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Identifier

000404259

Title

Investigation of the role of the HNF4a transcription factor in the pathogenesis of metabolic diseases using mouse models

Alternative Title

Διερεύνηση του ρόλου του μεταγραφικού παράγοντα HNF4a στη παθογένεση των μεταβολικών ασθενειών

Author

Σκέντερης, Νικόλαος Ταξιάρχης

Thesis advisor

Καρδάσης, Δημήτριος

Reviewer

Τσατσάνης, Χρήστος
Μπερτσιάς, Γεώργιος

Abstract

Metabolic Syndrome (MetS) is a cluster of several pathologies including obesity, hypertension, dyslipidemia (hypertriglyceridemia combined with low levels of high density lipoprotein cholesterol (HDL) and high levels of low density lipoprotein cholesterol (LDL)), and impaired glucose tolerance or insulin resistance, all of which increase the risk for developing cardiovascular disease. The etiology of MetS is poorly understood and effective therapies are urgently needed. Nonalcoholic fatty liver disease (NAFLD) is defined as a disorder with excess fat in the liver due to non-alcoholic causes. It has been proposed that NAFLD is the “hepatic manifestation” of MetS. Hepatocyte nuclear factor 4 alpha (HNF4a, NR2A1) is a member of the hormone nuclear receptor superfamily of transcription factors that is expressed mainly in the liver, intestine, kidney and pancreas. Mutations in the HNF4a gene cause Maturity Onset Diabetes of the Young (MODY) in humans whereas SNPs in the HNF4a locus have been associated with reduced HDL levels in GWAS. In mice, HNF4a is required for liver development and for controlling the expression of many genes involved in lipoprotein metabolism. The main goal of this thesis is to elucidate the role of HNF4a in the pathogenesis of metabolic diseases such as MetS and NAFLD. To achieve this goal, two mouse lines were used: a) the APOE*3Leiden.CETP transgenic mouse which is a well-established model for diet-induced MetS; and b) the HNF4a liver knockout mouse. Mice lacking hepatic HNF4a expression accumulate lipids (triglycerides, cholesterol) in the liver and can be used as a model of NAFLD. As a first step towards the achievement of our goal, three small hairpin RNAs (shRNAs) were designed and screened for efficient targeting of sequences in the mouse HNF4a mRNA. The most efficient shRNA was introduced into an appropriate plasmid vector under the control of the H1 or U6 promoters and the constructs were packaged into Adeno-Associated Viruses (AAVs). In the next step, the AAV-shHNF4a viruses will be used, along with control AAVs, for long term silencing of HNF4a in the livers of the APOE*3Leiden.CETP transgenic mice. The infected mice will be subjected to diet-induced MetS and this will be followed by extensive phenotypic and transcriptomic analysis of these mice. The transcriptomic analysis of the livers of HNF4a Liver KO mice has been completed in our lab and revealed significant changes in the expression of several genes involved in lipid and apolipoprotein biosynthesis and metabolism. The focus of this thesis was on the validation of novel genes with not yet established roles in High Density Lipoprotein metabolism which were found to be differentially expressed and could serve as direct targets of HNF4. Validation of the microarray data was performed by RT-qPCR and chromatin immunoprecipitation in mouse liver samples was used to verify the direct binding of HNF4a to the promoter regions of these genes. In summary, using appropriate mouse models we are investigating the contribution of HNF4a in the pathogenesis of metabolic diseases such as MetS and NAFLD and the identification of novel HNF4a target genes. The ultimate goal is to identify novel drug targets which could be explored therapeutically in the future for the treatment of these diseases.

Language

English

Subject

Metabolic syndrome

Καρδιολογικές ασθένειες

Μεταβολικό σύνδρομο

Issue date

2016-12-13

Collection

School/Department--School of Medicine--Department of Medicine--Post-graduate theses

Type of Work--Post-graduate theses

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