CONOSCENZE E ABILITÀ DA ACQUISIRE

Lo/la studente/essa dovrà:

-       Major aim of the course is to provide students with notions on basic principles and applications of the most important methodologies of molecular biotechnology with a particular emphasis to proteomics, also to the goal of designing therapeutic compounds and new diagnostic approaches. Students will have knowledge on major molecular targets to develop innovative therapeutic tools in oncology. Finally, starting from the biological problem, successful examples of drug development in oncology will be discussed

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Indicare i risultati di apprendimento attesi come riportati nei seguenti Descrittori di Dublino.

Capacità relative alle discipline:

-       Conoscenza e comprensione

-       Capacità di applicare conoscenza e comprensione

Capacità trasversali /soft skills

-       Autonomia di giudizio

-       Abilità comunicative

-       Capacità di apprendimento

PROGRAMMA/CONTENUTI DELL’INSEGNAMENTO

MODULO: Molecular Biotechnology

In this course students will learn the fundamental biochemical and biotechnological approaches to study proteins. A first part of the course focuses on the study of the proteome and the preparation of human originate samples (body fluids and tissues) for proteomic analysis and the basic methods for differential display protein analysis such as 1DE, 2DE, DIGE, ICAT, SILAC. Particular relevance is also given to the use of mass spectrometry for protein identification and analysis. The second part of the course is mainly focused on the description of chromatographic techniques to purify proteins: ion exchange, size exclusion, reverse phase, hydrophobic interaction, hydroxyapatite, chromatofocusing and multidimensional chromatography. Different approaches for protein/protein interactions are also described such as: calculation of kinetics parameters through SPR analysis, expression of protein in fusion with tag (e.g., FLAG, MBP, GST), co-immunoprecipitation and GST pull down, use of cross linkers to stabilize interactions, yeast two hybrid, TAP-Tag technologies, FRET and PLA.

Learning material will be provided to students during the course.

1.            The Proteome (3h)

Definition of ‘Proteome’ and the concept of ‘dynamicity’ of the Proteome. Description of the level of complexity: from Genome to Transcriptome and to Proteome.

The plasma Proteome: description of the fundamental proteins and concept of ‘dynamic range’ of protein expressed. Methodology to remove abundant proteins (e.g., albumin, immunoglobulin) from plasma Proteome: analysis of pros and cons. Use of plasma Proteome as diagnostic tool: definition of ‘sensibility’ and ‘specificity’.

2.            Methods on Proteomics research (2h)

Electrophoretic techniques (1DE, 2DE) and non-elettrophoretic approaches (proteolitic digestion and MS, chromatography) for the study of the Proteome. Comparative analysis of the two approaches.

Fundamental steps of proteomic techniques: sample preparation, proteins separation, structure and function analysis of proteins.

Source of biological samples: cells, tissues and body fluids and their processing. The ‘Laser Capture Microdissection’ (LCM): description of the method and its application for protein analysis.

3.            2-DE (6h)

Description of the method: use, dynamic range, pros and cons. Steps of the analysis: sample preparation, isoelectrofocusing (IEF), SDS-PAGE, gel staining, image acquisition, differential display analysis, and protein identification through MS.

•             Sample preparation: protein solubilization for 2DE analysis. Methods for cells and tissues lysis, buffers and protease inhibitors, removal of contaminants (DNA, lipids, salts, detergents, …). Protein precipitation: organic solvents and ‘salting out’.

•             IEF (first dimension): use of IPG strips, first dimension buffer composition, sample loading (rehydratation vs cup loaing). Troubleshooting: under-, over-focusing, and sample precipitation.

•             SDS-PAGE (second dimension): gel composition and definition of T and C parameters and the effect on proteins separation. Protocols for separation of low molecular weight proteins.

•             Gel staining: Coomassie, SyPro Ruby, silver nitrate. Comparison between the different staining procedures. Staining for specific protein PTMs.

•             Image acquisition: light and laser scanners, and software for 2D gel analysis.

•             Differential display analysis: definition of intra- and inter experiment analysis, normalization of signal and evaluation of background, removal of false positive. Spot picking for protein identification for MS analysis. Protein database and presentation of proteomic data.

Advanced methods for differential display analysis: DIGE, ICAT and SILAC.

4.            Mass Spectrometry for protein identification (4h)

Components of a mass spectrometer: ion sources (ESI: electrospray ionization, MALDI: matrix assisted laser desorption ionization), analyzer, detector. Sample preparation for ESI and MALDI. Interpretation of MS spectra: the concept of ‘mass-to-charge ratio’ (m/z). Characteristics of analyzer: m/z range, transmission, resolution, and accuracy. Analyzer: TOF (time of fly), quadrupole, FT-ICR (Fourier Transform - Ion Cyclotron Resonance), and Orbitrap.

MS approaches for the identification of peptides by ‘peptide mass fingerprinting’. Strategy for protein fragmentation: top-down vs bottom-up. Peptide fragmentation on gas phase: family of ions and determination of amino acid sequence by MS/MS analysis. Identification of protein PTMs.

5.            Chromatography techniques (14h)

Introduction to chromatography. Definition of ‘resolution’, ‘capacity’, ‘speed’, and ‘recovery’. Steps for protein purification: clarification, enrichment, intermediate purification, and polishing. Basic component for a chromatography: the column, the matrix, the stationary phase, and the mobile phase. Determination of the efficiency of protein purification and evaluation of the necessary amount of starting material (cells, body fluids, tissue, …). Matrix: characteristics of inorganic, synthetic and natural polymers. Definition of theoretical plates, permeability, column efficiency, and particle dimension (dP). Solid phase: derivatization of silica and polystyrene surfaces. The partition coefficient (KD). Definition of ‘selectivity’, ‘retention’, ‘efficiency’, and ‘resolution’. Circumstance leading to loss of resolution: ‘band spreading’ and ‘broadening’.

Presentation of all major chromatographic techniques, their use for protein purification, and discussion of practical applications presented in literature.

•             Ion exchange chromatography (IEC): characteristics of the method, working principle, and use for protein purification. Buffer concentration, effect of ionic strength and temperature on capacity. Most commonly used resins, and elution methods. Example of practical applications.

•             Size exclusion chromatography (SEC): characteristics of the method, working principle, and use for protein purification. The partition coefficient (KAV) and the determination of the efficiency of a column for SEC. Definition of void volume (V0), inner pore volume (Vi), total volume (Vt), and elution volume (Vr). Factors affecting the chromatographic resolution: particle dimension, flux, column length, loading volume, and viscosity. Example of practical applications.

•             Reverse phase chromatography (RPC): characteristics of the method, working principle, and use for protein purification. Parameters affecting the RP-HPLC: matrix, pore diameter, particle dimension, column length, column diameters, elution buffer, pH, flux, and temperature. Example of practical applications.

•             Hydrophobic interaction chromatography (IHC): characteristics of the method, working principle, and use for protein purification. Comparison between RP-HPLC and HIC. Most used adsorbent: butyl, phenyl, pencil and methyl groups. Parameters affecting the IHC: length of chain, pH, temperature, and presence of salts. Variation of ion concentration for binding and elution phases. Example of practical applications.

•             Affinity chromatography (AC): characteristics of the method, working principle, and use for protein purification. The matrix and its derivatization. Type of AC: Protein-ligand, Antibody, DNA, Covalent, Dye-ligand, and Metal-ion (IMAC). Description of the methods and example of practical applications.

•             Hydroxyapatite chromatography (HAP): characteristics of the method, working principle, and use for protein purification. Matrix and outline of elution. Example of practical applications.

•             Chromatofocusing: characteristics of the method, working principle, and use for protein purification. Comparison between IEC and chromatofocusing. Most used solid phases and buffers. Equilibration, elution, and dynamic range of the method. Example of practical applications.

•             Multi Dimensional Chromatography (MDLC): characteristics of the method, working principle, and use for protein purification. Definition of ‘orthogonality’ and ‘pick capacity’. MDLC of-line and on-line. Compatibility of mobile phase. Example of practical applications.

General protocols for recombinant protein expression in fusion with a tag and purification strategies. Comparison of ‘batch culture’ and ‘continuous culture’. Determination of protein purification efficiency.

6.            Functional proteomics (8h)

Description of methods for in vitro and in vivo analysis of protein/protein interaction. The use of chemical cross linkers (bifunctional, trifunctional, zero length): pros and cons.

•             Use of fusion proteins for studying protein/protein interaction in vitro: GST, HA, HisTag, FLAG, biotin.

•             Yeast two hybrid: characteristics of the method, working principle, and pros and cons. Experimental controls to remove false positives. Bacterial two hybrid to study protein/protein and protein/DNA interactions. Example of practical applications.

•             TAP-Tag technology: characteristics of the method, working principle, and example of practical applications.

•             Methods based on fluorescence: FRET, and PLA.

Methods for studying protein trafficking: photobleaching, photoactivation, and photoconversion.

7.            Surface Plasmon Resonance (SPR) (2h)

Description of the method and its use for the determination of kinetics parameters of interaction between two molecules. Definition of ‘analyte’, ‘ligand’, ‘mobile phase’, ‘sensor chip’, ‘direct immobilization’, and ‘capturing kit’. Multi-cycle vs single-cycle kinetics, and ‘bulk effect’. The sensorgram: interpretation and determination of association and dissociation constants.

8.            Use of radionuclide for protein analysis (1h)

The β radioactive decay. Radioactive isotope most used in biology and their characteristics: H3, C14, P32, S35, and I125. Quantities and units of ionizing radiation: Becquerel (Bq) and Curie (Ci). Determination of beta emitter dose. Safety and personal protection devices. Detection methods: direct autoradiography, fluorography, and use of intensifier screens. Description of alternative methodological approaches.

MODULO: Pharmacogenomics and Pharmaceutical Biotechnologies

This course teaches students how human health outcomes can be improved through the blending of the sciences of pharmacogenomics and pharmaceutical biotechnologies. The information is presented in two distinct educational units. The first unit confers upon students a broad perspective of the field of pharmacogenomics and provides them with insight into the growing importance that the field has acquired in clinical therapeutics and future drug design. The second unit focuses on how the principles of pharmacogenomics are applied to biotechnology for the development of drugs as example of building the molecular and pharmacologic bridge to improved health outcomes. Many therapeutic drugs on the current market are manufactured by biotechnology, such as antibodies, nucleic acid products as well as proteins and therapeutic hormones. These biotech biopharmaceuticals are developed through several stages that include: understanding the pharmacogenomic principles underlying health and disease and understanding the fundamental molecular mechanisms governing the function of related therapeutic biomolecules. In this course, each of these topics is explored and discussed how each applies to improving human health and quality of life.

Course Learning Objectives

After completing this course, the student will be able to:

• Explain the basic concepts of pharmacogenomics;

• Describe how genetic differences can affect the pharmacokinetics or pharmacodynamics of certain drugs;

• Explain the impact of selected genetic differences upon drug efficacy in certain diseases such as cancer, cardiovascular disease and diabetes;

• Describe the basic concepts of pharmaceutical biotechnology;

• Explain the advantages and disadvantages of production of peptides, proteins, and glycoproteins, in Gram-negative, Gram-positive, and yeast expression systems;

• Demonstrate a clear understanding of how biochemical pathways relate to biotechnological applications

• Explain how pharmaceutical biotechnology can be applied to improve human health outcomes;

• Describe how pharmacogenomics can be utilized to improve the efficacy of drugs while reducing potential adverse effects;

• Predict how scientists in pharmaceutical biotechnology can utilize pharmacogenomics to improve drug efficacy and improve human health outcomes for selected diseases.

Recommended Textbooks:

1. Pharmaceutical Biotechnology: Concepts and Applications Authors: Dr. Gary Walsh University of Limerick, Ireland. Publisher: John Wiley & Sons (Google eBook); 2013

2. Pharmacogenomics: an Introduction and Clinical Perspective Edited By: Dr. Joseph S. Bertino, Jr.Columbia University, New York, NY USA Publisher: McGraw-Hill Medical; London; 2013

3. Remington: The Science & Practice of Pharmacy, 21st Edition Editors: Drs. David B. Troy and Paul Beringer University of Sciences in Philadelphia, PA Publisher: Lippincott, Williams and Wilkins; 2006

Lecture Topics Outline

Unit 1: Pharmacogenomics (~6 weeks)

 Overview of pharmacogenomics (PG)

 Methodologies used in PG

 PG and pharmacokinetics

 PG and pharmacodynamics

 PG and improving human health

Unit 2: Pharmaceutical biotechnologies (~6 weeks)

 Definition of pharmaceuticals, biologics and biopharmaceuticals

 Overview of protein structure and protein post-translational modification (glycosylation, carboxylation and hydroxylation, sulfation and amidation)

 Recombinant production of therapeutic proteins and protein engineering

 Protein pharmacokinetics and tailoring of pharmacokinetic profile

 Protein mode of action and pharmacodynamics

 Sources of biopharmaceuticals

o Escherichia coli as a source of recombinant, therapeutic proteins;

o Expression of recombinant proteins in animal cell culture systems;

o Additional production systems- yeast, fungal production systems, transgenic animals, transgenic plants, insect cell-based systems

 Some influences that can alter the biological activity of proteins

 Cytokines and other growth factors as biopharmaceuticals

 Biotechnology and pharmacogenomics of therapeutic hormones (insulin and glucagon)

 The impact of genetic engineering and pharmacogenomics on vaccine technology

 Antisense oligonucleotides, their mode of action, and additional antigene agents: RNA interference and ribozymes.

MODULO: Molecular oncology

The course aims to discuss about the most important genetic and cellular alterations in human cancer. Examples of mutations and implication on the protein functions of the most

Relevant oncogenes and tumor suppressor genes for human cancer will be described and discussed

Hallmarks of cancer:

i)                    Cancer a genetic disease that affects the social relationships of the cells. The tyrosin-kinase receptors and the proliferative signalling. PI3K and BRAF as paradigmatic examples of genes frequently mutated in cancers and of interest for target therapies. Structure and functions of these enzymes. Mutations in the PI3K and cancer. BRAF and cancer. RAS, RAF and the activation of the kinase. BRAF mutations in melanoma.

ii)                   Oncogene Induced Senescence (OIS) and p16. OIS and melanoma, CDKN2A PTEN and LKB1 signalling. Overcoming OIS.

iii)                 Anti-proliferative master genes: p53 and pRB. Description of p53 regulation and mutations. Viral oncogenes. pRB structure and functions. Mechanisms controlling pRb phosphorylation. The phospho-code. New oncosuppressive functions of pRb.

iv)                 Density-dependent inhibition of proliferation. NF2/Merlin and RTK sequestration. LKB1 and anti-proliferative signals. LKB1 and cell polarity. The asymmetric mitosis. Mechanisms controlling LKB1 activation. LKB1 mutations and cancer. Kinases under LKB1 regulation the AMP kinase family.

v)                  Differentiation pathways and colon cancer. The model of colon cancer. The stem cell compartment. The morphogenetic pathways. WNT, NOTCH, BMPs e SHH. Description of the different signalling pathways and their dysregulation in cancer.

ATTIVITÀ DI APPRENDIMENTO E METODI DIDATTICI PREVISTI

L’insegnamento prevede:

lezioni frontali, seminari ed esempi pratici

MODALITÀ DI VERIFICA DELL’APPRENDIMENTO

L’esame consiste in

Molecular Biotechnology

Tema scritto con domande aperte su tematiche svolte a lezione

Pharmacogenomics and Pharmaceutical Biotechnologies

Presentazione orale

Molecular oncology

Tema scritto con 3-4 domande aperte su tematiche svolte a lezione

TESTI/BIBLIOGRAFIA

Costituiscono fonti di studio per l’esame:

Molecular Biotechnology

Il materiale didattico verrà fornito durante le lezioni

Pharmacogenomics and Pharmaceutical Biotechnologies

Il materiale didattico verrà fornito durante le lezioni

Molecular oncology

The biology of cancer of A. Weinberg second edition Garland Ed.

STRUMENTI A SUPPORTO DELLA DIDATTICA

I files PDF delle lezioni scaricabili presso il sito materiale didattico dell’ateneo

TESI DI LAUREA

Molecular oncology

Disponibili posti per svolgere la tesi di laurea su tematiche affini all’oncologia molecolare presso il laboratorio del prof. Claudio Brancolini.