CELL BIOCHEMISTRY AND BIOMOLECULAR TECHNIQUES
(objectives)
The course of CELLULAR BIOCHEMISTRY (MODULE A) AND BIOMOLECULAR TECHNIQUES (MODULE B) intends to provide students with (i) theoretical knowledge in the field of cellular biochemistry, deepening the mechanisms that regulate the cell cycle in eukaryotes, (ii) theoretical and practical knowledge in protein engineering, and iii) theoretical and practical knowledge of the major molecular biology and biochemical techniques applied to the study of genes, genomes, proteins and proteomes. Experimental approaches will be discussed, making use also of bioinformatics, to address complex biological questions in biochemistry and molecular biology. In detail, for MODULE A: The course intends to go into two themes of considerable scientific interest: 1) deepening the biochemical and molecular mechanisms of cell cycle control in eukaryotes with particular emphasis on the experimental approaches used for its elucidation; 2) protein engineering elements that allow the design in silico recombinant proteins by using bioinformatics tools and finally the expression and purification of recombinant proteins using both prokaryotic and eukaryotic organisms. This last part of the course includes practical laboratory related to the cloning of a eukaryotic gene and its expression in bacteria. For MODULE B: Specifically, it is intended to provide students with specific skills for the manipulation and analysis of nucleic acids and proteins (mutagenesis and genome editing techniques, differential proteomic), for the analysis of gene expression levels (qPCR, microarrays, differential transcriptomic) and gene expression regulation (study of epigenetic modifications and protein-DNA interactions), for the study of transduction signal pathways by protein-protein interaction analysis. The advances in the field of the sequencing of whole genomes and the application of biomolecular techniques in diagnostic field will be also discussed. Bioinformatics tools will be used for in silico prediction of interaction between biomolecules, or as complementary for the use of the discussed techniques (for input or output analysis). Finally, laboratory practical experiences will be organized to acquire techniques for studying nucleic acids and proteins.
b) EXPECTED LEARNING OUTCOMES 1) Knowledge and understanding At the end of the course the students will: MODULE A: To have in-depth knowledge of the biochemical and molecular basics of cell cycle control in eukaryotes. They will also learn the main techniques for the in silico design and expression of recombinant proteins in heterologous systems. In general, they will have developed the ability to understand the pivotal experimental approaches for acquiring knowledge. MODULE B: To know the basic techniques used in the field of fundamental and applied research. They will have an in-depth knowledge of molecular and advanced techniques and the related bioinformatics tools to support them; they will know the importance of statistical validation of the results of an experiment and of the controls that make an experiment scientifically reliable. 2) Applying knowledge and understanding At the end of the course the students will: MODULE A: Be encouraged to use the knowledge acquired for their application to specific problems, such as the design of new, more potent and / or more selective proteins for their use in various fields of interest (biomedical, agri-food, etc.). They will be able to put into practice the acquired knowledge to perform the planned experiments during the practical experience. MODULE B: Be able to use the acquired knowledge to evaluate and interpret the results of an experiment, identify its strengths and weaknesses and optimize it by evaluating the possible impact of variations in key experimental parameters; orient themselves among the main qualitative and quantitative methods to select the most suitable ones for studying the biological problem of interest; perform the experiments carried out during the practical part of the course. 3) Making judgements MODULE A and MODULE B: Students will be able to interpret and discuss the scientific papers presented during the course and be able to design and express new proteins with different characteristics. Students will have to acquire the ability to understand and critically discuss the experimental results obtained in the laboratory and use them as a starting point for planning subsequent experiments. 4) Comunication skills MODULE A: During the lessons students will be stimulated to discuss and compare different point of views in order to develop their communicative abilities that will be verified during the preliminary and final examinations at the end of training activities. MODULE B: Students should have the ability to convey the acquired knowledge in a clear and comprehensible manner, even to people who are not in the field, and must demonstrate the ability to present information also with schemes and formulas. 5) Learning skills MODULE A and MODULE B: Students will have to be able to describe scientific topics related to the course. This skill will be developed through the active involvement of students during class discussions and practical experiences during the hours dedicated to the experimental laboratory.
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Code
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119643 |
Language
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ITA |
Type of certificate
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Profit certificate
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Module: CELL BIOCHEMISTRY
(objectives)
The course of CELLULAR BIOCHEMISTRY (MODULE A) AND BIOMOLECULAR TECHNIQUES (MODULE B) aims to provide students with (i) theoretical knowledge in the field of cellular biochemistry, deepening the mechanisms regulating the cell cycle in eukaryotes, (ii) theoretical and practical knowledge in protein engineering, and iii) theoretical and practical knowledge of the major molecular biology and biochemical techniques applied to the study of genes, genomes, proteins and proteomes. Experimental approaches will be discussed, making use also of bioinformatics, to address complex biological questions in biochemistry and molecular biology. In detail, for MODULE A: The course aims to deepen knowledge of two topics of considerable scientific interest: 1) deepening the biochemical and molecular mechanisms of cell cycle control in eukaryotes with particular emphasis on the experimental approaches used for its elucidation; 2) protein engineering elements that allow the design of recombinant proteins by using bioinformatics tools and also the expression and purification of recombinant proteins using both prokaryotic and eukaryotic organisms. This last part of the course includes a practical laboratory related to the cloning of a eukaryotic gene and its expression in bacteria.
b) EXPECTED LEARNING OUTCOMES
1) Knowledge and understanding At the end of the course the students will: MODULE A: To have in-depth knowledge of the biochemical and molecular basics of cell cycle control in eukaryotes. They will also learn the main techniques for the in silico design and expression of recombinant proteins in heterologous systems. In general, they will have developed the ability to understand the pivotal experimental approaches for acquiring knowledge.
2) Applying knowledge and understanding At the end of the course the students will: MODULE A: Be encouraged to use the knowledge acquired for their application to specific problems, such as the design of new, more potent and/or more selective proteins for their use in various fields of interest (biomedical, agri-food, etc.). They will be able to put into practice the acquired knowledge to perform the planned experiments during the practical experience.
3) Making judgments MODULE A: Students will be able to interpret and discuss the scientific papers presented during the course and be able to design and express new proteins with different characteristics. Students will have to acquire the ability to understand and critically discuss the experimental results obtained in the laboratory and use them as a starting point for planning subsequent experiments.
4) Communication skills MODULE A: During the lessons, students will be stimulated to discuss and compare different points of view in order to develop their communicative abilities which will be verified during the preliminary and final examinations at the end of training activities.
5) Learning skills MODULE A: Students should be able to describe scientific topics related to the course. This skill will be developed through the active involvement of students during class discussions and practical experiences during the hours dedicated to the experimental laboratory.
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Language
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ITA |
Type of certificate
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Profit certificate
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Credits
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6
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Scientific Disciplinary Sector Code
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BIO/10
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Contact Hours
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40
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Laboratory Hours
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8
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Type of Activity
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Core compulsory activities
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Teacher
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CARUSO Carla
(syllabus)
Module A The cell cycle (3 CFU) General strategy and cell cycle phases (M, G1(G0), S and G2. Experimental systems for the study of the cell cycle: Xenopus leavis eggs, mammalian cells and yeast. Molecular regulation of MPF and SPF: mitotic and G1 cyclins. Cdks and cyclins in mammalian cell cycle. Cell cycle checkpoints. Hereditary and sporadic retinoblastoma and role of Rb in cell cycle regulation. UV-damaged DNA and role of p53. Oncogenes and oncoproteines.
Proteic engineering fundamentals (2 CFU) Recombinant protein expression in prokaryotic systems: principles and applications. Recombinant protein expression in eukaryotic systems: -Expression in Saccharomyces cerevisiae and Pichia pastoris; -Expression in plants.
Experimental laboratory (1 CFU) • Enzymatic digestion of pGEM-HEL plasmid and purification of the HEL gene from agarose gel; • Subcloning of the HEL gene in the expression vector pGEX-4T; • Transformation of competent BL-21 cells with the newly generated GST-HEL plasmid; • Expression of recombinant GST-HEL in BL-21 cells and analysis of the recombinant protein trough SDS-PAGE.
(reference books)
Module A
RECOMMENDED TEXTBOOKS Cell cycle Selected chapters from the following books: Murray A & Hunt T, The cell cycle, an introduction, Oxford University Press, New York. Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K & Walter P, Biologia Molecolare della Cellula, Zanichelli, 2016 (VI Edizione) Harvey Lodish, A Berk, C.A. Kaiser, M. Krieger, M.P. Scott, A. Bretscher, P. Ploegh, Paul Matsudaira Biologia Molecolare della Cellula, Zanichelli, 2009 (III edizione)
Elements of protein engineering Selected chapters from the following books: Glick & Pasternak, Biotecnologia Molecolare, Zanichelli Primrose, Twyman & Old, Ingegneria Genetica, Zanichelli Watson, Caudy, Myers & Witkowski, DNA Ricombinante, Zanichelli 2008 (II Edizione) Brown, Biotecnologie molecolari, Zanichelli 2007
The teaching resources will be available on the Moodle platform
Non-attending students are encouraged to contact the teacher for information on the program, teaching resources and how to assess their achievement.
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Dates of beginning and end of teaching activities
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From to |
Delivery mode
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Traditional
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Attendance
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not mandatory
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Evaluation methods
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Oral exam
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Module: BIOMOLECULAR TECHNIQUES
(objectives)
The course of CELLULAR BIOCHEMISTRY (MODULE A) AND BIOMOLECULAR TECHNIQUES (MODULE B) intends to provide students with (i) theoretical knowledge in the field of cellular biochemistry, deepening the mechanisms that regulate the cell cycle in eukaryotes, (ii) theoretical and practical knowledge in protein engineering, and iii) theoretical and practical knowledge of the major molecular biology and biochemical techniques applied to the study of genes, genomes, proteins and proteomes. Experimental approaches will be discussed, making use also of bioinformatics, to address complex biological questions in biochemistry and molecular biology. In detail, for MODULE B: Specifically, it is intended to provide students with specific skills for the manipulation and analysis of nucleic acids and proteins (mutagenesis and genome editing techniques, differential proteomic), for the analysis of gene expression levels (qPCR, microarrays, differential transcriptomic) and gene expression regulation (study of epigenetic modifications and protein-DNA interactions), for the study of transduction signal pathways by protein-protein interaction analysis. The advances in the field of the sequencing of whole genomes and the application of biomolecular techniques in diagnostic field will be also discussed. Bioinformatics tools will be used for in silico prediction of interaction between biomolecules, or as complementary for the use of the discussed techniques (for input or output analysis). Finally, laboratory practical experiences will be organized to acquire techniques for studying nucleic acids and proteins.
b) EXPECTED LEARNING OUTCOMES: 1) Knowledge and understanding MODULE B:To know the basic techniques used in the field of fundamental and applied research. They will have an in-depth knowledge of molecular and advanced techniques and the related bioinformatics tools to support them; they will know the importance of statistical validation of the results of an experiment and of the controls that make an experiment scientifically reliable. 2) Applying knowledge and understanding MODULE B:At the end of the course the students will be able to use the acquired knowledge to evaluate and interpret the results of an experiment, identify its strengths and weaknesses and optimize it by evaluating the possible impact of variations in key experimental parameters; orient themselves among the main qualitative and quantitative methods to select the most suitable ones for studying the biological problem of interest; perform the experiments carried out during the practical part of the course. 3) Making judgements MODULE B: Students will be able to interpret and discuss the scientific papers presented during the course and be able to design and express new proteins with different characteristics. Students will have to acquire the ability to understand and critically discuss the experimental results obtained in the laboratory and use them as a starting point for planning subsequent experiments. 4) Comunication skills MODULE B: Students should have the ability to convey the acquired knowledge in a clear and comprehensible manner, even to people who are not in the field, and must demonstrate the ability to present information also with schemes and formulas. 5) Learning skills MODULE B: Students will have to be able to describe scientific topics related to the course. This skill will be developed through the active involvement of students during class discussions and practical experiences during the hours dedicated to the experimental laboratory.
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Language
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ITA |
Type of certificate
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Profit certificate
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Credits
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6
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Scientific Disciplinary Sector Code
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BIO/10
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Contact Hours
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32
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Laboratory Hours
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16
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Type of Activity
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Core compulsory activities
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Teacher
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PROIETTI Silvia
(syllabus)
Theoretical part (32 hours) Introduction to nucleic acids and proteins handling: Methods for DNA, RNA and proteins isolation from biological samples and related analysis. Comparison between protein samples: differential-in-gel electrophoresis (DIGE). Methods for gene expression analysis: Real-time PCR. DNA microarray technology. RNA-Seq. Methods for protein-protein interaction analysis: Far-Western, Pull-down, yeast-two/three hybrid assay, Co-Immunoprecipitation, Tandem Affinity Purification (TAP) system, Phage Display, Bimolecular Fluorescent Complementation (BiFC), FRET. Methods for DNA/RNA-protein interaction analysis: Chromatin Immunoprecipitation assay (ChIP and ChIP-on-chip). Electrophoretic Mobility Shift Assay (EMSA). DNA pull-down. Southwestern. Yeast three-hybrid system. Methods for epigenetic modifications analysis : DNA Methylation analysis: Methylation-Sensitive Amplification Polymorphism (MSAP). Bisulfite (non methylation)-specific PCR and Methylation-specific PCR (MSP). ATAC-Seq. Methods for histone modifications analysis by ChiP. Mutagenesis techniques and genome editing: Site-directed mutagenesis and CRISPR-Cas9 System. Diagnostic application of PCR. Next Generation Sequencing: second and third generation sequencing platforms. Bioinformatics tools will be used for in silico prediction of interaction between biomolecules, or as complementary for the use of the discussed techniques (for input or output analysis).
Practical part (16 hours) Total RNA extraction, RT-qPCR. Genomic DNA extraction. DNA methylation analysis. Total protein isolation and protein-protein interaction assays.
(reference books)
Brown T.A. Gene cloning and DNA analysis: an introduction. 7th ed., 2016, Wiley-Blackwell. Watson J.D., Caudy A.A., Myers R.M., Witkowski J.A. Recombinant DNA, genes and genomes – a short course. 3rd ed., 2007, W.H. Freeman & Co. Lesk A.M. Introduction to Genomics. 3rd ed., 2017, Oxford University press.
Slides are available in the teaching platform. Handouts are provided by the teacher for practical activities. Non-attending students are encouraged to contact the teacher for information about the program, teaching materials, and the examination mode.
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Dates of beginning and end of teaching activities
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From to |
Delivery mode
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Traditional
|
Attendance
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not mandatory
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Evaluation methods
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Oral exam
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|
|
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