Prior attendance of the following courses:Introduction to Biochemistry Laboratory; Analytical Biochemistry Laboratory, Inorganic Biochemistry Laboratory, Biochemistry of Proteins, Nucleic Acids and Lipids, Fundaments of Physiology, General Physics, Statistic Methods, Biochemistry of Metabolism. The students should be proficient in written English in order to handle all the recommended bibliography.
Short protocols with bibliography are supplied for prior preparation. The students work in groups, autonomously but under supervision. Techniques are approached at the beginning of the class, and each group discusses the respective experimental strategy.
Students are encouraged to interpret the results during the classes, in order to correct trajectories and avoid artifacts. Laboratory procedures are adjusted accordingly. Methods for analysis are also discussed so that a proper analysis can be performed out of the class. Results and analyses are presented and discussed in "TP" classes.
Learning laboratory techniques that are essential for the study of cell physiology and biophysics.
Learning experimental strategies for interfering with cell function, namely at the levels of O2 production or consumption, membrane potential, membrane permeabilities, second messenger systems and exocytotic secretion. Implementing these strategies is expected to enhance the student's theoretical background in cell physiology and biophysics.
Students are also expected to:
1) enhance their ability to plan, organize and think critically in the laboratory, in order to foster problem solving and the adaptation to new situations;
2) enhance both their ability to engage in group work and their concerns with quality management and ethical issues in experimentation;
3) enhance their skills in data processing, data analysis and data presentation.
- Isolation of animal cells and subcellular fractionation of plant cells. Techniques: dissection, homogenization, differential centrifugation, cell counting, viability assessment and protein quantification (photometry).
- Mitochondrial and chloroplastidial bioenergetics by measurement of O2 consumption or production. Techniques: Clark electrodes and transmembrane potential recording.
- Study of intracellular pH (pHi) homeostasis by continuous pHi recording. Techniques: fluorometry using fluorescent indicators.
- Regulation of membrane potential (m.p.) by continuous m.p. recording. Techniques: tetraphenylphosphonium-selective electrodes.
- Neurosecretion studies by continuous recording of amperometric currents . Techniques: vitreous carbon electrodes.
- Data calibration and processing. Interpretation of the cellular responses and integration with the general physiology of the cell types under study.
- Oral presentation of results, discussion and peer evaluation.
Rosa Maria Moreira Alves dos Santos
Oral presentations: 20.0%
Laboratory work or Field work: 30.0%
J. Graham, D. Rickwood (1997) Subcellular Fractionation: A Pratical Approach, 1st ed., Oxford University Press.
D. Rickwood (1993) Preparative Centrifugation: A Pratical Approach, 1st ed., Oxford University Press
D.G. Nicholls and S. Ferguson (2002) Bioenergetics,3rd ed., Academic Press
J. Hau, and G.L. Van Hoosier (2002) Handbook of Laboratory Animal Science–Essential Principles and Practices. 2nd ed., CRC Press
N. Sperelakis (2012) Cell physiology source book: essentials of membrane biophysics. 4th ed., N. Sperelakis (ed.), Academic Press.
D.J. Aidley (1998) The The physiology of excitable cells. 4th ed., Cambridge University Press
D.L. Nelson and M.M. Cox (2013) Lehninger Principles of Biochemistry, 6th ed., W.H. Freeman