66609 - Biochemistry (B)

Learning outcomes

At the end of the course, the student will have to know: - the biological processes at the molecular level - the structure-function relationships of biomolecules with particular regard to protein - energy metabolism. They will have acquired  an integrated view of cell signaling and metabolism - the correct procedures for the work under sterile conditions and the main basic techniques for animal cell cultures; - the basics of structural biochemistry and enzymology; - the main methods for the determination of protein by spectrophotometry; - the use of experimental protocols for the calculation of enzymes 'specific activity . The students have also to know how to critically evaluate the results obtained through the drafting of a report on the experiments carried out.

Course contents

1st module (6 credits) Prof Romana Fato

Structure and properties of amino acids; protein structure: primary, secondary, tertiary and quaternary structure.

Fibrous and globular proteins. Hemoglobin and myoglobin. Structure of heme. Saturation curves and cooperativity.

Enzymes: Classification of enzymes. The enzymatic catalysis. Activation energy. Structure and general properties of enzymes. Cofactors and coenzymes.

Enzyme kinetics: Km, Vmax, kcat. The Michaelis-Menten equation. Effect of pH and temperature on enzyme activity. Inhibition of enzyme activity: reversible inhibition competitive and non-competitive. Regulation of enzyme activity: allosteric enzymes and cooperativity, positive and negative modulators; reversible, irreversible and covalent modifications.
 Lipids. Fatty acids, the triacylglycerols, the glycerophospholipids, sphingolipids, and steroids. The membranes. Movements of molecules across membranes: systems of simple diffusion, passive transport mediated, active transport primary and secondary; selective ion channels.
Carbohydrates.
Bioenergetics and metabolism: catabolism and anabolism. Free Energy Change and coupled reactions. Transfer of phosphate groups and ATP. The thioesters. The biological oxidation-reduction reactions. Flow regulation in the metabolic pathways.
Glucose metabolism. Glycolysis. The fate of pyruvate. The glycolysis regulation. The pentose phosphate pathway. Gluconeogenesis and glycogen metabolism: glycogen degradation  and synthesis of and their control. Gluconeogenesis: reactions and adjustment.

Tricarboxylic acid cycle its regulation and  interrelationships with other metabolisms (anaplerotic role).

Electron transport and oxidative phosphorylation: The electron carriers in the mitochondrial respiratory chain. Oxidative phosphorylation and ATP synthesis. Uncoupling regulation of oxidative phosphorylation and inhibition.

Lipid metabolism. β-oxidation of fatty acids. Training and use of ketone bodies. polyunsaturated fatty acids. Biosynthesis of fatty acids: reactions and adjustment. Elongation and desaturation of fatty acids. Biosynthesis of triacylglycerols and phospholipids. Cholesterol and regulation of metabolic pathway.

Degradation of amino acids and urea cycle. transamination and oxidative deamination  reactions. Protein degradation.

Signal transduction. cAMP-dependent signaling pathway. The inositol phosphate signaling pathway. Pathway that involves a receptor tyrosine kinase phosphorylation cascade.

Module 2 (3 credits)  Prof. Giovanna Farruggia (group A); Prof. Concettina Cappadone (group B)

Theoretical part:

     Organization of an animal cell cultures laboratory: description and use of major equipment used and behavior rules.
     Safety in cell cultures.
     The main types of crops and land used.
     The cryopreservation.
     Contamination.
     The theoretical basis of the centrifugal techniques, microscopic and flow cytometry in cell cultures.

Practice:

     Preparation of a growth curve using a suspension culture and determination of the time of duplication of the crop.
     Cell culture in membership.
     Preparation of slides for the direct growth of cultured cells on glass slides and staining selective for the observation in transmitted light microscopy.
     Freezing and thawing of animal cells.
     Preparation of cell lysates.

Laboratory of Biochemistry: measurement of enzyme activity, construction of the Michaelis-Menten curve, calculation of Km and Vmax. Study of the pH dependence of the enzymatic activity.

Readings/Bibliography

1st Module

Berg, Tymoczko, Stryer "Biochemistry"   Ed. Zanichelli .

Nelson, Cox "Lehninger Principles of Biochemistry"  Ed.Zanichelli.

Garret, Grisham "Biochemistry" 5th edition Ed. Piccinin

 

Biochemistry

Concepts and Connections . Dean R. Appling - Spencer J. Anthony-Cahill - Christopher K. Mathews Pearson ed.

2nd module
The student will be provided protocols with a detailed description of the procedures to be followed during the workshop.
Reference text: Freshney RI, Culture of Animal Cell Manual of Basic Techniques, 4th ed., Wiley-Liss, 2000

Teaching methods

1st module: lectures
2nd module: laboratory practice

Assessment methods

The learning assessment at the end of the course aims to assess the achievement of the following objectives:

1st module:

- Know the structure and function of the main biological macromolecules and the theoretical basis of enzymology.

- Knowing the cellular bioenergetics, the main metabolic pathways and mechanisms underlying their regulation and signal transduction.

- Know the basic molecular mechanisms of living systems, and the molecular logic of their adjustment.

 

2nd module:

-Knowledge of the main basic techniques of cell cultures

-Knowledge of the main methods for the determination of protein

-Knowledge of experimental methods for the measurement of enzyme activity

 

For the 2nd  module the exam will consists of an evaluation of a written report on a laboratory experience, for the 1st module it will be an oral examination. Registration on Alma Exams is required only for the oral test.

 

1st module of Biochemistry: The examination consists of an oral exam, which lasts 20-30 minutes, with three questions aimed at assessing the theoretical knowledge by the student on the structure and function of biological macromolecules, metabolism and its regulation.

 

2nd module of Biochemistry : evidence of evaluation consists of a written report on the experience gained in the laboratory. In the written report will be awarded a score between Poor, Fair, fair, good and excellent. The test is passed if the score obtained is adequate.

 

The final grade will be determined primarily by the grade obtained in the oral examination, obtaining a good / excellent assessment of the lab report will contribute to obtaining an assessment of excellence.

Teaching tools

1st Module: power point slides

2nd module: practical laboratory equipped with spectrophotometers and thermostatic baths. Computer laboratory with single-seat. Cell cultures laboratory.

Office hours

See the website of Romana Fato

See the website of Concettina Cappadone