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Seminario t.b final

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  1. 1. Mara Alejandra Maya Roldan Johan H. Muoz Ros 2015
  2. 2. INTRODUCTION
  3. 3. Pentose phosphate pathway PPP
  4. 4. Glycolisis
  5. 5. ENZIME: RELATION Ribulose 5 phosphate Ribose 5 phosphateR5Pi A Humans B T. brucei Atracttive drug tagger
  6. 6. ENZIME Ribose 5 phosphate isomerase (Rpi) catalyzes the interconversion: ribulose 5 phosphate (Ru5P) ribose 5 phosphate (R5P). Transformation from group aldose to ketose. Transition to the non oxidative branch of the PPP
  7. 7. The reaction mechanism involves an initial opening of furanose ring of R5P, followed by the aldolase-ketose isomerization, via a cis-enediolate high energy intermediate.
  8. 8. Trypanosoma Trypanosomatides protozoa. Monophyletic group of unicellular protist parasites. Different species infect vertebrates causing trypanosomiasis.
  9. 9. Trypanosoma Transmitted by invertebrates such as biting insects, or penetrate the skin. Complex life cycle. Has a variety of different ways in the invertebrate host, and guests vertebrate cells take a form feature called trypomastigote
  10. 10. Trypanosoma brucei Kingdom: Protist Phylum: Euglenozoa Class: Kinetoplastea Order: Trypanosomatid Genus: Trypanosome Species: T. brucei
  11. 11. Trypanosoma brucei Causes African trypanosomiasis (sleeping sickness) Subspecies: T. b. gambiensechronic tripanosomiasis slow start. T. b. rhodesiense, tripanosomiasis quick start. T. b. brucei, African (or nagana) animal trypanosomiasis.
  12. 12. unusual features Only large mitochondria with mitochondrial DNA condensed structure, in association with the basal body of the flagellum, unusually constitutes the mechanism of cell cytoskeleton organization Unique and remarkable cover variant surface glycoprotein (VSG) in order to avoid the host immune system
  13. 13. Due to the large difference between these two hosts, the cell undergoes complex changes to facilitate survival in the insect gut and blood of mammals.
  14. 14. VECTOR
  15. 15. Trypanosoma brucei
  16. 16. OBJECTIVE Investigate the importance of RpiB in T. brucei bloodstream form viability and infectivity.
  17. 17. MATERIALES Y MTODOS Declaracin tica Investgacin IBMC.INEB Parlamento europeo Acreditacin de la direccin veterinaria portuguesa
  18. 18. CULTIVO Cultivo Conjunto de tcnicas Mantenimiento, supervivencia, diferenciacin, crecimiento y funcin. Preservando sus propiedades Fisiolgicas Qumicas Genticas
  19. 19. Mediodecultivo Sustrato Nutrientes Condiciones abitica Mantenimiento, supervivencia, diferenciacin, crecimiento y funcin.
  20. 20. T.BruceiLister247 Procclica F. Sangre MEM- Pros HMI-9 FELOMICINA
  21. 21. PCR PCR Amplificacin directa de un gen o fragmento de DNA o indirecta de RNA Enzimas De 20-40 ciclos Pasos: Desnaturalizacin, alineamiento y sntesis
  22. 22. Se obtuvieron los genes de R5Pi DNA genmico Producto: * Aislacin * Purificacin * Clonacin * Secuenciacin
  23. 23. DNA Recombinante Molcula de DNA artificial que proviene de la unin de DNA = origen. Se emplea para la produccin de protenas.
  24. 24. Qu se necesita: 1. DNA donador 2. DNA receptor 3. Enzimas de restriccin. 4. Ligasas UNA VEZ OBTENIDO EL DNA RECOMBINANTE SE INSERTA EN UNA BACTERIA PARA LA PRODUCCIN DE PROTENA RECOMBINANTE
  25. 25. Ensayo enzimtico Ensayo enzimtico Actividad, inhibicin y cintica enzimtica Curva La reaccin de equilibrio Rapidez de formacin del producto o desaparicin del sustrato Hay 2 tipos Continuos Discontinuos
  26. 26. Km de R5P Km de Ru5P Mecanismo de inhibicin del 4_PEH
  27. 27. Inmunofluoresencia Inmunofluoresencia Tcnica inmunoqumica con marcador de fluorocromo Utiliza reaccin Ag-Ac Puede ser de 2 tipos: Directa Indirecta
  28. 28. Encontrar dnde se encontraba La R5Pi : DAPIDNA Aldolasa..Glicosoma R5Picitoplasma
  29. 29. Permeabilizacin con Digitonina Digitonina Digitalis purpurea Detergente Ataca el colesterol Permeabiliza las membranas cells.
  30. 30. F.sangre Permeabilizacin con Dig. Acceder a contenidos citoplasmticos de las cells. Sobrenadante se midi por W.B: *Enolasa *Aldolasa *R5Pi
  31. 31. Transgnicos Tambin llamados OMG. Organismo vivo creado a partir de la manipulacin de sus genes. MEJORAR CONDICIONES
  32. 32. Tcnica Aislar segmentos de DNA . Introducir ste en el material hereditario de otro. Ejemplo
  33. 33. Creacin Cells con RNAi Cells con complementacin funcional T.brucei T.cruze
  34. 34. Northern Blot Reconocimiento de secuencias especficas de RNA Cmo se realiza? 1.Aislar RNA 2.Electroforesis. 3.Fijacin a medio slido. 4. Hibridacin. 5. Separacin. 6. Autoradiogrfia RNA presente en la Forma sangunea
  35. 35. Western Blot Identificacin de protenas. Cmo se hace? 1. Electroforesis (SDS/PAGE) 2. Transferencia al filtro con Ac. 3. Identificacin de protenas Cuantificacin protenas Parsitos en sangre Protena recombinante
  36. 36. RESULTS Figura 1
  37. 37. Figura 2
  38. 38. Figura 2
  39. 39. Figura 3
  40. 40. Figura 4
  41. 41. DISCUSSION AUTHOR CONCLUSION AGREE DISAGREE Stern Al Et al TbRpiB has the ability of using bothe R5P or Ru5P as subtrates, but with remarkable preference for Rub5P . Jensen BC Et al The levels of TbRPIB mRNA in logarithic phase procyclic forms compared to bloodstream forms are higher. X. Colasante C Et al Make a proteomic analysis that failed to find TbRpiB enzime in purified glycosomes. . Ong HB Et al TbRpiB is not the fist protein reported as dispensable under estandar laboratory culture condictions but crucial for parasities growth in tHe animal host. . .
  42. 42. CONCLUSIONS Is very important Identify and know the metabolics pathways, its enzymes or obligatory steps to use as pharmacological targets. To study the importance of an enzyme, we have to make a lot of techniques to identify its action, origin, cinetic profile, location, concentration and the mechanism of inhibition
  43. 43. In the study is corraborated use of RpiB as a drug target. Consider non only the possiblity of using it against African sleeping sickness, but tuberculosis, a real affliction of our enviroment. The odds must be open to new investigations, searching for that drug target in multiple pathological agents.
  44. 44. para
  45. 45. BIBLIOGRAFA Martinez Sanchez, Lina Maria. Biologia molecular.7.ed.Medellin: UPB. Fac.Medicina. Castao, Mara Eugenia. Cultivos celulares. Biognesis, principios de la virologa.