Chronic environmental exposure to inorganic arsenic is widely associated with human disease. Low human arsenic secondary methylation efficiency (SME), represented by high urinary monomethylarsonic acid (%uMMA) and low urinary dimethylarsinic acid to monomethylarsonic acid ratio (uDMA/uMMA), has been consistently associated with increased risk of arsenic-related diseases. Therefore the determination of factors modulating arsenic SME acquires particular importance. The aims of the present study are to identify novel factors of variability in arsenic secondary methylation, and to test for potential factors influencing arsenic SME for which there is equivocal literature support. A population of 808 subjects was recruited from northwest Mexico environmentally exposed to arsenic. The mean total urinary arsenic in the population was 171 μg/L. Great interindividual variability in %uMMA excretion was observed (0.85% - 40.5%). Three intronic polymorphisms in arsenic (3+ oxidation state) methyltransferase (AS3MT), the key gene in the metabolism of arsenic, were confirmed to be associated with increased arsenic SME in this study. Further analysis of this genomic region showed a large block of linkage disequilibrium (LD) comprising these three genetic variants and other 43 intronic polymorphisms within AS3MT and four additional genes. Genetic association analysis showed that all linked polymorphisms in this region except one were significantly associated with higher arsenic SME. The existence of this long region of LD associated with arsenic SME underscores the complexity of association studies involving any of these linked polymorphisms since there is no certainty of which polymorphism or gene is the causative of the association. In addition, a strong positive association between body mass index (BMI) and arsenic SME was observed in females but not in males. This association was replicated in two independently recruited populations of adult women. Moreover a unique finding of this study is the association between higher genetically estimated indigenous American (AME) ancestry and increased arsenic SME in this ancestrally admixed Mexican population. These results establish the importance of genetic and phenotypic factors in the efficiency of arsenic secondary methylation. Furthermore this study has identified several arsenic-associated risk factors that should be carefully considered in future studies seeking to better understand disease susceptibility in arsenic-exposed populations.

Clonally derived liver tumours and an ovarian cyst developed in mice following EIAV and FIV delivery in utero. LAM PCR and 454 sequencing was used to retrieve proviral insertion sites. TaqMan analysis revealed gene expression changes in lentiviral infected tumours. STRING and IPA networks identified links between genes flanking the lentivirus provirus and oncogenic pathways supporting the role of insertional mutagenesis in Hepatocellular Carcinoma (HCC). Global methylation analysis demonstrated increased relative methylation levels in lentivirus (EIAV, FIV, and HIV) infected normal and tumour samples. This provided strong evidence for host defence against lentivirus infection by epigenetic means. Microarray data showed altered expression of Dnmt1 and Dnmt3b and TaqMan analysis revealed specific changes in Dnmts levels when compared to uninfected liver. The evidence found for involvement of DNA methylation associated with lentivirus infection and possibly tumour development required that this study be repeated in vitro. DNA methylation was investigated at early time points after lentivirus and retrovirus infection in HepG2 cells. Results revealed sharp increases in global methylation and Dnmt levels at 24 and 30hrs post infection. E2F targets play a key role in the regulation of gene expression and aberrations result in the development of cancer. Of the 94 E2F target genes analysed 77.7% were involved in DNA damage and repair mechanisms, 21.3% were known oncogenes or shown to exert oncogenic activity and 80.9% were categorised as HCC target genes. The fact that all lentiviral/retroviral vectors used in this study were found to cause changes in methylation and gene expression in vivo and in vitro suggests that these vectors, at least in the mouse, are genotoxic. Findings here support the use of the fetal animal model to identify vector genotoxicity and the mechanisms of lentiviral vector-induced tumorigenesis. This model may be a valuable tool to evaluate the safety of lentiviruses for gene therapy.

Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.

Breast cancer it is the most common type of cancer and leading cause of cancer mortality among women worldwide. Women who inherit mutations in the breast cancer 1 susceptibility gene (BRCA1) are five times more likely to develop breast cancer than women who do not. However, only ~5-10% of breast cancer cases are due to germline mutations in tumor suppressor genes. There are currently no targeted therapies available triple negative breast cancers (TNBC), which often lack BRCA1 expression. BRCA1 is epigenetically silenced by the activated aryl-hydrocarbon receptor (AhR), suggesting that dietary antagonists of the AhR may inhibit BRCA1 silencing. Genistein is an isoflavone abundant in soy foods and its high consumption levels is thought to underlie the lower prevalence of breast cancer in Asian countries compared to Western countries. The hypothesis of this work is that genistein antagonizes AhR-dependent epigenetic silencing of BRCA1. To test this hypothesis we first determined the capacity of genistein to prevent AhR-dependent silencing of BRCA1 in estrogen receptor-alpha (ERα) expressing cells, with wild-type BRCA1 and inducible AhR (MCF-7). We also determined the effectiveness of genistein in reversing silencing of BRCA1 in ERα-negative cells with hypermethylated BRCA1 and constitutively active AhR (UACC-3199). The effect of genistein on BRCA1 promoter methylation and markers of cell proliferation was also determined in both cell lines.

While obesity is a world leading health problem, the most efficient treatment option for severely obese patients is Roux-Y gastric bypass (RYGB) surgery. However, there are large inter-individual differences in weight loss after RYGB surgery. The reasons for this are not yet elucidated and the role of genetics in weight loss-regulation is still not fully understood. The main aim for this thesis was to investigate the effects of common obesity-associated genetic variants and their effect on weight loss and food intake. We examined if the weight loss two years following RYGB surgery depends on the  FTO genotype, as well as pre-surgery vitamin D status. For FTO AA-carriers, the surgery resulted in a 3% per-allele increased excess BMI loss (EBMIL; P=0.02). When split by vitamin D baseline status, the EBMIL of vitamin D deficient patients carrying AA exceeded that of vitamin D deficient patients carrying TT by 14% (P=0.03). No such genotypic differences were found in patients without pre-surgery vitamin D deficiency. As the influence of individual single nucleotide polymorphisms may be small, we identified a novel method to combine SNPs into a genetic risk score (GRS). Using the random forest model, SNPs with high impact on weight loss after RYGB surgery were filtered out. An up to 11% lower EBMIL with higher risk score was estimated for the GRS model (p=0.026) composed of seven BMI-associated SNPs (closest genes: MC4R, TMEM160, PTBP2, NUDT3, TFAP2B, ZNF608 and MAP2K5). Pre-surgical hunger feelings were found to be associated with EBMIL and the SNP rs4846567. Before surgery, patients filled out the Three Factor Eating Questionnaire and were genotyped for known BMI and waist-hip ratio (WHR) associated SNPs. Patients with the lowest hunger scores had up to 32% greater EBMIL compared to the highest scoring patients (P=0.002). TT-allele carriers of rs4846567 showed a 58% lower hunger feelings. TT- carriers also showed a 51% decrease in disinhibition, but no significant impact on cognitive restraint was observed. Due to the association of eating behaviour and weight loss, acute effects on DNA methylation in response to a food intake intervention of a standardized meal were also investigated. After food intake, 1832 CpG sites were differentially methylated compared to the baseline after multiple testing correction. When adjusted for white blood cell fractions, 541 CpG sites remained. This may be interpreted as that the immune system is playing an active role in the response to food intake and highlights the dynamic nature of DNA-methylation. These findings will contribute to a better care for morbidly obese patients. Post-surgical treatment may be optimized so that patients with a less favourable genetic profile may receive additional support for weight loss and weight management. This may be considered as a step in the transition towards personalized medicine.

Many haematological malignancies are associated with widespread alterations of the transcriptional and epigenetic programmes. Changes in DNA methylation provide the clearest example of epigenetic changes, but the mechanism(s) underlying such changes is unknown. To investigate this I studied DNA methylation across an ~80kb segment of the genome which is not known to be mutated in haematological malignancies. Methylation was perturbed in 35-100% of samples of DNA from individuals with a wide range of haematological malignancies but not in non-malignant haematological disorders. DNA methylation was comprehensively assessed by Southern blot analysis, classical bisulphite sequencing and using a newly developed capture bisulphite sequencing protocol. The results were also compared with analysis by MeDIP, an immunoprecipitation-based technique. These analyses provide methylation status at various levels including individual CpG resolution. This showed both gain and loss of methylation at CpG dinucleotides. Of interest, hypomethylation was most frequently seen in intergenic regions corresponding to transcription factor binding sites and areas of increased chromosome accessibility. These observations suggested that hypomethylation of the genome in haematological malignancies could arise from aberrantly expressed DNA binding proteins which, recruited to sequences in regions of open chromatin, would protect the underlying CpG dinucleotides from the methylation machinery. This, in turn, could lead to passive demethylation accumulating with increasing cell divisions. This hypothesis was tested with electrophoretic mobility shift assays using oligonucleotides representing the DNA underlying one such region. This showed that, compared to nuclear extracts from the lymphocytes of normal individuals, those from patients with CLL were enriched for a protein which binds to oligonucleotides containing the underlying sequence. Using a mass spectrometry approach, I identified a variety of proteins that may bind such regions and account for their passive demethylation in haematological malignancies.

Recently, a genome-wide analysis identified DNA methylation of the HIF3A (hypoxia-inducible factor 3A) as strongest correlate of BMI. Here we tested the hypothesis that HIF3A mRNA expression and CpG-sites methylation in adipose tissue (AT) and genetic variants in HIF3A are related to parameters of AT distribution and function. In paired samples of subcutaneous AT (SAT) and visceral AT (VAT) from 603 individuals, we measured HIF3A mRNA expression and analyzed its correlation with obesity and related traits. In subgroups of individuals, we investigated the effects on HIF3A genetic variants on its AT expression (N = 603) and methylation of CpG-sites (N = 87). HIF3A expression was significantly higher in SAT compared to VAT and correlated with obesity and parameters of AT dysfunction (including CRP and leucocytes count). HIF3A methylation at cg22891070 was significantly higher in VAT compared to SAT and correlated with BMI, abdominal SAT and VAT area. Rs8102595 showed a nominal significant association with AT HIF3A methylation levels as well as with obesity and fat distribution. HIF3A expression and methylation in AT are fat depot specific, related to obesity and AT dysfunction. Our data support the hypothesis that HIF pathways may play an important role in the development of AT dysfunction in obesity.

Interferon γ is an important T-cell helper type 1 (Th1) cytokine involves in antimicrobial immunity. It is a part of the inflammatory immune response in the site of infection. However, for its proper function, the regulation of immunity is necessary to avoid injury of the tissue caused by long-term inflammation. While interferon γ triggers expression of proinflammatory genes, it also regulates genes which inactivate immune response. The B7-H1 molecule belongs among these inhibitory regulators. Furthermore, antitumour effect of interferon γ is well-known as well. After extensive experiments, interferon γ was tested as an immunotherapeutic drug against melanomas in clinical trials. However, the trials had to be terminated prematurely because of unsuccessful results. It started to be clear that interferon γ could have also a protumour effect. Interferon γ upregulates the expression of B7-H1 molecule which aids tumour in escape from immunity. The B7-H1 molecule possesses a binding site for interferon regulatory factor 1 (IRF-1) in its promoter region. This IRF-1 is induced by interferon γ - JAK/STAT signalling pathway. In our previous research, we observed interferon γ induced DNA demethylation of promoters in genes that are involved in antigen presenting machinery. Additionally, DNA methylation of...

Human papillomavirus is connected with induction of cervical carcinoma as well as for some other anogenital carcinomas and subset of carcinomas of head and neck. Presence of viral E6 and E7 oncoproteins may induce cell transformation, higher load of oncoproteins is caused by the regulatory E2 protein inactivation. Aims of recent study are mechanisms of E2 protein inactivation. One option is integration of viral DNA into the host genome, which is located into the E2 gene region. Some carcinomas, where virus with extrachromosomal form was presented, were found. It appears that epigenetic changes can play the role in the development of this type of tumors, especially DNA methylation or mutation in the regulatory region of the virus. The methylation degree analysis on samples of tonsillar carcinomas with extrachromosomal and integrated form of the virus was conduced, as well as viral load of both groups was compared and the expression of E6 and E7 gene was confirmed. The results of methylation analysis showed increased methylation of the virus with integrated DNA. Mutations in the E2 protein binding sites are not revealed. The expression of the viral oncogenes were confirmed in all tumors regardless of the form of the viral genome. The mechanism of tumors induction, especially for virus with...

La tomate (Solanum lycopsersicum), qui forme un clade monophylétique restreint au sein de la large famille des Solanacées, est utilisée comme modèle pour l’analyse du génome, et le développement du fruit. A ce jour, de nombreux efforts ont été consacrés à l'analyse de la diversité génétique des espèces de tomate. Cependant peu de travaux ont porté sur l'analyse de la diversité épigénétique, alors qu’il est aujourd’hui admis que les processus épigénétiques jouent un rôle essentiel dans la diversité phénotypique. Dans un premier temps, le niveau de méthylation de l'ADN a été comparé dans les feuilles et les fruits de différentes variétés de tomates sauvages et cultivées. Puis la famille des gènes Enhancer of zeste (E (z)) a été analysée. Chez la tomate, cette famille comprend deux gènes fonctionnels ainsi qu’un pseudogène. Finalement la stabilité épigénétique reste un facteur majeur pouvant avoir un impact essentiel sur les stratégies de sélection végétales. En outre nous avons fait une caractérisation fine des différents aspects du développement du fruit et de la maturation. / Tomato (Solanum lycopsersicum) which forms a small monophyletic clade within the large Solanaceae family has been chosen as a model system for studying the Solanaceae genome, fruit development and ripening. At that time, many efforts have been devoted to the analysis of the genetic diversity of tomato species, little work has focused on the analysis epigenetic diversity in this clade, although there is a general agreement that epigenetic processes play essential role in the phenotypic diversity in animal and plant system. As first step, DNA methylation level was analyzed in leaves and fruits of various wild and cultivated tomato species.Additionally, the Enhancer of zest (E(z)) gene family has been analyzed. In tomato, the E(z) family consists in two functional genes (SlEZ1, SlEZ2) and in a pseudogene (SlEZ3). In addition, the epigenetic stability is an important consideration that could have a significant on strategies for crop breading. Finally, we made a fine characterization of the different aspects of fruit development and ripening. / All’interno della grande famiglia delle Solanacee è stato scelto il pomodoro (Solanum lycopsersicum) come sistema modello per studio dello sviluppo e maturazione del frutto. Molti sforzi sono stati fatti per analizzare la diversità genetica delle specie di pomodoro, pochi lavori invece riguardano l’analisi della diversità epigenetica, sebbene ci sia accordo sul fatto che processi epigenetici giochino un ruolo essenziale nella diversità fenotipica dei sistemi animali e vegetali. Inizialmente è stato analizzato il livello di metilazione del DNA in foglie e frutti delle diverse specie di pomodoro selvatico e coltivato. Inoltre, è stata analizzata la famiglia genica Enhancer of Zeste (E (z)). In pomodoro la famiglia E(z) consiste di 2 geni funzionali SlEZ1, SlEZ2 e di uno pseudogene SlEZ3. Inoltre la stabilità epigenetica è importante in quanto può avere un impatto sulle strategie di miglioramento genetico delle specie coltivate. Infine è stata condotta una attenta caratterizzazione dei meccanismi cellulari dello sviluppo del frutto e della sua maturazione.