BIOCHEMISTRY - APPLIED BIOCHEMISTRY
Learning outcomes of the course unit
At the end of the course, the students should demonstrate the understanding of the overall rationale of metabolism, of the structure and function of biological macromolecules and of the mechanisms that allow chemical reactions in living organisms, their regulation and coordination. In details:
1. Knowledge and understanding:
- Know and understand the function and structure of biological macromolecules (mono-, disaccharides and polysaccharides, amino acids, proteins and nucleotides, cofactors and prosthetic groups)
- Know and understand the main metabolic pathways
- Know and understand at the molecular level the mechanism of the enzyme reactions described in the course
2. Applying knowledge and understanding
- describe the structure of any protein or macromolecule
- solve simple biochemical problems
3. Communication skills
- explain the topics of the course effectively with the appropriate scientific terminology
4. Learning skills.
- learn new catalytic mechanisms from simplified scientific literature\textbooks
- learn new metabolic pathways from simplified scientific literature\textbooks.
Upon completion of the course, the student should have gained insight into the main biochemical laboratory techniques. He\she should be able to address simple
biochemical problems and analyse simple instrumental data.
1. Knowledge and understanding. At the end of the course, the student should be able to:
- Know and understand the main biochemical methodologies described in the course
- Know and understand the principles of each methodology in molecular details
- Know the applications of the techniques described in the course
2. Applying knowledge and understanding. At the end of the course, the student should be able to:
- Identify the most suitable technique to obtain functional or structural pieces of information on macromolecules
- solve simple problems on the application of the techniques
3. Communication skills. At the end of the course, the student should be able to:
- Discuss the topics described in the course in a clear, concise and effective way.
- report experimental scientific results
4. Learning skills. At the end of the course, the student should be able to:
- make connections between the various topics of the course
- Understand the potential of research in biochemistry.
General and Inorganic Chemistry, Organic chemistry. For the module of Applied Biochemistry, the attendance of the Biochemistry module is required.
Course contents summary
The course consists in two teaching modules (Biochemistry and Applied Biochemistry).
The biochemistry module will focus on the structure and function of biological macromolecules, on the principle of cell metabolism, on the metabolism of carbohydrates and lipids and on the principles of protein and nucleic acids metabolism.
The module of Applied Biochemistry will cover the main recombinant DNA techniques and the methods used for the expression of proteins in recombinant form, their purification and characterization.
- Principles of living organisms.
- Molecular and macromolecular components of cells.
- Structure, dynamics and function of proteins.
- Myoglobin and hemoglobin.
- Allostery and allosteric models.
- Enzymes and coenzymes.
- Mechanisms of catalysis, inhibition and regulation.
- Lipids and biological membranes.
- Principles of metabolism and bioenergetics.
- Glycolysis, gluconeogenesis and their regulation.
- Degradation and synthesis of glycogen.
- Degradation and synthesis of fatty acids.
- Ketone bodies.
- Krebs cycle.
- Oxidative phosphorylation and synthesis of ATP. Pentose phosphate pathway
- Degradation and synthesis of amino acids, urea cycle.
- Degradation and synthesis of purines and pyrimidines.
- Principles of system biology.
PART I: RECOMBINANT DNA AND ITS APPLICATIONS
-Recombinant DNA technology: restriction endonucleases, DNA ligase, cloning vectors, plasmids as cloning vectors, host organisms, bacterial cultures, selection of recombinant clones, -complementation, PCR, RT-PCR, site-directed mutagenesis, genomic and cDNA libraries. DNA sequencing
- Expression of recombinant proteins: expression vectors, cell strains, lac and T7 promoters, fusion proteins. Protein therapeutics: insulin and recombinant vaccines.
PART II: PURIFICATION OF PROTEINS AND ELECTROPHORESIS
- Protein purification: extraction techniques; centrifugation; ultrafiltration, dialysis, diafiltration; fractionation methods; chromatography (hydrophobic interaction, ion exchange, size exclusion, affinity).
-Electrophoretic techniques: principles and applications to biological macromolecules. Protein electrophoresis (native, SDS-PAGE and their applications). Two-dimensional electrophoresis. DNA electrophoresis and restriction analysis with its applications).
PART III: CHARACTERIZATION AND IDENTIFICATION OF PROTEINS
- Absorption spectroscopy: principles and instrumentation. Lambert-Beer law. Chromophores of biological interest. Protein absorption spectra. Determination of protein concentration.
- Fluorescence emission spectroscopy: principles (fluorescence emission, Stokes shift, fluorescence quantum yield), instrumentation and applications (fluorophores of biological interest).
-Circular dichroism: principles (linearly polarized radiation, circularly polarized radiation, eliptically polarized radiation and elipticity). Applications to the study of protein structure
- Enzymatic activity assays: continuous methods and discontinuous methods. Direct, indirect and coupled assays. Chromogenic substrates.
- Immunochemistry: antibodies (structure, specificity, labeling). Production of monoclonal and polyclonal antibodies. Immunoassays: RIA and ELISA. Applications: pregnancy tests and tests for the identification of viral infections (HIV and Covid-19). Blotting techniques
- introduction to proteomics
For both modules, the material used in the lectures will be provided on the Elly platform.
- Nelson e Cox
I PRINCI DI BIOCHIMICA DI LEHNINGER
- Appling, Anthony-Cahill e Mathews
BIOCHIMICA: Molecole e Metabolismo
- Metodologie Biochimiche. A cura di Carmela Bonaccorsi di Patti, Roberto Contestabile e Martino Luigi di Salvo. Zanichelli
- Metodologie Biochimiche e Biomolecolari. Mauro Maccarrone. Zanichelli
The textbooks listed above are available only in the Italian version.
- Principles and Techniques of Biochemistry and Molecular Biology. Wilson & Walker. Cambridge University Press.
The course will take place as indicated by the University for the second semester, in compliance with the legal provisions that will be in force for the containment of COVID-19 (in-person, online synchronous, online asynchronous, or mixed).
The teaching activities will be carried out through lectures, using slides that will be loaded on the Elly platform.
The teaching activities will be carried out through lectures with the help of slides that will be made available weekly to the students on the Elly website.
Lessons will be integrated with classroom exercises aimed at improving the student's ability to apply theoretical knowledge to real-life problems.
The teacher will show, when possible, videos of the techniques treated in the course, in order to provide students with practical indications useful for understanding the applications of the methods.
Assessment methods and criteria
The exam consists of the two partial exams of Biochemistry, (written test and oral examination) and Applied Biochemistry (oral examination). The final mark will be the average of the evaluation of the two modules. The student needs to pass the exams of both modules in the same session for the exam to be valid.
The exam consists of a written test followed, after about a week, by an oral interview. If the regulations for the containment of the Covid-19 pandemic will allow it, the examination will be carried out in person. The 30-minute written test consists of 3-4 open-ended questions that will require schematic answers. It will assess the knowledge of biological macromolecules, the mechanisms of enzyme reactions and the reactions of the metabolic pathways. The candidate's ability to solve a biochemical problem will also be evaluated. The oral examination, open only to the students that passed the written test, will focus on the entire content of the course. The final mark for the Biochemistry module will be an overall evaluation of the two tests.
The exam is an oral interview and takes place in the same session as the Biochemistry exam. The examination will be carried out in person, if allowed by the regulations for the containment of the Covid-19 pandemic.
Questions will cover the entire program and the student will also be asked to solve a problem. The scoring, on a scale 18-30, will take into account the level of analysis, the ability to critically apply the knowledge, the appropriateness of the scientific language and the autonomy in the discussion.
Students diagnosed of a specific learning disorder (SLD), certified in accordance with law 170/2010, will be granted the means allowed by law.