|a||1||II semestre||Massimo Delledonne|
|b||1||II semestre||Paola Dominici|
|c||1||II semestre||Alejandro Giorgetti|
|d||1||II semestre||Daniela Cecconi|
|e||2||II semestre||Alessandra Maria Bossi|
The course is divided into 4 interdisciplinary laboratory modules focused on a topic of biological relevance. The main purpose of the course is to offer the student tools to focus on the problem, by using different highly complementary techniques. The GENETICS module aims to provide expertise on experimental approaches and bioinformatics analysis necessary to identify genetic variants associated with specific pathological conditions and their validation. The module of PROTEIN ENGINEERING aims to provide to the students with specific information on the principles and techniques used in protein engineering, with particular reference to the production of recombinant proteins in heterologous systems (construction and expression of foreign gene in prokaryotic and eukaryotic host cell). The BIOINFORMATICS module aims to introduce the computational methods used today to predict the effect of variants associated with diseases on the structure/function of proteins. At the end of the course, the student must demonstrate that he is able to use state-of-the-art computational methods to predict the effect of mutants from the sequence and structure of proteins. The module of EXPRESSIONAL PROTEOMICS aims to acquire laboratory skills for the preparation of an experiment in differential proteomics. The experiment can be aimed at the comparison of a pathological sample with a control sample for the identification of potential biomarkers of clinical use, or aimed at the comparison of a cellular sample treated or not with a drug for the recognition of the mechanism of molecular action of the drug itself.
Definition of recombinant protein. Introduction to protein engineering. Acquisition of the required information (theoretical and experimental) to carry out the process of engineering of a protein function/structure. Production of recombinant proteins. Experimental approaches to study and modulate the protein functionality. Protein characterization (Site directed mutagenesis, Gel electrophoresis, Tryptophan (Trp) fluorescence, ANS Fluorescence, Limited proteolysis). Examples of application of protein engineering.
The module will be entirely developed in a computer laboratory. The module is based on the seminal article: Predicting the Effects of Amino Acid Substitutions on Protein Function by Pauline C. Ng and Steven Henikoff and published in: Annual Review of Genomics and Human Genetics. The techniques reviewed in the article will be briefly introduced to the students. Then the students will put their hands on the problem by using those methods to assess the effects on mutants on human Calmodulin. The methods include: Sequence based methods: - Sift - PolyPhen - Panther - PSEC Structure based methods - Analyse the wild-type structure usgin the Chimera program - Introduce the mutants - Analyse the lost/gain interactions upon mutation - Study of the electrostatic potential on the surface of the protein (wild-type and mutated) Annotation based methods: - iHop - Pfam
The expressional proteomics module includes key issues for a proteomics laboratory, for example, methods for protein quantification before a proteomic analysis, separation of proteins by two-dimensional electrophoresis, the detection of the proteomic profile by different staining (colorimetric and/or fluorescent), image acquisition of proteomic profiles, and an introduction to identification of deregulated proteins by mass spectrometry.
The functional proteomics module focuses on the use of biomimetic approaches for selective recovery of protein classes, for the proteomic analysis. The experimental design is: In silico design of the best epitope target in a defined protein. Preparation of the biomimetic material. Functional characterization of the biomimetic material. Application of the biomimetic material for the selective enrichment of biological samples and analysis 2DE of the enriched fraction. In silico modelling of the protein corona.
The verification of the acquisition of concepts and protocols inherent to the thematics of the research inspired laboratory , will be through a global exam, subdivided into 10 open questions based on the 5 modules (2 questions for bioinformatics; 2 for biochemistry; 2 for expressional proteomics, 2 for genetics and 3 for functional proteomics) to be replied in 3 hours.
All the questions aims at verifying acquisition of the knowledge of the practicals and of the inherent theories discussed over the course.