Search

Current transplant immunosuppression regimens have numerous limitations. Recent evidence suggests histone deacetylase inhibitors (HDACis) may represent a class of drug with immunosuppressive properties. This study initially assessed a panel of experimental and established HDACis, identifying a novel HDAC6-specific inhibitor (KA1010) to compare with cyclosporin and the pan-HDACi suberoylanilide hydroxamic acid in models of alloreactivity. Proliferation and MLR-based assays were used to determine the immunosuppressive effect of compounds, and a murine model of allogeneic skin transplantation was adopted to assess the in vivo effects of HDAC6 inhibition. KA1010 displayed superior inhibitory effects on the activation of PBMCs using in vitro models of transplantation. In a one-way MLR, KA1010 (5μM) reduced parent cell proliferation from 92% to 64% (p=0.001). A two-way MLR, adopting IFN-γ production as a marker of alloresponse, resulted in up to 91% reduction. Dose-response curves revealed dose-dependent profiles with a greater potency of HDACis over CyA. Mice treated with KA1010 displayed no significant features of skin allograft rejection upon histological analysis at 70 days and graft survival of upto 80%. Immunological assessment, revealed a significant increase in regulatory T cells (from 18% to 25%, p=0.0002) and a corresponding reduction in CD4\(^+\) T cells (from 58% to 42%, p=0.0009). HDAC6 may represent an optimal target for future immunosuppressant therapeutics with a particular role in transplantation. In this thesis, we have demonstrated a superior immunosuppressive effect of KA1010 over both CyA and SAHA, in the models of allotransplantation adopted.

I have analyzed the interactions between SR proteins and splicing components that are bound at the 5’ or 3’ splice site using fluorescence resonance energy transfer (FRET) microscopy. The SR proteins interact with the U1 snRNP-associated 70 kDa protein (U170K) at the 5’splice site and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) at the 3’ splice site. These interactions have been extensively characterized biochemically in the past, and are proposed to play roles in both intron and exon definition. We employed FRET acceptor photobleaching and fluorescence lifetime imaging microscopy (FLIM) to identify and spatially localise sites of direct interactions of SF2/ASF, and other SR proteins, with U2AF35 and U1-70K in live cell nuclei. These interactions were shown to occur more strongly in interchromatin granule clusters (IGCs). They also occur in the presence of the RNA polymerase II inhibitor, DRB, demonstrating that they are not exclusively co-transcriptional. FLIM data have also revealed a novel interaction between HCC1, a factor highly related to the large subunit of the U2AF splicing factor, with both subunits of U2AF that occur in discrete domains within the nucleoplasm but not within IGCs. These data demonstrate that the interactions defining intron and exon definition do occur in living cells in a transcription-independent manner.

The population genetic structure of three rare and declining bumblebee species, Bombus muscorum, B. humilis, and B. sylvarum was examined using microsatellite markers. We find evidence of significant genetic structuring in both oceanic island populations of B. muscorum and in fragmented mainland populations of B. jumilis and B. Sylvarum. In the former species we find that all populations greater than 10km apart are significantly differentiated from one another suggesting dispersal above this distance is infrequent. Evidence of genetic bottlenecks was found in populations of both B. muscorum and B. humilis. All three species show reduced genetic diversity relative to the common B. pascuorum and fragmented UK populations of B. sylvarum show reduced genetic diversity relative to a continental population. Effective population sizes were found to be low in B. humilis and B. sylvarum, especially so in the latter species. Diploid male production was observed in all three rare species, but was infrequent. We propose that diploid male production is not an appropriate measure of inbreeding depression for social Hymenoptera. Why some bumblebee species have declined in the face of agricultural intensification while others have not is not well understood. Diet breadth of rare species was examined as a possible explanation of this. To some extent all three rare species studied show a narrow diet breadth. We hypothesize that this is because as these species emerge late, they must specialize on plants with higher quality pollen in order to raise the brood more quickly. Following losses of floral diversity, agricultural intensification has than affected these species more greatly.