Content of lectures I. Introduction: History of mutation genetics. Classification of mutation types. Genotoxic stress. II. Spontaneous mutations: Types of spontaneous mutations. Hypermutation. Instability of expanded trinucleotide repeats. Oxidative stress. III. Induced mutations: Mutagens: sources and mechanisms of acting (UV, ionizing radiation; chemical and biological mutagens). IV. Mutagens and carcinogens in human environment: Mutagen vs. carcinogen, genotoxic and non-genotoxic carcinogens, co-carcinogens, procarcinogens. The most often environmental mutagens and carcinogens, mechanisms of their acting and their sources. Chemotherapeutic agents - mechanisms, treatment. V. Metabolism of mutagens and promutagens: Detoxification and toxification of mutagens. Metabolic differences among species, strains, and experimental systems. Effect of phytochemicals on metabolism of mutagens and promutagens. VI. Mutagenicity testing 1: Test system, comparison of their sensitivity and validity. Microbial systems. Mutagenesis assays in cultured mammalian cells. VII. Mutagenicity testing 2: Cytogenetic tests. Plant, Drosophila, and mammalian systems. Testing and monitoring human populations. VIII. Laboratory mutagenesis: Random and targeted mutagenesis - chemically, radiation, transposons, site-directed mutagenesis, homologous recombination, zinc-finger nucleases, Delitto perfetto, gene trapping, gene knockout, gene knockdown (RNAi), GAL4/UAS system. IX. Cell response to DNA damage: DNA damage recognition. Cell cycle checkpoints, their molecular components. Signal pathways. Role of p53. Apoptosis. X. Mechanisms of DNA repair: NHJE (non-homologous end-joining), SSA (single strand annealing), HR (homologous recombination), BER (base excision repair), NER (nucleotide excision repair), enzymatic photoreactivation, GGR (global genome repair), XPC complex, TCR (transcription coupled repair), role of RNA polymerase II, DAR (domain associated repair), coordinating of DNA repair processes with cell cycle. XI. Chromatin structure during DNA repair and carcinogenesis: DNA repair with respect to chromatin dynamics, histones, post-translation modifications of histones. Chromatin structure as a signal for cell cycle checkpoints. Chromatin changes during carcinogenesis, chromatin role and epigenetic gene silencing during tumor suppression. Chromatin components and its modifiers as modern biomarkers of genotoxic stress and carcinogenesis. XII. Genotoxic risk in humans: Genetic diseases with DNA repair defects. Syndromes of premature aging. Telomeric instability: role in cell proliferation, genome stability, carcinogenesis, and aging. Effect of genetic polymorphism, life style and exposition to genotoxic agents in environment, effect of life style character on a rate of genotoxic stress, effect of diet, epidemiological and laboratory studies of a diet composition, effect of antioxidants. Environmental health monitoring data on mutagenic stress in regions of Czech Republic. XIII. Gene therapy: Viral and non-viral vectors. Controlled gene transfer using natural and synthetic polymers. Immune response to gene therapy (TLR), risk of gene therapy, perspectives, DNA vaccines in immunotherapy and tumor treatment, clinical studies, ethic aspects. Content of practicals Work with mutant strains of Drosophila, techniques for maintenance of Drosophila laboratory strains and preparation of crosses, identification of marker mutations, balancer chromosomes. Mutagenicity testing of several mutagen and potential mutagen agents using Drosophila somatic mutations, statistic analysis. Oxidative stress testing.
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