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Lecturer(s)
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Küpper Hendrik, prof. Ph.D.
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Course content
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Content of practicals:Basics, selected important methods, with emphasis on applications in bioinorganic chemistry and biophysics of plants, and special emphasis on methods for analysis in vivo, in situ and methods developed or improved in the Küpper group (e.g. FKM, GPS-fitting, QISH). The projects of this course originate from the current research of our department as described on our homepage (http://www.umbr.cas.cz/~kupper/AG_Kuepper_homepage.html). And as far as this is possible for a six-week project, they are independent subprojects, each supervised by a member of the department. The research of the department mainly deals with mechanisms of active uptake of metals (with terrestrial hyperaccumulator plants as model organisms), mechanisms of metal toxicity (with sensitive water plants as models), and mechanisms of metal detoxification (in hyperaccumulators, sensitive plants as well as algae). Main methods applied in the department: 2.2.1. Analytical Methods (a) Two-dimensional (imaging) and spectrally resolved measurements of photosynthesis biophysics via in vivo chlorophyll fluorescence kinetics in the FKM (see above). (b) In vivo kinetic UV/VIS-absorption and fluorescence spectroscopy with subcellular resolution in the FKM. (c) Two-dimensional (imaging) quantitative measurements of reactive oxygen species and metal uptake in vivo via specific fluorescent dyes in the FKM. (d) Quantitative mRNA in situ-hybridisation (QISH) for analysing cell-specific gene regulation (see above). (e) UV/VIS-Spectroscopy for pigment analysis via fitting of GPS (see above). (f) Metal analysis via atomic absorption (F-AAS and GF-AAS) and inductively coupled plasma mass spectrometry (ICP-MS). (g) Measurement of reactive oxygen species via chemoluminescence. (h) Biochemical activity assays of metal transport proteins after re-constitution into artificial lipid vesicles (proteoliposomes). (i) Identification of metal binding to proteins via protein chromatography and subsequent metal analysis and mass spectrometry (metalloproteomics). (j) SDS-PAGE, native PAGE & Western Blots for quality control & protein identification. (k) UV/VIS-spectroscopy of metal-ligand charge transfer bands and of electrochromic dyes for analysis of metal binding and metal transport in proteins (l) Data analysis. You will learn how to work with scientific data analysis software (Origin, SigmaPlot, others depending on the individual project) incl. data import from various sources and how to make publication-ready graphs, aspects of statistics that are important for research in biochemistry and biophysics (e.g. nonlinear regression, ANOVA). 2.2.2. Preparative Methods (a) Hydroponic cultivation of plants in chemically defined nutrient solution with continuous automatic media exchange and simulation of natural light/temperature cycles (b) Tissue digestion by acids for metal analysis via AAS/ICP-spectroscopy (c) Machine-assisted low temperature (< 80°C) tissue homogenisation for protein isolation (d) Preparative high-performance liquid chromatography (HPLC) (e) Isolation and purification of integral membrane proteins (e.g. via metal affinity FPLC) (f) Methods for reproducible work with extremely low (10 7 to 10 10 M) concentrations of metals (--> how to avoid and remove contaminations)
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Learning activities and teaching methods
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Monologic (reading, lecture, briefing), Dialogic (discussion, interview, brainstorming), Laboratory, Project-based learning
- Class attendance
- 180 hours per semester
- Preparation for exam
- 10 hours per semester
- Preparation for credit
- 20 hours per semester
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Learning outcomes
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Building on the previous lecture series, in the course students will be practically introduced to bioinorganic chemistry and biophysics, as well as selected research topics, such as metal uptake & transport, metals as essential trace elements e.g. for active centres of proteins (incl. effects of metal deficiency), mechanisms of metal toxicity, metal resistance/detoxification. They will furthermore learn various methods employed in this kind of research, as well as getting training in advanced data analysis, interpretation and presentation of research data.
Students are practically acquainted with the scientific methods of studying the uptake and transport of metals, with metals as basic trace elements, with mechanisms of metal toxicity, resistance to metals and detoxification.
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Prerequisites
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Prerequisites - other information about course preconditions (a) Previous completion of the course "Bioinorganic Chemistry and Plant Biophysics I" (b) completed bachelor's or master's degree in Biology, Biochemistry or Biophysics
KEBR/201
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Assessment methods and criteria
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Oral examination, Seminar work
- Combined examination: At the end of the course, each participant writes a final report in the format of a scientific publication and explains his/her project to the other course participants in an oral presentation resembling a short lecture at a scientific conference.
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Recommended literature
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Andresen E, Küpper H (2013) Cadmium Toxicity in Plants. In: Cadmium: From Toxicity to Essentiality, Chapter 13, Volume 11 of series "Metal Ions in Life Sciences". (Eds: Sigel A, Sigel H, Sigel RKO). Springer Science + Business Media B.V., Dordrecht; pp. 395-414..
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Leitenmaier B, Küpper H (2013) Compartmentation and complexation of metals in hyperaccumulator plants. Frontiers in Plant Science, doi: 10.3389/fpls.2013.00374.
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Reviews: Küpper H, Kroneck PMH (2005) Heavy metal uptake by plants and cyanobacteria. In: Metal Ions in Biological Systems, Band 44, Kapitel 5. (Hrsg.: Sigel A, Sigel H, Sigel RKO). Marcel Dekker, Inc., New York; pp. 97-142.
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