence of epigenetic markers, this thesis study is divided of two parts: In the first part, European wheat populations coming from a set of seven farmer and one modern varieties, each of which was grown on seven farms (distributed across Europe) for three years, were used to study their short term response to contrasting agro-climatic conditions in Europe by analysing their phenotypic and genotypic variations. For the second part the effect of vernalization on the DNA methylation profile of theVRN-A1 gene in winter wheat was studied as a first step towards the development for the epigenetic marker in this gene.The results from the first part of the study revealed that conservation history of these farmer varieties strongly influenced the genetic diversity and fine genetic structure. Ex situ conserved farmer varieties showed low genetic diversity and simpler structure whereas in situ conserved farmer varieties and mixtures revealed higher level of genetic diversity and complex genetic structure. Genetic and phenotypic spatio-temporal differentiation depending upon the level of diversity and structural complexity of the farmer variety was observed. The traditional varieties tend to become more differentiated than the modern variety arguing in favour of use of these diverse traditional (farmer) varieties in organic and low input agriculture systems. Interestingly, a significant phenotypic differentiation for varieties with very low genetic diversity has also been observed in this study, which gives indication of a possible role of epigenetic variation in the process of evolution.From the second part of the study (effect of vernalization on the DNA methylation profile of the VRN-A1 gene), it was found that in addition to the detection of gene body methylation across the VRN-A1 gene, we identified a region within intron 1 that shows significant increase in DNA methylation in response to vernalization treatment that is positively correlated with the gene expression. Although the role of this shift in gene regulation is still unclear due to time limitations in the thesis and the small number of genotypes analysed, this study will provide a good material towards future identification of new epialleles and the development of epigenetic markers to study the epigenetic variability of these populations.This study at large provides useful knowledge on the understanding of farmers' varieties evolutionary response to be used in the development of different breeding and conservation approaches for organic agriculture, taking into consideration of the importance of within population diversity, to satisfactorily address the problems of organic agriculture.

[Truncated abstract] Introduction: Breast cancer is a heterogeneous disease, resulting in very different outcomes for women with apparently similar tumour characteristics. In order for patients to have optimal treatment, a better understanding of the molecular nature of their disease is required. Aims: The aims of this thesis were: 1) To determine whether methylation of RARβ2, ER, CDH1, BRCA1, CCND2, p16 and TWIST genes are associated with phenotypic features of breast cancer and the prognostic significance of methylation of these genes. 2) To investigate for possible associations between the frequency of methylation at RARβ2, CDH1, ER, BRCA1, CCND2, p16 and TWIST genes and the presence of germ-line variants in the TS, MTHFR, MS, CBS, MTHFD1 and DNMT3B genes, as well as for possible correlations between these polymorphisms and clincopathological features of breast cancer including patient outcome. 3) To determine whether PIK3CA mutations determined clinical phenotype and the prognostic significance of PIK3CA mutations in a large and well characterized cohort of breast cancer patients. Methods: A large and well characterized series of primary breast tumours were selected for methylation of RARβ2, ER, CDH1, BRCA1, CCND2, p16 and TWIST genes using MSP, and for polymorphisms in TS, MTHFR, MS, CBS, MTHFD1 and DNMT3B genes using PCR, PCR-RFLP and PCR-SSCP. Mutations to PIK3CA were detected using F-SSCP. Results and Conclusions: Methylation frequencies ranged from 11% for CCND2 to 84% for ER. More frequent hypermethylation was observed in tumours with poor histological differentiation compared to those with well/moderate differentiation, as well as trends for association with larger tumour size and mutant TP53. Tumours with ER and CDH1 methylation were associated with significantly lower hormone receptor levels, younger age at diagnosis and the presence of mutant p53. TWIST methylation is firstly reported to be associated with significantly older patient age at diagnosis and larger tumour size. Our data suggests that gene methylation may be linked to various pathological features of breast cancer. However, there appears to be little support for a distinctive CpG island methylator phenotype in breast cancer.