The physiological adaptations enabling the terrestrial mite Alaskozetes antarcticus (Michael) (Acari: Cryptostigmata) to inhabit the maritime Antarctic environment at Signy Island, South Orkney Islands, were investigated by experiments on animals cultured in the U.K. Log10 individual oxygen consumption rate was linearly related to log10 live weight and metabolic rate (oxygen consumption per unit live weight) was related to the reciprocal of the absolute temperature by the Arrhenius equation. Alaskozetes possesses a higher metabolic rate than comparable temperate species, measured at the same temperature, and is thereby enabled to remain active at temperatures that would immobilize temperate forms. A low activation energy for certain reactions may be responsible for metabolic rate elevation. Alaskozetes lacks the ability to regulate its metabolic rate in response to diurnal or seasonal temperature fluctuations. This is considered to enable the maximal utilization of higher temperatures for activity, feeding and growth and the conservation of metabolic reserves at low temperatures. Starvation, food materials, oxygen concentration and length of culture period are among the other factors affecting oxygen consumption rates in Alaskozetes. Freezing is fatal in Alaskozetes; winter survival occurs by means of supercooling and absence of ice formation in the tissues. The capacity to supercool extensively is dependent on the absence of gut contents containing efficient ice nucleating agents. Low acclimation temperatures prevent feeding and thereby promote supercooling. Supercooling ability is also increased by glycerol production in response to low temperature or low relative humidity, since glycerol concentration and supercooling points (temperatures of spontaneous body freezing) are linearly and inversely related. The ability to avoid freezing by supercooling and the use of low temperature as a cue for the development of increased cold tolerance are of considerable survival value in the field. The concept of adaptation is discussed with illustrations drawn from the results of the present research on Alaskozetes.

Bovine CL were collected on days 5, 10, and 15 of the oestrous cycle following synchronised oestrus. In addition, CL were collected following prostaglandin-induced luteolysis. <i>In situ</i> hybridisation detected luteal expression of IGF-I, -II and the type 1 IGF receptor mRNA throughout the oestrous cycle. IGF-I mRNA concentrations varied significantly during the cycle, increasing from low levels in the early luteal phase (day 5) to high levels in the late luteal phase (day 15). Concentrations were maximal 48 hours after exogenous prostaglandin. In contrast, there was no significant effect of day of the oestrous cycle on IGF-II and the type 1 IGF receptor mRNA concentrations in the corpus luteum. IGF-II mRNA expression was localised to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. In addition, the relative abundance of IGF-II mRNA was greater than that of IGF-I mRNA. mRNA encoding the type 1 IGF receptor was widely expressed, in a pattern suggestive of large and small luteal cell expression. The actions of the IGFs are modulated by their association with members of a family of IGF-specific binding proteins (IGFBPs), which regulate the transport of IGFs and their presentation to specific receptors. <i>In situ</i> hybridisation detected mRNA encoding IGFBP-2, -3, and -4 in the bovine corpus luteum throughout the luteal phase. IGFBP-2 and -4 mRNA concentrations were low within the corpus luteum, and showed no temporal variation. In addition, a subset of large vessels in the periphery of the CL showed moderate to intense hybridisation for IGFBP-2 mRNA. IGFBP-3 mRNA concentrations were high throughout the luteal phase, and expression was localised to small luteal cells and cells of the luteal vasculature. These data demonstrate for the first time that the bovine CL is a site of IGF production, reception and regulation, and further support the hypothesis that the IGF system is important in regulating luteal function in the cow.

Ovaries were studied on luteal days 10, 18 and 22 (corresponding to the mid luteal phase, functional luteal regression and structural luteal regression respectively), and also 12 and 24 hours after administration of either PGF2a or GnRH antagonist. Decreased progesterone concentrations indicative of functional luteal regression were apparent 12 hours later. Analysis of haematoxylin and eosin stained sections of corpora lutea indicated that the administration of PGF2a or GnRH antagonist resulted in apoptosis, and also in the formation of cytoplasmic vacuoles in steroidogenic cells. Apoptosis in corpora lutea was further investigated by 3' end labelling DNA extracted from corpora lutea, and by <i>in situ</i> 3' end labelling of sections of ovarian tissue. Apoptosis was found to occur after induced luteolysis and in naturally regressing corpora lutea but only after progesterone had decreased to follicular phase values. Therefore the decline in progesterone characteristic of functional luteal regression was not caused by the apoptotic cell death of steroidogenic cells. However, apoptosis played a role in structural luteal regression. Ubiquitin is expressed only by live cells undergoing a process of non-apoptotic cell death. Ubiquitin expression was only found in PGF2a, but not in GnRH antagonist treated luteal tissue, suggesting three possible explanations: that the cells in GnRH antagonist treated animals were dead prior to the collection point of 12 hours, or that the cells were not in a cell death pathway, or that cell death was occurring via different mechanisms in PGF2a and GnRH antagonist treated animals. The importance of the vasculature in luteal regression was investigated by labelling endothelial cells with an antibody against von Willebrand Factor VIII Antigen. Endothelial cell numbers remained constant after administration of luteolytic agents, indicating that induced luteal regression was not effected by vascular changes. Similarly, the vascualture did not change during functional regression in untreated animals. Vascular remodelling, however, occurred during structural luteal regression, when the vasculature changed from an extensive network of small capillaries to a system comprised of a lower number of larger blood vessels.

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The major outer membrane protein (MOMP) of <I>Chlamydia</I> shares several biochemical properties with classical porin proteins. To directly test the "porin channel" hypothesis at the molecular level, MOMP was reconstituted into planar lipid bilayers where it gave rise to "triple-barrelled" channels which were modified by an anti-MOMP neutralising monoclonal antibody. These observations are consistent with the well characterised homo-oligomeric nature of MOMP previously revealed by biochemical analysis, and the "triple-barrelled" behaviour of other porins. MOMP channels were weakly anion selective (P<SUB>Cl</SUB>/P<SUB>K</SUB> ~ 2) and permeable to ATP. They may therefore be a route by which <I>Chlamydia</I> can take advantage of host nucleoside triphosphates, and explain why some anti-MOMP antibodies neutralise infection. In order to undertake more detailed studies of the MOMP structure/function relationship, recombinant MOMP from both <I>C. psittaci</I> and <I>C. pneumoniae</I> have been cloned and expressed. The recombinant proteins were functionally reconstituted in planar lipid and analysed at the single channel level. Both form porin-like ion channels that are functionally similar to the native protein. The <I>C. psittaci</I> recombinant porin was modified by the same anti-MOMP neutralising monoclonal antibody that effected the native protein. In contrast to the native protein, both recombinant <I>C. psittaci</I> (P<SUB>Cl</SUB>/<SUB>K</SUB> ~ 0.38) and <I>C. pneumonia </I>(P<SUB>Cl</SUB>/P<SUB>K</SUB> ~ 0.49) proteins were marginally cation selective. This is the first time native function has been demonstrated for recombinant chlamydial MOMP and will have an important impact on the future development of subunit vaccines.

Microneme proteins (MICs) belong to a protein family that is actively involved in the invasion of host cells by apicomplexan parasites. Among these proteins, MIC2 is a member of the Thrombospondin-related anonymous (TRAP) family of adhesive proteins, which are highly conserved within the apicomplexans. The other microneme protein M2AP, known to facilitate the transport of MIC2 through the secretory network, is also essential to the virulence of the parasite. Most importantly, MIC2 and M2AP form a multimeric functional complex that presents in the cell invasive stages and serves as the prototype of other TRAP family members. This thesis describes the NMR assignments and two solution structure calculations of M2AP and a Thrombospondin type-1 repeats (TSR-1) domain. M2AP is composed exclusively of beta strands and, despite no sequence similarity, demonstrates clear structural similarity to the galectin-like domain from MIC1. And TSR-1 repeats from MIC2 demonstrate two typical TSR-like-fold domains. Interactional studies reveal that the M2AP binds both the first and the last pair of TSR-1 repeats of MIC2. The results on structure–function relationship of MIC2 and M2AP reinforce the critical role of TRAP protein in the successful invasion of cells by T. gondii and reveal potential therapeutic targets that may be used to control toxoplasmosis.

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