Tumor necrosis factor alpha (TNFalpha) is a key proinflammatory cytokine which is produced primarily by macrophages. Although this cytokine is very beneficial to the host when released in small amounts or in localized fashion, abnormally high levels of TNFalpha can however be very detrimental. The biological effects of this cyokine is thus dependent on its timing, location and extent of release. In recent years major interest has been placed on the AU rich elements (ARE) present in the 3' untranslated region of the TNFalpha mRNA as it plays a pivitol role in the posttranscriptional control of TNFalpha protein production. / We have previously shown that a protein binding site within this main ARE (positioned between bases 1291 and 1320) has the ability to bind to three protein complexes (A,B,C). In this study we describe a second AU rich element which also has protein binding capabilities. This second protein binding site (located 158 bases downstream from the first ARE) is 31 nucleotides long and contains a single AUAUUUAU sequence. Fractionation experiments have enabled us to show that a protein complex (complex D) present in both nuclear and cytoplasmic cell compartments can interact with this second binding region. The binding of this second ARE to this complex is readily competed for by other AU rich elements present in the mRNA of certain protooncogenes and cytokines such as c-fos and IL-1beta. The importance of binding of macrophage proteins to the ARE in the posttranscriptional regulation of TNFalpha was evidenced by the fact that polymorphisms (GAU trinucleotide insertional mutation) in the main ARE (positioned between bases 1291 and 1320 of the 3 ' untranslated region) of TNFalpha mRNA results in the decreased binding of macrophage protein complexes to the element. The GAU insertional mutation in the main ARE hinders the binding of a protein named HuR to the region. This protein is a nucleo-cytoplamic shuttling protein previously shown to play a prominent role in the stability and translatability of mRNA containing ARE. In order in elucidate and further our understanding of signalling mechanisms which regulate the function of proteins binding to ARE we verified if priming of macrophages with IFNgamma is important in the posttranscriptional regulation of TNFalpha mRNA. We show that priming of macrophages with IFNgamma prior to LPS treatment posttranscriptionally upregulates TNFalpha production by increasing the stability of the message and the levels of mRNA present on translationally active polyribosomes. The involvement of IFNgamma in the posttranscriptional regulation of / Overall data presented in the thesis will further our understanding of how ARE in the 3'UTR of the TNFalpha mRNA posttranscriptionally regulates the expression of TNFalpha.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37648 |
| Date | January 2001 |
| Creators | Di Marco, Sergio. |
| Contributors | Radzioch, Danuta (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001863569, proquestno: NQ78676, Theses scanned by UMI/ProQuest. |
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