No description available.

No description available.

A particularly intriguing aspect of the erythroblast enucleation process, which creates an anucleate immature erythrocyte called a reticulocyte, is how the sorting of proteins between the nascent reticulocyte and the extruding nucleus occurs. A large-scale proteomic analysis was conducted on isolated pure populations of reticulocytes and extruded nuclei, and the results for key proteins of interest confirmed using western blotting. Many erythroid membrane and cytoskeletal proteins partitioned predominantly or exclusively to the reticulocyte. In contrast, nuclear proteins, endoplasmic reticulum proteins and a contingent of membrane proteins were enriched in the extruded nuclei. This confirms enucleation as a key remodelling event in the development of an erythrocyte where important proteins needed for the erythrocyte are retained, but unwanted or excess proteins are removed with the nucleus. During this thesis lentiviral transduction methodology was further developed to achieve both knockdown of cytoskeletal proteins and overexpression of cytoskeleton fragments to disrupt the erythroid cytoskeleton and elucidate the role membrane-cytoskeletal interactions play in membrane multi-protein complex assembly and stabilisation during erythropoiesis. shRNA mediated knockdown of ankyrin achieved the most striking results with >95% reduction in ankyrin levels. The expression profiles of key erythroid membrane and cytoskeletal proteins following ankyrin knockdown were monitored throughout terminal differentiation by flow cytometry and western blotting. Ankyrin is required for the stabilisation of members of the ankyrin complex throughout terminal differentiation, and these dependencies become established at different stages for different members within the complex. The rhesus subcomplex members Rh, RhAG and CD47 were depleted from the basophilic erythroblast stage in the absence of ankyrin, with additional loss during enucleation. Protein 4.2 also exhibited early depletion at the basophilic stage. Interestingly, band 3 and GPA levels were comparable to the control throughout the early stages of terminal differentiation when ankyrin was depleted, but then exhibited decreased retention with the reticulocyte during enucleation. RhAG loss at the basophilic stage was shown to be due to enhanced internalisation and turnover. The work presented here has greatly increased our understanding of the assembly of membrane multi-protein complexes and the protein sorting processes that occur during human terminal differentiation and enucleation, and has provided an explanation for the secondary protein deficiencies observed in Hereditary Spherocytosis where the attachment of membrane proteins in the bilayer to the underlying cytoskeleton is disrupted.

Platelets are vital in haemostasis to prevent bleeding after injury. Uncontrolled platelet activation, however, leads to thrombosis. Platelet granule secretion is critical in thrombus formation and, due to the wide variety of granule contents released, also plays a role in many other physiological and pathological processes. The signalling molecule PKC is key in secretion of both dense and a-granules, but the downstream pathways that link it to secretion still need to be defined. A regulator of a-granule release, specifically, is ADP, which acts on the P2Y12 receptor after being secreted by dense granules. How it may control the release of functionally different cargoes is poorly understood. I first identified an extensive range of candidate platelet PKC substrates through proteomics screening using an anti-phosphopeptide antibody-based approach. Among these, I validated cytohesin-2, an Arf-GEF not previously characterised in platelets. This study demonstrated that, upon platelet activation, PKC phosphorylation of cytohesin-2 relieves the basal suppression of dense granule release, shedding new light on how PKC regulates secretion. I next examined the role of ADP in a-granule secretion with a specific focus on whether P2Y12 can induce differential cargo release, considering the implications for the clinical use of the P2Y12 blocker, clopidogrel. I investigated this using a P2Y12 antagonist and the Munc13-4-1 - mouse model where dense granule secretion is ablated and therefore autocrine ADP is not released. Results revealed that the secretion of certain a-granule contents is more dependent on ADP than others and that, in the absence of ADP signalling, granules may release contents without complete fusion with the plasma membrane. Taken together, this thesis provides new findings on the regulation of dense and a-granule secretion, which can be used in the design of improved therapies for thrombosis and other disorders where platelet granule contents play a role.

No description available.

No description available.

Elucidation of the mechanisms underlying the reciprocal phenomena of HPC homing and mobilisation is the objective of this thesis, and may allow the development of novel approaches to mobilisation regimens and/or <I>ex vivo</I> HPC manipulation. Accurate and reproducible assays for quantitative and qualitative studies of HPC were established. These included an immunocytometry based assay of CE34 antigen expression, dual colour immunocytometry studies of cell adhesion molecule, lineage and activation marker expression by HPC derived from different sources, and three colour immunocytometry of adhesion molecule expression within HPC subsets. A chromium<SUP>51</SUP>-labelling technique was developed as a functional assay with which to examine the adhesion of haematopoietic cell lines to extracellular matrix components, stromal and endothelial tissues in culture. A variety of techniques for adhesion blockade were explored. A protocol for high-purity enrichment of HPC was developed, and the feasibility of applying the chromium<SUP>51</SUP> adhesion assay to these cells was examined. CD34 immunocytometry was confirmed as a valid method for defining HPC populations. Marrow HPC were found to express nine adhesion molecules, two of which were reduced by circulating cells. HPC derived from different sources showed variation in lineage and activation marker expression, and HPC subsets displayed differences in adhesion molecule expression. Haematopoietic cell lines adhered to fibronectin and thrombospondin, but not to other extracellular matrix components. Blockade of fibronectin adhesion was effected by divalent cation chelation, synthetic peptides and chondroitinase ABC. Cell line adhesion to stromal and endothelial tissue cultures was demonstrated, but highly-enriched HPC labelled poorly with chromium<SUP>51</SUP>.

Human Serum Albumin (HSA) is a plasma protein of great significance with an ability to bind numerous endogenous and exogenous ligands, having a single polypeptide chain of 585 amino acids constructed in three domains of relatively equal size. Although HSA's existence has been known for many years, its characterisation and ability to bind ligands still remain enigmatic. Only in 1992 was the crystal structure of HSA reported (Carter and Ho, 1992). Effective crystallisation and the determination of meaningful crystallographic data and structure had proven difficult. The 1992 report concerned HSA crystals grown using zero gravity conditions. The objective of this project is the characterisation of recombinant and native HSA using Ultraviolet (UV) &amp; circular dichroism spectroscopy (CD) and involved HSA conformational changes, which altered the ability to bind or off-load ligands. Changing environment, together with the use of characteristic "marker ligands," influences binding to provide a handle that can be utilised and monitored. This enables the assignment of binding sites and the study of perturbating conditions. Changing conditions such as pH, temperature, ionic strength and solvents in the presence and absence of ligands were employed to extract further information on this elusive protein. Fragments of recombinant HSA were also used (namely domain I and domain I + II) under identical conditions as the whole protein in order to help elucidate and assign binding sites.

The thesis describes studies on the control of ion movements across membranes of human red blood cells. The first part deals with whether active and passive movements of sodium and potassium are influenced by changing the potential difference (p.d.) across the membrane. Replacement of external chloride with non-penetrating anions changed the chloride gradient and, as chloride was passively distributed, the p.d. was calculated. With low external chloride the intracellular chloride concentration was greater than that outside, indicating reversal of the sign of the membrane potential from -9 to +30 mV. Ouabain-sensitive (pump) sodium and potassium fluxes were little affected. Potassium efflux and ouabain-insensitive sodium efflux were increased ten- and three-fold respectively, whilst ouabain-insensitive potassium influx was halved and sodium influx unaffected. Thus, the main effect of reversing the chloride gradient was a marked increase in passive outward movements of sodium and potassium ions. Part Two describes a study of chemical reactions of the sodium pump enzyme in relation to the regulation of active sodium and potassium movements. The main aspect was to see whether p-nitrophenylphosphate (pNPP) hydrolysis by human red blood cells, ghosts and fragmented membranes was mediated by the sodium pump. In cells, pNPP uptake was inversely proportional to external chloride and the uptake mechanism was similar to that for inorganic phosphate. Hydrolysis of pNPP by both cells and ghosts was insensitive to external potassium and ouabain. Hydrolysis by fragmented membranes was partly sensitive to potassium and ouabain. The main finding was that there was a marked decrease in enzymic activity on raising the ionic strength of the medium, whereas there was no effect on ATPase activity. It is this effect, as well as the presence of a large amount of intracellular phosphatase, which accounts for the absence of a ouabain-sensitive component of pNPP hydrolysis when cells or ghosts are incubated in physiological electrolyte media.

The classic view of the renin-angiotensin system is that of a circulating, systemic hormonal cascade regulating blood pressure and extracellular fluid volume. However, increasing evidence has led to the suggestion that functional renin-angiotensin systems comprising some or all of the components of the enzymatic pathway may exist within several individual organs or tissues. The purpose of the work presented in this thesis was (i). to confirm renin- angiotensin system gene expression in various tissues (ii). to examine the effects of inhibition at different levels on expression (iii). to investigate the effects of experimental post-infarction heart failure on tissue renin-angiotensin systems and to see whether ACE inhibitor treatment, which improves this condition, influences gene expression. In the work presented, first a systematic study of rat tissues by Northern blotting and RT-PCR demonstrated the expression of mRNA for components of the renin-angiotensin system in several tissues, supporting the concept of their existence and resolving controversy over the potential for expression in certain tissues. Subsequently, both acute and chronic pharmacological blockade of the cascade by an ACE inhibitor demonstrated differential regulation of the renin-angiotensin system in different tissues. In farther studies, blockade of the cascade at three different levels in the marmoset confirmed differential regulation of specific components in different tissues. In contrast to previous reports, induction of experimental heart failure in rats followed by administration of an ACE inhibitor resulted in a 1.6 fold increase in renal angiotensinogen mRNA and a change in cardiac ACE mRNA was not seen with heart failure. A reduction in mortality was also associated with ACE inhibitor treatment. The results obtained raise interesting questions both of the role of tissue renin- angiotensin systems in contributing to the pathophysiology of disease states and mediating the effects of pharmacological blockade of the renin-angiotensin system, and more generally regarding their organisation.