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Tesi etd-04032008-181658


Thesis type
Tesi di dottorato di ricerca
Author
VALLEGGI, SIMONA
URN
etd-04032008-181658
Title
XENON INDUCED GENE EXPRESSION ANALYSIS IN ANIMAL MODELS
Settore scientifico disciplinare
MED/08
Corso di studi
ONCOLOGIA SPERIMENTALE E MOLECOLARE
Commissione
Relatore Dott. Cavazzana, Andrea
Parole chiave
  • rat brain
  • Rab14
  • Prothymosin alpha
  • PI5P4K beta
  • nitrous oxide
  • neuroprotection
  • Hif 1-alpha
  • gene transcription
  • anesthesia preconditioning
  • ADNP
  • SAP 102
  • Similar to DNER
  • suppression subtractive hybridization
  • xenon
Data inizio appello
17/04/2008;
Consultabilità
parziale
Data di rilascio
17/04/2048
Riassunto analitico
Xenon, a rare noble gas discovered by Ramsay and Travers in 1898, has medicinal use in radiological and perioperative clinical settings. A possible mechanism for Xenon&#39;s salubrious properties in the perioperative period (including anesthesia, analgesia and organ protection) have been attributed either to a non-competitive inhibition of NMDA receptors or to an activation of potassium channels. Interest in Xenon has evolved from the recent description of its neuroprotective properties in several models of acute neurological injury. Xenon&#39;s neuroprotective effect has been reproduced both in vitro and in vivo at sub-anaesthetic doses, suggesting that Xenon may find clinical utility for current unmet medical needs such as stroke or neonatal asphyxia. Xenon&#39;s neuroprotection is long lasting when used either as a post-injury treatment or in the setting of preconditioning. The molecular mechanisms underlying Xenon-induced neuroprotective effects have not yet been elucidated. For this reason exploring at molecular level the effects of Xenon exposure, may contribute to partially elucidate the molecular mechanisms involved in Xenon-induced neuroprotection. To gain further insight in Xenon’s salubrious effects, we decided to adopt a global approach, by isolating and cloning the mRNAs differentially expressed in rat brains after Xenon exposure compared to either air or nitrous oxide, another inhalatory anaesthetic agent that does not exhibit preconditioning or organ protective effects. This approach allowed us to identify Xenon’s “genetic signature” that has yielded possible insights into the molecular mechanisms involved in Xenon-induced neuroprotection. In order to achieve our goal we adopted the Suppression Subtractive Hybridization (SSH) method. SSH is an efficient technique, used to identify significant changes in gene expression comparing two cell populations differing for one character: in our case the Xenon treatment. The study has been performed on two paired sets (the experimental and the validation sets) of animals (7 days old, Sprague Dawley rats). Each set has been subdivided in two groups: the control group has been exposed to air (75% nitrogen and 25% oxygen), the treated one has been exposed to Xenon (75% Xenon and 25% oxygen). Under the same conditions, a third set of four animals (7 days old, Sprague Dawley rats), has been treated with nitrous-oxide (25% oxygen and 75% nitrous oxide) to <br> <br><br>control for the anaesthetic state produced by Xenon. RNA Poly(A)+ isolated from control and <br>treated rat brains of the experimental set, has been used to performed SSH. Once evaluated subtraction efficiency, the secondary PCR products of the forward library were cloned and subjected to differential screening. Clones showing an on/off hybridization signal have been sequenced by the dideoxy chain terminator method and analyzed at the National Center of Biotechnology Information server (http://www.ncbi.nlm.nih.gov), at the European Bioinformatics Institute server (http://www.ebi.ac.uk) and at the Rat Genome Database (http://rgd.mcw.edu ). In this way we in silico selected the main target of our interest. Selected sequences were submitted to two steps data validation. As first step we performed a semi-quantitave RT-PCR on messenger RNAs used for library construction; as second step we performed a Relative Real-Time PCR on total RNAs obtained from the validation set. Relative Real-Time PCR data, were then statistically analyzed with REST 2005. A p value &lt;0.05 will be considered significant. In order to verify if the Xenon induced gene transcription could be shared by other noble gases, the expression of Xenon’s induced transcripts has been further investigated by relative Real Time PCR, on total RNA isolated from another set of animals (7 days old, Sprague Dawley rats) treated with three other different noble gases: Argon (75% Argon and 25% oxygen), Krypton (75% Krypton and 25% oxygen), Neon (75% Neon and 25% oxygen) and the paired air-control cases (75% nitrogen and 25% oxygen). Next we investigated if the Xenon induced up-regulation of validated genes was followed by a parallel increase in their pair gene products. Proteins subcellular localization was analyzed by immunohistochemistry on paraffin-embedded brains of a new set of animals, treated according to the same protocol and sacrificed 24 hours after exposure. Sections will be stained with available commercial antibodies. Difference in protein-expression level was studied by Western blot technique.<br>
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