Knowledge of basic techniques and methodologies of applied biochemistry. Applications of biochemical and molecular investigation in human samples. Knowledge of laboratory analytical and diagnostic tools in the field of biochemistry applied to the clinic. Applications of biochemical and molecular methodologies for the study of pathophysiological parameters in humans for diagnostic purposes Production of recombinant human proteins for therapeutic purposes
Biochimica clinica essenziale.
Elisabetta Albi, Tommaso Beccari, Samuela Cataldi
Ed Zanichelli ISBN 978-88-08-72057-3
Metodologie biochimiche
Maria Carmela Bonaccorsi Di Patti, Roberto Contestabile, Martino Luigi Di Salvo
Ed Zanichelli ISBN: 9788808712448
Learning Objectives
The course aims to educate the student on the general principles of applied and molecular biochemistry. The course provides theoretical and scientific skills for the study of biochemical parameters in biological samples also in relation to the different human pathophysiological states.
Prerequisites
The course requires knowledge of general and molecular biochemistry.
Teaching Methods
classroom lessons
Type of Assessment
The final examination consists of an interview designed to ascertain knowledge of basic biochemical and molecular techniques and knowledge of the application of biochemical and molecular techniques for monitoring pathophysiological parameters in humans. Reasoning ability and use of specialized vocabulary will be evaluated. The final grade will result from the combination of these contributions, which are considered important indicators of the adequacy of the level of learning achieved and the attainment of the intended learning outcomes of the course.
Course program
Spectrophotometry. Laws of light absorption. Colorimetric assays. Spectrofluorimetry: principles and applications. Luminescence: principles and applications. Preparation and handling of biological samples. Cell disruption; preparation and purification of cell organelles. Centrifugation by separation of cell organelles, purification of nucleic acids. Outline of eurariotic cell cultures and bacteria. The enzymatic kinetics. Equation by Michaelis-Menten. Enzymatic kinetics applications and practice. Methods of dosage of enzymes. Immunochemical techniques. Production of mono and poly-clonal antibodies. Applications: RIA and ELISA assays, immunohistochemistry. Electrophoretic techniques: concept of electrophoretic mobility and relationship between mobility, charge, applied electric field, dimensions. Relationship between isoelectric point and mobility of proteins Electrophoresis for proteins in native or denaturing conditions. Isoelectrofocusing, mono and bidimensional electrophoresis. Western-blotting. Preliminary procedures for protein purification general considerations, fractional precipitation of proteins (salting in and salting out). Chromatographic techniques: generalities and definitions. Characteristics and applications of chromatography: ion exchange, size exclusion (with examples of determining the molecular weight of proteins), affinity chromatography (with examples of ligands and general strategies for covalent binding of ligands to the stationary phase. Examples of strategies of protein purification. Recombinant DNA technology. Basic techniques to DNA cloning. Techniques for the identification of nucleic acids and DNA. Microarrays. Amplification of DNA by Polymerase Chain Reaction (PCR), RT-PCR. Nucleic acid electrophoresis . Production and purification of recombinant proteins and examples of pharmacological applications. Human recombinant antibodies. ScFv and hVH.
Applications of biochemical methodology to study human biochemical parameters
• Hemoglobinopathies. Serum proteins. Coagulation profile and coagulation .
• Lipids and lipoproteins. Dyslipidemias. Lipid profile and cardiovascular risk. Laboratory measurements of the various lipids.
• Carbohydrates and diabetes. Blood sugar regulation. Diabetes mellitus. Metabolic syndrome. Laboratory methods. Glucometers.
• Cardiac function markers. Myoglobin. Creatine Kinase isoenzymes. LDH. Troponine. Less common markers.
• Endocrinological assays. Pancreatic disorders. Adrenal glands. Thyroid function tests. Hypothalamus.
• Liver function markers and interpretations. Transaminases. Jaundice. Measurements of bilirubin and other dosages.
• Renal function tests. Measurement of creatinine at other related dosages. Creatinine. Urea and uric acid.
• Congenital errors of metabolism. Disorders of amino acid metabolism. Disorders of carbohydrate metabolism: galactosemia, glycogen accumulation. Lactose intolerance. Disorders in urea metabolism. Mitochondrial disorders, lysosomes. Iron metabolism.
• Introduction to tumor markers and examples.
• Assay of drugs of abuse in urine and other biological fluids. Ethanol assay.