APPLIED BIOCHEMISTRY

To confer knowledge and to acquire skills related to the rationale of the various methodologies and instrumental techniques involved in the biochemistry and biomolecular experimentation. To provide the cognitive tools to monitor developments in molecular genetics and its protocols for the identification, cloning with its vectors, sequencing and gene expression that have revolutionized the understanding of biological processes at the molecular level, contributing to the interdisciplinary character of the course.

Lectures will be held. Theoretical and practical exercises and in-depth seminars are planned.

Principles of biochemical research. Preparation and manipulation of biological samples. Use and preparation of buffer solutions. Cell Disruption; preparation and purification of cellular organelles; centrifugation: centrifuges, rotors, differential centrifugation and isodensity balance with application examples (separation of cellular organelles, purification of nucleic acids).

Biological experimentation models: whole Animals, perfused organs, tissue sections. Buffer solutions. Cell cultures. The laboratory for cell cultures. Sterilization. culture methods: primary cultures, secondary, cell lines. Culture medium. Separation and analysis of cell populations. Cell count. Cryo-preservation. Flow cytometry, FACS and MACS.

Electrophoretic techniques: General principles. Zone electrophoresis on cellulose acetate; serum proteins. Gel electrophoresis: agarose, polyacrylamide gel electrophoresis (PAGE and SDS-PAGE). Isoelectric focusing (IEF). Two-dimensional electrophoresis (applications to the study of the proteome). Methods of detection and quantitative assessments (colorations "gel" and following blotting). Capillary electrophoresis.

Chromatographic techniques for the purification, separation, identification and quantitative analysis of low molecules (metabolites, drugs, hormones, etc.) and high (nucleic acids and proteins) PM: General principles. I note on TLC. Ion exchange chromatography. Exclusion chromatography, affinity chromatography. HPLC and GC (stationary phases, detectors and key applications).

UV-Visible: Bullets on the nature of electromagnetic radiation spectrometry. UV-VIS spectrophotometry (principles, instrumentation and applications). Absorption spectra. Beer-Lambert law and its quantitative applications. Concept of line calibration. Spectrophotometric assay of the protein concentration. Application of spectrophotometry in enzyme assays.

Spectrofluorimetry (general principles and applications) and Chemiluminescence. Flow cytometry and "cell sorting". Elements of luminometry.munochemical techniques. The structure of the antibodies, antigen-antibody reaction. Production of antisera, monoclonal antibodies. Analysis methods: immunoprecipitation reaction in free phase and gel immunodiffusion; radioimmunoassay RIA and IRMA; immunoassays: EMIT, ELISA and PEIA. hormonal assays: methods and applications.

Electrochemical techniques. an oxygen electrode; studies on mitochondrial respiration. Biosensors.

Radioisotope methods. Principles, instrumentation and applications. Calls on the types of radioactive decay. Energy and speed of radioactive decay. Detection and measurement of radioactivity. quenching and counting efficiency. Autoradiography. radioisotopes in biochemistry applications. Application of radioisotopes in clinical analysis.

Techniques of mass spectrometry for the qualitative and quantitative analysis of low molecules (metabolites, drugs, hormones, etc.) and high (nucleic acids and proteins) molecular weight. general principles and instrumentation (sources and analyzers).

Techniques for the study of proteins: methods for sequencing, the determination of secondary and tertiary structure, and of post-translational modifications and mutations. proteomic Background.

Methods for marking biological molecules: Bullets on the types of radioactive decay. Energy and speed of radioactive decay. Detection and measurement of radioactivity by scintillation. Autoradiography. Marking with non-radioactive systems.

Molecular biology techniques. Recombinant DNA technology: general principles. Polymerase Chain Reaction (PCR): principles and diagnostic applications. Identification of specific sequences of DNA and RNA: Southern hybridization (Southern blotting) and applications in the diagnosis of genetic diseases. Northern blotting and applications in the study of gene expression. evaluation of gene expression: the DNA microarray technique.

The course includes practical exercises in the laboratory mandatory on some of the topics covered in the course.

Wilson K., Walzer J.: Biochimica e biologia molecolare: principi e tecniche. Ed. Raffaello Cortina Editore.

Ninfa A.J., Ballou D.P.: Metodologie di base per la biochimica e la biotecnologia. Ed. Zanichelli.

D.L. Nelson, M.M. Cox: I Principi di Biochimica di Lehninger, VII Edizione 2018, Ed. Zanichelli.

Voet D., Voet J.V., Pratt C.W. Fondamenti di Biochimica. Edizione IV. Ed. Zanichelli.