BIOCHEMISTRY

9 CFU - 1° Semester

Teaching Staff

VITTORIA RITA ANNAMARIA SPINA - Module BIOCHIMICA MEDICA - BIO/10 - 4 CFU
VINCENZA BARRESI - Module Molecular Diagnostic Technologies - BIO/12 - 5 CFU

Learning Objectives

BIOCHIMICA MEDICA
Course Aims

To obtain advanced knowledge and competences in Medical Biochemistry, related to Biotechnology and its applications to Medicine.
Molecular Diagnostic Technologies
The Molecular Diagnostic Technologies have broad application in the biomedical field and the course aims to give the student the knowledge of methods and tools to be used for the detection of gene mutations and genomic alterations necessary for diagnostic, prognostic and predictive of response before the start of targeted therapeutic protocols

Detailed Course Content

BIOCHIMICA MEDICA
The main metabolisms, metabolic interrelations and cellular and mitochondrial bioenergetics are summarized
Blood glucose and glucose uptake in cells: main transporters and insights on the role of insulin. Stages of anaerobic glycolysis and reactions.
Enzymes and mechanism of action. Thermodynamic and kinetic regulation. Enzymatic defects and enzyme inhibitors.
Fate of pyruvate in the cytosol: lactic and alcoholic fermentation, with description of the reactions and enzymes involved. Energy balance.
Pyruvate dehydrogenase and cycle of tricarboxylic acids, flow regulation, energy balance, anaplerotic reactions.
Gluconeogenesis. Coordinated regulation of glycolysis and gluconeogenesis.
Blood glucose and glucose homeostasis.
Glycogen metabolism.
Pentose cycle.
Fat metabolism: catabolism and synthesis of fatty acids and their regulation; energy balance.
Main reactions in the metabolism of amino acids. Mitochondria: structure and dynamics. Mitochondrial oxidative phosphorylation: electrochemical potential and protonmotor force in the mechanism of phosphorylation of ADP. Structure and catalytic mechanism of the ATP synthase complex.

Functional biochemistry
Metabolic characteristics of liver, muscle, adipose tissue, brain. Glucose homeostasis. Diabetes.

Biochemistry of cellular communication
Generality. Hormone receptors; ligand-receptor interaction; molecular characteristics of the receptors. Receptors for steroid hormones and their mechanism of action. Membrane receptors. GTP-binding proteins: Gi, Gs, Gq, and their activation. Role of beta and gamma dimers. Intracellular effectors regulated by GTP-binding proteins: adenylate cyclase phospholipase C (PLCbeta), phosphatidylinositol 3-kinase. Adenylate cyclase and cAMP. CAMP-dependent protein kinase (PKA). Examples of PKA phosphorylated substrates: consequences on metabolism and gene transcription. Inositol phosphates and diacylglycerol, calcium. NO, cGMP and PKG. Receptors with tyrosine kinase activity. Activation and transphosphorylation mechanism. Formation of supramolecular signaling complexes: role of the domains SH2 and SH3. Effectors activated by tyrosine kinase receptors. Activation of MAP kinases. Cell activation by insulin: Ras dependent and Ras independent.

Biochemistry of hormones
Classification, biosynthetic mechanisms and their control. Pituitary and hypothalamic hormones; thyroid hormones; hormones of the cortical and medulla of the adrenal gland; pancreatic hormones; parathormone and calcitonin; hormones of male and female gonads, eicosanoids. Biochemical correlations and metabolic effects.

Cell cycle
Cycline and kinase cyclin employees
Progression in the various phases of the cycle and related molecular events.
Regulation: cicline localization, role and characterization of p53 and Rb.
Oncogenes and oncosuppressors.
Apoptosis.

Molecular bases and biochemical mechanisms associated with the inflammatory process
[The course in the classroom will be integrated with the following laboratory exercises] - [Teaching activity].

Fundamental principles of cell culture technology.
Introduction to basic practical knowledge for setting up and maintaining cell cultures in sterile conditions.
Organization of a cell culture laboratory: sterility, pH, temperature, culture media, sera.
Maintaining in culture primary, immortalized and tumor cell lines.
Basic techniques of cell culture: suspension culture, monolayer culture, 3D culture.
Count and plate a known amount of cells, freeze and defrost cells, acquire and present digital images and movies of their cells in culture. Trypsinization of cells and preparation of cell subcultures.
Protocols for the evaluation of proliferation and cell viability.

Extraction of proteins from cultured cells: preparation of lysis buffer and cell lysis. Protein electrophoresis. Polyacrylamide gel. Electrophoresis. Electrophoretic running system.
Western blot.
Molecular Diagnostic Technologies
Biological samples: types, blood and biological samples, processing and storage.

Extraction and purification of nucleic acids from human cells by chromatographic columns for gel filtration, ion exchange and affinity. Purification of nuclec acids with magnetic beads.

Quantitative assay for nucleic acids using spectrophotometric and fluorimetric methods.

Technologies based on the complementarity of the purine and pyrimidine nucleotides.

Synthesis of cDNA by reverse transcription.

Polymerase chain reaction (PCR). Qualitative PCR or End Point and quantitative PCR or Q-Real Time PCR. Programs for the design of primers to be used for PCR reactions.

Evaluation of gene expression in real time by the method of 2-ΔΔCT. for Allelic discriminationusing the Real Time PCR. Restriction and Modification enzymes.

Agarose gel electrophoresis for the separation of nucleic acids. Capillary electrophoresis for acid nucleic sequencing and analysis of microsatellite.

Protein extraction and analysis. Electrophoresis of proteins. Western blot.

DNA- and RNA-microarrays: Preparation of samples of DNA or RNA for the global analysis of the genome and transcriptome. Molecular analysis of the human karyotype microarray genomic photolithography-based probes to "Single Nucleotide Polymorphism-SNP" and "Copy Number Variation-CNV". Using algorithms and softwares for the interpretation of the data (CN state, Allele difference). Transcriptome analysis. Microarray expression to oligonucleotides obtained by photolithography synthesis. Array-CGH. Using algorithms and softwares for data interpretation (RMA and SAM).

Application of the "microarray" technology for the study of solid and hematological tumors.

Acid Nucleic First-generation sequencing technology, second generation and third generation. Methods for the preparation of libraries for the exome analysis and specific regions using a multiplex. Methods of clonal amplification. Techniques for the detection of incorporated nucleotides. Bioinformatics and statistical methods for interpreting data obtained from second-generation sequencing platform (NGS).

Implementation of the first generation sequencing techniques for the detection of mutations with prognostic and predictive significance of response to therapy. Application of second generation technologies for the analysis of solid and hematological tumors.

Detection of chromosomal aberrations by MLPA technology (Multiplex Ligation-dependent Probe Amplification).

Human cell cultures.

Textbook Information

BIOCHIMICA MEDICA
1. Biochimica con aspetti clinici di Thomas M. Devlin.

Casa ed. Idelson-Gnocchi.

2.Principi di Biochimica Medica

G. Meisenberg- W.H.Simmons III edizione

Casa ed. Minerva

3. I Principi di Biochimica di Lehninger

Casa ed. Zanichelli (4° edizione)

4. Biochimica per le discipline biomediche J.W. Baynes M.H. Dominiczak

Casa ed. Ambrosiana (2° edizione)
Molecular Diagnostic Technologies
1. Additional material supplied by the teacher; 2) Fundamental Molecular Biology; Lizabeth A. Allison, Editore: John Wiley & Sons Inc; 3) Online courses: http://www.dsf.unict.it/index.php?page=progetto-phar-in; "Riparazione del Dna e mutagenesi nel cancro" (D.F. Condorelli); Mismatch repair (MMR) instabilità dei microsatelliti (V. Barresi); "Tecnologie avanzate per la Bio-Medicina Genomica" (Vincenza Barresi).

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