Teaching code

4S02696

Credits

12

Coordinator

Paola Dominici

Language

Italian

Also offered in courses:

• Introduction to biochemistry of the course Bachelor's degree in Bioinformatics

Moodle Seminars 0

The teaching is organized as follows:

Learning outcomes

Biochemistry is a branch of life sciences which deals with the study of chemical reactions and their organization in living cells and organisms. It takes into account the studies related to the nature of the chemical constituents of living matter, their transformations in biological systems and the energy changes associated with these transformations. Students will be guided through the course to understand the relationships between structure and function of macromolecules, and the regulatory strategies, with particular focus on metabolic pathways. The experimental activities are aimed at understanding the rationale behind the basic biochemical protocols developed to investigate the macromolecules and their functions. Pratical knowledge of basic biochemical techniques by means of lessons and experiments. Students will gain a thorough competence on the main technique used in biochemistry labs, in particular they will learn isolation, identification and structure- function relationship of macromolecules, with a focus on proteins.

Program

------------------------
MM: BIOCHIMICA
------------------------
The properties that characterize living systems Biological molecules: Hierarchy among biological molecules: simple molecules as units of complex structures Water, pH and ionic balances Amino acids Acid-base properties of amino acids. Proteins: biological functions and primary structure Peptide bond. Architecture of protein molecules The three-dimensional structure of proteins: The secondary structure: description of helices and folded sheets. Ramachandran's graphs Globular proteins: tertiary structure and functional diversity Folding models. Factors determining the secondary and tertiary structure Information and thermodynamics of the survey. Conformational diseases. Quaternary structure of proteins Lipids and membranes: Fatty acids. Triacylglycerols. Membranes: Fluid mosaic model. Membrane transport Nucleotides and nucleic acids. DNA and RNA. Properties of nucleotides. Stability and formation of the phosphodiester bond. DNA denaturation and renaturation. Enzymes: catalytic power, specificity and regulation. Enzymatic kinetics: Free energy of activation and action of catalysts. The Michaelis-Menten equation. Steady state assumption. The kinetic parameters. Linear graphic methods. Enzymatic inhibition: irreversible and reversible. Competitive and non-competitive. Control mechanisms of enzymatic activity. Oxygen transport and storage: the role of hemoglobin and myoglobin. Structure-function relationship of Mb and Hb Hemoglobin: cooperative binding and allosteria. Models for the transition to allosteria in hemoglobin: the symmetrical model and the sequential model. Homotropic and heterotropic effectors. Bioenergetics. Basic thermodynamic concepts. Entropy and free energy. The course of a reaction, the variation of standard free energy. Effect of pH and cancentration on standard free energies. The importance of coupled processes in living systems. ATP and high-energy compounds: group transfer potential. The energy charge. The metabolism. The central metabolic pathways and energy metabolism. Existence of independent degradative and biosynthetic pathways. Oxidations as a source of metabolic energy. Main metabolism control mechanisms. Glycolysis: General aspects. The importance of coupled reactions in glycolysis. reactions of glycolysis. The metabolic fates of NADH and pyruvate The cycle of tricarboxylic acids: The connection phase: the oxidative decarboxylation of pyruvate. The reactions of the cycle. The cycle of TCAs as a source of intermediates for the biosynthetic pathways. Anaplerotic reactions. Electron transport and oxidative phosphorylation: Reduction potentials. The complexes of the electron transport chain. The thermodynamic approach to the chemoosmotic coupling hypothesis. ATP synthase. Shuttle systems for the transport of cytosolic NADH into mitochondria. Gluconeogenesis, glycogen metabolism and hormonal regulation. Hormones and second messengers: cAMP and G. The beta-oxidation of fatty acids with an equal number of saturated C atoms. The pentose phosphate pathway: reactions and control. Nitrogen metabolism: Transamination reactions; Fate of the carbonaceous skeleton of amino acids; fate of the amino group; urea cycle. NUCLEIC ACIDS: how the structure carries information. Structural levels of nucleic acids (DNA, RNA). DNA denaturation. TRANSFER OF INFORMATION DNA replication. Replication enzymology: structure / function of DNA polymerases. DNA replication fidelity.
------------------------
MM: BIOCHIMICA ANALITICA
------------------------
------------------------ MM: teoria ------------------------ Protein purification Ionic properties of amino acids and proteins. Isoelectric point. Sample preparation. Methods of cell disruption and production of the initial crude extracts. Protein solubilization methods. Saline swabs. Fractionation and precipitation techniques. Filtration, dialysis, sample concentration. Spectroscopic techniques. Properties of electromagnetic radiation. Light-matter interaction. States and processes involved in the phenomena of absorption, emission and decay. Absorption spectroscopy in ultraviolet and visible. Qualitative and quantitative aspects of light absorption. Colorimetric and spectroscopic methods applied to the determination of protein concentration. Spectrophotometers. Fluorescence and emission spectroscopy. Intrinsic and extrinsic fluorophores. Green fluorescent protein Spectrofluorimeters. Fluorescence resonance energy transfer (FRET). Circular dichroism. Chromatographic techniques. Principles of chromatography. The chromatogram. Parameters that determine the chromatographic performance. Van Deemter equation. Column chromatography: ion exchange, molecular exclusion, affinity, hydrophobic interaction and their applications. Electrophoretic techniques. General principles and electrophoretic mobility. Support materials. Nucleic acid electrophoresis. Protein electrophoresis. SDS PAGE. Electrophoresis in native conditions. Protein coloring on gel. Protein blotting (western blotting). Isoelectrofocusing. Basics of: Capillary electrophoresis. Two-dimensional electrophoresis gel. ------------------------ MM: laboratorio ------------------------ 1. Determination of the concentration of an unknown protein by absorption at 280 nm and by Bradford colorimetric method. 2. Determination of the kinetic parameters, Michaelis-Menten constant, turnover number, and inhibition constant of the acid phosphatase enzyme using the Lineweaver-Burk graphical linearization method. 3. Determination of the absorption spectrum of the pyridine coenzyme NADH (reduced form) and determination of the molar extinction coefficient of NADPH. 4. Determination of the molecular weight of an unknown protein by molecular exclusion chromatography. 5. Protein separation by electrophoresis under denaturing conditions (SDS-PAGE) followed by visualization of the bands by Coomassie Blue staining 6. Transfer of proteins onto the nitrocellulose membrane by electroblotting followed by immunodetection of the proteins for the identification of one or more proteins by exploiting the specificity of binding with an antibody (WESTERN BLOT). ------------------------ MM: laboratorio ------------------------ 1. Determination of the concentration of an unknown protein by absorption at 280 nm and by Bradford colorimetric method. 2. Determination of the kinetic parameters, Michaelis-Menten constant, turnover number, and inhibition constant of the acid phosphatase enzyme using the Lineweaver-Burk graphical linearization method. 3. Determination of the absorption spectrum of the pyridine coenzyme NADH (reduced form) and determination of the molar extinction coefficient of NADPH. 4. Determination of the molecular weight of an unknown protein by molecular exclusion chromatography. 5. Protein separation by electrophoresis under denaturing conditions (SDS-PAGE) followed by visualization of the bands by Coomassie Blue staining 6. Transfer of proteins onto the nitrocellulose membrane by electroblotting followed by immunodetection of the proteins for the identification of one or more proteins by exploiting the specificity of binding with an antibody (WESTERN BLOT).

Bibliography

Examination Methods

------------------------
MM: BIOCHIMICA
------------------------
The objective of the exam is to verify the level of knowledge and depth of the topics of the course program and the reasoning skills developed by the student. The evaluation is expressed out of thirty (minimum grade 18). The final exam (written for Bioinformatics students) will focus on all the topics of the program. The student will have to answer a series of open-ended questions, demonstrating that they understand and be able to use the fundamental concepts of each topic. The final exam (oral for Biotechnology students) will focus on all the topics of the program. The student will have to answer a series of questions, demonstrating that they understand and be able to use the fundamental concepts of each topic.
------------------------
MM: BIOCHIMICA ANALITICA
------------------------
------------------------ MM: teoria ------------------------ The final (written) exam will focus on all the topics of the program. The student will have to demonstrate that they understand and be able to use the fundamental concepts of each topic. ------------------------ MM: laboratorio ------------------------ The written exam of Analytical Biochemistry will contain questions related to laboratory experiences ------------------------ MM: laboratorio ------------------------ The written exam of Analytical Biochemistry will contain questions related to laboratory experiences