BF/0035/EN - ADVANCED BIOLOGICAL METHODOLOGIES
Anno Accademico 2022/2023
FLAMINIA CESARE MARINCOLA (Tit.)
- Modalità d'Erogazione
- Lingua Insegnamento
|[60/71] BIOLOGIA CELLULARE E MOLECOLARE||[71/10 - Ord. 2021] Advanced cellular studies||9||80|
The course is taught in English
At the end of the course, the student will have:
1) a general overview of the main techniques applied in structural biology and will develop clear awareness about concepts and critical steps in data collection, processing, and validation.
2) an advanced knowledge about the applications of spectroscopic techniques, the analysis and interpretation of spectroscopic data.
3) an indispensable knowledge to understand and apply the basic notions of mass spectrometry and proteomic techniques in biochemistry and biomedical research.
KNOWLEDGE AND UNDERSTANDING
The course introduces students to:
1) a general overview of the main techniques used for studying macromolecular complexes. Particular emphasis on proteins and nucleic acids will be given.
2) the main concepts of the main spectroscopic techniques used in the biological field
3) the main concepts of mass spectrometry and techniques applicable to different proteomic approaches to study proteins in several biological samples.
At the end of the course, the awareness will be developed in such a way that:
1) the main critical aspects related to a given macromolecular system will be identified and taken into account while developing an experimental strategy.
2) students will be able to understand the use of spectroscopic techniques for the study of the structure and reactivity of biological systems
3) students will be able to understand the basic notions of mass spectrometry and proteomic techniques and their application in the biochemistry and biomedical research.
The course will provide:
1) basic notions essential to design an experimental strategy for macromolecular complexes studies. In particular, the learned knowledge will make it possible to understand the most suitable techniques to be chosen. In this respect, the strategy chosen will be a conscious consequence of the critical aspects and properties of the experimental system studied.
2) the tools to use the acquired knowledge in the spectroscopic field for acquiring information on the structure and reactivity of molecules of biological interest.
3) the tools to use the acquired knowledge in the proteomic field for qualitative and quantitative characterization of proteins and peptides of biological interest.
AUTONOMY OF JUDGMENTS
At the end of the course, the student will be able:
1) to develop the ability to choose an experimental strategy in complete autonomy and will be able to perform a preliminary data validation in structural biology.
2) to formulate their own judgment on spectroscopic data, mass spectrometry-based proteomics and discuss them logically based on the interpretation of the available information.
The course will provide students with the ability to explain the main concept laying at the base of the main techniques exploited to study macromolecular complexes in biology, spectroscopic, and mass spectrometry data. This will imply the use of appropriate technical language both written and spoken.
ABILITY TO LEARN:
Acquisition of adequate scientific knowledge on the main techniques used in structural biology with a particular focus on spectroscopic techniques, crystallography/cryo-EM and mass spectrometry-based proteomics. Concepts will be acquired through lectures and specific scientific books as well as recent publications in the field. Indications on how to find updated sources will be provided showing how to proceed in building a bibliography and in consulting databases.
Fundamental knowledge of organic chemistry, physics, and biochemistry.
Mass spectrometry-based proteomics module (16 h of classroom lectures and 12 h of Laboratory experiences):
* Proteomics: aims, structural, expression and functional proteomics, application fields.
* Mass spectrometry (MS) applied on protein study. Basic concepts of MS. Ionization sources (MALDI, ESI, and AI). Low and high resolution mass analyzers.
* Gel-free and gel-based proteomic approaches. Principles of chromatography: reverse phase chromatography (RP-HPLC). Electrophoretic separations: SDS-PAGE, Two-dimensional electrophoresis (2D PAGE). Spot picking and processing.
* Bio-informatics tools for analysis of mass spectrometry data.
* MS Methods for protein identification. Peptide Mass Fingerprinting. Tandem mass spectrometry, principles and application for protein sequencing, de novo sequencing.
* Bottom-up, shut-gun, middle-down and top-down proteomics. Imaging mass-spectrometry.
* Quantitative strategies, label-free and label-based, relative and absolute, SILAC, AQUA-peptide, XIC, SIM, SRM, MRM and PRM.
* Laboratory: 2-DE electrophoresis of protein mixture. Spot picking and protein digestion. Mass spectrometry analysis. Interpretation of data by Proteome Discoverer software. Manual inspection and interpretation of mass spectrum.
Structural Biology module (16 h of classroom lectures and 12 h of Laboratory experiences):
X-rays and Neutrons sources, Three-dimensional crystallization of proteins and crystals diffraction analysis by X-rays and Neutrons, advantages and limits of Neutron vs X-ray diffraction; Structure and dynamics of proteins studied by X-ray Free Electron Laser, laser sources, pump probe systems. Small Angle X-ray Scattering and Small Angle Neutron Scattering and coupled with Size Exclusion Chromatography. Extended X-ray Absorption Fine Structure and X-ray Absorption Near Edge Structure. Electrons sources, Transmission and Scanning Electron microscopes, Two-dimensional crystallization of proteins and crystals diffraction analysis by Electrons (Electron crystallography). Cryogenic electron microscopy, Single Particles Analysis, cryo-electron tomography and subtomogram averaging; Protein structure determination by Nuclear Magnetic Resonance Spectroscopy; Analysis of extended structures (e.g. cells, organelles, membranes) by Atomic force microscopy. Practicals: Quality check of proteins samples by Size Exclusion Chromatography and Native electrophoresis, oligomeric profiles, mono-dispersion of the components; Two-dimensional and Three-dimensional crystallization of proteins.
Spectroscopy Module: (24 h of classroom lectures)
* Electromagnetic spectrum. Electromagnetic radiation (emr): the classical and quantum-mechanics model. Absorption and emission of emr: basic principles
* UV-visible spectroscopy. Priciples. Chromophore. Lambert-Beer Law. UV spectra of proteins and nucleic acids. Applications of UV-visible spectroscopy for the study of biological systems.
* Linearly polarized light and circularly polarized light. Optical activity and circular dichroism (CD). CD spectra of protein and DNA. Conformational studies by CD spectroscopy.
* Fluorescence spectroscopy. Singlet and triplet states. Radiationless and radiation transitions. Internal conversion. Intersystem crossing. Quencing. Quantum yield. FRET.
* IR spectroscopy. Molecular Vibrational modes. The harmonic oscillator. The anharmonic oscillator. Selection rules. The IR spectrum. IR spectroscopy applications to the study of biological systems. Reading and analysis of a scientific paper.
* Raman spectroscopy. Basic principles. Selection rules. Resonance Raman spectroscopy
* Nuclear Magnetic Resonance spectroscopy (NMR). Basic principles. Larmor frequency. Chemical shift. Spin-spin splitting. NMR spectroscopy applications to the study of biological systems. Reading and analysis of a scientific paper.
Lectures will be prevalently held in classrooms, also integrated with online teaching resources, by using specific online platforms managed by the University of Cagliari.
The teaching method includes classroom lectures with oral presentations (56 h), during which the students are guided to the understanding of the basic and applicative concepts of Spectroscopic Methodologies, Structural Biology and Mass spectrometry-based proteomics and laboratory experience (24 h).
Each lesson will be structured as follows:
- introduction: this includes a clear presentation of the objectives, the key ideas, and their relation to the objectives of the entire course. The fundamental aims of the introduction are to consolidate attention, reinforce motivation, and provide an overview of what will be subsequently developed.
- development: this presents in detail the contents and highlights the links among ideas or key points.
- conclusion or summary: this aims at reinforcing the learning of the lesson content.
The evaluation of the student's learning is done through an oral evaluation* that is conducted to verify:
(a) the acquisition of the basic concepts in structural biology, mass spectrometry and proteomics;
(b) the understanding of how X-rays, electrons, spectroscopic and proteomic techniques can be exploited to study biological macromolecules;
(c) the ability of the student to expose complex concepts in a clear way, adequately using the technical-scientific language;
(d) the ability to design an experimental strategy to acquire specific information from biological samples;
(e) the ability to use the acquired knowledge to proactively solve new problems.
* For needs due to an epidemiological emergency, the exam could be carried out remotely via Teams.
In the evaluation of the exam and in the assignment of the final mark, the following aspects will be taken into account:
(1) level of knowledge of the contents;
(2) ability to apply the theoretical concepts:
(3) personal qualities (critical spirit, ability to self-evaluate);
(4) exhibition mode (expressive capacity: appropriate use of the specific language of the discipline; logical skills and consequentiality in the connection of content; ability to link different topics finding common points and to establish a consistent overall design; the ability of synthesis also through the use of the symbolism proper of the material and the graphic expression of notions and concepts, in the form, for example, of formulas, schemes, equations)
Consequently, the judgment will be express with a mark (in thirtieths) that can be:
a) Sufficient (from 18 to 20): The candidate demonstrates little knowledge acquired, superficial level, many gaps; expressive abilities modest, but still sufficient to support a coherent dialogue, logical and consequential in the fitting of the subjects of the elementary level; poor capacity for synthesis and ability to graphic expression rather stunted, lack of interaction with the teacher durations interview.
b) Moderate (from 21 to 23): The applicant demonstrates the discreet acquisition of knowledge but lack of depth, a few gaps; expressive abilities more than sufficient to support a coherent dialogue; acceptable mastery of the scientific language, logical and consequential in the fitting of the arguments of moderate complexity, more than enough capacity for synthesis and the ability to graphic expression acceptable.
c) Good (from 24 to 26): The candidate demonstrates a wealth of knowledge rather large, moderate depth, with small gaps; satisfactory mastery of the expressive capabilities and significant scientific language; abilities dialogical and critical well detectable, good capacity for synthesis and ability to graphic expression more than acceptable.
d) Outstanding (from 27 to 29): The candidate demonstrates a wealth of notions very extensive, well depth, with marginal gaps; remarkable powers of expression and high mastery of scientific language; remarkable capacity dialogue, good competence and relevant aptitude for logic synthesis, high capacity for synthesis and graphic expression.
e) Excellent (30): The candidate demonstrates a wealth of very extensive and in-depth knowledge, gaps irrelevant, high capacity and high mastery of scientific language; excellent ability dialogical and aptitude to make connections between different subjects, excellent ability to synthesize, and very familiar with the expression graphics.
The praise “30/30 cum laude” is attributed to the candidates clearly above the average, and whose expressive, conceptual, logical, and notional limits are completely irrelevant as a whole.
The final grade is obtained by computing the weighted average of the grade obtained in the three modules.
Materials for studying the subject (reviews and publications) will be provided at the lesson.
** For the "Mass spectrometry-based proteomics" module (free choice among the following texts):
Peter Wyatt Proteomics: Principles, Techniques and Analysis. Syrawood Publishing House
Edmond De Hoffmann, Vincent Stroobant Mass Spectrometry: Principles and Applications-Wiley
Throck Watson O. David Sparkman. Introduction to Mass Spectrometry: Instrumentation, Applications and Strategies for Data Interpretation, Wiley-Backwell
Josip Lovric’: Introduciong Proteomics. Wiley-Backwell
** For the "Spectroscopy" module:
Gordon G. Hammes “Spectroscopy for the Biological Sciences”, Wiley
Robert M. Silverstein, Francis X. Webster, David J. Kiemle Spectrometric Identification of Organic Compounds, Wiley
• The slides of the lectures will be available for students
• Teachers are available every day by appointment (via e-mail) for further information or clarification on the topics discussed in the lectures.