In vivo and in vitro experimental models. Generation of monoclonal antibodies by DNA recombinant technologies. New vaccination strategies. The "omics":
genomics, proteomics, metabolomics, metagenomics and transcriptomics. Methods for gene expression manipulation. Gene therapy. DNA editing. NGS sequencing of DNA.
As a general basis, Molecular Cell Biology by Alberts et al., And Zanichelli, or any other good molecular and cell biology textbook. Due to the innovative nature of the topics covered, the course is continuously updated with teaching material provided by the teacher.
Learning Objectives
Acquisition of the knowledge of the most advanced techniques for the study of basic cellular and molecular biology, with aims of application in translational medicine.
Prerequisites
Bachelor's degree with the achievement of a good knowledge in biochemistry, molecular biology and cellular physiology.
Teaching Methods
Lectures and laboratory experiences.
Type of Assessment
Oral exam at the end of the course
Course program
The scientific method. Experimental animal models. Mammalian cell cultures from solid and liquid tissues. In vivo and in vitro models of angiogenesis. The role of TME in cancer. Humanized mouse models and their application in oncology and pharmacodynamics: BRGSF-a2-HIS and BLTS. Natural and artificial stem cell models: ESC, PSC, iPSC. 3D cell cultures. Differences among embryonic, adult and induced tissue stem cells. The Yamanaka factors. Applications of organoids in precision medicine. Applications of 3D cultures in the study of tumor metabolism, response to anticancer therapies and the determination of cancer stem cells: culture- and patient-derived tumor-spheres. Perspectives for 3D bioprinting. Generalities on the mechanisms of the cell-mediated immune response. Optimization of immunization techniques to obtain antibodies directed against specific antigens for applications in cell biology. New vaccination strategies: the COVID19 paradigm. Generations of mono- and polyclonal antibodies. Monoclonal antibodies in anticancer therapy: examples. Generation of humanized monoclonal antibodies by phage display and recombinant DNA techniques. Application of antibodies in cell biology: western blotting; immunofluorescence for cell sorting. Confocal fluorescence microscopy. Immuno- and co-immunoprecipitation. The ChIP. Alternative methods to immunofluorescence for the determination of biomolecules: conjugated phalloidin and DNA base intercalators. Examples of immunofluorescence image analysis obtained by confocal microscopy. Proteomics: two-dimensional electrophoresis and MS techniques. The BLAST system in the determination of ESTs. Principles of recombinant DNA. Enzymes and vectors. Vector modules necessary for gene cloning. Linkers and adapters. Gene expression vectors for formammalian cells: plasmid, retroviral (lentiviral) and adenoviral vectors. Pseudotyping of the viral envelope. Recombinant virus package. Lipofection. DNA libraries: differences between expression and genomic libraries. Construction of a genomic library. CONTIG alignment and sequencing. Next Generation Sequencing techniques (e.g. ILLUMINA). Examples of bioinformatic data processing banks and tools. The study of the transcriptome. Construction of an expression library: purification of mRNA, reverse transcription and adaptation of cDNAs for cloning into a plasmid. Preparation of the genome and transcriptome for sequencing by NGS. Single cell sequencing. The DNA barcoding. The Chip-seq. The double hybrid system in yeast. Transcriptome analysis: real time RT-PCR and RNA-seq. The Hidden Genome: History of the Discovery of RNAi. Molecular basis and significance of RNAi. Differences between siRNA and miRNA in expression regulation. Techniques for the introduction and expression of RNAi in mammalian cells. Experimental applications of the RNAi in silencing of gene expression. The lncRNAs: structure and functions. The ChiRP. The CLIP. Animal transgenesis. The mammary gland as an animal bioreactor. The tissue-specific promoters. Techniques for animal transgenesis: microinjection method in the nucleus; method of manipulating embryonic stem cells. The engineered mouse as an experimental model for the study of human pathologies. Gene knock-in and knock-out by gene targeting. Positional cloning for the determination of hereditary disease-linked genes. Determination of polymorphic alleles. Use of STR polymorphisms for gene mapping. Validation of the correlation between the pathology and mutations in the identified DNA sequence. NGS in the screening of mutations and in the generation of computer biobanks. The Cancer Genome Atlas (TCGA) paradigm. Gene therapy for gene replacement using recombinant viral vectors. Corrective gene therapy: the discovery of CRISPR systems in bacteria and the development of the CRISPR/CAS9 editing system.
Lab experience: DNA extraction from mammalian cells. High definition fluorescence microscopy: labeling of cells with fluorophores-conjugated probes or antibodies and/or DNA intercalators. Actin staining with rhodaminated phalloidin. Observation, acquisition and analysis of images for the observation of biological phenomena (eg: apoptosis; EMT; subcellular translocations of proteins, analysis of the cytoskeleton, etc.)