ADVANCED BIOCHEMISTRY AND BIOCHEMICAL METHODS
- Overview
- Assessment methods
- Learning objectives
- Contents
- Full programme
- Bibliography
- Delivery method
- Teaching methods
- Contacts/Info
The background in biochemistry and analytical chemistry acquired during a Bachelor in the class of chemical science and technology is sufficient for a fruitful learning.
The exam will have the format of a journal club. Each student will make a choice of a transdisciplinary article provided by the teacher that will focus on the topics of the course. The student will present the contents of the chosen article and a brief analysis of the state of the art as well as a critical analysis of the results and of the conclusions drawn by the authors by carefully examining the body of evidences provided in the article and in the supplementary materials.
Knowledge and ability to understand the relationships among molecular/crystal structure of biological macromolecules, their complexes and complexes between macromolecules and small molecules of physiological and pharmaceutical interest.
Knowledge and ability to apply preparative and analytical separation techniques used to investigate biological systems.
Knowledge and ability to apply most common techniques in molecular biology to engineer cell systems.
PART I – Structural biochemistry (2 ECTS): Structure and function. Structure analysis. Analysis of structural families of pharmaceutical interest.
PART II – Basic biochemical methods (2 ECTS): Physical methods. Immunochemical methods. Optical methods.
PART III – Separation and analysis of protein mixtures (3 ECTS): Protein chromatography. Protein electrophoresis. Mass spectrometry of peptides and proteins.
PART IV – Nucleic acids (1 ECTS): Molecular biology methods. Genomics, transcriptomics and next-generation sequencing.
PART I – Structural biochemistry (2 ECTS)
Structure and function: Principles of protein x-ray crystallography. Principles of protein NMR spectroscopy. Principles of cryoelectron microscopy. Structural organization of proteins. Cooperativity and allostery. Denaturation and folding.
Structure analysis: Principal data banks for protein sequence and structure. Hierarchical classification of proteins. Some examples of the main protein folds. Visualization and analysis of three-dimensional models. Computational modelling of protein structures using Homology Modeling and Fold Recognition. Simulation of ligand-receptor interaction through Monte-Carlo Simulated Annealing techniques. Practical session in computer room.
Analysis of structural families of pharmaceutical interest: Immune system proteins. Ligand-gated channels. G-protein coupled receptors. Tyrosine kinase receptors.
PART II – Basic biochemical methods (2 ECTS)
Physical methods: Sedimentation and centrifuges. Subcellular fractionation. Analytical centrifuge.
Immunochemical methods: Antibodies. Immunodiffusion. Immunoprecipitation. RIA. ELISA. Co-immunoprecipitation. Atomic force microscopy.
Optical methods: Colorimetric methods for quantitative and kinetics analysis. Cell viability. Cytofluorimetry. Ligand-receptor affinity. Circular dichroism of proteins. Fluorescence Resonance Energy Transfer (FRET). Raman and Resonance Raman. Surface Plasmon Resonance. Optical microscopy. Immunofluorescence. Immunocytochemistry.
PART III – Separation and analysis of protein mixtures (3 ECTS)
Protein chromatography: Precipitation, salting out and desalting. Gel filtration chromatography. Ion exchange chromatography. Hydrophobic (HIC) and hydrophylic (HiliC) chromatographies. Affinity chromatography. Reverse phase chromatography of peptide mixtures. Determination of peptide sequence (Edman).
Protein electrophoresis: Principles of gel resolution. Supports (cellulose acetate, agarose gel, polyacrylamide gel). Native PAGE and SDS-PAGE. Western blotting. Isoelectrofocusing. Two-dimensional electrophoresis. Capillary electrophoresis.
Mass spectrometry of peptides and proteins: Ion sources compatible with biomolecules (MALDI, ESI). Principal types of analyzers (TOF, quadrupole, ion trap, orbitrap). Fragmentation techniques (CID, ECD). Data-dependent acquisition vs. data-independent acquisition. Shotgun proteomics and peptide identification. Quantitative proteomics. Isotopic labeling. Multiple reaction monitoring.
PART IV – Nucleic acids (1 ECTS)
Molecular biology methods: Genetic engineering. Cloning. Transfection. DNA and RNA extraction. Southern blot and Northern blot. DNA polymerase chain reaction. Quantitative RT-PCR.
Genomics, transcriptomics and next-generation sequencing: Sanger sequencing. DNA microarrays. Next-generation sequencing. Single molecule sequencing. Epigenetics and epigenomics.
M Williamson, Come funzionano le proteine, Zanichelli, 2013
M Duranti, Introduzione allo studio delle proteine, Zanichelli, 2016
MC Bonaccorsi di Patti et al., Metodologie Biochimiche, Casa Editrice Ambrosiana, 2012
Classroom lectures are integrated with practical sessions, visits to state-of-the-art laboratories, journal clubs and group work. The calendar is defined together with the students to guarantee that all students will have the possibility to take part to work sessions.
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