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Ethanol-induced changes in G-proteins gene expression

Ethanol-induced changes in G-proteins gene expression. UEL Research & Knowledge Exchange Conference 2013 Stefano Casalotti Oghenetega Umukoro School of Health, Sport and Bioscience. More informative title:.

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Ethanol-induced changes in G-proteins gene expression

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  1. Ethanol-induced changes in G-proteins gene expression UEL Research & Knowledge Exchange Conference 2013 Stefano Casalotti OghenetegaUmukoro School of Health, Sport and Bioscience

  2. More informative title: Investigations of cellular and molecular effect of alcohol in a Drosophila (fruit fly) model.

  3. Journalistic style Alcohol addiction: Fruit flies are used to investigate the effect of alcohol on the brain

  4. Brain scientists enjoy getting young flies drunk

  5. Tax payers’ money ‘wasted’ on drunken flies

  6. Ethanol-induced changes in G-proteins gene expression • Alcohol addiction • Changes in gene expression • G-proteins

  7. Alcohol

  8. Addiction

  9. What is addiction ? Addiction Tolerance Craving Withdrawal Relapse

  10. Tolerance Following multiple stimulation: • Reduced response to the same stimulus Or: • The need for a higher stimulus to obtain the same response

  11. Tolerance to alcohol in flies Sedation Time Exposure

  12. What is the mechanism of tolerance ? Stimulus Response Stimulus Stimulus Stimulus Response Stimulus

  13. What has changed at…. ? …brain level? …network level? …cellular level?

  14. Neurochemical Transmission Pre-synapse Neurotransmitter e.g Dopamine DA DA DA Post-synapse Dopamine Receptors

  15. Neurochemical Transmission Pre-synapse Dopamine DA DA DA Post-synapse Dopamine Receptors

  16. Neurochemical Transmission Pre-synapse Dopamine DA DA DA Post-synapse Dopamine Receptors

  17. Neurochemical Transmission Pre-synapse Drugs of Addiction DA DA DA Post-synapse

  18. Acute and chronic drug effectand gene expression

  19. Acute/ chronic effect Which gene should be studied?

  20. G proteins a b g GTP

  21. Molecular effect of alcohol Alcohol Cell membrane fluidity Receptors (e.g. GABA-R) ?? Dopamine release Dopamine Receptors/ G-proteins

  22. Quantification of G-Proteins gene expression=measurement of mRNA for G-proteins Method: Real Time ,Reverse Transcriptase Polymerase Chain Reaction q-RT-PCR

  23. Primer design 6 Gα : Gs, Go, Gq, Gi, Gf and cta 3 Gβ : Gβ13F, Gβ5 and Gβ76C 2 Gγ: Gγ1 and Gγ30A

  24. Primer specificity

  25. RNA dose dependancy

  26. Changes in G protein expression in ethanol treated drosophila (acute effect) 2.0 1.5 1 0.5 0. Relative Gene Expression Control Treated Gq Gb5 Gb76C

  27. Type of changes Acute effect (naïve flies) Acute effect (‘cronic’ flies) Cronic effect (‘cronic’ flies)

  28. System complexity G proteins Alpha as ai ao ….. Beta b1 b2 b3 ….. Gamma g 1 g2 g3 ……. G protein coupled Receptors Dopamine R Opiate R Serotonin R GABA Glutamate Etc ,etc Effectors Adenylatecyclase PLC K+ channels

  29. Rationale for studying G proteins gene expression Effect A Effect B a a b b g g GTP GTP Protein X Protein Y Tolerance Normal

  30. System variability(are all flies equal?) 2.0 1.5 1 0.5 0. Relative Gene Expression Control Treated Gq Gb5 Gb76C

  31. Onset of sedation induced by alcohol Time (sec)

  32. Onset of sedation induced by alcohol Time (sec) Early Female Flies Late Female Flies

  33. Onset of sedation induced by alcohol Time (sec) Early Males Flies Late Males Flies

  34. ‘Behavioural’ sub-groups of flies

  35. Genetic differences(Genetic crosses) F1 Early F1 Late Early female Early male Late female Late male

  36. Changes in gene expression on a variable genetic background Gene Expression Ethanol

  37. Other ‘addiction’ research at UEL Substance use and misuse ‘ network’ SUM-UEL

  38. SUM Psychological wellbeing Poverty Genetics Substance Use and Misuse Crime Education (lack of) • Peer • pressure • Physical • Addiction • Experimentation

  39. Clinical Interventions Cognitive Studies Ethnographic Studies UEL SUM Network Toxicology Pharmacology Training Education Child Development Social Policies

  40. Get in touch SUM ‘network members’ • Dr Daniel Briggs LSS • Dr. Stefano Casalotti. HSB • Dr Olivia Corcoran HSB • Dr. Lynne Dawkins Psychology • Prof. Angela Harden. HSB • Stephanie Lynch Psychology • John McCarthy. Student Services • Prof. Mark McDermott Psychology • Dr . Olive McKeown . HSB • Prof. Derek Moore Psychology • Dr Winston Morgan. HSB • Dr Barbara Pendry. HSB • Dr Alice Sampson LSS • Dr. Kirstie Soar . Psychology • Dr. John Turner Psychology • OghenetegaUmukoro HSB Contact: John Turner Turner2@uel.ac.uk Stefano Casalotti stefano1@uel.ac.uk

  41. Thank you • OghenetegaUmukoro (PhD Student) • UEL – Start up-grant

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