Research inspired laboratory (2017/2018)



Course code
4S003669
Credits
6
Coordinator
Alejandro Giorgetti
Academic sector
BIO/10 - BIOCHEMISTRY
Language of instruction
English
Teaching is organised as follows:
Activity Credits Period Academic staff Timetable
a 1 II sem. Alessandra Astegno

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b 1 II sem. Alejandro Giorgetti

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c 2 II sem. Daniela Cecconi

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d 2 II sem. Alessandra Maria Bossi

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Learning outcomes

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MM: a
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The credit is designed to provide basic knowledge related to protein engineering, with particular reference to the production and biochemical characterization of recombinant proteins. Objectives • To understand construction and expression of foreign gene in prokaryotic and eukaryotic host cells. • To understand recombinant protein production. • To acquire fundamental elements at molecular level concerning protein structure-function relationships • To acquire the required information (theoretical and experimental) to carry out the process of engineering of a protein function/structure.
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MM: b
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The bioinformatics module will provide an overview of amino acid substitution (AAS) prediction methods using in silico techniques. At the end of the course, the student should able to use state of the art techniques to assess the effect of disease-associated mutants into the structure/function of proteins.
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MM: c
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The DIFFERENTIAL EXPRESSION PROTEOMICS course aims to enable students to acquire laboratory skills for the preparation of an experiment of differential proteomics. The experiment can involve the comparison of a pathological sample with a control for the identification of potential biomarkers having clinical utility; or the comparison of a drug-treated sample with a control for understanding the molecular mechanism of action of the drug molecule.
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MM: d
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The FUNCTIONAL PROTEOMICS module aims to enable students to acquire laboratory skills related to a targeted proteomics approach. Topics of the module include the design and preparation of biomimetic materials and their applicaiton to the selective capture of the target protein from biological samples.

Syllabus

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MM: a
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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.
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MM: b
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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 intorduced 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 are: 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
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MM: c
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The practical experiences include 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.
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MM: d
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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.

Assessment methods and criteria

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 4 modules (2 questions for bioinformatics; 2 for biochemistry; 3 for proteomics and 3 for functional proteomics) to be replied in 2.5 hours.
All the questions aims at verifying acquisition of the knowledge of the practicals and of the inherent theories discussed over the course.

Reference books
Activity Author Title Publisher Year ISBN Note
a Keith Wilson, John Walker-Edizione italiana a cura di M.S. Pilone e L. Pollegioni Biochimica e biologia molecolare - Principi e tecniche - Le bioconoscenze e le biotecnologie in laboratorio Raffaello Cortina Editore 2006
a Wilson, Walker Principles and Techniques of Biochemistry and Molecular Biology Cambridge University Press 2010
b Stefano Pascarella e Alessandro Paiardini Bioinformatica Zanichelli 2011 9788808062192

STUDENT MODULE EVALUATION - 2017/2018