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CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL FARMACO AA 2010-11 Adriana Maggi LEZIONE 13. INGEGNERIA ANIMALE APPLICAZIONI. I SISTEMI REPORTER POSSONO ESSERE UTILIZZATI PER LA GENERAZIONE DI ANIMALI IN CUI SI POSSANO MISURARE EVENTI MOLECOLARI SPECIFICI IN TEMPO REALE?.

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slide1

CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL FARMACO AA 2010-11

Adriana Maggi

LEZIONE 13

INGEGNERIA ANIMALE

APPLICAZIONI

slide2

I SISTEMI REPORTER POSSONO ESSERE UTILIZZATI PER LA GENERAZIONE DI ANIMALI IN CUI SI POSSANO MISURARE EVENTI MOLECOLARI SPECIFICI IN TEMPO REALE?

slide4

Functional Magnetic Nuclear Resonance

Nuclear imaging

MicroPET

Positron emission tomography

Micro SPECT

single-photon emission

computerized tomography

Bioluminescence

Optical imaging

Fluorescence

Ultrasound

2/06

MULTI-MODALITY MOLECULAR IMAGING IN LIVING SMALL ANIMALS

slide9

LA GENERAZIONE DI UN TOPO REPORTER

La scelta del:

sistemadiingegneriaanimale

reporter bioluminescenza, PET, NMR

costrutto

reporter

slide11

GENE

B-Galattosidasi (Batterica)

Ben caratterizzata, stabile, rilevazione automatizzabile

Utile per studi di tipo anatomico-funzionale

Luciferasi (Lucciola)

Alta attività specifica, mancanza di attività endogena (basso bkg)Richiede l’aggiunta di cofattore, O2, ATP.

Ligandi per sostanze radiomarcate

Difficoltà nel generare i ligandiradiomarcati

Utilizzabili per PET

fluorescent proteins (GFP, varianti RFP, BFP)

Monomerico, non richiede substrato, assente nei mammiferi, varianti con diversa l di fluorescenza. Bassa sensibilità per mancanza di amplificazione

I geni reporter

Presenza di attività endogena in cellule di mammifero, enzima tetramerico (risposta non lineare)

Applicabile a studi di bioluminescenza in vivo

Applicabile a studi di fluorescenza in vivo

slide12

LA GENERAZIONE DI UN TOPO REPORTER

La scelta del:

sistemadiingegneriaanimale

reporter bioluminescenza, PET, NMR

costrutto

reporter

slide13

Studio dellafunzionedipromotore

reporter gene

reporter gene

slide14

I sistemi reporter nello studio di rilascio/sintesi di trasduttori del segnale (complementazione)

slide16

+/-

+/-

ERE 2x

INSULATOR

( MAR )

firefly luciferase

INSULATOR

( MAR )

TK

light

luciferin + ATP = oxyluciferin + AMP +

ERE-Luc reporter mouse

ERE

The ERE-Luc reporter mouse: a model

tostudyof ER transcriptionalactivity

kinase-dependent

activation

Ciana et al., 2001

slide17

Evaluation of ER transcriptional activity

20 min.

i.p. of D-luciferin

5 min. after the acquisition

slide18

13.5

14.5

15.5

16.5

18.5

ERE-Luc mouse

dpc (day post conception)

ER istranscriptionallyactive at day 14.5 pc

slide19

ectoderm

endoderm

mesoderm

13.5

14.5

16.5

18.5

12.5

P1

dpc (day post conception)

post-natal day 1

ERE-Luc mouse

imaging

IHC

F

C

D

A

B

C – intestine 20x

D – bone 40x

E – heart 40x

F – forebrain 10x

G – moustache 40x

H – skin 20x

G

E

H

16.5 dpc

16.5 dpc

GP Rando, 2007

slide20

6

6

4

4

bone

bone

ovaries

ovaries

4

4

2

2

2

2

0

0

0

0

9

9

20

20

brain

brain

uterus

uterus

7

7

15

15

5

5

10

10

3

3

5

5

1

1

0

0

LUCIFERASE ACTIVITY (RLU)

4

4

12

12

thymus

thymus

hypothalamus

3

3

2

2

6

6

E

M

D

P

1

1

0

0

0

0

20

20

16

16

intestine

intestine

liver

liver

12

12

10

10

8

8

4

4

0

0

P

E

M

D2

0

0

P

P

E

E

M

D2

D

P

P

E

E

D

D2

ERE-Luc mouse

Luciferase activity in adult, cycling females

/ml)

ESTRUS

S

50

5

pg

0

40

(

4

E2 (pg/ml)

0

30

3

Estradiol

0

20

2

0

10

10

day 1

M

day 2

D

day3

P

day 4

E

Ciana et al, Nature Ned., 2003

slide21

Pregnancy

&

Embryo

Development

19.5

DAY 1

18.5

DAY 10

DAY 18.5

LIFE

17.5

DAY 16.5

CYCLE

16.5

DAY 15.5

15.5

DAY 14.5

14.5

13.5

Immature

DAY 13.5

Mice

12.5

AdultMice

ERE-Lucmicetounderstand ER involvement in mammalsphysiopathology

SucklingMice

slide22

THE COMPLEXITY OF

ESTROGEN ACTION

THE COMPLEXITY OF ESTROGEN TARGETS

  • REPRODUCTIVE SYSTEM
    • - male and femalegonads
    • - hypothalamus and pituitary
  • SKELETAL SYSTEM
  • VASCULAR SYSTEM
    • - endothelium
    • smoothmusclecells
  • RESPIRATORY SYSTEM
  • IMMUNE SYSTEM
  • NERVOUS SYSTEM
    • - central
    • - peripheral

G-protein,

IP3K...

SP1

NFKB

AP1

ERE

slide23

ER COMPLEXITY OF ACTION and A NEW CLASS OF DRUGS: Selective Estrogen Recepor Modulators

CNS

cardiovascular

reproductive

growthfactors

P

bone

SP1

NFKB

AP1

tissue-specific

coregulators

ERE

slide24

ESTROGEN REPLACEMENT THERAPY

The efficacy of SERMs on estrogen receptor transcriptional activity was measured in a model of surgical menopause (ovx mice)

SERMs ability to replace the natural hormone was evaluated by comparison with ER activity in healthy, cycling mice

slide25

Measuringbioluminscence

in the ERE-Luc reporter mouse

Thymic area

Hepatic area

Bioluminescenceafter

6h treatment with

15b-estradiol (50ug/kg)

Reproductiveorgans

(mammari glands and

vagina)

Intestine

Muscle-Skeletal System

slide26

days of treatment

pellet

CONTROLS

REFERENCE DRUG

DRUG OF INTEREST

In vivo analysis of photon emission

Manual

Automatic

Rando et al. 2009

slide27

REPORTER MICE TO STUDY DRUG ACTION “IN VIVO”

VAGINA

CHEST

Photonemission

Luciferaseenzymaticactivity

Vehicle

PCUD 3mg/kg

BZA 10mg/kg

BZA 10mg/kg + PCUD 3mg/kg

Raloxifene 10mg/kg

Biserni et al, in preparation

slide28

EffectsofSERMs on ER activity – in vivo imaging

CHRONIC treatment (21 days)

**

°

Vehicle

PCUD 3mg/kg

BZA 10mg/kg

BZA 10mg/kg + PCUD 3mg/kg

Raloxifene 10mg/kg

Biserni et al, in preparation

slide29

EffectsofSERMs on ER activity – in vivo imaging

CHRONIC treatment (21 days)

Vehicle

PCUD 3mg/kg

BZA 10mg/kg

BZA 10mg/kg + PCUD 3mg/kg

Raloxifene 10mg/kg

Biserni et al, in preparation

slide30

TAIL

LIMB

ACUTE

CHRONIC

Rando et al, Mol. Endocrinol. 2010

slide31

HEPATIC AREA

ABDOMEN

ACUTE

CHRONIC

Rando et al, Mol. Endocrinol. 2010

slide32

SERCHING FOR NOVEL MODALITIES TO MEASURE

THE EFFICACY OF SERMs

N° peaks, amplitude, frequency

AUC

slide33

GENITAL AREA

SKELETAL AREA

*

*

*

*

*

A

Peaks/21d

*

*

*

*

Amplitude

B

*

Period (d)

C

*

*

*

*

*

AUC

*

D

Rando et al, Mol. Endocrinol. 2010

*

*

*

*

E

Potency

*

*

slide34

PHENETICS OF DRUG ACTION

THE APPLICATION OF AGGLOMERATIVE HIERARCHICAL CLUSTERING

(AGGLOMERATIVE NESTING version 1.02 )

slide35

DEGREE OF FUNCTIONAL CORRELATION AMONG THE PARAMETERS SELECTED

a

b

Rando et al, Mol. Endocrinol. 2010

slide36

Space-temporal analysis of drug action in living animals

clustering data to generate novel families of compounds

Genital area

Skeletal area

Rando et al, Mol. Endocrinol. 2010

slide37

C

Reverse Medicinal Chemistry

A

Genital area

B

Skeletal area

Cl

Rando et al, Mol. Endocrinol. 2010

slide38

CONCLUSION 1

Adding the time dimension to the study of drug activity leads to a novel ability to define drug efficacy

slide39

INTACT

OVX

Biserni et al. in preparation

slide40

TISSUE SPECIFIC EFFECT OF OVX ON

THE AMPLITUDE AND FREQUENCY OF ER ACTIVITY

slide41

CONCLUSION 2

The possibility to measure in vivo the activity of estrogenic compounds on their target, may lead to the identification of novel and more efficacious therapies for the post-menopause

slide42

University of Milan

Center of Excellence on

Neurodegenerative Diseaseas

Paolo Ciana

ElisabettaVegeto

GianpaoloRando

Valeria Benedusi

Sara Della Torre

Cristina Vantaggiato

Cristian Ibarra

BalajiRamachandran

Andrea Biserni

Monica Rebecchi

Clara Meda

Collaborators at Milan University:

Paola Campadelli

David Horner

Funding: EU Strep EWA EWA LSHM-CT-2005-518245

EU IP CRESCENDO LSHM-CT-2005-018652

EU NoE DIMI LSHB-CT-2005-512146

NIH RO1(AG027713)

slide43

The real impact of molecular engineering on drug discovery

“The whole is more than the sum of its parts “

Aristotle (384 BC – 322 BC)

Methapysics

MODERN PHARMACOLOGY

NEEDS TO REVISIT ANIMAL MODELS

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