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Ribosome-inactivating proteins (RIPs) are found in a wide variety of plant species. They possess an RNA N-glycosidase activity whereby the removal of a specific adenine residue from 28 S RNA renders a eukaryotic ribosome inactive. Type II RIPS contain both an active polypeptide and a sugar-binding polypeptide. Type I RIPs are composed of a single polypeptide functionally homologous to the active type II polypeptide. This thesis describes studies of the gene sequences of RIPs representative of each class: Ricin, a type II RIP from the castor oil plant (Ricinus communis h.), and saporin, a type I RIP from soapwort (Saponaria officinalis L.). Two ricin gene sequences were isolated from a Ricinus genomic library and partially characterised. One gene was a badly damaged ricin-like pseudogene whilst the other was shown to encode an active polypeptide. A second ricin sequence encoding an active polypeptide was isolated using Polymerase Chain Reaction (PGR) DNA amplification. The specificity of PGR amplification was investigated using the ricin and related agglutinin gene sequences. Partial amino acid sequence data derived from protein sequencing of saporin-6 was used to synthesise degenerate inosine-containing oligonucleotides. These directed the PGR amplification of part of the saporin coding sequence from genomic DNA. The product was used as a saporin-specific hybridisation probe. Southern analysis of Saponaria genomic DNA indicated that saporin sequences comprised a small multigene family. Three independent saporin containing genomic clones were isolated from a Saponaria genomic library. Two clones were truncated whilst the third contained a complete saporin coding sequence. The saporin and ricin coding sequences were expressed in vitro and shown to inhibit protein synthesis. Aniline cleavage assays of ribosomal RNA extracted from ribosomes exposed to the products of the RIP coding sequences were carried out. These indicated that the polypeptides encoded by the RIP gene sequences had specific RNA N-glycosidase activity.

This thesis investigated the helical structure of core histone octamers to discover interacting surfaces and their relevance to the compaction of nucleosome arrays into the chromating fiber.

Evidence suggesting that Hsp72 is actively participating in cellular signalling as well interacting with immune system dynamics has been increasing. This is true in healthy, stressed and diseased cells but to different degrees. Modulation of the plasma membrane association and secretion in the extracellular environment by different types of stressors is the key event that leads to different degrees of immune system activation. Hence a better understanding of the mechanisms of Hsp72 secretion and association with plasma membrane is crucial. This thesis investigated the tissue source and mechanism of Hsp72 surface presentation to plasma membrane structures and release in relation with different cellular and physiological stressors. In vivo models confirmed that different tissue types determine specific Hsp72 responses following the same stress and increase serum Hsp72 dependant on intensity and duration of the stress. Diseases models confirm that Hsp72 responses in specific cell populations is related to disease progression, while in vitro models clearly showed that there are multiple mechanisms of secretion and surface presentation, dependent on the nature of the stressor as well as the intensity and duration. This observations clearly change the view of extracellular Hsp72 as a danger signal and lead to a revision of the original danger model. It also suggests that manipulation of Hsp72 translocation through the different pathways involved may prove effective therapeutically.

Although a number of HSPs have been shown to be up-regulated in a wide range of human cancers, the full significance of this remains to be determined. The localisation of HSPs seems to be critical in determining their role in cancer cell survival; High intracellular levels (iHsp) appear to be advantageous to the tumour cell, inhibiting key steps in apoptosis, while in some circumstances, surface expression (sHsp) appears to be detrimental to the cell, aiding immune recognition by various effector cells. Consequently, clarifying the importance of HSP cellular location in the cancer setting may lead to the development of novel therapies based upon manipulation of HSP localisation. This thesis had two major aims; (1) to investigate the cellular localisation of HSPs in leukocytes from patients with both myelocytic and lymphocytic malignancies in order to establish relationships between apoptosis and stage of disease (2) to study the synergistic effect of four chemotherapeutic drugs with membrane fluidising agents, compounds which have the potential to modulate HSP localisation. Hsp90 and Hsp27 expression was shown to be restricted to the inside of peripheral blood leukocytes, while Hsp72 was localised both intracellularly and on the cell surface. In CLL, iHsp90 and iHsp27 levels were found to be significantly higher than in control subjects, while surface and intracellular Hsp72 was shown to be expressed either at very high levels or at very low levels. Furthermore, iHsp90 levels were found to be associated with stage of disease, while iHsp27 levels were shown to negatively correlate with levels of apoptosis. CLL patients with stable disease were found to express higher levels of iHsp72 than patients with progressive disease. However, in AML and MDS, levels of all HSPs in peripheral blood were found to be similar to those seen in control subjects, but disease patients showed a much wider range of expression. In AML, levels of sHsp72 positively correlated in all cell types, an observation not made in MDS patients or control subjects. HSP localisation was shown to be affected by membrane fluidising agents, with a movement of Hsp72 and Hsp60 to the cell surface. This effect was not due to proteotoxicity and supports data implicating the cell membrane in the regulation of HSP responses. This manipulation of HSP localisation and the increase in membrane fluidity resulted in increased sensitivity of CLL cells to three chemotherapeutic agents and points to the possibility that manipulation of membrane fluidity, may have significant value in the development of new treatment regimes.

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No description available.