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III Encuentro JIP 2017 - Charla 2. Jacob Lorenzo Morales

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III Encuentro JIP 2017 - Charla 2. Jacob Lorenzo Morales

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III Encuentro JIP 2017 - Charla 2. Jacob Lorenzo Morales

  1. 1. Investigación en Amebas de Vida Libre: desde la organización unicelular al “tejido productivo”
  2. 2. Inicios: Aislamiento y caracterización morfológica amebas de vida libre
  3. 3. ¿Quiénes éramos? • Dr Basilio Valladares • Dr Enrique Martínez • Dr José E. Piñero • Jacob Lorenzo-Morales  doctorando  Ramón y Cajal…
  4. 4. ¿quiénes somos ahora y qué hacemos? • Molecular characterization of Free Living Amoebae from environmental and clinical sources • Diagnostic Service for National Health System • Discovery of therapeutic agents • 4 cellular models: T. cruzi, Leishmania spp., Acanthamoeba spp., N. fowleri • Senior Lecturer: Dr José E. Piñero • Associate Lecturer: Dr. Jacob Lorenzo-Morales • 3 Postdocts: Dr. María Reyes, Dr. Atteneri López & Dr Ines Sifaoui • PhD students: Miss Carolina Wagner, Miss Olfa Chiboub, Mr Carlos Bethencourt • Master students: Miss Ana B. García , Mr Aitor Rizo
  5. 5. • Molecular characterization of Free Living Amoebae from environmental and clinical sources • Diagnostic Service for National Health System • Discovery of therapeutic agents • 4 cellular models: T. cruzi, Leishmania spp., Acanthamoeba spp., N. fowleri • Senior Lecturer: Dr José E. Piñero • Associate Lecturer: Dr. Jacob Lorenzo-Morales • 3 Postdocts: Dr. María Reyes, Dr. Atteneri López & Dr Ines Sifaoui • PhD students: Miss Carolina Wagner, Miss Olfa Chiboub, Mr Carlos Bethencourt • Master students: Miss Ana B. García , Mr Aitor Rizo
  6. 6. COLLABORADORES • Tunisia  University of Cartague; University of Sfax • Algeria  University of Constantine • Mexico  UNAM, Cinvestav, ITSON • Costa Rica Universidad Central de Costa Rica • UK, France, Ireland, Italy, Germany, etc • CDC, USA • Venezuela  UCV • Poland  MUW • Malaysia  Sunway University • RICET • Inst. de Salud Carlos III • Univ. De Granada • Hospital Ramón y Cajal, Madrid • Univ. De Salamanca
  7. 7. WHAT WE DO HERE... • Gene silencing • Active molecules  natural, synthetic  Discovery of therapeutic agents • Toxicity assays • Apoptosis • Traslational medicine
  8. 8. • Biological activity Cytotoxicity assay Murine macrophage Cytotoxicity detection kit (LDH) Leishmanicidal activity againts intracelular amastigote Macrophage + Infective promastigotes AlamarBlue Leishmanicidal activity in vitro Promastigotes AlamarBlue + + Molecules Wash + Molecules 24h 0,05% SDS 30” + Molecules 24h 72h Supernatants + Kit reactive AlamarBlue AlamarBlue 72h IC50 IC50 CC50
  9. 9. • Cell death determination Plasma membrane Permeability Promastigotes Sytox Green Changes in the mitochondrial membrane potential (ΔΨm) Promastigotes JC-1 Mitochondrial Membrane Potential Assay Kit Analysis of ATP levels Promastigotes CellTiter-Glo® Luminescent Cell Viability Assay Sytox Green + ADN Sytox Green
  10. 10. Analysis of the expression of 35 apoptosis-related proteins L. donovani promastigotes Human Apoptosis Array Proteome Profiler Pixel quantification (Fiji software) Statistical analysis (SigmaPlot software) (** p<0,01) Search and evaluation of molecular targets
  11. 11. Analysis of 7 protein-kinases phosphorilation L. donovani promastigot es Human Phospho- Kinase Array 1 Proteome Profiler 96 Search and evaluation of molecular targets Pixel quantification (Fiji software) Statistical analysis (SigmaPlot software) (** p<0,01)
  12. 12. MUCHAS GRACIAS PROF. BASILIO VALLADARES
  13. 13. Leptomyxa valladaresi, 2017

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