Úvod do molekulárnej medicíny MUDr. Ing. Bc. Peter Celec, PhD.

2 Linus Carl Pauling (1901 – 1994) Nobel prize 1954 – Chemistry 1962 – Peace

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

4 Basics

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Francis Crick –

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James Watson –

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11 Molecular Medicine Based on molecular knowledge Based on XNA Proteomics Genomics Molecular interactions Regulations Signalling pathways Gene therapy

12 What we will NOT talk about... XNA structure Monogenic disorders Immunology Carcinogenesis

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

18 Ligands Hydrophilic Small molecules Larges proteins Hydrophobic Steroids Thyroxine Retinoids

19 Receptors G-protein coupled Tyrosine kinases Ion channels Intracellular

20 Signalling pathways

21 Phosphate

22 G-protein coupled

23 G-protein coupled Animation

24 Second messangers cAMP cGMP IP 3 DAG Ca ++

25 Gene „The gene is by far the most sophisticated program around.“ Bill Gates

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

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

30 GR – Glucocorticoid Receptor FKBP – FK506 (tacrolimus)-Binding Protein HSP – Heat Shock Protein GRE – Glucocorticoid Response Element

31 c-Fos – FBJ (Finkel MP, Biskis BO, Jinkins PB) OsteoSarcoma virus AP-1 – Activator Protein 1 TGACTCA

32 FAP FAP – c-Fos AP-1 binding site IP-1 – Inhibitory Protein 1

33 NFκB

34 FasL – Fas Ligand TRAF – TNFα Receptor Associated Factor NIK – NFκB Inducing Kinase CBP – CREB Binding Protein NFkB

35 MAP kinases GEF – Guanine Nucleotide Exchange Factor

36 MAP kinases Animation

37...but...

38 Signalling pathways

39 Responses???

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42 Apoptosis

43 Apoptosis Videos

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46 Autophagy MacroautophagyMicroautophagy

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49 Oxygen sensing

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 (Karcag, Hungary) b The Nobel Prize in Chemistry 2004 "for the discovery of ubiquitin-mediated protein degradation"

56 Ubiquitin

57 Ub E1 Ub E2 Ub E3 Ub 26S proteasome degradation Target Ub Ub Ub Ubiquitin pathway Ubi-activating EUbi-conjugating EUbi-ligase

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 

59 Molecular diagnostics

60 Kary Banks Mullis (1944 – ) Nobel prize 1993 – Chemistry

61 PCR

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63 PCR Expensive?

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65 PCR Analyzed by Electrophoresis

66 Real-time PCR Quantitative PCR Fluorescent detection of PCR products in real time

67 RFLP

68 RFLP & VNTR Animation

69 Blotting Western Southern Northern

70 Western Blot

71 Western Blot Animation

72 Southern Blot

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74 Southern Blot Animation

75 RT PCR Animation

76 FISH Procedure

77 FISH

78 Frederick Sanger (1918 –) Nobel prize 1958 – Chemistry 1980 – Chemistry

79 Sequencing Animation

80 Microarray

2006 Ústav patologickej fyziológie MUDr. Ing. Peter Celec, PhD. Génová terapia

DefiníciaPrincípyRozdeleniaVektoryKlinikaProblémy

Definícia Všetky prístupy na liečenie chorôb, ktoré využívajú nukleové kyseliny a génové inžinierstvo

Princípy génovej terapie Náhrada génu Zabitie cieľovej bunky Oprava mutácie Cielená inhibícia génovej expresie

Princípy génovej terapie Náhrada génu

Princípy génovej terapie Zabitie cieľovej bunky

Princípy génovej terapie Zabitie cieľovej bunky

Princípy génovej terapie Zabitie cieľovej bunky indukciou imunitného systému

Princípy génovej terapie Oprava mutácie

Princípy génovej terapie Cielená inhibícia génovej expresie

Princípy génovej terapie RNA interferencia

Rôzne delenia génovej terapie Podľa funkčnej nukleovej kyseliny – DNA based – RNA based

Rôzne delenia génovej terapie Podľa typu ovplyvnených buniek – Somatická – Zárodočná

Rôzne delenia génovej terapie

Podľa prístupu – In vivo – Ex vivo

Rôzne delenia génovej terapie Podľa prístupu

Vektory VírusovéNevírusovéBakteriálne

Vírusové vektory RetrovírusAdenovírus Adeno-associovaný vírus HerpesvírusAlfavírus...

Nevírusové vektory Nahá alebo obalená DNA Rôzne fyzikochemické transportné systémy

Nevírusové vektory PlazmidyPCR produkty Injekcia nahej DNA Elektroporácia Gene gun Lipoplexy Polyplexy Oligodeoxynukleotidy

Bakteriálne vektory Baktofekcia – Listeria – Escherichia – Salmonella Alternatívna génová terapia

Baktofekcia

Alternatívna génová terapia

Klinika 1990 – prvá klinická štúdia Severe combined immunodeficiency (SCID) Retrovírusový vektor Integrácia do chromozómu Ex vivo

Klinika

Problémy GT Etické Prenos génu – Účinnosť – Špecificita – Bezpečnosť – Regulácia expresie

September 11, 2001 February 15-16, 2001