ECTS - University of Pisa
Lo studente avrà acquisito conoscenze sulla realtà del laboratorio dal punto di vista organizzativo e soprattutto in merito all' operatività.
Verranno approfonditi i principi che stanno alla base dei test di laboratorio impiegati in biochimica clinica.
Biologia molecolare: lo studente avrà acquisito le conoscenze di base sulla struttura, funzione e manipolazione degli acidi nucleici
The student will have acquired knowledge of the reality of the laboratory from an organizational point of view and above all with regards to operations.
The principles underlying laboratory tests used in clinical biochemistry will be studied in depth.
Molecular biology: the student will have acquired basic knowledge on the structure, function and manipulation of nucleic acids
La verifica delle conoscenze sarà oggetto della valutazione dell'elaborato scritto previsto in sede d'esame.
The knowledge check will be subject to the evaluation of the written document foreseen during the exam.
lo studente sarà in grado di comprendere i principi che stanno alla base dei test eseguiti in laboratorio ed individuare eventuali azioni correttive per risoluzione delle problematiche.
the student will be able to understand the principles that underlie the tests performed in the laboratory and identify any corrective actions to resolve problems.
La verifica delle capacità avverrà in aula con attraverso un confronto diretto tra docente e studenti.
Tale verifica sarà occasione per chiarire eventuali dubbi e risolvere criticità.
The verification of skills will take place in the classroom with a direct comparison
between teacher and students. This verification will be an opportunity to clarify any doubts and resolve critical
issues.
Lo studente potrà acquisire conoscenze sulle principi che stanno alla base dei test esguiti in laboratoriorio di biochimica clinica e biologia molecolare. Sviluppare consapevolezza per risoluzione di problematiche inerenti il corretto svolgimento del proprio lavoro ed attendibilità dei risultati prodotti.
The student will be able to acquire knowledge on the principles that underlie
the tests carried out in the laboratory of clinical biochemistry and molecular biology.
Develop awareness for the resolution of problems inherent in the proper performance of
one's work and reliability of the results produced.
La verifica dei comportamenti avverrà in aula con attraverso un confronto diretto tra docente e studenti.
Tale verifica sarà occasione per chiarire eventuali dubbi e risolvere criticità.
Verification of behavior will take place in the classroom with a direct comparison
between teacher and students. This verification will be an opportunity to clarify any doubts and resolve critical
issues.
Per seguire il corso non è necessaria alcuna conoscenza iniziale.
No initial knowledge is required to follow the course.
Lezioni frontali con ausilio di slide, filmati e quando possibile piccola strumentazione trasportabile.
Frontal lessons with the aid of slides, videos and whenever possible small transportable instruments.
METODOLOGIE ANALITICHE
BIOLOGIA MOLECOLARE
Cenni storici. Gli acidi nucleici, struttura e funzioni. Organizzazione del genoma eucariotico, procariotico e mitocondriale. La replicazione del DNA. Paradosso C, K, N. Sistemi di riparazione del DNA. Tipi di RNA e loro funzioni. La trascrizione. Operone Lac e operone Trp. Regolazione della trascrizione nei procarioti: controllo epigenetico e fattori di trascizione. Cos’è l’imprinting. Electrophoretic mobility shift assay (EMSA) e superschift. Maturazione dell’mRNA (capping, poliadenilazione , splicing ed editing) e dell’rRNA. Il codice genetico. La traduzione. I miRNA, DROSHA, DICER, RISCH. Metodi di estrazione degli acidi nucleici (fenolo /cloroformio, Tryzol, salting out, kit commerciali con biglie magnetiche e colonnine). La precipitazione del DNA. western blotting cenni Qualità e quantificazione degli acidi nucleici: metodo spettrofotometrico e controllo elettroforetico. Elettroforesi degli acidi nucleici su gel di agarosio e su gel di poliacrilammide. Pulsed-field gel electroforesys (PFGE). L’ibridazione: curva di denaturazione del DNA, temperatura di melting. Tipi di sonde e fattori che influenzano l’iridazione. Il Southern blotting. Il Nothern blotting. La PCR. Touchdown PCR. Nested primer PCR. Come disegnare i primer. Tipi di contaminazioni possibili e organizzazione ottimale di un laboratorio di biologia molecolare. Reverese Trasciptase PCR. Real Time PCR. SYBR green, sonde specifiche (sonde Taq man, sonde FRET, molecular beacon). Analisi del segnale: curve di amplificazione e curve di melting. Quantificazione relativa e assoluta. Cenni sul modulo HRM. Enzimi di restrizione. La transfezione stabile e transiente. Metodi per transfettare. Tipi di vettori per il clonaggio: plasmidi, cosmidi, fagi, cromosoma batterico e di lievito. Il clonaggio del cDNA tramite enzimi di restrizione e topo cloning vector. Fase di screening mediante inattivazione inserzionale con doppio antibiotico e mediante inattivazione della beta-galattosidasi (colonie bianche/blu). Subclonaggio in vettori di espressione. Il sequenzaimento del DNA: il metodo di Sanger. I polimorfismi. Ibridazione in situ. PCR e RT-PCR in situ. Microarray. Editing genomico: CRISPR/Cas9.
BIOCHIMICA CLINICA
Aspetti generali di biochimica clinica. Preparazione del campione biologico. Principali alterazioni del campione biologico e approccio sistematico alla gestione del campione biologico. Bioregolatori: elettroliti e molecole volatili. Alterazione del bilancio idroelettrolitico, significato clinico e metodi di determinazione. Equilibrio acido base nel sangue, disordini acido-base e metodi di determinazione (emogasanalisi). Esame emocromocitometrico: caratteristiche principali e approccio clinico. Anemie, coagulazione e approccio alle principali alterazioni. Vitamine e valutazione delle principali alterazioni. Metabolismo dei Carboidrati. Studio di ipo- ed iperglicemie. Metodi di misurazione della glicemia. Lipoproteine e lipidi. Studio delle dislipidemie e disturbi aterosclerotici. Valutazione biochimica-funzionale del fegato (bilirubina totale diretta e indiretta, AST, GOT, LDH, ALP, γGT).Valutazione biochimico-funzionale del rene (GRF, PRF, FG, FF, creatinina, acido urico, urea). Analisi delle urine. Aspetti generali dell'endocrinologia: ruolo dell'ipotalamo, Ipofisi, Tiroide, Paratiroide, Gonadi, Surrene, Corticosurrene e Pancreas. Marcatori tumorali: ruolo e utilizzo nella pratica clinica
ANALYTICAL METHODOLOGIES
1-Introduction to laboratory organization. Pre analytical and inherent problems.
Urgent and routine exams. Decentralized analysis. Practical examples and critical issues.
Quality control: Internal and external quality control. Types of errors.
Accuracy and precision. Specificity and sensitivity 2-Specificity and sensitivity. Repeatability and reproducibility. Interlaboratory and intralaboratory checks. Trust limits. Control cards. 3-How to set up a quality control with serum pools; practical example. Westgard rules. Corrective actions in case of card out of control. Centrifugation techniques (sedimentation principles). Types of centrifuges and rotors. Differential centrifugation. Centrifugation in density gradient, isopycnic and zonal. Ultracentrifugation. 4-Chromatographic techniques: General principles. Selectivity and efficiency. Number of theoretical dishes. Column chromatography, thin layer, paper. Adsorption chromatography. Distribution chromatography. Ion exchange chromatography. Exclusion chromatography. Affinity chromatography. Gas chromatography. HPLC. Notes on glycated hemoglobin. Examples of instrumentation. Interpretation of chromatographic peaks. Immunochemical techniques: Introduction to Antigens and Antibodies. Antigen-antibody binding. Immunoprecipitation techniques - simple radial immunodiffusion and double immunodiffusion. Competitive Elisa, Elisa double antibody method. RIA - liquid and solid phase separation methods. IRMA Elements of radiation protection. 5-Immunofluorescence, direct and indirect. Practical example of how an ELISA test is performed; meaning of the standard curve; how to interpret absorbance values, trace any concentrations of the analytes sought. Electrophoresis, basic principles. Capillary electrophoresis. Interpretation of results. 6- Fundamentals of spectrophotometry. Wavelength and frequency. Monochromatic and polychromatic light. Quantitative and qualitative analysis. Emission and absorption spectra. Transmittance, absorbance and Lambert and Beer law. Basic principles. General information on spectrophotometers. 7-Electrophoresis and capillary electrophoresis. Optical microscopy. 8- Visit to the Clinical Chemical Analysis Laboratory 1, Dr. Pellegrini, agreed with the tutor, where practical examples relating to quality control were made (how a control is inserted on the instrument, how it is evaluated, card interpretation, corrective actions). Display of control cards on instruments. External quality control. The evaluation report. View of the instrument for the determination of glycated hemoglobin. Mechanical components, operating principle. Quality control. Visualization and interpretation of chromatograms. Traced with anomalies. Possible solutions to problems. Vision of the instrument for capillary electrophoresis. Visit to the University Microbiology laboratory - Dr. Barnini, for practical training for the use of the optical microscope. Preparation of a fresh glass slide. Microscopic observation.
MOLECULAR BIOLOGY
Background. Nucleic acids, structure and functions. Organization of the eukaryotic, prokaryotic and mitochondrial genome. DNA replication. Paradox C, K, N. DNA repair systems. Types of RNA and their functions. The transcription. Lac operon and Trp operon. Regulation of transcription in prokaryotes: epigenetic control and transcription factors. What is imprinting. Electrophoretic mobility shift assay (EMSA) and superschift. Maturation of mRNA (capping, polyadenylation, splicing and editing) and rRNA. The genetic code. Translation. I miRNA, DROSHA, DICER, RISCH. Nucleic acid extraction methods (phenol / chloroform, Tryzol, salting out, commercial kits with magnetic beads and columns). The precipitation of DNA. Western blotting nods. Nucleic acid quality and quantification: spectrophotometric method and electrophoretic control. Nucleic acid electrophoresis on agarose gel and polyacrylamide gel. Pulsed-field gel electroforesys (PFGE). Hybridization: DNA denaturation curve, melting temperature. Types of probes and factors that influence iridation. Southern blotting. Nothern blotting. PCR. Touchdown PCR. Nested primer PCR. How to draw primers. Types of possible contamination and optimal organization of a molecular biology laboratory. Reverese Trasciptase PCR. Real Time PCR. SYBR green, specific probes (Taq man probes, FRET probes, molecular beacon). Signal analysis: amplification curves and melting curves. Relative and absolute quantification. Notes on the HRM module. Restriction enzymes. Stable and transient transfection. Methods for transfecting. Types of vectors for cloning: plasmids, cosmids, phages, bacterial and yeast chromosome. Cloning of cDNA by restriction enzymes and mouse cloning vector. Screening phase by insertional inactivation with double antibiotic and by inactivation of beta-galactosidase (white / blue colonies). Subcloning in expression vectors. DNA sequence: the Sanger method, next generation sequencing (illuminates). Polymorphisms. In situ hybridization. PCR and RT-PCR in situ. Microarray. Genomic editing: CRISPR / Cas9.
CLINICAL BIOCHEMISTRY
Main background in clinical biochemistry. Biological sample preparation. Main biological sample alterations. Systematic approach to biological sample handling. Bio-regulators: electrolytes and volatile molecules. Alterations of the hydro-electrolytic balance, clinical significance and methods of determination. Acid-base balance in the blood, acid-base disorders and methods of determination (blood gas analysis). CBC examination: main features and clinical approach. Anemias, coagulation and approach to the main alterations. Vitamins and evaluation of the main alterations. Carbohydrate metabolism. Evaluation of hypo- and hyperglycemia. Blood glucose measurement methods. Lipoproteins and lipids. Dyslipidaemia and atherosclerotic disorders. Biochemical-functional assessment of the liver (direct and indirect total bilirubin, AST, GOT, LDH, ALP, γGT). Biochemical-functional assessment of the kidney (GRF, PRF, FG, FF, creatinine, uric acid, urea). Urine analysis. General aspects of endocrinology: role of the hypothalamus, pituitary gland, thyroid, parathyroids, gonads, adrenal and adrenocortical glands and Pancreas. Tumor markers: role and use in clinical practice
Materiale didattico fornito: materiale praparato dai docenti.
Biologia molecolare si consiglia in più:
BIOLOGIA MOLECOLARE terza edizione , F. Amaldi, P. Benedetti,G. Pesole e P. Plevani. Casa Editrice Ambrosiana. Distribuzione esclusiva Zanichelli
Didactic material provided: material prepared by the teachers.
Molecular biology is recommended in addition:
MOLECULAR BIOLOGY third edition, F. Amaldi, P. Benedetti, G. Pesole and P. Plevani.
Ambrosiana Publishing House. Zanichelli exclusive distribution
Elaborato scritto: Tre domande a risposta aperta per ogni modulo.
Written paper: Three open-ended questions for each module.
