Analysis of gene- and protein expression in an Alzheimer model of Drosophila melanogaster
Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
Alzheimer’s disease is a common and very costly disease in today’s society. The hallmarks of the disease are the formation of two proteinaggregates, amyloid plaques containing Aβ-peptides and neurofibrillary tangles containing hyperphosphorylated tau protein. The formation ofneurofibrillary tangles is thought to be promoted by amyloid formation and is why the cellular events surrounding the formation and interactionsof the Aβ-peptide is a prime target for Alzheimer’s research. In this thesis, the gene of the highly aggregation prone form of Aβ-peptide, the Aβ1-42, has been inserted in a Drosophila melanogaster to promote expression in the central nervous system through the use of the Gal4-UAS system.Gene expression analysis was done using a RNA purification kit, translating the RNA into cDNA using RT-PCR and the levels were analyzed usingquantitative real-time PCR. For protein expression analysis the immunological techniques of dot blot and western blot were used combined withan immunoprecipitation step using magnetic beads. A fibrillation experiment was also performed to look into the potential seeding effect onamyloid formation from the Aβ1-42 expressing Drosophila using fluorescence spectroscopy.The aim for this thesis was to look into expression of the Aβ1-42 gene and the impact of ageing on expression levels. Another aim was to try andseparate and detect soluble Aβ-peptide species from tissue homogenates of Drosophila.No amplification could be detected in the quantitative real-time PCR, most likely due to concentration issues of the reaction components. For thisreason gene expression could never be quantified nor could the effect of ageing and gene expression be looked into. Insoluble aggregates but nosoluble Aβ-peptide species could be detected or separated from the tissue of the Drosophila. No seeding effect on the amyloid formation could bestatistically determined by the fibrillation experiment, but interesting quenching effects on the total quantum yield of Aβ fibrils in the presence ofbrain homogenates were noted.
Place, publisher, year, edition, pages
2009. , 58 p.
Drosophila melanogaster, Amyloid, Aβ1-42 peptide, Western blot, q-PCR
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:liu:diva-20728ISRN: LITH-IFM-A-EX--09/2077--SEOAI: oai:DiVA.org:liu-20728DiVA: diva2:235807
UppsokPhysics, Chemistry, Mathematics
Berg, Ina, PhD student
Hammarström, Per, Professor