Programme

The course is divided into two parts: a theoretical component and an experimental component. The theoretical part consists of classroom lectures and focuses on the main methodologies and techniques used in biochemistry and molecular biology laboratories. The experimental part takes place in teaching laboratories, where students will perform experiments related to the topics covered during the lectures.
- Biochemical methodologies in scientific research. Basic equipment in the biochemistry laboratory (glassware, scales, stirrers, thermostated systems, pH meters, hoods, centrifuges).
- Volumetry and weighing. Preparations of solutions, dilutions, buffer systems.
- Production of recombinant proteins: cloning, mutagenesis, expression systems in bacteria, yeasts, mammalian cells, insect cells. Fusion proteins.
- Methods for protein purification: separation techniques. Extraction of proteins from the biological source. Methods of fractionation of protein mixtures. Homogenization of cells with potter, ultraturrax, sonication, continuous pressure homogenizers (1st Laboratory experience).
- Protein purification with chromatographic techniques: gel filtration, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, hydrophobic interaction chromatography, reverse phase chromatography. HPLC applications (with laboratory experience) (2nd laboratory experience).
- Quantification of proteins. (3rd Laboratory experience)
- Electrophoresis. Polyacrylamide gel electrophoresis in native or denaturing conditions (SDS-PAGE) detection techniques, quantitative densitometric analysis of bands, determination of molecular weight, gradient electrophoresis, preparative electrophoresis, isoelectric focusing, two-dimensional electrophoresis. (4th Laboratory experience)
- Immunochemical techniques: antibodies, western blotting, ELISA and RIA, immunoprecipitation. (5th Laboratory experience)
- Spectroscopic methods for the study of proteins. UV-VIS absorption spectroscopy. Fluorescence spectroscopy. Polarized light spectroscopy. Advanced spectroscopies. (6th Laboratory experience)
- Study of enzymes and proteins. Enzymatic kinetics: calculation of Km, Ki, Ka. Study of the type of inhibition: competitive, non-competitive, uncompetitive and mixed. Use of substrate analogues, effect of pH and temperature (7 ° Laboratory experience).
- Dosage of metabolites. Acid or basic deproteinization. Metabolite assay by spectrophotometric, spectrofluorimetric methods, ATP assay, glucose. Use of HPLC in the dosage of metabolites.
- Biochemical methods for the analysis of molecular interactions. Ligand protein analysis. Protein interaction analysis.
-Crystallographic structures
- Fundamentals of Bioinformatics: Biological Databases, Sequence Analysis, and Applications in Biochemistry.
Laboratory sessions are mandatory

Core Documentation

- Keith Wilson and John Walker: Biochimica e biologia molecolare: principi e tecniche. EDs. 2019 Raffaello Cortina.
- M.Maccarrone. Metodologie Biochimiche e Biomolecolari, Ed. Zanichelli Ed.
- Bonaccorsi, Contestabile, Di Salvo. Metodologie biochimiche, Ed. Zanichelli

Type of delivery of the course

The Biochemical Methods Module (Prof.ssa Alessandra di Masi) is divided into 3 credits of frontal teaching and 1.5 credits of exercises. Frontal teaching takes place using power point presentations and can be provided both in person and on a telematic platform depending on the pandemic situation. The laboratory exercises will all be carried out in presence.

Attendance

Attendance is NOT required for in-class lectures. Attendance is required for at least 75% of the lab sessions.

Type of evaluation

The exam will consist of two parts: 1. Written exam, to be held in the classroom on the scheduled exam date, consisting of 20 multiple-choice questions and open-ended questions. 2. Oral presentation, delivered using PowerPoint, based on a scientific article assigned by the instructor and related to one or more biochemical techniques covered in the Biochemical Methodologies course. During the presentation, students are required to describe the scientific question addressed in the study, explain how researchers used biochemical techniques to answer it, and critically discuss the results obtained. Students should also highlight the strengths and limitations of the methodologies used, and, where appropriate, compare them with other experimental approaches covered in the course. The discussion may include follow-up questions from the instructor aimed at further assessing the student’s understanding of biochemical methodologies and their ability to evaluate their applicability critically. The final grade will be calculated as the arithmetic mean of the marks obtained in the two assessments. The evaluation will consider the following criteria: -appropriateness, accuracy, and completeness of the acquired knowledge and skills; - correct use of scientific language and disciplinary terminology; - clarity of presentation and communication skills; - analytical and critical thinking skills; - ability to synthesize information; - ability to integrate and connect different topics covered in the course. The final grade will be assigned according to the following scale: 18–20/30: Sufficient 21–23/30: Fair 24–26/30: Good 27–29/30: Very good 30/30: Excellent 30/30 with honours: Excellent with honours, awarded to students demonstrating outstanding preparation, excellent critical thinking, and a high level of mastery of the subject.