[Truncated abstract] Cerebral ischaemia results from severe reductions of cerebral blood flow during the occlusion, rupture or constriction of cerebral blood vessels, cardiac arrest, or periods of prolonged systemic hypotension. Subsequent brain damage is due to a sequelae of events culminating in cell death and loss of neurological function. The resultant cell death occurs via many pathways and can take on many molecular and biochemical characteristics, many of which are activated or influenced by gene expression. To investigate the molecular mechanisms that influence either ischaemic brain cell death or survival, this project aimed to identify genes differentially expressed in the hippocampus in a rat model of global cerebral ischaemia. A combined strategy of subtraction hybridisation and differential screening identified several genes with unique expression in the ischaemic hippocampus. Northern blot analysis further verified differential expression of heat shock cognate protein (HSC70), heat shock proteins 90 and 110 (HSP90, HSP110), TrkB receptor (TRKB), sodium calcium exchanger 1 protein (NCX1) and a cDNA clone that, at the time, showed no sequence homology to any known genes. The novel cDNA clone was referred to as B5- A. Following confirmation of differential gene expression, the remainder of the thesis focused on NCX and B5-A, as the roles of the heat shock proteins and TRKB had been extensively investigated following cerebral ischaemia. A temporal profile of protein expression of the three NCX subtypes (NCX1, NCX2 and NCX3) was examined following damaging (8 min) and non-damaging (3 min) periods of global cerebral ischaemia. Western analysis indicated that NCX2 and NCX3 protein levels did not change following non-damaging ischaemia, whereas NCX1 protein was up-regulated at 6 and 12 hours after ischaemia. In contrast, following the damaging ischaemia, NCX1 proteins levels remained stable, NCX2 was down-regulated and NCX3 was up-regulated. The remainder of the study focused on investigating the gene corresponding to the cDNA clone B5-A, a gene now known as FAM21. Northern analysis using B5-A cDNA as a probe detected a 4.2kb transcript in normal brain tissue, as well as, a 3.6kb transcript only detectable in ischaemic brain tissues. This was an interesting finding as it suggested that the FAM21 gene was subject to alternative splicing. However, Western blot analysis of brain tissue detected only a 130kDa protein, which was within the size range predicted for FAM21 (145kDa), and no additional bands were observed in ischaemic tissue. The transcribed region of the rat gene corresponding to the B5-A cDNA, was cloned and sequenced, and was found to correspond to a highly conserved, newly identified gene, FAM21. Bioinformatics revealed that full-length FAM21 mRNA consisted of a 4,269bp transcribed sequence containing 31 exons and encoding a 1,328 amino acid protein. Analysis of amino acid sequence did not reveal any functional motifs predicting subcellular location or domains indicative of function...
|Qualification||Doctor of Philosophy|
|Publication status||Unpublished - 2013|