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Seminario molecular


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Seminario molecular

  2. 2. GENERAL OBJETIVE <ul><li>Demonstrate how  Prothrombin, Apolipoprotein A-IV and Haptoglobin concentrations in CSF has an important role in the HD evolution and compare these concentrations in HD and control patients  . </li></ul>
  3. 3. INTRODUCTION <ul><li>HD is a hereditary, degenerative brain disorder. It is caused by an unstable CAG trinucleotide. </li></ul><ul><li>Today there are others proteins involved in HD development and symptoms and its important to clarify through proteomics studies in CSF wich are the concentrations (up or down) and if this levels of biomarkers are correlated in HD. </li></ul>
  4. 4. PROTHROMBIN <ul><li>Plasma protein synthesized in the liver. </li></ul><ul><li>It plays an important function in coagulation process by reaction with the thromboplastin enzyme , with Ca ++ cation and V activate factor as coenzymes. </li></ul><ul><li>In HD prothrombin  pro inflammatory agent : DISEASE STATUS BIOMARKER </li></ul>Tromboplastina Ca++ Xa Va
  5. 5. APOLIPOPROTEIN AIV (APO AIV) <ul><li>Glycoprotein synthesized by the human intestine and liver </li></ul><ul><li>The formation of chylomicrons acts as a signal for the induction of Apo AIV synthesis </li></ul><ul><li>Alters the activity of the enzymes (LPL and LCAT) of lipoprotein metabolism and cholesterol efflux from extrahepatic tissues </li></ul><ul><li>Is involved in the long-term regulation of both food intake and body weight </li></ul>
  6. 6. HAPTOGLOBIN <ul><li>Protein produced by the liver </li></ul><ul><li>Haptoglobin binds free hemoglobin (Hb) released from erythrocytes with high affinity and thereby inhibits its oxidative activity </li></ul><ul><li>The haptoglobin-hemoglobin complex will then be removed by the reticuloendothelial system </li></ul><ul><li>It prevents loss of iron through the kidneys and protecting the kidneys from damage by hemoglobin </li></ul>
  7. 7. HUNITNGTON DISEASE - HD <ul><li>Progressive neurodegenerative disorder with autosomal dominant i nheritance : </li></ul><ul><li>- Movement Disorders. - Cognitive impairment. - Psychiatric disorders. </li></ul>
  8. 8. MATERIALES Y METODOS <ul><li>LCR y suero extraido de 9 HD ( 5 hombres- 4 mujeres). </li></ul><ul><li>LCR y suero de 18 pacientes tipo control (7 hombres- 11 mujeres). </li></ul><ul><li>Cuatro grupos: </li></ul><ul><li>1 Electroforesis de dos dimensiones. </li></ul><ul><li>3 Cuantificacion de protrombina, Apo-A IV y haptoglobina. </li></ul>PREPARACION DE LAS MUESTRAS
  9. 10. ELECTROFORESIS DE DOS DIMENSIONES (2-DE ) <ul><li>  Técnica de alta resolución cuyo objetivo es la separación de mezclas de proteínas </li></ul><ul><li>Punto isoeléctrico (isoelectroenfoque ). </li></ul><ul><li>ánodo es ácida y la del cátodo es alcalina </li></ul><ul><li>Masa molecular por  electroforesis gel de poliacrilamida </li></ul>
  10. 11. DIGESTION EL GEL <ul><li>Forma parte de la preparación de las muestras para la espectrometría de masas </li></ul><ul><li>Decoloración. </li></ul><ul><li>Reducción y alquilación. </li></ul><ul><li>Corte de la proteína serina proteasa tripsina </li></ul><ul><li>Extracción del péptido generado . </li></ul>
  11. 12. ESPECTROMETRIA DE MASA <ul><li>Técnica experimental que permite la medición de iones derivados de moléculas </li></ul><ul><li>Se basa en el fenómeno conocido como desbastado ( sputtering , en inglés) de partículas centradas en un blanco, que son bombardeadas por iones, átomos o moléculas. Dependiendo del intervalo de energía de la partícula primaria, ocurren colisiones. </li></ul><ul><li>El patrón de fragmentación resultante así como los iones residuales constituyen el espectro de masas </li></ul>
  12. 13. WESTERN BLOT <ul><li>Busco proteínas especificas </li></ul><ul><li>Uso ac </li></ul><ul><li>Uso técnica SDS-PAGE </li></ul><ul><li>Separación de proteínas por electroforesis en gel </li></ul>
  13. 14. ELISA <ul><li>Ensayo por inmunoabsorción ligado a enzimas </li></ul><ul><li>Fosfatasa alcalina—peroxidasa de rabano </li></ul><ul><li>Detección ag-ac </li></ul><ul><li>3 tipos: </li></ul><ul><li>Ensayo tipo sandwich </li></ul><ul><li>Ensayo competitivo </li></ul><ul><li>Ensayo indirecto </li></ul>
  14. 15. RESULTADOS <ul><li>Representative Western blots of prothrombin (72 kD) and albumin (64 kD) in CSF and serum of HD patients and their corresponding controls. </li></ul>
  15. 16. <ul><li>Representative Western blots of Apo A-IV (46 kD) and albumin (64 kD) in CSF and serum, respectively, of HD patients and their corresponding controls (C). </li></ul>RESULTADOS
  16. 17. <ul><li>Qalb, CSF haptoglobin, and serum haptoglobin concentration in HD patients. </li></ul>RESULTADOS
  17. 18. DISCUSSION AUTOR OPINION YES NO Bjorkqvist M. The mutant huntingtin may enchance the ability of microglia to produce proinflamatory mediators (IL-6, IL-8 and TNF-a) and contribute to neurodegeneration in HD. Djoussé L. In HD, weigth loss is a characteristic feature in advanced as well early stage. Fang Q, Strand In proteome analyses several the neuronal proteins were found to be reduced Underwood BR A serum metabolic profile indicative of a pro-catabolic phenotype in early HD
  18. 19. CONCLUTIONS <ul><li>The proteomic studies in CFS in patients HD and control patients are very important to determinate if the increase levels of Haptoglobin, Apolipoprotein A-IV and Prothrombin are correlated with HD development. </li></ul><ul><li>It is important to recognize this proteins like specifics biomarkers in HD; thats why the concentrations of each one were quantified in CSF and serum; thus indirectly evaluate the BBB integrity. </li></ul>
  19. 20. CONCLUTIONS <ul><li>To be able to correlate the levels of these proteins with other mental diseases such as Alzheimer's, multiple sclerosis, among others; allow us to determine whether the activity of proteins mentioned in the study are a common phenomenon in a variety of neurodegenerative-tive disease or are specifically for HD. </li></ul><ul><li>Becouse of the intact values of BBB and the increase levels of proteins, it suggest that they are produced in the CNS itself and that the relation within de concentrations of proteins in CSF has nothing to do with the concentration of these in serum. </li></ul>
  21. 23. BIBLIOGRAFIA <ul><li>MARTINEZ, L. Biología Molecular. ED UPB. Segunda edición. Medellín. 2011. </li></ul><ul><li>Yen-Chu Huang. Increased Prothrombin, Apolipoprotein A-IV, and Haptoglobin in the Cerebrospinal Fluid of Patients with Huntington’s Disease . Plos One. Vol 6, January 2011. </li></ul><ul><li> </li></ul>