Aktuelle Masterarbeitsthemen der Forschungsgruppe Molekular- und Systemtoxikologie
Current Master's Theses Projects of the Research Group in Molecular & Systems Toxicology
|MSc Pharmazie||MSc Drug Sciences||Bemerkungen|
Investigating Drug liver injury through AKR1D1 and SRD5A1
The liver is the central organ responsible for the selective uptake, metabolism and excretion of endogenous and exogenous compounds, including drugs. Drug induced liver injury (DILI) can be caused by various chemical and can present as an array of different pathologies, dependent on the specific function of the liver that is impaired. Numerous drugs have been shown to cause liver injury but the manifestations of drug-induced hepatotoxicity are highly variable, ranging from asymptomatic alterations of liver enzymes to fulminant hepatic failure. AKR1D1 and SRD5A1 are two important enzymes involved in bile acid synthesis from cholesterol in the liver. The disruption of their function has been associated with different liver pathologies including inflammation, NASH/NAFLD, fibrosis, cancer and infections.
In the present project, we aim to investigate a selected range of drugs screened by bioinformatics tools for potential inhibitory effects towards AKR1D1 and SRD5A1. Upon establishing activity assays for these two enzymes the most promising hits will be investigated for their potential to inhibit their enzyme activities and the consequences for bile acid homeostasis.
Methods: RNA/DNA/proteins extractions and quantification, bacterial transformation and eukaryotic cell culture, cloning, DNA amplification (mini/maxiprep), transfection, cell line culture and maintenance, enzyme activity assays, western blot, PCR/qPCR, protein purification.
Sensing NADPH in the endoplasmic reticulum
Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential cofactor for many reactions in living organisms. It supports reducing capacity, macromolecular biosynthesis, superoxide and nitric oxide production and detoxification of drugs and xenobiotics. Perturbations in the NADPH metabolism lead to impaired cellular function causing increased risk of inflammation, cancer, diabetes mellitus and neurodegeneration.
The existing methods for NADPH measurements in the endoplasmic reticulum are often invasive and hence do not allow measurements in living cells; in addition they are prone to sample oxidation during the process. Other methods like Fluorescence lifetime imaging can distinguish between NADPH and NADH fluorescence in living cells; however, specialized equipment is needed and may not be readily accessible. In response to these challenges, Tao et al (2017) have recently developed a genetically encoded fluorescent sensor capable of sensing NADPH in the cytoplasm and mitochondria of the living cell.
The aim of this master project will be to modify the existing sensor to ensure appropriate expression and functionality in the endoplasmic reticulum. This will include a site directed mutagenesis based screening of different forms of the sensor to find the optimal form for the unique environment of the endoplasmic reticulum.
Methods: Bacterial and eukaryotic cell culture, DNA purification, cloning and site-directed mutagenesis, fluorescence microscopy and imaging.
LC-MS/MS methods as a tool for biomarker discovery
Bile acids are involved in several important functions in the liver and the intestine. The formation of bile acids is important for the regulation of the maintenance of cholesterol homeostasis. They are regulatory molecules for a number of metabolic processes, excretion of endogenous and exogenous toxic substances. Bile acids and their derivatives are potential therapeutic agents for treating metabolic diseases of the liver. On the other hand, oxysterols are oxidised forms of cholesterol or of its precursors. They are involved in neurodegenerative disease (e.g. Huntington's, Parkinson's and Alzheimer's diseases) and in cancer. Disease, especially those involving oxidative stress, may alter the equilibrium, and oxysterols have to potential to serve as diagnostic markers.
Methods for the quantification of various bile acids and oxysterols are essential for understanding their mechanisms of action in vivo, and valuable for diagnosing rare diseases of cholesterol biosynthesis and metabolism as well as for detecting drug-induced toxicity. Liquid chromatography- mass spectrometry is considered a gold standard technique in Analytical Chemistry in both Academia and Industry.
The aim of the master project will be to optimize extraction protocols for the detection and quantification of bile acids and oxysterols in biological matrices. The newly established method will be applied to assess samples from in vivo studies. The master student will be able to learn the basics of chromatography and mass spectrometry techniques and data analysis.
Methods: LC-MS/MS, Solid-Phase Extraction, Liquid-Liquid Extraction, Derivatization.
|Cristina Gómez Castellà||no||yes|
Involvement of retinoic acid-related orphan receptor gamma in long-chain fatty acid synthesis regulation (NO LONGER AVAILABLE)
The retinoic acid-related orphan receptor gamma (RORγ) is a nuclear receptor that is involved in the regulation of proliferation of different types of cancer. We are interested in elucidating the role of RORγ on long-chain fatty acid synthesis in cancer cell lines, which is partially mediated via the 17β-hydroxysteroid dehydrogenase type 12 (17β-HSD12). Recently, silencing of 17β-HSD12 in breast cancer cells showed significant effects on cell proliferation and migration . This project aims to study the role of RORγ in cancer cell lines and its influence on the long-chain fatty acid synthesizing enzyme 17β-HSD12. The results of this study will provide further valuable insight into the mechanisms of cancer cell physiology.
Methods: cell culture, western blotting, quantitative PCR, luciferase reporter assays, gene knockdown, cell proliferation and migration assays.
 Tsachaki M. et al. manuscript submitted