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The use of flow injection analysis with fluorescence detection was evaluated using the host-guest phenomenon between the cyclodexnins and DL-Iysine and Lserine. Auorescence enhancement, kinetic and equilibrium studies were recorded and the effect of pH and time on fluorescence were also observed. Rhodamine isothiocyanate was conjugated to insulin. Insulin and dye were mixed in different ratios, and the dye : insulin ratio was detennined for each conjugate. These conjugates were checked for immunoreactivity. Insulin-biotin and antibody-iminobiotin conjugates were also prepared. Insulin : biotin ratio was also determined. An insulin-biotin avidin-Texas Red complex was also prepared. Each was checked for immunoreactivity. Protein G-agarose, protein A-controlled pore glass(CPG), streptavidin-agarose, and avidin D-agarose-biotin-antibody solid phase immunoreactors were used in flow injection immunoassay of insulin. In these immunoassays, antibody, insulin and labelled insulin were incubated in vitro and then injected onto the immunoreactor. A binding buffer carried the sample through the immunoreactor and a fluorescent detector. An acidic buffer then eluted the components of the sample bound to the immunoreactor, which were then measured. An assay range for insulin was developed in each solid phase assay.

The Life Marker Chip (LMC) instrument is an antibody assay-based system which will attempt to detect molecular signatures of Life in the Martian subsurface as part of the payload on board the European Space Agency (ESA) ExoMars mission rover, currently scheduled for launch in 2018. The LMC will have the ability to detect up to 25 different molecular targets of different origins that are associated with meteoritic in-fall, extinct or extant Life, prebiotic chemistry and spacecraft contamination. Regolith / crushed rock samples will be collected for the LMC by the rover and subjected to solvent extraction to extract organic molecules for analysis by the immunoassays. One of the key stages in the development of the LMC is the selection of antibodies to be used in the flight instrument. The challenge lies in the nature of the molecules or classes of molecules that are LMC targets and the need for antibodies that remain functional in the extreme conditions during a planetary exploration mission, especially the radiation environments. The work described within focuses on two main aspects of the search for LMC-relevant antibodies; the effect of space radiation on antibody performance [in the form of both ground-based and Low Earth Orbit (LEO)-set studies] and the development of ―customised‖antibodies against some of the molecules that are being investigated as potential LMC targets. The need to study the effects of space radiation on antibodies arose due to lack of any heritage of their use in interplanetary missions. For all the antibodies in the LMC, the ability to resist inactivation due to space radiation seen during a Mars mission will be a prerequisite. The objective of the ground-based radiation studies was to expose a number of LMC-relevant antibodies to simulated Mars mission radiation in the form of proton and neutron radiation which are the components of the mission radiation environment that are expected to have the dominant effect on the operation of the LMC. Cont/d.

In their early development and before their antibodyforming mechanism is fully established the young of mammals depend entirely on the maternal supply of antibodies. The passive transfer of immunity acquired by the young from its mother can occur before birth, after birth, or both. In man, rabbit and guinea-pig, for example, the transfer of immunity from mother to young occurs entirely prenatally. In the ungulate, horse and pig the passive transfer of immunity occurs postnatally, whilst in dog, rat and mouse it occurs both pre- and postnatally. The difference between these groups in the mechanism of antibody transfer from mother to young was earlier thought to be due to the difference in the number of layers intervening between the maternal and the foetal circulations (Kuttner and Ratner, 1923). In other words the structure of the placenta was the decisive' factor in the mechanism of the transfer of passive impunity to the young. According to Grosser's (1909; 1927) classification, there are four types of mammalian placental structure. (1) the epitheliochorial with six intervening layers, (2) the syndesmochorial with five, (3) the endotheliochorial with four and (4) the haemochorial placenta with three. These were later modified by Mossnan (1926), who added a fifth type, the haemoendothelial with one. Since animals with epitheliochorial and syndesmochorial placentae transfer antibody entirely postnatally to their young, whereas in those with haemochorial and haemoendothelial placentae transfer is mainly prenatal (with the exception of rats and mice where transfer is mainly postnatal), and in those with endotheliochorial placentae both prenatal and postnatal transfer is considerable, it was perhaps not surprising that a'purely physical hypothesis of antibody transfer should be generally accepted. But in 1946 Hartley showed that the human placenta was selective in transferring antibody, so, that refined horse antibody was not transferred to the foetus at all, while human. antibody passed freely. Shortly after, in a series of papers, Brambell and his colleagues (1949, 1950,1951,1952) showed that there was considerable doubt whether in rabbits antibody is transferred via the placenta at all. They suggested that antibody circulating in pregnant rabbits is secreted into the uterine cavity, whence it passes via the yolk-sac splanchnopleur into the foetal circulation. Moreover they showed that the yolk-sac splanchnopleur is a highly selective membrane, differentiating between -globulins not by molecular size but by*species of origin. In particular Brambell, Hemmings and Oakley (1959) showed that rabbit antibody digested with pepsin was less readily transferred to the foetus, notwithstanding its lower molecular weight, than the unmodified antibody. When rabbit -globulin wap digested with papain (Brambell, Hemmings, Oakley and Porter, 1960) to yield Porter's fractions 1, II and III and these fractions were injected into the rabbit uterine cavity, fraction III was transferred to the foetus almost as well as the unmodified -globulin, fraction I and II much less well. In the Guinea-pig the route of antibody transfer from mother to young was also shown to be via the yolksac splanchnopleur and the vitelline circulation of the foetus (Barnes, 1957). The present work was undertaken to investigate by the technique of intra-uterine injections of antitoxic sera used by Brambell and colleagues the selectivity of the yolk-sac splanchnopleur of the guinea-pig foetus for homologous and various heterologous antitoxins and for the antibody fragments obtained by the peptic digestion of the antitoxin molecule.

Neutrophils are at the front line of defence in humans against attack by invading pathogens. They undergo complex behaviours, many of which are controlled by changes in the concentration of cytosolic free calcium ions (Ca2+). However, the control and mechanisms of these behaviours are still the subject of much research and debate. Ca2+ is known to be important in many different cell systems. There are a plethora of Ca2+ signalling events in the "Ca2+ toolkit", but not all cells have each element. It is not fully understood elements of the toolkit that neutrophils employ to make up their well- characterised global Ca2+ changes. This thesis is an investigation into the involvement of Ca2+ in neutrophil behaviours that underlie much of their functionality, particularly focussing on subcellular Ca2+ release events (puffs) and Ca2+ signalling during neutrophil spreading on surfaces and during phagocytosis. Reports were published by Petty et al of a travelling zone of elevated Ca2+ (z-wave) in neutrophils. However, research laid out in this thesis demonstrates that these z-waves are questionable in neutrophils. Instead, conventional Ca2+ puffs and waves were seen and characterised in response to fMLP stimulation and uncaging IP3. Neutrophils undergo dramatic, rapid morphological changes during Ca2+ signals triggered by both fMLP and uncaging IP3, representative of behaviour during adhesion and extravasation. It was shown that the high concentration of Ca2+ under the plasma membrane that results from Ca2+ influx activated the protease calpain, which released the membrane from the cytoskeleton for rapid shape change. Similar signals and behaviours are seen when neutrophils are stimulated by binding of beta3-integrins. Indeed, adhesion to ICAM-1 expressing cells and phagocytosis resulted in global Ca2+ signals and Ca2+ influx accompanied by rapid shape change. However, no Ca2+ puffs were seen in these latter experiments, implying that the signalling in these conditions occurs through a different pathway, possibly PIP3. In conclusion, neutrophils utilise conventional Ca2+ signalling events (release puffs and influx waves) not z-waves to control many of their functions. They are capable of producing Ca2+ puffs, but the lack of ER means this is unlikely to be a primary signalling pathway. Binding through jS-integrins does trigger Ca2+ signals, possibly through the PIP3 pathway, but not store release. The characterisation of Ca puffs and the functionality of the Ca2+-activated protease calpain in these conditions are novel and merit future investigation. The findings in mis thesis enhance the general understanding of how neutrophil behaviours are regulated by Ca2+ and provide a new methodology for further research.

The published articles [p. 268-278] are not available in the electronic version of this thesis due to third party copyright restrictions. The full version can be consulted at the University of Leicester Library.