Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen

(1)

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

(2)

INTRODUCTION INTO

MOLECULAR MEDICINE

Editors: László Nagy and Bálint László Bálint

Written by: Bálint L. Bálint L., Bertalan Meskó, László Nagy, Árpád Lányí, Beáta Scholtz, Lajos Széles and Tamás Varga

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

(3)

TÁMOP- 4.1.2-08/1/A-2009-0011 A projekt az Európai Unió támogatásával az Európai Szociális Alap társfinanszírozásával valósul meg.

Introduction to Molecular Medicine: Basics

László Nagy

(4)

History I.

1869 F. Miescher isolated an acidic substance from cell nuclei and named it nuclein

1944 O. Avery: The DNA of Pneumococcus contains the genetic information 1950 E. Chargaff A-T, G-C ratios are equal

1953 R. Franklin, M. Wilkins, J. Watson, F. Crick DNA double helix

1956 A. Kornberg DNA polymerase, later: mRNA, plasmids, bacterial genes, DNA modification

1966 V. McKusick Mendelian Inheritance in man

1970 H. Temin, D. Baltimore Reverse transcription

(5)

Introduction to Molecular Medicine

History II.

1960’es: Restriction enzymes, ligases H. Smith, D. Nathans, W. Arber

1972 P. Berg recombinant DNA

1976 Cloning of the first eukaryotic gene (beta globin) T. Maniatis DNS probes, hybridisation

1975 solid phase hybridisation E. Southern, Southern blot 1977 DNA sequencing F. Sanger, W. Gilbert

1978 Split genes W. Gilbert, P. Sharp, R. Roberts

1980 D. Botstein DNA polymorphism as marker,

1987 R. Conn mitDNA, Afrikan origin of Mankind

(6)

History III.

Modern times 1980-2000

Functional cloning thalassemies Positional cloning

1986 Chronic granulomatosis disease S. Orkin

1987 Duchenne muscular dystrophy

(7)

Genes identified by positional cloning

Cystic fibrosis

Neurofibromatosis 1,2 Testis factor

Fragile X

Familiars Adenomatosis Polyposis Myotonic dystrophy

Huntington disease

DNA repair defects, ataxia teleangiectasy Bloom syndrome

BRCA

Hemochromatosis

(8)

Polymerase Chain Reaction PCR

1985 K. Mullis, R. Saiki Cetsu Corporation, California Nobel prize 1993

DNA amplification is virtually unlimited.

Human Genome Program 1990-2000

1990-1995 HUGO

1996-2000 HGP

(9)

Genomic Information Is Freely Available

(10)

(11)

The Genome

Beáta Scholtz

(12)

Circular DNA in mitochondria

DNA organized in

chromosomes in nucleus

Localization of genetic material

(13)

Human chromosomes

(14)

30X 10 000X 400 000X

The structure of the chromatin in the cell

(15)

myglassshallnotpersuademeiamoldsolongasyouthandthouareofo nedatebutwhenintheetimesfurrowsibeholdthenlookideathmydays shouldexpiateforallthatbeautythatdothcovertheeisbuttheseemlyr aimentofmyheartwhichinthybreastdothliveasthineinme:howcanit henbeelderthanthouartothereforelovebeofthyselfsowaryasinotfo rmyselfbutfortheewillbearingthyheartwhichiwillkeepsocharyaste ndernurseherbabefromfaringillpresumenotonthyheartwhenminei sslainthougavstmethinenottogivebackagai n

My glass shall not persuade me I am old, So long as youth and thou are of one date;

But when in thee time's furrows I behold, Then look I death my days should expiate.

For all that beauty that doth cover thee, Is but the seemly raiment of my heart, Which in thy breast doth live, as thine in me:

How can I then be elder than thou art?

O! therefore love, be of thyself so wary As I, not for myself, but for thee will;

Bearing thy heart, which I will keep so chary As tender nurse her babe from faring ill.

Presume not on thy heart when mine is slain, Thou gav'st me thine not to give back again

MBP H3K9 MET HP1

TSA, BUTYRATE, Valproic acid AZACYTIDINE

JMJ ACTIVATORS, Pargyline

DNA is not a linear structure

MBP

H3K9 MET HP1

(16)

HG

Nuclei

> 3000 Mb

Mitochondria 16 kb

Coding Non coding

regions

Genes

Structure of the Human Genome

Non-gene regions

Known function

Regulatory

Unknown function

Repetitive 1-5%

45-50%

(17)

Repetitive regions: Pseudogenes

exon

intron DNA

mRNA

Transcription

DNA

Reverse transcription

(18)

The study of human genome

Bálint L. Bálint

(19)

Fred Sanger

A, G, T, C

Sanger sequencing

(20)

1. Chromosome:

> 250 mil bp

2. DNA ffragments:

150 k bp 3. Cloning into bacterial

libraries

4. Identification of inserts

5. Large inserts are further fragmented

6. Small fragments are

sequenced

7. Bioinformatic assembly

Hierarchic “shotgun” sequencing

(21)

Can this be simpler?

*Without bacteria!

*Amplify DNA in a clonal way only with enzymes!

(22)

“454” sequencing technology

DNA library

1 DNA/ bead

Oil/water emulsion microreactors

Beads on a plate

Millions of parallel sequencing

reactions

(23)

Genes and diseases

László Nagy

(24)

Genes might be present on both strands

(25)

Mobile genetic elements

(26)

Mobile genetic elements 2

(27)

(28)

Deletions in Duchenne and Becker muscular distrophy

(29)

(30)

(31)

Defects caused by mutations in the collagen genes

(32)

Mutations

Deletion Insertion

Single nucleotide replacement missense

non-sense

splice site

framshift

dynamic

(33)

Loss of function Gain of function Haploinsufficiency Dominant negatíve Null allele or amorph

Hypomorph Neomorph

Hypermorph Antimorph

Consequences of mutations:

(34)

Ways to destroy the function of a gene

Deletion: the whole gene or parts Insertion

Changing its structure translocation inversion

Changing promoter activity mutation

methylation

Changing mRNA stability polyA mutation

non-sense mediated decay

Changes in Splicing

donor site inactivation acceptor site inactivation change in splicing enhancer new spicing site

Frame shift Stop codon

Essential AA change

Change in posttranslational modification

Change in subcellular localisation

(35)

Nuclear receptors: beyond the waterfalls

Lajos Széles

(36)

General mechanism of Nuclear Receptor activity

DNA Ligand

N C

AF-2 AF-1

F E

D C

A/B

Ligand metabolism Direct ligand

Nuclei

RXR

5’- AGGTCA -3’ ^DR

„empty” receptor

Endogenous ligand

n

(37)

Groups of NRs

Endocrine receptors

“adopted” orphan receptors

Orphan receptors Ligand High affinity

hormones

Low affinity metabolites

unknown ER

PR AR GR MR

RAR TR VDR EcR

RXR PPAR LXR FXR

PXR/SXR CAR

SF-1 LRH-1 SHP TLX

NGFI-B ROR ERR HNF-4

COUP-TF

(38)

(39)

General mechanisms III

There are more than 300 cofactors!!!

HDAC inhibitors can be used in antitumor therapies

(epigenetic therapies) SAHA (Vorinostat=suberoylanilide hydroxamic acid,

CTCL (T sejt lymphoma)

(40)

Microarrays can be used to map the activity of

nuclear receptors

(41)

RXR a RAR

a RXR

a ^LXR ^a RXR

a

9-cis RA

diferentiation

Lipids

PPAR

g

diferentiation Lipid metabolism

Inflamation Diabetes (T2D)

Oxysterols

Cholesterol metabolism

9-cis RA, rexinoids

ATRA

Inflamation atherosclerosi s

RXR

a VDR

Retinoids Vitamin D

diferentiation

Bone metabolism

osteoporosis

immunedeficiencies

RxR heterodimers

(42)

(43)

Personalized Genetics

Bertalan Meskó

(44)

ApoE and Alzheimer’s disease ?

Apolipoprotein E

↓ 2 SNP

↓

4 variant (E1-4)

↓ 4 AA

↓

E4 susceptibility

↓

E2 less susceptible

(45)

Genes

Monogenic disease Poligenic diseases

e.g haemophylia

E.g. Diabetes

(46)

First steps

Syncumar (Warfarin, Coumadin) Two genes:

 CYP2C9

 VKORC1

(47)

(48)

(49)

Immunodeficiencies

Árpád Lányi

(50)

Environment

Self

Non-self Dangerous ,

Pathogens

Immunesystem

Tolerance

Immune response

The immunesystem as a black box

(51)

(52)

Immune deficiencies

Inherited

Immune genes are mutated Increased susceptability

towards infections

Specific to some pathogens known since 1950mainly

due to the usage of antibiotics

Aquired

Infectious AIDS

Other viruses Malnutrition Iatrogenic

Drugs

Radiioactivity

(53)

Inherited Immunedeficiencies

Mutation in a recessive gene: dominant ones are eliminated by selection

Autosomal genes

Homozygous children are affected Heterozygous are the carriers

X linked

Males are affected Womens are carriers

Mutation of IFNγ receptor - dominanat

Uncontrolled infection caused by the non pathogenous

strain Bacilus Calmette Guerlin (Mycobacterium used for

vaccination)

(54)

(55)

(56)

(57)

(58)

Types of inherited immune deficiencies

B-cell deficiency

-Extracell. Bakt. infection

B cell development

(XLA, IgA deficiency)

B – T interaction

CD40 ligand, hiper IgM

T cell DEFICIENCY SCID,

T cell development

IL-7/Jak3

Citoszkeleton

Thymus epithelial cells development

DiGeorge syndrome

Purine metabolism problems

DNA repair problems

MHC II synthesis problems

(59)

• Complement system

Extracellular bacterial infection

• Cellular surface and soluble factors

• C3

• C1 – C4

• Complement inhibitory factors

Phagocytic System

• CD18 adhesion

• NADPH oxidase

• Vesicular fusion

The Complement and the Phagocytic systems

(60)

Molecular mechanism of cancer 1.

Tamás Varga

(61)

Lifetime Probability of Developing Cancer, Men, 2002-2004*

Site Risk

All sites ^† 1 in 2

Prostate 1 in 6

Lung and bronchus 1 in 13

Colon and rectum 1 in 18

Urinary bladder ^‡ 1 in 27

Melanoma 1 in 41

Non-Hodgkin lymphoma 1 in 46

Kidney 1 in 59

Leukemia 1 in 67

Oral Cavity 1 in 71

Stomach 1 in 88

(62)

All sites 50 54 66

Breast (female) 75 79 89

Colon 51 59 65

Leukemia 35 42 50

Lung and bronchus 13 13 16

Melanoma 82 87 92

Non-Hodgkin lymphoma 48 53 64

Ovary 37 40 45

Pancreas 2 3 5

Prostate 69 76 99

Rectum 49 57 66

Urinary bladder 74 78 81

Site 1975-1977 1984-1986 1996-2003

Trends in Five-year Relative Survival (%)* Rates, US, 1975-2003

(63)

US Mortality, 2005

 1. Heart Diseases 652,091 26.6

 2. Cancer 559,312 22.8

 3. Cerebrovascular diseases 143,579 5.9

 4. Chronic lower respiratory diseases 130,933 5.3

 5. Accidents (unintentional injuries) 117,809 4.8

 6. Diabetes mellitus 75,119 3.1

 7. Alzheimer disease 71,599 2.9

 8. Influenza & pneumonia 63,001 2.6

 9. Nephritis* 43,901 1.8

 10. Septicemia 34,136 1.4

Rank Cause of Death No. of

deaths

% of all

deaths

(64)

(65)

(66)

Molecular mechanism of cancer 2.

Tamás Varga

(67)

(68)

(69)

(70)

(71)

(72)

(73)

OBESITY

-molecular mechanisms-

Bálint L. Bálint

(74)

(75)

(76)

(77)

Afferent signals 1. Fatty acid receptor in tongue 2. GI peptides are satiety signals

3. Leptin is a key afferent signal from the adipose cell

4. A dip in plasma glucose level precedes the meals

The central controler 1. Integration is taking place in medial hypothalamus

2. Signals are monoamines: histamine, serotonine, dopamine, norepi.

3. Neuropeptid Y, Agouti related pept, ghrelin, orexin and opioids stimulate feeding

4. A-MSH, cocaine amphetamine and corticotropine releasing hormone

decrease feeding

(78)

Efferent signals

1.Growth hormone, testosterone and estrogene control fat distribution

2.Glucocorticoids modulate adipose diffferentiation

3. Insulin is essential for fat cell differentiation and

lipogenesys/lipolysis

(79)

(80)

Pharmaceutical treatment of obesity:

1. Thyroid extract 1893

2. Dinitrophenol 1920 -cataract and neuropathy 3. Amphetamine 1930

4. Thyroid+digitalis+diuretics+amphetamine=Rainbow pill (caused sudden death)

5. Aminorex -apetite suppressor 1970= pulmonary hypertension 6. Very low calorie diets - poor quality protein caused death