Learning outcomes of the course unit
In this course, students will be provided with the basic knowledge and understanding needed to perform experimental analyses in biochemistry and to properly interpret the results of such analyses.
In the first part of this course, students will be introduced to the structure and the multiple functions of proteins, emphasizing the fundamental role of these macromolecules in the cellular processes.
Moreover, the students will become familiar with the main classes of small biomolecules (sugars, lipids, amino acids, nucleotides) and will learn the basic metabolic pathways through which these molecules are degraded and synthesized.
In the second part of the course, the students will receive a systematic preparation on the main techniques used in the biochemistry laboratory for the identification, isolation and characterization of biological macromolecules.
Finally, they will be informed about the themes, the strategies and major techniques employed in the field of proteomics.
Basic courses in general and organic chemistry
Course contents summary
Structure and function of proteins.
The amino acids. The peptide bond. The three-dimensional structure of proteins: primary, secondary, tertiary and quaternary structures. Protein folding. Hemoglobin: an allosteric model protein. Enzymes: catalytic strategies and regulation mechanisms of enzyme activity. Proteases: evolution and specificity. A model enzyme: chymotrypsin.
Foundations of bioenergetics: DG0 and DG0' and RealDG. ATP, the prototypic “high energy” molecule. Other “high energy” molecules, including NADH, FADH2.
Glicolysis and fermentations; piruvate dehydrogenase complex; citric acid cycle; pentose-phosphate pathway; gluconeogenesis; glycogen metabolism.
Metabolism of lipids and its regulation: fatty acids degradation (beta-oxidation) and fatty acids biosynthesis. Degradation and utilization of ketone bodies.
Degradation of amino acids and the urea cycle. Oxidative phosphorylation: the mitocondrial chain of electron transporters; proton gradients and the biosynthesis of ATP.
Protein basic properties related with Applied Biochemistry: isoelectric point (pI), protein solubility as a function of pH and ionic strength, "unfolding" of proteins in the presence of denaturing agents (acidic pH, heat and detergents) .
Subcellular fractionation by using centrifugation techniques.
Strategies used for the purification of proteins obtained from natural sources or in recombinant form.
Basic methodologies dedicated to proteins either for analytical or preparative purposes: electrophoretic and chromatographic techniques.
Antibodies: structure-function relationships and immune response. Preparation of monoclonal and polyclonal antibodies. Immunochemical methods. Outline on the diagnostic and therapeutic use of antibodies.
Enzyme technology. Outline of methods used for the determination of enzyme activity. Steady state enzyme kinetics (Michaelis-Menten model): determination of kcat, Km and kcat / Km, and relationships with enzyme properties. Reversible and irreversible enzyme inhibitors, and their biotechnological relevance (drugs, pesticides etc.). Examples of the biotechnological use of enzymes.
Outline of radioisotopes as biological tracers.
Methods of analysis of protein-ligand interactions.
Proteomics - general concepts.
Systematic Proteomics and related basic methodologies. Proteomic studies of post-translational modifications.
Differential and functional proteomics: detailed analysis of some examples of proteomics approaches aimed at addressing biological problems.
Biochemistry. David L. Nelson e Michael M. Cox I Principi di Biochimica di Lehninger Zanichelli, Bologna
Applied Biochemistry.and Proteomics: Bonaccorsi, Contestabile, Di Salvo, Metodologie Biochimiche, Casa Editrice Ambrosiana
The course will be based on formal classroom lessons.
Formal classes will focus on the properties of proteins and enzymes, as well as on the description of the main metabolic pathways, main biochemical methods and main topics of proteomics.
Assessment methods and criteria
The acquired knowledge and the ability to use in practice will be verified through a written test concerning structure and function of proteins and metabolism, and an oral final test concerning topics of applied biochemistry and proteomics. The same weight will be given to the two tests, in order to be able to formulate a final overall vote.
In these tests it will be evaluated the ability to apply knowledge and understanding, as well as the autonomy of judgment and the ability to expose ideas and analysis with clarity and properties.
If a student will not pass a test (either written or oral) for three times, the student will be invited to support a new oral test, after having evaluated together, through an interview, the critical issues that have led to the failure of each part of the exam.