The impact of paternal metabolic health on sperm DNA methylation and fetal growth

Low birth weight is associated with cardiovascular disease and T2DM in later life. Paternal obesity and T2DM have been associated with an increased risk of fathering low birthweight offspring. Obesity is associated with epigenetic changes in blood, but few studies have replicated DNA methylation differences found in obese subjects. Animal studies have shown that obesity and insulin resistance are associated with DNA methylation changes in sperm, which in turn could mediate intergenerational effects. Such findings are lacking in humans. My PhD explored the association between paternal metabolic traits and the birth weight of his offspring. I then investigated whether DNA methylation signatures in spermatozoa of obese fathers could underlie any observed association with his offspring birthweight. First, I performed a prospective cohort study of 500 mother-father-offspring trios to identify paternal metabolic traits associated with an increased risk of fathering low birth weight offspring. Out of 390 trios, including 64 obese men and 48 growth restricted offspring, I did not discover any significant paternal metabolic traits associated with fathering low-birthweight offspring. However, I found that paternal (own) birth weight is associated with the birth weight of his offspring. This suggests that paternal genetic factors are more influential in determining his offspring’s growth in utero than are factors acquired during his lifetime. Second, I performed a systematic review of studies that had investigated DNA methylation in human sperm. From this review, I summarised current knowledge and generated recommendations for future research. I then performed the largest characterisation of matched human sperm and blood samples to date using the most comprehensive DNA methylation profiling array, the MethylationEPIC array. Results showed that the DNA methylomes of sperm and blood are highly discordant and in effect completely uncorrelated. Future studies of intergenerational effects will have to study germ cells, rather than blood. Lastly, I attempted to validate previously-identified DNA methylation signatures associated with male obesity. Despite comparing 96 well-characterised obese men with 96 lean men, I was unable to replicate any previously identified differentially methylated CpG sites associated with obesity, in their blood. In a linear regression model, I identified two CpG sites, cg07037944 and cg26651978, as being suggestive of an association with BMI. These results will contribute to a larger cohort study of 1000 obese and 1000 lean men that aims to identify a robust and reproducible DNA methylation profile associated with obesity. In conclusion, this thesis did not prove my pre-determined hypotheses. However, it does present findings which advance our understanding of the intriguing possibility that acquired parental metabolic phenotype may influence offspring birthweight through intergenerational inheritance of epigenetic marks

Rare mendelian forms of obesity and diabetes and their implications for treatment outcomes

Obesity and diabetes are becoming epidemic health issues worldwide. In recent decades, a considerable amount of work has been done to study the pathogenicity underlying those diseases, which has led to valuable insights into the genetic basis, treatment and prevention of obesity and diabetes. Nevertheless, despite our more detailed pathophysiological understanding of the rare forms of diabetes and obesity than of more common polygenic forms, we still know little about their prevalence and implications outside specialised genetics services. In the present work, I have explored the contribution of Mendelian forms of obesity in individuals with severe obesity. Initial segregation analyses of families with an obese proband, led to the identification of an oligogenic mode of inheritance for obesity. This was followed by re-analysis of pre-existing whole exome sequencing data from 91 individuals with extreme obesity, which revealed an additional 21 possible causative variants in known monogenic/syndromic obesity genes and three further cases of oligogenic inheritance. In addition, 11 candidate variants were identified in genes suggested by rodent models of obesity and/or diabetes, but not previously reported in humans. To further expand the analysis, a unique custom genotyping array focusing on, obesity and diabetes mellitus (T2D, and monogenic forms of diabetes) was designed to be applied to a larger number of samples (N=2068). Application of the array led to the identification of a total of 161 potential causative variants in 40 monogenic obesity/syndromic obesity genes, with a putative diagnostic yield of 11%. Initial analysis suggests that having one of these putative Mendelian forms of obesity resulted in no statistical difference in percentage weight loss at 2 years post-surgery and diabetes remission. Our first analysis on obesity indicates that the use of a custom-designed genotyping array for specific rare diseases may be an advantageous first level screening strategy in terms of cost and time. The work presented here also suggests that the true prevalence of Mendelian forms of obesity among bariatric surgery patients is likely to be high - this presents a significant unmet need for genetic analysis and follow-up.  Open Acces

Marine artificial light at night:An empirical and technical guide

The increasing illumination of our world by artificial light at night (ALAN) has created a new field of global change research with impacts now being demonstrated across taxa, biological ranks and spatial scales. Following advances in terrestrial ecology, marine ALAN has become a rapidly growing research area attracting scientists from across all biomes. Technological limitations, complexities of researching many coastal and marine ecosystems and the interdisciplinary nature of ALAN research present numerous challenges. Drawing on expertise from optical oceanographers, modellers, community ecologists, experimental and molecular biologists, we share practical advice and solutions that have proven useful for marine ALAN research. Discussing lessons learnt early on can help in the effective and efficient development of a field. The guide follows a sensory ecology approach to marine light pollution and consolidates physics, ecology and biology. First, we introduce marine lightscapes highlighting how these differ from terrestrial ones and provide an overview of biological adaptations to them. Second, we discuss study design and technology to best quantify ALAN exposure of and impacts on marine and coastal organisms including molecular tools and approaches to scale-up marine ALAN research. We conclude that the growing field of marine ALAN research presents opportunities not only for improving our understanding of this globally widespread stressor, but also for advancing fundamental marine photobiology, chronobiology and night-time ecology. Interdisciplinary research will be essential to gain insights into natural marine lightscapes shaping the ecology and evolution coastal and marine ecosystems

Differential Global Effects of Selective Estrogen Receptor Modulators on Estrogen Receptor Binding and Transcriptional Regulation

Ph.DDOCTOR OF PHILOSOPH

Selenium-gene interactions involving microRNAs

PhD ThesisSelenium (Se) is an essential nutrient for health. In mammals Se is incorporated into ~25 selenoproteins in the form of the amino-acid selenocysteine encoded by the UGA codon through a complex interacting with selenocysteine insertion sequence (SECIS) in the 3’Unstralated Region. The selenoproteins have functions in antioxidant defence and redox control, thyroid hormone metabolism and mitochondrial metabolism. Previous scientific work has found that Se also affects a group of downstream targets. The aim of my work is to investigate whether expression of selenoproteins or the downstream targets affected by Se is regulated through epigenetic mechanisms involving microRNAs, a small non-coding RNA species that regulates a gene or groups of genes by binding to the mRNA 3’UTR. Gut epithelial Caco-2 cells were grown in either Se deficient or Se-supplemented medium for 72h. RNA extracted and miRNA expression analysed using a custom-designed human genome V2 Agilent 8x15K array. In addition, global mRNA transcriptome expression was analysed using an Illumina HumanRef-8 v3 microarray. Se supply increased the expression of thirteen miRNAs and 53 mRNAs the observed differences were confirmed by real time PCR. miR-185 was selected as a further target of investigation because of its high sensitivity to Se. Bioinformatic analysis of the Se susceptible miRNAs and Se sensitive genes was carried out using miRWalk, MicroCosmo, microRNA.org, miRBase and microRNA.org algorithms and Ingenuity Pathway Analysis. This identified miR-185 recognition elements in the Se-sensitive mRNAs for Glutathione peroxidise 2 (GPX2), Glutathione peroxidise 3 (GPX3), and Selenophosphate Synthetase 2 (SEPHS2). Expression of GPX2 and SEPHS2 was altered by miR-188 with a specific anti-miR and affected differently according to miRNA exposure. The data suggests that these mRNAs are targets of miR-185. In conclusion, the experiment indicates that miRNA expression as regulation of target genes is regulated by Se supply in Caco-2 cells.National Council of Science and Technolog

DNA methylation changes associated with acquired platinum resistance in ovarian cancer

Despite high responses to initial chemotherapy most patients with ovarian cancer (OC) relapse and inevitably die from their disease. Aberrant DNA methylation is frequently seen in ovarian tumours and may provide biomarkers of clinical outcome or insight into mechanisms of chemoresistance. We firstly performed Differential Methylation Hybridisation (DMH) to identify loci that gained methylation between 34 matched cisplatin sensitive and resistant OC tumour cell lines. Differentially methylated loci identified were further validated by Methylation Specific PCR (MSP) and bisulphite pyrosequencing. Selected loci were further investigated for association with clinical outcome in primary OC tumour samples and matched tumour samples from patients' pre- and post-chemotherapy. Frequent increased methylation of a CpG island at the NR2E1 gene was identified in this experiment. Increased methylation correlated with decreased gene expression and could be reversed following treatment with a demethylating agent. Increased methylation at NR2E1 was observed between matched pre- and post-treatment tumour pairs. A novel biostatistical method, methylation linear discrimination analysis (MLDA), was next used to identify differentially methylated loci in sensitive and resistant A2780 human ovarian cell lines. Eight of nine loci identified were validated by MSP. A locus at the SP5 gene was further investigated by pyrosequencing and found to show a very high level methylation in most cell lines and ovarian tumours. Increased methylation correlated with decreased gene expression and this could be reversed using decitabine treatment. Knockdown of SP5 expression caused increased apoptosis. DMH was next used to identify loci that gained methylation between 3 in vivo derived matched sensitive and resistant cell lines. KIAA1383, a gene of unknown function, was identified and methylation shown to correlate with response to chemotherapy and progression-free survival (PFS) in patients with OC. Over-expression was found to attenuate the response to cisplatin, in the PEA2 cell line, as measured by cell cycle analysis

The Role of Genes and Environment on Fetal Growth

Fetal growth is influenced by the in utero environment and genetic factors inherited from both parents. Poor fetal growth leading to low birth weight is associated with insulin resistance and type-2 diabetes in later life. The fetal programming of adult disease hypothesis suggests that growth-restricted fetuses make enduring physiological adaptations that predispose to diabetes in later life. The fetal insulin hypothesis suggests that poor fetal growth and diabetes are two phenotypes of genetically determined insulin resistance. Under these circumstances, an insulin resistant fetus cannot optimise insulin-mediated growth and is predisposed to diabetes in later life. Environmental and genetic influences come together through epigenetic modifications, for example DNA methylation, that alter gene expression without altering the nucleotide sequence. The first aim of this thesis was to investigate whether men who fathered pregnancies complicated by fetal growth restriction had an insulin resistant phenotype at the time of the index pregnancy. A case-control study showed that men who fathered growth-restricted offspring have pre-clinical insulin resistance and are more likely to smoke than fathers of normal grown offspring. This observation supports the concept that an insulin resistant genotype inherited from a father could manifest as poor fetal growth in offspring. I then investigated the mechanisms through which paternal insulin resistance might be inherited by a growth-restricted fetus. I studied DNA extracted from the cord blood of growth-restricted offspring using whole exome sequencing to identify novel gene variants and those known to be associated with type-2 diabetes. I validated findings with Sanger sequencing and Taqman genotyping in all family members. Using the Illumina Human 450 BeadChip, I found marked differences in genome wide DNA methylation of fetal cord blood and placental samples from growth restricted compared with normal grown offspring. Future work is aimed at investigating the functional consequences of genetic and epigenetic differences to identify targets for treatment and prophylaxis against fetal growth restriction and diabetes

Microarray-based expression profiling : improving data mining and the links to biological knowledge pools

Having identified differentially regulated genes, the final and most labour intensive part of the analysis process is drawing biological conclusions and hypothesises about the data. A novel solution is presented which combines experimental data with a curated annotation sources along with analysis tools to assist the researcher in exploring the information contained within their dataset.EThOS - Electronic Theses Online ServiceGBUnited Kingdo