|
Lecturer(s)
|
-
Doležal Petr, RNDr. Ph.D.
-
Kodrík Dalibor, prof. RNDr. CSc.
-
Košťál Vladimír, prof. Ing. CSc.
-
Okrouhlík Jan, Mgr. Ph.D.
|
|
Course content
|
Each lecture will provide the necessary theoretical background followed by practical laboratory exercises. Content of practicals 1. Isolation and purification of neurohormones (Dalibor Kodrík) Isolation of adipokinetic hormones (AKH) from brains of the firebug, Pyrrhocoris apterus - brain dissection, methanol extraction of peptides, HPLC (Waters) separation of peptides using C18 reversed phase column, identification and semi-quantification of AKH with synthetic standards. 2. Use of ELISA method for identification of peptide hormones (Dalibor Kodrík) Identification and quantification of AKH in samples of CNS of the firebug, Pyrrhocoris apterus - brain dissection, methanol extraction of peptides, quantification with competitive ELISA using Pyrap-AKH antibody and AKH synthetic standards. 3. Phase transitions of body water in insects - supercooling, freezing, vitrification (Vladimír Košťál). Haemolymph sampling, osmolality measurements (Vapro 58520, Wescor). Assessment of supercooling and freezing points (Ministat 240cc, Huber). Free and bound water determination, measurement of the glass transition temperature (DSC4000, Perkin Elmer). Changes in properties caused by nucleation bacteria and ice crystals. 4. Membrane phase status and fluidity measurements (Vladimír Košťál). Extraction of phospholipids from insect tissues (Folch extraction procedure and SPE separation). Determination of molar concentration of phospholipids (phosphorus-molybdate method). "Artificial cell" preparation (phospholipid vesicles, SUV). Measurements of SUV fluidity with polarization fluorometer (Infinite F200Pro, Tecan). Changes in membrane fluidity related to the composition of membranes. 5. Metabolic rate measurements in vertebrates and invertebrates (Jan Okrouhlík, Petr Doležal) Measurement of oxygen consumption in larvae of aquatic insects using chemical and fluorescence based oxygen sensitive probes. Measurement of CO2 production with infrared spectrophotometry in open and closed circuits (Qubit Systems, Sable Systems, LI-COR Biosciences). Application of doubly labeled water (DLW) to measurement of metabolic rate in vertebrates. 6. Thermoregulation and heat production (Jan Okrouhlík, Petr Doležal) The mechanisms of thermoregulation in insects and mammals (cooling, warm-up behavior, muscle activity, hormonal stimulation etc.) - infrared camera experiments.
|
|
Learning activities and teaching methods
|
Monologic (reading, lecture, briefing), Demonstration, Projection, Laboratory
- Class attendance
- 30 hours per semester
- Preparation for exam
- 10 hours per semester
|
|
Learning outcomes
|
To review modern methods of animal physiology, mainly in relation to their practical importance, which is achieved by selection of model organisms. Information on ecology and management (if applicable) of model rganisms will be provided together with the methodical approach.
The course will focus on mastering a wide range of laboratory methods used in animal physiology such as HPLC, ELISA, osmolarity measurement, differential scanning calorimetry, supercooling point measurement, respirometry and metabolism measurement, thermoregulation etc.
|
|
Prerequisites
|
Special course for Ph.D. study field Physiology and Developmental Biology. Brief knowledge of courses provided during the Master's program of Physiology and Developmental Biology is expected.
|
|
Assessment methods and criteria
|
Development of laboratory protocols, Interim evaluation
At least 80% of practical laboratory exercises are required to pass the exam. The exam will be conducted orally and all methodologies demonstrated will be discussed.
|
|
Recommended literature
|
-
Literatura u vedoucích jednotlivých úloh.
|