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No description available.

No description available.

No description available.

No description available.

No description available.

Despite the technological advances of the twentieth century, we are not yet able to produce artificial limbs which "mimic" perfectly their natural counterparts. In general, artificial limbs are not as dextrous as human limbs, the control is unnatural and there is no proper feedback by which the user can assess the status of the prosthesis. In this thesis the problems related to upper-limb prostheses are considered. The use of a special material known as Shape Memory Alloy (SMA) is investigated towards producing improved joint actuators for small artificial prostheses such as those required by young children. SMA actuators can be very lightweight, their motion is silent and smooth and yet they are capable of delivering considerable power per unit of weight. The Shape Memory phenomenon and the many challenges involved in its application are discussed. The detailed design of an SMA joint actuator for a hand mechanism in an above-elbow prosthesis for young children is given. To assist the design and construction of both the artificial hand and the actuator, a mathematical model was developed and incorporated in a computer program simulating the forces and movements within the hand. The model was used to optimise the hand mechanism and specify the required joint actuator. Suitable SMA elements were identified through laboratory tests. The hand mechanism was constructed and the actuator, control systems and power source were attached to it. Tests were performed to investigate the characteristics of the complete device. The results show that, although SMA actuators must be designed and used with great care, they do offer a viable and more natural alternative to conventional actuators such as pneumatic devices and electric motors in certain applications.

The aim of this thesis was to investigate and characterize the effects of isoenzyme- selective phosphodiesterase (PDE) inhibitors on a range of biochemical and functional parameters in bovine airway smooth muscle in order to further our understanding of 'cross-talk' between different second messenger pathways in this tissue. Cyclic nucleotide levels were measured after selective or non-selective PDE isoenzyme inhibition under conditions of basal and stimulated adenylyl and guanylyl cyclase activity. These data led to the conclusion that PDE IV plays an important role in the regulation of agonist-stimulated increases in cAMP levels, since under conditions of isoprenaline stimulation, incubation with the PDE IV-selective inhibitor rolipram resulted in an increase in cAMP from a control level of 20 2.7 to 50.3 4.0 pmol/mg protein. This response was potentiated in a synergistic manner by simultaneously inhibiting PDE III with ORG 9935. Co-inhibition of PDE III/IV also resulted in a significant increase in basal cAMP levels in tracheal smooth muscle slices and a marked decrease in the rolipram EC50 for cAMP accumulation in isoprenaline-stimulated slices from 205 102 to 7.3 3.0 muM. This suggested that metabolism of cAMP by PDE III is also functionally important in this tissue at least when PDE IV activity is compromized. Evidence was also obtained to suggest that cGMP is metabolized by PDE V, since incubation with zaprinast resulted in a 47% increase in basal cGMP values; however, where cGMP levels were elevated a greater increase in cGMP accumulation was seen in the presence of the nonspecific PDE inhibitor, IBMX (24.3 1.6 pmol/mg protein) compared to that seen with zaprinast (15.2 1.6 pmol/mg protein) suggesting that cGMP metabolism by other PDEs plays a significant role under these conditions. The effects of selective PDE inhibition on agonist-stimulated inositol phosphate accumulation was then investigated. Stimulation with maximally-effective concentrations of carbachol and histamine for 30 min resulted in 36- and 10-fold increases in inositol phosphate accumulations. The response to carbachol (1-100 muM) was largely unaffected by increases in cAMP accumulation caused by isoprenaline or PDE inhibition, however both manipulations inhibited the response to histamine (100muM) by approximately 80%. Again the inhibitory effects of rolipram were potentiated by ORG 9935 such that the EC50 value for rolipram-mediated inhibition was decreased from 120 27 to 1.5 0.9 muM. Such results suggest that PDE III/IV inhibition may be effective in relaxing airway smooth muscle. Consequently it was established that whilst rolipram could inhibit smooth muscle contraction stimulated by histamine or sub-maximal concentrations of methacholine, co-inhibition of PDEs with rolipram and ORG 9935 resulted in a much more potent anti-spasmogenic action. Furthermore, in contrast with the effects of rolipram alone the PDE III/IV inhibitor combination also significantly inhibited phasic contractions generated by either agonist. For example rolipram/ORG 9935 completely abolished the ability of 30uM histamine to cause a phasic contractile response. In common with previous suggestions that the membrane hyperpolarizing actions of cAMP-elevating agents may be responsible, at least in part, for the relaxation of trachealis muscle, experiments reported here suggest that PDE inhibition causes membrane hyperpolarization, possibly through increasing the open-state probability of the high- conductance, calcium-activated potassium channel, and this action may account for the mechanism whereby selective PDE inhibitors can inhibit phosphoinositide turnover and possibly as a consequence relax airway smooth muscle.

The generic term 'esterases' broadly represents enzymes that hydrolyse aliphatic or aromatic esters. and within this definition is a group of esterases that show a preference for short acyl chain esters such as a-naphthyl acetate, propionate or butyrate. These are ti often collectively referred to as 'non-specific esterases' (NSE), but a more accurate and descriptive designation is according to the substrate used. The demonstration of esterases by azo-dye techniques has found particular applications in haematological cytochemistry, and, with specific reference to normal myeloid cells, a-naphthyl acetate esterase (ANAE) 0 cytochemical reactions of moderate-strong diffuse type are typically associated with cells of monocyte/macrophage lineage. In contrast. granulocytes at all levels of morphological differentiation are cytochemically ANAE negative. Isoelectric focusing (IEF) studies of ANAE isoenzymes have further revealed the existence of two species with apparent differences in lineage affiliation. The first (ComEst) is expressed by both granulocytes and monocytes, and comprises a series of isoenzymes with isoelectric points (p1) ranging from 6.3 to 7.9, whereas a second (MonEst) species that is specifically expressed by haemopoietic cells of monocytic/macrophage origin is seen on IEF zymograms as a series of isoforms within a relatively narrow pI range of 5.5-6.2. These present studies were undertaken in order to extend what is currently known of the cellular, kinetic and molecular features of the two main myeloid esterase species. It was considered that these investigations were necessary to clarify the nature of atypical cytochemical reactions in leukaemic and dyshaemopoietic myeloid cells, to establish whether or not the two species were related or distinct enzymes, to gain further insights into their possible functional role(s), and to provide molecular details of relevance for the longer term aim of cloning MonEst protein in particular. Studies reported here of normal myeloid cells confirmed the lineage affiliation of the two main esterase species, and analysis of a large number of acute myeloid leukaemias also resolved the nature of atypical ANAE cytochemistries. Abnormally increased focal and granular reactions of myeloid blasts was shown to be due to over-expression of ComEst, and the lack of ANAE cytochemical staining in a significant proportion of monocytic leukaemias was shown to result from a failure to synthesise MonEst. As a prelude to the biochemical purification of myeloid esterases, the ComEst and MonEst species were also investigated to determine their chromatographic characteristics. This involved an evaluation of a wide range of column gels including ion-exchange, hydrophobic interaction, affinity, and gel filtration. The purification protocol resulting from these evaluations successfully permitted the purification of ComEst to a highly enriched state and MonEst to homogeneity. Subsequent molecular and kinetic analyses revealed that enzymatically active MonEst exists in its native state as an apparent trimer which, under non-reducing conditions, dissociates to inactive 63 kDa monomers. In contrast, native ComEst was shown to be a 68 kDa monomer which retained enzymatic activity following SDS treatment, and was not dissociated under reducing conditions. Lectin affinity studies confirmed that both esterase species were glycoproteins but differed in that MonEst contained oligomannosidic-type glycan(s) whilst ComEst contained a mixture of fucosylated and non-fucosylated biantennary N-acetyllactosamine-type glycan(s). Neuraminidase, a-mannosidase, a-L-fucosidase, and endoglycosidase H were shown to have no effect on the pI distribution of individual ComEst or MonEst isoforms, but endoglycosidase treatment did reduce the Mr of MonEst from 63 to 60 kDa. Enzyme kinetic studies also revealed that purified ComEst preferentially hydrolysed esters of short acyl chain length (C2 and C3) whilst MonEst hydrolysed esters of higher acyl chain length (butyrate > propionate > acetate). However, MonEst failed to hydrolyse a wide range of natural and synthetic peptidase substrates thus tending to exclude its functional role in peptide processing. Possible differences in reaction mechanisms of the two esterase species were also evaluated by examining the inhibitory effects of representative enzyme inhibitors which demonstrated that serine and histidine residues were required. for MonEst but not ComEst activity. N-terminus amino acid sequencing of purified MonEst indicated almost complete identity with human alveolar macrophage esterase. differing only in a Val-Thr substitution at position 12, and close similarities with rabbit liver carboxylesterase. In summary, substrate and inhibitor studies strongly suggested that the MonEst and ComEst species should be classified as carboxylesterases (EC 3.1.1.1) and acetylesterases (EC 3.1.1.6) respectively and that, together with distinct differences in their molecular and biochemical characteristics, it is concluded that these are unrelated myeloid enzymes which share only the ability to hydrolyse a-naphthyl acetate. Although yet to be established, the kinetic and molecular differences reported here may have fundamental relevance with respect to the biofunctional role(s) of these enzymes.