III Encuentro JIP 2017 - Charla 2. Jacob Lorenzo Morales

1. Investigación en Amebas de Vida Libre: desde
la organización unicelular al “tejido
productivo”

3. Inicios: Aislamiento y caracterización morfológica amebas de vida libre

4. ¿Quiénes éramos?
• Dr Basilio Valladares
• Dr Enrique Martínez
• Dr José E. Piñero
• Jacob Lorenzo-Morales  doctorando  Ramón y Cajal…

5. ¿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

6. • 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

7. 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

8. WHAT WE DO HERE...
• Gene silencing
• Active molecules  natural, synthetic  Discovery of therapeutic agents
• Toxicity assays
• Apoptosis
• Traslational medicine

9. • 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

10. • 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

11. 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

12. 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)

13. MUCHAS GRACIAS PROF. BASILIO VALLADARES

14. Leptomyxa valladaresi, 2017