The aims of the course is to provide students with the tools  to understand the methods for the study of proteins (enzymes) and metabolites in animal and vegetable cells. With the lab experiences, the students will be able to assay the activity of an enzyme, to purify a protein and measure the concentration of a metabolite as well as to perform an electrophoresis.

The scientific process: the hypothetic deductive method. The code of ethics for animal and human experimentation.

In vivo studies: diet changes. drug administration, transgenic  and knock-out animals

In Vitro studies: perfused organs, tissue slices, primary cultured cells, immortalized cell lines.

Centrifugation techniques. Separations of cells and subcellular organules by isopycnic and differential centrifugation

Red Blood cells lysis and preparation of the haemolysates. Buffers and antiproteolitic cocktails, reducing and stabilizing compounds.

Omogenization of tissues by means of potter, ultraturrax. French Press, sonication.

Measurement of enzymatic activity: spectrophotometric. Fluorimetric and radiochemical methods. Determination of the kinetic characteristics: Km. Ki. pH curves. Thermal stability. Protein studies and quantification: absorbance at 280 e 260 nm, method of Bradford. Biureto, Lowry.

Protein purification by conventional chromatography: gel filtration, Ionic exchange, affinity, immunoaffinity. Hydrophobic interactions. High performance liquid chromatography (HPLC) by riverse and direct phase.

Protein primary structure determination by means of the Edman degradation.

Electrophoresis on polyacrylamide gel under native and denaturing conditions (SDS-PAGE), densitometric analysis of the protein bands. Determination of protein molecular weight. Preparative electrophoresis, isoelectrofocusing. 2-D Electrophoresis. Capillary Electrophoresis.

Metabolite assays: deproteinization methods. Assay of metabolites by spectrophotometric. fluorimetric and radiochemical methods.

Advanced spectroscophy: Infra red spectroscopy. NMR, EPR, Mass Spectrometry.

Immunochemical techniques: production of monoclonal and plyclonal antibodies, immunotitration curves, immunocytochemical detection of proteins. Direct and indirect ELISA systems. Radiochemical methods (RIA).
 

The student must have passed Biochemistry (second year)

The Dublin Descriptors will be the teaching goals

The teaching material prepared by the lecturer in addition to recommended textbooks (such as for instance slides, lecture notes, exercises, bibliography) and communications from the lecturer specific to the course can be found inside the Moodle platform › blended.uniurb.it

Students will perform the following lab experiments: assay of enzymatic activity and metabolite, purification of a protein, SDS-PAGE with the help of the Dr Giordano Serafini. Dr Paola Ceccaroli e Roberta Saltarelli. The students will receive the protocols of the lab experiments and /or  the electronic file

Teaching

The course will consist of lecturers and lab experiences. Intermediate tests to evaluate the level of  knowledge will be proposed monthly.

Attendance

Attendance of lectures is not mandatory, but students are warmly requested to participate in the lab experiences

Assessment

Oral examination (can be either in English or Italian)

Disability and Specific Learning Disorders (SLD)

Students who have registered their disability certification or SLD certification with the Inclusion and Right to Study Office can request to use conceptual maps (for keywords) during exams.

To this end, it is necessary to send the maps, two weeks before the exam date, to the course instructor, who will verify their compliance with the university guidelines and may request modifications.