ECTS - University of Pisa
Il corso fornisce conoscenze avanzate in microbiologia e biologia molecolare necessarie per affrontare lo studio dei microrganismi in ambito bio-medico, industriale e agrario.
The student who successfully completes the course will be able to demonstrate a solid knowledge of basic molecular methodologies used for generation of recombinant strains, monitoring and analyze phylogenetic relationships, and producing new molecules and recombinant vaccines.
Durante l'esame orale, lo studente dovrà essere capace di dimostrare la sua conoscenza del materiale del corso ed essere capace di discutere gli argomenti trattati appropriatamente e con proprietà di espressione.
During the oral exam the student must be able to demonstrate his/her knowledge of the course material and be able to discuss the reading matter thoughtfully and with propriety of expression.
Methods:
Acquisizione delle basi teoriche sull’organizzazione, la replicazione e l’evoluzione dei genomi microbici e delle metodologie applicabili alla generazione di ceppi ricombinanti, rilevamento di agenti infettanti ed analisi dei rapporti filogenetici, produzione di nuove molecole e vaccini ricombinanti. Il bagaglio culturale acquisito sarà sufficiente per attività post-lauream indirizzate verso una professione presso centri di ricerca.
Per garantire un buon apprendimento, è consigliata:
Presenza alle lezioni
Preparazione di un report orale
Partecipazione alle discussioni
Studio individuale
Studio di gruppo
Studio con supporto ICT
Delivery: face to face
Learning activities:
Attendance: Advised
Teaching methods:
Scopo del corso è fornire le nozioni teorico-pratiche di metodologie molecolari microbiologiche. Verranno approfondite: (i) basi molecolari del trasferimento genico e generazione di ricombinanti; (ii) criteri di sistematica molecolare dei microrganismi ed analisi delle loro relazioni filogenetiche; (iii) sistemi molecolari innovativi per la rilevazione di batteri, virus e funghi da campioni biotici e non; (iv) Strategie per la produzione di farmaci antimicrobici e vaccini ricombinanti.
The course is aimed at providing theoretical and practical knowledge of molecular microbiological methodologies. Particular attention will be paid to learning: (i) molecular bases of gene transfer and generation of recombinant strains; (ii) criteria for molecular systematic of microbes and analyses of their phylogenetic relation; (iii) molecular tools for monitoring bacteria, viruses, and fungi in biotic and non-biotic samples; (iv) strategies for production of new antimicrobial molecules and recombinant vaccines.
Genetic information in prokaryotes. Genome packaging. Pathogenicity islands. Bacterial integrons. Comparative genomics. The gene mutation. Types of mutants. Molecular basis of mutations. Chemical and physical mutagens. IS and transposons. Mutagenesis by transposition. Mutagenesis with cassette. Characterization of mutant strains: stability analysis, complementation. Plasmids. Types of plasmids: functions. Plasmid replication and replication source functions. Control of the number of copies, specificity of the host, incompatibility groups. Plasmid partition. History of plasmid vectors. Second generation of plasmid vectors (pUC series). Background-TOPO zero plasmids. Expression plasmids. Reporter plasmids. Genetic transfer in bacteria. Molecular mechanisms of conjugation and transduction. The natural transformation in bacteria. Induction of competence. Details about B. subtilis and S. pneumoniae. Molecular mechanisms of transformation. Details about B. subtilis, S. pneumoniae, N. gonorrhoeae, V. cholerae. Artificial transformation: mechanisms. Calculation of transformation efficiency. Preparation of plasmid DNA: growth and lysis of bacteria, mild purification. Accurate purification of plasmids. Qualitative and quantitative analysis of plasmid DNA. Methods of DNA electrophoresis. Electrophoresis on agarose gel, polyacrylamide gel. Electrophoresis in pulsed field: principles, apparatuses, applied methodologies. Evaluation of protein-protein interactions in vivo and in vitro. Optimizing a PCR reaction. RT-PCR, PCR multiplex, nested PCR, RAPD-PCR, quantitative PCR and realtime PCR. Methods of sequencing and interpretation of results. Methods of classification of bacteria. Determinative microbiology. DNA/DNA hybridization, sequencing of specific genes. The MALDI-TOF MS system for bacterial classification and typing. Phenotypic typing methods: biotyping, serotyping, phage typing. Amplification, MLEE and MLST. Diagnostic procedures: direct and indirect examinations. Methodologies in conventional microbiology. Particular biological samples: CFS, blood, urine and feces for microbiological research. Antibiotic chemotherapy: introduction and classification criteria of antibacterial drugs. Beta lactam antibiotics, glicopeptides, aminoglycosides, tetracyclines, macrolides, chloramphenicol, quinolones, rifamycin, polymyxins, sulfamides. Molecular mechanisms of antibiotic resistance. Role of antibodies in immune response to infecting agents. Primary and secondary response to an antigenic stimulus. Serological diagnosis. Antibody title concept. Neutralization, precipitation and agglutination reactions. Complement fixation reaction, EIA, immunofluorescence, western blotting. Evaluation of the immune response to mycobacterial antigens. Molecular methods in diagnostic microbiology. Probes. Target amplification techniques: PCR, TMA, SDA. Amplification of probe molecules: Qbeta replicasi, LCR. Signal amplification methods: branched DNA. Vaccines: characteristics and types. Conventional vaccines. Recombinant vaccines. New generations of vaccines. Environmental microbiology techniques. Techniques based on cultivation: enrichment, cultivation, identification. Techniques based on cell marking: fluorescent colors, live-dead, cytofluorimetry. Amplification techniques for the evaluation of microbial biodiversity. Metagenomic techniques. Measurements of microbial activity in the environment.
Esame orale finale
