dizzines zziness CHRONIC KIDNEY DISEASE WORLDWIDE 10 - 14% R$ 1.4 - - PowerPoint PPT Presentation

CHRONIC KIDNEY DISEASE (CKD)

Cardiovascular disease Anemia

Strict treatment

4 h hours s each 2 a a 3 t times imes a w week

quea easi sines ess

dizzines zziness

CHRONIC KIDNEY DISEASE WORLDWIDE R$ 1.4 billion

• r

US$ 570 million

10 - 14%

Equipment & Infrastructure

1

No easily handled devices

2

Sensitivity

3

WHY!? Where these expenses come from?!

Equipment & Infrastructure

1

No easily handled devices

2

Sensitivity

3

1 2 3

Detection Module

 Quorum

rum sen ensin ing g system

 Identifies Cys C

Detection Module

The Cys C Biomarker

 More sensiti sitive e to small changes in the Glomerular Filtration Rate  - Concent centrat ation ion less s sucept eptib ible e to factor such as:

Detection Module

How it works ?

• 1. Cleavage of the Linker
• 2. AIP (Auto inducer peptide) is released

and binds to the ComD receptor

• 3. ComE phosphorilation

4.

lasR

3. 1. 2.

lasR

nM CatS CysC CatS CysC Ki 008 . ] : [ ] ][ [  

• 4. LasR expression

Diagnosis Module

How do we discriminate between increased and normal Cys C concentrations?

Increase creased levels Cys C Prognosi gnosis for CKD

We already have the means to...

production

qteE (BBa_K1521000)

qteE

RBS SpoVG

 Pseudomonas aeruginosa

 Responsible for creating

expres ressi sion

• n thres

resholds holds

 Const stitu tuti tive expression

• Imunoblots. Espressing

qteE from the beggining

• f the culture reduces

LasR accumulation.

• Hamp

mpers LasR ability to promote downstream gene expression

SIEHNEL R, A unique regulator controls the activation threshold if quorum-regulated genes in Pseudomonas aeruginosa. Proc

• cee

eedin ings s of the Nati tion

• nal Academy

y of Sciences ces USA, 2010, 107(17):7916-7921.

Diagnosis Module

Plac c promo moter er + qteE

• Manipulate the expression rate

through IPTG G induction

• Set the appropria

priate e thresh shol

• ld

d for Cys discrimination

Response Module

 Indu duced ced by by

 , practical mesurement reasons Control Device 1 Example from Interlab Study

High High Levels Levels of

• f

Cystatin Cystatin C LasR LasR

I N P U T

R E E S S P P O N N S S E E

GFP GFP

D I I A A G G N O O S S I I S S :

High High Levels Levels of

• f

Cystatin Cystatin C LasR LasR

I N P U T

R E E S S P P O N N S S E E

GFP GFP

D I I A A G G N O O S S I I S S :

High High Levels Levels of

• f

Cystatin Cystatin C LasR LasR

I N P U T

R E E S S P P O N N S S E E

GFP GFP

D I I A A G G N O O S S I I S S :

SICK

Normal Levels Normal Levels of

• f

Cystatin Cystatin C LasR LasR

I N P U T

R E E S S P P O N N S S E E

GFP GFP

D I I A A G G N O O S S I I S S :

Normal Levels Normal Levels of

• f

Cystatin Cystatin C LasR LasR

I N P U T

R E E S S P P O N N S S E E

GFP GFP

D I I A A G G N O O S S I I S S :

Normal Levels Normal Levels of

• f

Cystatin Cystatin C LasR LasR

I N P U T

R E E S S P P O N N S S E E

GFP GFP

D I I A A G G N O O S S I I S S : HEALTHY

Important

 One crucial step for the feasibility of our project is the proper barrier er set et up

After construction

IPTG O.D.  Biodetector calibrat

bration ion

• Standardized Cathepsin S

solution

• Cys C concentration mimiking

normal blood levels

Chassi

Assembly Map

Mathematical Modelling

Chemical Kinectics

   

A  dt A d

 Ordinary differential equation  Evolution of the concentration in time

• Initial conditions
• Other parameter for dynamic strength

 Our chemical reactions are those that regulate:

• Inducer/repressor binding/unbinding on promoter
• Enzyme activity and its interaction with inhibitor
• Reporter gene production (GFP)

The CAOS !!!

 ComE limit concentration experiments (Imperial College Team 2010)  Tokyo team Plac IPTG induction equation

 Considering the formation of a LasR-QteE complex

Creation of the Expression Barrier

lasR

gfp

PlasR PVeg Result Control

Creation of the Expression Barrier

lasR

gfp

PlasR PVeg

qteE

PVeg Control Result

Next steps

 Complementary experiments with extra circuits:

• Objective: Characterize the LasR:QteE interaction (ratio and

induced response)

gfp

PlasR

lasR

Plac

qteE

Plac

lasR

Pveg

qteE

Pveg

In a near Future

 After better characterization of the qteE threshold property, it can be used for:

• Creation of mul

ultiple iple st standar ndardi dize zed d thres eshold

• lds for more

qua uantit itati tive biological system

• Coor
• rdina

dinati tion

• n of cellular pathways; conc

ncentrat entration ion trigge gers

Microfluidics

• 1. Easy

sy to Use

• 2. Low

Sample Usage

• 3. More

accurat rate

and reproducibl ducible 4.

. Less ss

dependent

• n costly

stly

detection

instruments

• 5. Chea

eaper per devices

Transform complex biodetection system into user-friendly devices

1.

• 1. Spores
• 2. Activation medium +

IPTG

• 3. Standardized

Cathepsin S Solution

• 4. Sanitation solution

Reaction Chamber

1.

• 1. Spores
• 2. Activation medium +

IPTG

• 3. Standardized

Cathepsin S Solution

• 4. Sanitation solution

Reaction Chamber

1.

• 1. Spores
• 2. Activation medium +

IPTG

• 3. Standardized

Cathepsin S Solution

• 4. Sanitation solution

Reaction Chamber

1.

• 1. Spores
• 2. Activation medium +

IPTG

• 3. Standardized

Cathepsin S Solution

• 4. Sanitation solution

Reaction Chamber

1.

• 1. Spores
• 2. Activation medium +

IPTG

• 3. Standardized

Cathepsin S Solution

• 4. Sanitation solution

Reaction Chamber

1.

• 1. Spores
• 2. Activation medium +

IPTG

• 3. Standardized

Cathepsin S Solution

• 4. Sanitation solution

Reaction Chamber

How to use it

1) Press buttons 1 and 2 (spores activation) 2) Add blood sample in the reaction chamber 3) Seal reaction chamber with extra membrane 4) Press 3 5) Repeatedly press the membrane to mix the reaction chamber content 6) Wait for diagnosis 7) Interpretate result 8) Press 4 9) Ready to discard

The brazilian Jamboree

Brasil-SP team

Policy and Practices

Policy and Practices

Scient ntif ific ic Development elopment Social al Developmen

• pment

It’s Time !

Thank you all !!

Sponsors