The course is part of the Integrated Course: “Biochemistry-Applied Biochemistry”. The educational objective of the course is to present and critically discuss the most advanced techniques used in biochemical research, a research-based discipline, and their main applications. The course will enable the student to understand the general criteria of biochemical experimentation, biological materials, principles, and equipment used for preparative and analytical purposes in the biochemical laboratory. The course content is constantly updated to render students familiar with the analytical approach to the various problems encountered in modern research laboratories, both basic and applied.
The course aims to provide students with the basics of the main biochemistry and molecular biology techniques utilized in basic and applied research laboratories. The most modern biochemical methodologies are presented and critically discussed with the educational aim of providing knowledge of techniques useful in the separation, identification, and analysis of biomolecules for research and applied applications. The course content is constantly updated. The students' learning outcomes will be the theoretical knowledge and understanding of the main biochemical techniques and the ability to expound on them and identify their applicability in solving problems typical of biochemical laboratories with language property.
The course consists of classroom lectures. The lectures are developed with the support of computer-based material and, as far as possible, organized in a participative manner. Supporting and in-depth materials (ppt presentations with exhaustive images and films on the techniques exhibited, links to sites of interest, bibliography, etc.) will be made available on Aulaweb.
Any Student with documented Specific Learning Disorders (SLD), or with any special needs, shall reach out to the Lecturer(s) and to the dedicated SLD Representative in the Department ( Prof. Luca Raiteri, Luca.Raiteri@unige.it ) before class begins, in order to liaise and arrange the specific learning methods and ensure proper achievement of the learning aims and outcomes. VERY IMPORTANT: any request for compensatory tools and adaptations in the exam MUST be done within 10 working days before the date of the exam according to the instructions that can be found at https://unige.it/disabilita-dsa/comunicazioni
PROGRAMME/CONTENT - Applied Biochemistry- (5 CFU) Programme
Buffer solutions and pH: choice of buffers for biochemical use.
Cell cultures.
Biological samples. Homogenization: choice of methods and media, with special emphasis on ionic and non-ionic detergents: CMC and chemical properties.
Centrifugating techniques: principles of centrifugation; angular velocity, rpm, RCF, sedimentation coefficient. Centrifuges: types and rotors. Differential centrifugation: separation and analysis of subcellular fractions. Density gradient centrifugation: zonal and isopycnic.
Spectroscopic techniques: UV/VIS spectrophotometry. Lambert-Beer law. Protein concentration determination by Bradford method. NADH/NADPH absorption spectra. Enzyme activity assay of the main enzymes of clinical interest: LDH, succinic DH.
Spectrofluorimetry (general principles and applications) and Luminometry. ATP content assay using the Luciferin/Luciferase method.
Mention of cytofluorimetry and 'cell sorting'.
Chromatographic techniques: general principles and applications: analytical and preparative methods. Resolution and theoretical plates. Types of low-pressure column chromatography: ion exchange, exclusion (gel filtration). Affinity chromatography, and immunoaffinity. HPLC.
Electrophoretic techniques: general principles. Apparatus for different media (cellulose acetate, Agarose, polyacrylamide). Electrophoresis of proteins on polyacrylamide gels under native conditions and in the presence of sodium dodecyl sulfate (SDS-PAGE). PM determination of proteins. Western blotting in the study of proteins. Gel and nitrocellulose protein staining methods. Isoelectrofocusing (IEF) and isoelectric point determination of proteins. Two-dimensional electrophoresis (2-DE). Detection by Coomassie Brillant Blue and Blue Silver.
Protein purification techniques. Purification protocols. Specific activity. Fractional protein precipitation: with ammonium sulfate (salting-out); by heat. Edman sequencing. Dialysis and ultrafiltration: principles.
Electrochemical techniques. Oximetry; Oxygen electrode; examples of Oxygen consumption studies.
Radioisotopic methods. Principles, and instrumentation for measuring radioactivity. Mention of types of radioactive decay. Autoradiography. Mention of applications of radioisotopes in biochemistry: study of metabolic pathways. RIA.
Immunochemical techniques. Antibody structure, IgG-like structural motif. Polyclonal (antisera) and monoclonal antibodies. Immunoprecipitation; enzyme immunoassays: ELISA.
'-omics' techniques and Mass Spectrometry. Multi-omics techniques in Systems Biology: Proteomics, lipidomics and metabolomics. Genomics and transcriptomics. Sequencing and analysis of proteins by mass spectrometry; modern spectrometers such as the Quadrupole Mass Spectrometer.
Molecular biology techniques. Recombinant DNA technology: general principles. DNA sequencing. Identification of DNA and RNA sequences: hybridisation (Southern blotting and Northern blotting). mention of the CRISPR_Cas9 Technique.
Recommended texts for the module: "Applied Biochemistry":
Ricevimento: The lecturer receives students by appointment to be arranged by e-mail at the following address: Isabella.Panfoli@unige.it
ISABELLA PANFOLI (President)
MARIA GRAZIA SIGNORELLO
The examination consists of an oral interview and covers the program's topics.
The lecturer's examination of the students will consist of specific questions aimed at testing adequate knowledge and understanding of the biochemical techniques described during the Course, and the ability of the student to discuss their detailed application with effectiveness and using appropriate scientific vocabulary.
The teaching material can be downloaded on the AulaWeb platform of the current academic year, without any access key.
