logo SBA

ETD

Archivio digitale delle tesi discusse presso l’Università di Pisa

Tesi etd-11242020-114033


Tipo di tesi
Tesi di laurea magistrale
Autore
IACONO, ELENA
URN
etd-11242020-114033
Titolo
L'inibizione farmacologica dell'amidasi acida idrolizzante l'N-aciletanolammina blocca la replicazione di ZIKV e COXB5
Dipartimento
BIOLOGIA
Corso di studi
BIOLOGIA MOLECOLARE E CELLULARE
Relatori
relatore Dott. Lai, Michele
Parole chiave
  • autofagia
  • colture cellulari
  • NAAA
  • virus
Data inizio appello
09/12/2020
Consultabilità
Non consultabile
Data di rilascio
09/12/2060
Riassunto
Palmitoylethanolamide (PEA) is an endogenous lipid with anti-inflammatory and analgesic functions that is synthesized by the cell, starting from the conversion of membrane phospholipids into phosphatidylethanolamine (PE).
PEA inhibits mast cell degranulation, preventing the release of pro-inflammatory chemical mediators. PEA is also an agonist of nuclear receptors activated by peroxisomal proliferators (PPAR), to which it binds allowing the transcription of genes such as PPAR, which stimulate the -oxidation of fatty acids and activate the biogenesis of peroxisomes.
PEA action is inhibited by the enzyme N-Acylethanolamine-hydrolyzing Acid Amidase (NAAA) which hydrolyzes the bond between amine and lipid. The enzyme localizes in endosomal vesicles, where low pH values (4.5-5) promote its proteolytic activation.
Several studies have shown that endogenous PEA levels tend to decrease during the inflammatory process in patients with rheumatoid arthritis and osteoarthritis. In parallel, the accumulation of PEA exerts antinociceptive effects in mouse and rat models with acute and chronic pain. Although the mechanism of action of PEA and all its targets are not well known, the modulation of its levels represents a new and effective therapeutic strategy for the treatment of neuropathies and inflammations.
The aim of this thesis project is to evaluate the effects of modulation of PEA levels during viral infections of the central nervous system.
Based on these considerations we have selected two neurotropic viruses, responsible for serious neurological inflammation: Zika and Coxsackie B5 (ZIKV and COXB5).
ZIKV infection can cause microcephaly in infants or Guillain-Barrè syndrome in adults, as well as COXB5 which, in addition to mild gastrointestinal forms, can be responsible for aseptic meningitis.
In order to increase PEA levels within endosomal vesicles, we have decided to inhibit the enzyme responsible for its degradation (NAAA) that locates within them. The enzyme inhibition was obtained using recently developed drugs that are currently in the early stages of clinical trials for the treatment of chronic pain.
We have infected human adenocarcinomic alveolar epithelial cells (A549) with both selected viruses and evaluated viral replication using Real Time PCR, Western Blot and plaque assays, using NAAA enzyme inhibitors at different concentrations.
The molecular and cellular assays performed allowed us to highlight a dose-dependent reduction in intracellular viral proteins for both viruses and a decrease in viral load in the supernatants of cells treated only for ZIKV. We also observed an increase in the autophagic process in the presence of inhibitors that could support the antiviral activity of the studied compounds. Currently, we are reproducing the above experiments on a line of neuroepithelial stem cells of the cerebral cortex (NCX-NES), which are the natural target of ZIKV.
In conclusion, this thesis demonstrates a correlation between NAAA inhibition and the cells susceptibility to viral infections of ZIKV and COXB5. The results obtained differ depending on the virus used, but are in line with their mechanism of entry: ZIKV enters the cell only through endocytosis, while COXB5 also uses other routes, for this reason the viral inhibition observed for Zika virus is more effective. The use of two different viruses has allowed us to shed light on the possible mechanism of action of NAAA inhibition in the contrast of viral infection.
File