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| Semester | Frühjahrsemester 2013 |
| Angebotsmuster | Jedes Frühjahrsem. |
| Dozierende |
Verena Christen (verena.christen@unibas.ch)
Karl Fent (karl.fent@unibas.ch) Denise Kratschmar (denise.kratschmar@unibas.ch) Alex Odermatt (alex.odermatt@unibas.ch, BeurteilerIn) |
| Inhalt | SPECTROSCOPY IN TOXICOLOGY: (A. Odermatt / D. Kratschmar) The rat hepatoma cell line H4IIE is an accepted cell model for basic mechanistic toxicology. 1) H4IIE cells will be used as a model to evaluate the cytotoxic effect of ethanol and hydrogen peroxide (H2O2) in a standardized and accepted assay for mitochondrial activity and cytotoxicity (MTT Assay). 2) Nrf2 (nuclear factor (erythroid-derived 2)-like 2) is a key transcription factor regulating a plethora of detoxifying enzymes and antioxidant genes involved in drug metabolism and defence against oxidative stress. The action of Nrf2 and the protective effects of known Nrf2 Inducers can be studied in transactivation assays. The experimental setting is based on a stably transfected H4IIE cell line expressing an Nrf2-dependent luciferase reporter gene. Practical experience: Cultivation of mammalian cells under normal culture conditions (5% CO2, 37°C, 98% humidity). Improvement of the knowledge of cell viability (tryphan blue exclusion) and a standard cell counting procedure (Neubauer haemocytometer). Induction of oxidative stress and cytotoxicity in H4IIE cells by ethanol and hydrogen peroxide. Molecular biology related calculations (molarity) and handling of different cell culture formats as well as standard laboratory equipment. Toxicological insight into mechanisms underlying toxic effects of the well-known toxicants ethanol and hydrogen peroxide. Spectroscopic (UV/VIS) protein and formazan determination. Evaluation of the cytotoxic active concentration for ethanol and hydrogen peroxide in H4IIE cells. Transactivation of Nrf2 measured via reporter gene analysis. Deeper insight into enzymatic reactions (luciferase) and detection of activity via luminescence. MAGING IN TOXICOLOGY: (A. Odermatt / D. Kratschmar) Enhanced production of mitochondrial reactive oxygen species (ROS) leads to oxidative stress, an underlying mechanism for apoptosis and cellular damage. Cellular imaging technologies offer the opportunity to visualize the production of ROS and subsequently apoptosis in real time and/or as an endpoint stage. Students will use the sophisticated Cellomics technology to visualize three different fluorescence colours simultaneously. Using Cellomics, oxidative stress, nuclear condensation, cell permeability and viability and mitochondrial activity and membrane potential will be assessed. Practical experience: Cellular imaging and cytotoxic effects on different cellular compartments (nuclei, mitochondria, cytosol). Important calculations and experiment assessment. CELLULAR DETOXIFICATION SYSTEMS (K. Fent / V. Christen) Cellular Detoxification systems play an important role in the protection against xenobiotics. Some xenobiotics are known to alter the activity of these cellular defense systems. During the ecotoxicological risk assessment of potential environmental pollutions, it is important to investigate the effects of these substances on the cellular detoxification system. Beside these specific effects of xenobiotics, general cytotoxic effects and induction of cellulat stress responses triggered by xenobiotic exposure are also important to investigate. Additional to in vitro experiments, in vivo fish experiments are important to conduct environmental risk assessment of chemicals. Practical experience: Cultivation of human hepatoma cells and zebra fish liver cells under normal conditions. Determination of cytotoxicity of environmental chemicals and investigation of oxidative stress induction and DNA damage. CYP1A and CYP3A assays (part of the cellular detoxification system): determination of enzyme activity after exposure to xenobiotics. Development of fish gonads: investigation of the different developmental stages of fish gonads under the microscope. Disection of adult zebrafish and rainbow trouts. POTENTIIAL EFFECTS OF XENOBIOTIC EXPOSURE: (K. Fent / V. Christen) Potential effects of xenobiotic exposure are alterations in gene expression. Apoptotic gens or stress genes can be up-regulated by toxic chemicals or genes belonging to the hormone systems can be altered by hormonal active chemicals (endocrine disrupters).To investigate possible changes in gene expression, human and fish cells are exposed to substances followed by RNA isolation, reverse transcription and real time PCR. Practical work: Exposure of cells to test substance, isolation of RNA, reverse transcription of RNA into cDNA , real time PCR for selected target genes, data analysis. |
| Lernziele | Cell culture, cytotoxicity, hepatotoxicity, oxidative stress, antioxidant reponse pathway, spectroscopic methods (UV/VIS, luminescense), cellular imaging, fluorescence microscopy, RNA isolation, reverse transcription, real time PCR, ecotoxicological in vitro test systems, in vivo fish experiments |
| Bemerkungen | The examination consists of a written report/questions on underlying theoretical aspects of the performed experiments, representation of the experimental steps as well as critical interpretation of the results obtained. During the course essential questions and calculations have to be successfully solved to proceed. |
| Weblink | ADAM |
| Unterrichtssprache | Englisch |
| Einsatz digitaler Medien | kein spezifischer Einsatz |
| Intervall | Wochentag | Zeit | Raum |
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Keine Einzeltermine verfügbar, bitte informieren Sie sich direkt bei den Dozierenden.
| Module |
Modul Practical Training (Master Toxikologie) (Pflicht) Vertiefungsmodul Toxikologie (Master Pharmazeutische Wissenschaften) |
| Leistungsüberprüfung | Lehrveranst.-begleitend |
| Hinweise zur Leistungsüberprüfung | Friday, 24.05.2013, 13.15 - 17.00, PR 0094 |
| An-/Abmeldung zur Leistungsüberprüfung | Anmelden: Belegen; Abmelden: Dozierende |
| Wiederholungsprüfung | keine Wiederholungsprüfung |
| Skala | 1-6 0,5 |
| Wiederholtes Belegen | beliebig wiederholbar |
| Zuständige Fakultät | Philosophisch-Naturwissenschaftliche Fakultät, studiendekanat-philnat@unibas.ch |
| Anbietende Organisationseinheit | Departement Pharmazeutische Wissenschaften |