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

CHRONIC KIDNEY DISEASE (CKD) Anemia Cardiovascular disease 2 a a 3 t times imes a w week 4 h hours s each quea easi sines ess Strict treatment dizzines zziness

CHRONIC KIDNEY DISEASE WORLDWIDE 10 - 14% R$ 1.4 billion or US$ 570 million

WHY!? Where these expenses come from?! 1 Equipment & Infrastructure 2 No easily handled devices 3 Sensitivity

1 Equipment & Infrastructure 2 No easily handled devices 3 Sensitivity

3 1 2

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 1 . 2 . AIP (Auto inducer peptide) is released and binds to the ComD receptor 3 . ComE phosphorilation 4 . LasR expression lasR 2 . [ CysC ][ CatS ]   Ki 0 . 008 nM CysC CatS [ : ] 3 . 4 . lasR

Diagnosis Module Increase creased Prognosi gnosis levels Cys C for CKD We already have the means to... production How do we discriminate between increased and normal Cys C concentrations?

qteE (BBa_K1521000) RBS qteE SpoVG  Pseudomonas aeruginosa Imunoblots. Espressing qteE from the beggining of the culture reduces LasR accumulation.  Responsible for creating expres ressi sion on thres resholds holds Hamp mpers LasR ability to promote  downstream gene expression  Const stitu tuti tive expression SIEHNEL R, A unique regulator controls the activation threshold if quorum-regulated genes in Pseudomonas aeruginosa . Proc ocee eedin ings s of the Nati tion onal 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 old d for Cys discrimination

Response Module  Indu duced ced by by  , practical mesurement reasons Example from Interlab Study Control Device 1

I N P U T R E E S S P P O N N S S E E High Levels High Levels of of LasR LasR GFP GFP Cystatin C Cystatin D I I A A G G N O O S S I I S S :

I N P U T R E E S S P P O N N S S E E High Levels High Levels of of LasR LasR GFP GFP Cystatin C Cystatin D I I A A G G N O O S S I I S S :

I N P U T R E E S S P P O N N S S E E High Levels High Levels of of LasR LasR GFP GFP Cystatin C Cystatin SICK D I I A A G G N O O S S I I S S :

I N P U T R E E S S P P O N N S S E E Normal Levels of Normal Levels of LasR LasR GFP GFP Cystatin C Cystatin D I I A A G G N O O S S I I S S :

I N P U T R E E S S P P O N N S S E E Normal Levels of Normal Levels of LasR LasR GFP GFP Cystatin C Cystatin D I I A A G G N O O S S I I S S :

I N P U T R E E S S P P O N N S S E E Normal Levels of Normal Levels of LasR LasR GFP GFP Cystatin C Cystatin S : HEALTHY D I I A A G G N O O S S I I S

Important  One crucial step for the feasibility of our project is the proper barrier er set et up After construction IPTG  Biodetector calibrat bration ion  Standardized Cathepsin S solution  Cys C concentration mimiking O.D. normal blood levels

Chassi

Assembly Map

Mathematical Modelling

Chemical Kinectics  Ordinary differential equation     d A  A dt  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 P Veg P lasR gfp lasR Control Result

Creation of the Expression Barrier P Veg P Veg P lasR qteE gfp lasR Control Result

Next steps  Complementary experiments with extra circuits:  Objective: Characterize the LasR:QteE interaction (ratio and induced response) P veg P lac lasR qteE P lasR gfp P veg P lac lasR qteE

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 olds for more qua uantit itati tive biological system on of cellular pathways; conc  Coor ordina dinati tion ncentrat entration ion trigge gers

1. Easy sy to Use 2. Low 5. Chea eaper per Sample devices Usage Microfluidics 4 . . Less ss 3. More dependent accurat rate on costly stly and detection reproducibl ducible instruments

Transform complex biodetection system into user-friendly devices

1. 1. Spores 2. Activation medium + IPTG 3. Standardized 4. Sanitation solution Cathepsin S Solution Reaction Chamber

1. 1. Spores 2. Activation medium + IPTG 3. Standardized 4. Sanitation solution Cathepsin S Solution Reaction Chamber

1. 1. Spores 2. Activation medium + IPTG 3. Standardized 4. Sanitation solution Cathepsin S Solution Reaction Chamber

1. 1. Spores 2. Activation medium + IPTG 3. Standardized 4. Sanitation solution Cathepsin S Solution Reaction Chamber

1. 1. Spores 2. Activation medium + IPTG 3. Standardized 4. Sanitation solution Cathepsin S Solution Reaction Chamber

1. 1. Spores 2. Activation medium + IPTG 3. Standardized 4. Sanitation solution Cathepsin S 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 Social al Developmen opment Scient ntif ific ic Development elopment

It’s Time !

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