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Úvod do molekulárnej medicíny MUDr. Ing. Bc. Peter Celec, PhD.
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2 Linus Carl Pauling (1901 – 1994) Nobel prize 1954 – Chemistry 1962 – Peace
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3 „Life is a relationship between molecules, not a property of any one molecule. So is therefore disease, which endangers life. While there are molecular diseases, there are no diseased molecules.“ Linus C. Pauling, 1962
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4 Basics
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Francis Crick 8.6.1916 – 28.7.2004
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James Watson 6.4.1928 –
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11 Molecular Medicine Based on molecular knowledge Based on XNA Proteomics Genomics Molecular interactions Regulations Signalling pathways Gene therapy
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12 What we will NOT talk about... XNA structure Monogenic disorders Immunology Carcinogenesis
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13 Genome ( DNA ) Transcriptome ( RNA ) Proteome ( protein ) Metabolome ( pathways ) Genome
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15 Information processing Communication Intercellular Intracellular
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17 Signalling pathways
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18 Ligands Hydrophilic Small molecules Larges proteins Hydrophobic Steroids Thyroxine Retinoids
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19 Receptors G-protein coupled Tyrosine kinases Ion channels Intracellular
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20 Signalling pathways
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21 Phosphate
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22 G-protein coupled
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23 G-protein coupled Animation
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24 Second messangers cAMP cGMP IP 3 DAG Ca ++
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25 Gene „The gene is by far the most sophisticated program around.“ Bill Gates
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26 Gene expression regulation
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28 Jak – Janus (Roman god of gates and doors) Kinase Jak – Just Another Kinase STAT – Signal Transducers and Activators of Transcription APS – Adaptor protein with Pleckstrin homology and Src homology 2 domains C-Cbl - Casitas B-lineage Lymphoma EPO – Erythropoietin
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29 Tyk2 – Tyrosine kinase 2 mTOR – mammalian Target Of Rapamycin MAPK – MAP (Microtubule Associated Protein) Kinase MAPK – MAP (Mitogen Activated Protein) Kinase ISRE – Interferon-Stimulated Response Element GAS – IFN-Gamma-Activation Site
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30 GR – Glucocorticoid Receptor FKBP – FK506 (tacrolimus)-Binding Protein HSP – Heat Shock Protein GRE – Glucocorticoid Response Element
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31 c-Fos – FBJ (Finkel MP, Biskis BO, Jinkins PB) OsteoSarcoma virus AP-1 – Activator Protein 1 TGACTCA
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32 FAP FAP – c-Fos AP-1 binding site IP-1 – Inhibitory Protein 1
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33 NFκB
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34 FasL – Fas Ligand TRAF – TNFα Receptor Associated Factor NIK – NFκB Inducing Kinase CBP – CREB Binding Protein NFkB
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35 MAP kinases GEF – Guanine Nucleotide Exchange Factor
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36 MAP kinases Animation
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37...but...
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38 Signalling pathways
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39 Responses???
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42 Apoptosis
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43 Apoptosis Videos
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46 Autophagy MacroautophagyMicroautophagy
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49 Oxygen sensing
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50 HIF – Hypoxia Inducible Factor IGFR – Insulin like Growth Factor Receptor EGFR – Epidermal Growth Factor Receptor HER2 – Human Epidermal growth factor Receptor 2 PI3K – PhosphatidylInositol-3-Kinase AKT – serine/threonine protein kinase B FRAP – FKBP rapamycin associated protein PH – Prolin Hydroxylases HDAC – histone deacetylase VHL – von Hippel-Lindau tumour suppressor protein FIH – factor inhibiting HIF-1α B – elongin B C – elongin C C2 – cullin 2 HRE – hypoxia response element GLUT – glucose transporter ARNT – Aryl hydrocarbon Receptor Nuclear Translocator
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52 Angiogenesis
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55 Aaron Ciechanover Avram Hershko Irwin Rose 1/3 of the prize Israel 1/3 of the prize Israel 1/3 of the prize USA Technion – Israel Instutute of Technology Haifa, Israel Technion – Israel Institute of Technology Haifa, Israel University of California Irvine, CA, USA b. 1947b. 1937 (Karcag, Hungary) b. 1926 The Nobel Prize in Chemistry 2004 "for the discovery of ubiquitin-mediated protein degradation"
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56 Ubiquitin
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57 Ub E1 Ub E2 Ub E3 Ub 26S proteasome degradation Target Ub Ub Ub Ubiquitin pathway Ubi-activating EUbi-conjugating EUbi-ligase
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58 Ub ATP AMP + PPi E1 Ub E1 C E2 C Ub E3 C Ub Protein K Ub E3 Protein K K E2 Ub E3 Ub Protein K E3 E2 C Ub Protein Ub AMP + PPi Protein K K K K K K K K Proteosynthesis Antigen presentation H A B C1 C2 D E Ub Protein Ub ATP Protein Ub Protein Ub I F G J
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59 Molecular diagnostics
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60 Kary Banks Mullis (1944 – ) Nobel prize 1993 – Chemistry
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61 PCR
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63 PCR Expensive?
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65 PCR Analyzed by Electrophoresis
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66 Real-time PCR Quantitative PCR Fluorescent detection of PCR products in real time
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67 RFLP
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68 RFLP & VNTR Animation
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69 Blotting Western Southern Northern
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70 Western Blot
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71 Western Blot Animation
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72 Southern Blot
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74 Southern Blot Animation
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75 RT PCR Animation
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76 FISH Procedure
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77 FISH
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78 Frederick Sanger (1918 –) Nobel prize 1958 – Chemistry 1980 – Chemistry
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79 Sequencing Animation
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80 Microarray
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2006 Ústav patologickej fyziológie MUDr. Ing. Peter Celec, PhD. Génová terapia
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DefiníciaPrincípyRozdeleniaVektoryKlinikaProblémy
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Definícia Všetky prístupy na liečenie chorôb, ktoré využívajú nukleové kyseliny a génové inžinierstvo
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Princípy génovej terapie Náhrada génu Zabitie cieľovej bunky Oprava mutácie Cielená inhibícia génovej expresie
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Princípy génovej terapie Náhrada génu
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Princípy génovej terapie Zabitie cieľovej bunky
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Princípy génovej terapie Zabitie cieľovej bunky
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Princípy génovej terapie Zabitie cieľovej bunky indukciou imunitného systému
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Princípy génovej terapie Oprava mutácie
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Princípy génovej terapie Cielená inhibícia génovej expresie
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Princípy génovej terapie RNA interferencia
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Rôzne delenia génovej terapie Podľa funkčnej nukleovej kyseliny – DNA based – RNA based
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Rôzne delenia génovej terapie Podľa typu ovplyvnených buniek – Somatická – Zárodočná
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Rôzne delenia génovej terapie
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Podľa prístupu – In vivo – Ex vivo
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Rôzne delenia génovej terapie Podľa prístupu
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Vektory VírusovéNevírusovéBakteriálne
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Vírusové vektory RetrovírusAdenovírus Adeno-associovaný vírus HerpesvírusAlfavírus...
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Nevírusové vektory Nahá alebo obalená DNA Rôzne fyzikochemické transportné systémy
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Nevírusové vektory PlazmidyPCR produkty Injekcia nahej DNA Elektroporácia Gene gun Lipoplexy Polyplexy Oligodeoxynukleotidy
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Bakteriálne vektory Baktofekcia – Listeria – Escherichia – Salmonella Alternatívna génová terapia
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Baktofekcia
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Alternatívna génová terapia
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Klinika 1990 – prvá klinická štúdia Severe combined immunodeficiency (SCID) Retrovírusový vektor Integrácia do chromozómu Ex vivo
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Klinika
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Problémy GT Etické Prenos génu – Účinnosť – Špecificita – Bezpečnosť – Regulácia expresie
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September 11, 2001 February 15-16, 2001
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