Programme

Biochemical Methods Module (Prof. Alessandra di Masi)

- 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.

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

Slides of each lesson will be made available.

Type of delivery of the course

The Biochemical Methods Module (Prof. Alessandra di Masi) is divided into 3 credits of frontal teaching and 1.5 credits of exercises. Frontal teaching takes place through the use of 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.

Type of evaluation

The exam will consist of a multiple choice test that will take place in the classroom on the day of the exam session. This written exam will need to be supplemented as follows: 1. by means of a written report on the activity carried out in the Bioinformatics module (Prof. Polticelli), according to the methods agreed with the teacher; 2. with a short presentation in the classroom of a maximum of 10 minutes, using the Power Point software. The oral presentation will focus on a scientific article regarding biochemical techniques illustrated during the Biochemistry module (Prof. di Masi) and will be assigned by the teacher. The student will have to illustrate how the researchers used the biochemical technique/techniques to answer a scientific question. It will also be the student's task to discuss the potential and limits of the techniques used, and for this reason, during the presentation, it is possible that other methodological approaches illustrated during the course may also be discussed together with the teacher. The final mark will be given by the average of the marks obtained during the three tests listed above. The final grade will evaluate the following parameters: a) Appropriateness, correctness and congruence of the acquired knowledge and skills; b) Appropriate use of the specific language of the discipline; c) Expressive capacity; d) Logical skills; e) Ability to synthesize; f) Ability to link different topics. Consequently, the judgment may be: a) Sufficient (from 18 to 20/30) b) Fair (21 to 23) c) Good (from 24 to 26) d) Excellent (from 27 to 29) e) Excellent (30)

Programme

- Bibliographic and biological databases
- Sequence alignment and sequence homology search programs
- Molecular modelling
- Molecular docking

Core Documentation

Tramontano, Bioinformatica, Zanichelli

Type of delivery of the course

This module of the course comprises four hours of lectures and ten hours of practical exercises in a computer lab.

Type of evaluation

The evaluation will be based on a report on the practical activities carried out by the students and an oral examination.