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18430 -
Applied omics sciences
(objectives)
TRAINING OBJECTIVES
1) Homics is a set of biomolecular disciplines that belongs to the life sciences and which is divided into different themes (genomics, transcriptomics, proteomics, metabolomics).
The main objective of the course is to enable the student to face the study of the main analytical techniques and instruments by deepening his knowledge of modern omic analyzes, essentially applied to a modern vision of early diagnosis. The student will be prepared to face the search for new diagnostic markers in clinical and non-clinical analyzes. At the end of the lectures, during the scheduled workshop (24h), students are given the opportunity to work individually on practical and practical topics. In this way, the student will acquire the ability to analyze the different protein expression starting from protein extracts and the possibility of applying modern analytical tools.
b) EXPECTED LEARNING RESULTS:
1) Knowledge and understanding:
Having acquired a good analytical ability in the field of omics and biotechnology
2) Knowledge and applied comprehension skills:
Knowing how to integrate the knowledge acquired in the individual disciplines into an interdisciplinary knowledge necessary to face any complex problem in the biotechnology sector, in particular being able to apply the knowledge of mass spectrometry to the various applications related to biotechnology.
3) Autonomy of judgment
Students will have to develop the ability to process complex and / or fragmentary information and to arrive at original and autonomous ideas and judgments capable of finding and critically selecting sources of bibliographic data, databases, and the
scientific literature. The autonomy of judgment is developed through the critical study of scientific articles.
4) Communication skills:
The student will be able to hold a critical public discussion on the topics covered in particular will be able to apply proteomics and metabolomics techniques for any scientific topic of interest .. Will be able to work in a team in the field of design and execution of experimental protocols as laboratory credits are foreseen.
5) Ability to learn:
The student will have the ability to identify, apply and develop innovative techniques in the relevant field of
work autonomously.
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TIMPERIO Anna Maria
( syllabus)
COURSE CONTENT
- Definition of "omics" sciences
Description of the program and of the modalities of the examination
Safety rules to be adopted in scientific laboratories
- Proteomics definition: expression and functional proteomics, proteomic analysis strategies.
Native electrophoresis (BLUE-NATIVE gel), single and bi-dimensional (1D-GEL, 2D-GEL)
Iso-electro-focusing (IEF)
Reverse phase chromatography RP-HPLC
- Applications of proteomics for the study of tumor cells and for early diagnosis for the presence of biomarkers (examples)
- Laboratory 1
Protein extraction from material to be defined
Determination of protein concentration (Brendford method)
Use of the spectrophotometer
- Laboratory 2
Mono-dimensional electrophoresis (1D-SDS-PAGE)
Cutting bands from the gel and digestion in trypsin (Label Free)
- Laboratory 3
Reverse phase HPLC: construction of a gradient
Determination of slope and study of the chromatogram
- Metabolomics definition: Sample preparation techniques for metabolome analysis.
Chromatographic and mass spectrometric methods used to characterize metabolites that function as biomarkers
Examples of metabolomics studies in clinical research: Application of metabolomics in the study of autism
- Lipidomics defining major lipids and determining determination in mass spectrometry
determination of the red cell membranes
- Foodomic definition. determination of flavonoids, vitamins in foods
- Laboratory 3
Extraction of metabolites according to the method of (Bligh & Dyer)
- Mass Spectrometry: General
Main sources, analyzers and detectors
Analysis of proteins, peptides, metabolites, lipids in intact mass
Tandem mass: meaning and purposes
- Laboratory 4
Determination of the intact mass of a protein
Determination of amino-acid sequence with mass spectrometry
Determination of the main lipid classes
Determination of flavonoids
Statistics: Normalization and equalization, missing values.
o Probability and null hypothesis
o Descriptive statistics
Programs and websites for the identification of proteins, metabolites and lipids.
o Sequence database (MAVEN, SEQUEST, MASCOT, LIPID SEARCH, LIPID GATWAY, N and METABOANALYST)
o Algorithms for the "de novo sequencing" software PEAKS.
Complexity reduction:
• Subcellular fractionation
o Lysis of cells and homogenization of tissues
o Separation of subcellular fractions
o Proteomics, metabolomics and lipidomics of the membrane
o Proteomics, metabolomics, lipidomics of organelles and intracellular compartments
Immunoprecipitation
• Immunoprecipitation to reduce the complexity of a mass spectrometric sample
• Functional Proteomics and Immunoprecipitation
Systems biology
• Protein networks and meta-analysis
o Elements of graph topology
o Construction of a protein network (non-directional graph).
o Statistical validation of a protein network
o Over-representation analysis
o Hierarchical analysis of proteomic data (directional graphs).
or Meta-analysis
• Ontological classification and pathway
o Gene ontology (GO)
o Structure of GO
o Types of annotations in GO
o Browsing in ontologies: Quick GO and other GO browsers
o Ontological classification and pathway: specific applications
( reference books)
Power point slides shown in class by the teacher will be provided in PDF format.
The books or articles in magazines from which some specific topics are taken will be indicated by the teacher during the lesson.
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6
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BIO/11
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24
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24
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Core compulsory activities
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ITA |
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18431 -
Genetic toxicology
(objectives)
To be completed
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MOSESSO Pasquale
( syllabus)
1) Introduction to Genetic Toxicology
Origin and history of Genetic Toxicology. Chemical structure and morphological organisation of genetic material. Definition and classification of mutations: Gene mutations: base pair substitutions, insertions, deletions; reversion and suppression of mutations; phenotypic effects of gene mutations. Fluctuation test and spontaneous mutation in bacteria (neo Darwinian theory). Chromosome mutations: structural (chromosomal aberrations) and numerical (aneuploidy, polyploidy).
2) Spontaneous DNA alterations
Mis-incorporation of bases that can arise during DNA replication (mismatch, tautomerization of bases) and “proof-reading” mechanism for correction of errors. Deamination of bases, spontaneous loss of bases and oxidative damage to DNA. Molecular mechanisms of insertion and deletions of bases.
3) Environmental agents which damage DNA
Physical mutagens: Ionizing electromagnetic radiations (X-rays, γ-rays, betatron-synchrotron radiations; Ionizing subatomic particles (alpha particles, beta particles neutrons). Non-ionizing electromagnetic radiations (UV-A, UV-B, UV-C); UV and ozone. Electromagnetic fields (microwaves, HF, ELF etc.). Direct and indirect action of ionizing radiations on DNA and “S-independent” mechanism of induction of chromosomal aberrations. Chemical mutagens: Direct and indirect chemical mutagens (metabolic activation); alkylating agents; environmental pollutants in the cities [car exhaust, sulphur and nitrogen oxides, polycyclic aromatic hydrocarbons (PAH), formaldehyde, asbestos]; Food additives and contaminant of aliments (aflatoxins, ochratoxins nitrosamines, heterocyclic aromatic amines, phenols of vegetal origin, etc); therapeutic agents (mitomycin-C, bleomycin, nitrogen mustards, inhibitors of DNA topoisomerases I and II, cytostatic agents); pesticides. “S-dependent” mechanism of chemical mutagens. Biological mutagens: microbial and viral pathogens; transposable elements.
4) Systems of defence and cell response to DNA damage
Antioxidant and non-antioxidant systems of defence (superoxide dismutase, catalase, peroxidase, α- tocopherol, vitamin-C, carotenoids, flavonoids, polyphenols etc). Detoxification systems of xenobiotics: (phase I and phase II enzymes); DNA repair systems: fotoliase, alchiltransferases, “nucleotide excision repair” (NER), “base excision repair (BER), “mismatch repair” (MMR), “SOS” repair, recombinational repair: repair of DNA single strand breaks (SSB) and DNA double strand breaks (HR e NHEJR); DNA damage, activation of cellular “checkpoints, restoration of wild type condition, induction of mutation, apoptosis and genomic instability.
5) Phenotipic effects of somatic and germinal mutations
Mutation and cancer (activation of proto-oncogenes and deactivation of tumor suppressor genes); Mutation and cellular aging; Mutations and genetic diseases (Down, Klinefelter, Edwards, Xeroderma pigmentosum, Ataxia telangiectasia, Bloom syndromes).
6) Assays for detection of genotoxicity
In vitro short-term mutagenicity tests: Gene mutation assay in bacteria(Salmonella typhimurium reversion assay in or Ames test); Gene mutation assay in mammalian cells (loci HPRT or TK+/-); Cytogenetic tests in mammalian cells (chromosomal aberrations, sister chromatid exchanges, micronuclei). In vivo short-term mutagenicity assays: Test of chromosome mutation in somatic cells (analyses of cells in metaphase); micronucleus test in bone marrow erythrocytes of rodents; Comet assay; Unscheduled DNA synthesis (UDS).
7) Biomonitoring of human populations
Biomarkers of exposure (mutagenic activity of mutagens or their metabolites in the urines; determination of chemical adducts to proteins or DNA); Biomarkers of effects: cytogenetics changes (chromosome aberrations, micronuclei, aneuploidy) in peripheral blood lymphocytes or cells of buccal mucosa; Biomarkers of susceptibility: differential DNA repair capability among different individuals; polymorfism of metabolic enzymes.
8) Evaluation and regulation of mutagenic risk
Testing strategies and prediction of somatic effects (cancer, genetic diseases) following exposure to genotoxic agents; Food safety and strategies for risk assessment; identification of threshold values of exposure; regulatory aspects.
9) Practise exercise (obligatory):
Evaluation of mutagenic compounds (selected by students) in the following mutagenicity assays:
• Cytogenetic analyses of chromosome aberrations, sister chromatid exchanges (SCE’s) and micronuclei in mammalian cells in vitro;
• Analyses of DNA breakage in human lymphocytes by the alkaline “Comet assay”.
( reference books)
L. Migliore “Genomica e Mutagenesi Ambientale”
Additional books (Available in the university library):
A.P. LI “Genetic Toxicology”
D.H. Phillips and S. Venitt “Environmental Mutagenesis”
R.H. Burdon “Genes and Environment”
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6
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BIO/18
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40
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8
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Core compulsory activities
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ITA |
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18436 -
Chemistry of bioactive substances
(objectives)
OBJECTIVES
The course introduces the concepts and experimental approaches to the chemistry of bioactive substances by consolidating the principles gained under the organic chemistry course, focusing on biogenesis, synthesis, chemical structure and pharmacological properties of bioactive substances . In the first part of the course, the concept of the "pharmacophore theory" will be introduced as a minimal structural unit characterized by a specific biological and clinical activity. Bioactive substances, both of synthetic and natural origin, will be classified according to their main pharmacophores. The student will learn to recognize the pharmacophore even in the context of complex molecular structures. In the second part of the course, critical tools will be provided to associate certain pharmacophorees to specific pharmaceutical and pharmacological applications, with particular attention to the molecular action mechanism by with which the bioactive substances act in the body. The student will be able to understand the natural origin of bioactive organic substances and their possible industrial applications by receiving specific training on the design, development and evaluation of new drugs. In addition, due to the knowledge of molecular action mechanisms, the student may associate the use of bioactive substances with specific nutraceutical, cosmeceutic and cosmetic products, including restrictions on the use of potentially toxic substances and the possibility of their use after functional and structural improvement. This knowledge will enable the student to deal with a professional career within the pharmaceutical, nutraceutical and cosmeceutical industry.
B) EXPECTED LEARNING RESULTS
• Knowledge and understanding: Knowledge of the principles that define the minimum structural unit of an organic, natural or synthetic molecule, to have a certain biological activity (pharmacophore theory). Knowledge of the relationship between the type of pharmacophore present in an organic molecule and the pharmaceutical and pharmacological activity. Knowledge ot the molecular-level of the action mechanism of the major families of bioactive substances, with particular attention to substances with antioxidant, antiviral, anti-inflammatory and antitumour activity. Knowledge of the key steps for the design of a drug, and procedures for its clinical validation and use. Knowledge of the origin and distribution in nature of the main families of biologically active natural organic substances.
• Applied knowledge and understanding: In addition to the knowledge gained through the bioactive substance chemistry study, students will be able to apply theoretical concepts acquired during the course in solving practical exercises based on the teacher's request to present possible schemes for the design of a drug, having the initial indication of the target of action at the molecular level and knowing the type of pathology against which the treatment therapy is to be developed. In this case, students will also have to apply their previous knowledge of chemistry and biology for complete resolution of the problem.
• Making judgements: At the end of the course, the student will have acquired the necessary training for full autonomy of judgment on the possibility of using a certain organic substance of natural or synthetic origin for the therapy of a certain pathology . The student will then be able to link the acquired knowledge of biochemistry, molecular biology, enzymology, physiology and genetic to the design of a substance applicable in the pharmaceutical, nutraceutical and cosmeceutical fields.
• Communication skills: students will be continuously and consistently invited to participate actively in the lesson in order to deepen the topic and to collect proposals for possible solutions in the case of complex phatological scenarios. In this activity, students will be called upon to meet in order to support their ideas. The educational pitch is aimed at increasing the communicative skills and the ability to know how to work and to confront a group, all aimed at consolidating the acquired concepts.
• Learning Skills: Students' learning abilities will be evaluated during the course of the course by tests that will allow to individually monitor the maturation state of the knowledge, highlighting the student's ability to return the aquired cencepts.
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BOTTA Lorenzo
( syllabus)
General Section
Definition of drug.
Pharmacokinetics: absorption, distribution, metabolism and elimination. Physico-chemical properties of the drug that influence each of these phases.
Bioavailability and bioequivalence concepts. Cell membrane properties. Fatty acids: classification, properties and biological role.
Pharmacodynamics. Receptor: definition and characteristics. Classification of receptors. Receptor site and its specificity. Allosteric and accessory sites. Ligand-receptor interaction: role of the chemical bond in the receptor interaction. Ionotropic receptors: structure and characteristics.
Drug targets: Proteins, Enzymes, Receptors and Nucleic Acids: structure and function. Receptors and signal transduction.
Receptor activation mechanisms: ionotropic; voltage-dependent and ligand-dependent; receptor activated by phosphorylation. G protein coupled receptors: structure and activation of the G protein cycle. Role of the α portion of the G protein. Effectors of the α portion and effects mediated by Gs, Gi, Gq. Structure and functional groups of the main endogenous ligands of ionotropic and metabotropics receptors: Gaba, Glycine, Aspartate, Glutamate, Acetylcholine, Adrenaline, Noradrenaline, Serotonin, Dopamine, Histamine. Protein kinase receptor. Transmembrane single strand GTPase receptor.
The pharmacophore and the molecular outline of a drug. Concept of affinity and intrinsic activity. Definition of agonist, partial agonist, inverse agonist, antagonist.
Cellular excitability. Mechanism of propagation of the impulse. Chemical synapses: structure, role of vesicles, mechanisms of synthesis and storage of the mediator, release of the mediator. The postsynaptic receptors. Mechanism of presynaptic reuptake of the mediator.
Characteristics of the receptor site of the main neurotransmitters: Serotonin, Dopamine, Histamine, Acetylcholine, Noradrenaline.
Drug-receptor interactions. Electronic interactions. Bonds involved in the drug-receptor complex: covalent bond, ionic bond, hydrogen bond, charge transfer complexes, Van der Waals forces and other interactions. Steric interactions: steric effects in the drug-receptor complex.
Computational Chemistry: Molecular Modelling (conformational analysis, identification of the 3D pharmacophore), Docking, Virtual Screening, Homology Modelling, Pseudoreceptors, De novo drug design.
Formulation and delivery of drugs: principles and use of specific carriers (liposomes, micelles, antibodies, lignin).
Special Part
Antiviral agents: nucleic acids structure and properties, drugs against DNA and RNA viruses, antisense oligonucleotides, broad spectrum drugs, vaccines
Antitumor agents: drugs that act on nucleic acids (intercalants, topoisomerase poisons, alkylating and metalizing agents, chain terminator), antisense therapy, drugs that act on enzymes (adrenergic antagonists, antimetabolites), antibodies and antibody-drug conjugates.
( reference books)
Chimica farmaceutica di Patrick L. Graham
Chimica farmaceutica di Alberto Gasco, Fulvio Gualtieri, Carlo Melchiorre
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6
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CHIM/06
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48
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Core compulsory activities
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ITA |
