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The principal aim of the work presented in this thesis was to investigate the host-pathogen interaction in Toxoplasma gondii infected cells and to elucidate which proteins and pathways were involved in the host response to infection. Toxoplasma gondii is of clinical importance for both human and veterinary disease. There are currently no drugs available to treat the chronic stage of disease. The transcriptional response of host cells to Toxoplasma infection has previously been investigated using microarrays. Proteins are the functional molecules in the cell and the protein expression response is likely to differ from the transcriptional response due to gene expression control and post translational modifications. The advantage of a global proteomic expression profiling study of Toxoplasma gondii infected cells is that, in principle, all possible host responses could be identified without pre-supposing a specific mechanism. Advanced proteomic methods were developed and used to investigate the host protein response to infection with Toxoplasma gondii. A conventional gel-to-gel approach was used in concert with difference gel electrophoresis (DIGE) to investigate the protein expression in infected cells. This part of the work concluded that the patterns of protein expression in infected cells showed clear differences from that of the non-infected cell. This resulted in the unambiguous identification of one hundred and fifty seven host proteins which changed in expression during infection with Toxoplasma gondii. Many proteins not previously implicated in response to infection were identified. The results from the proteomic studies were compared with the transcriptional study in order to determine the gene to protein correlation in Toxoplasma gondii infected cells. A weakly positive correlation between gene and protein expression was determined. To investigate whether the host response is uniquely tailored to an individual pathogen or whether cells have a common response to infection, the protein response to a second intracellular parasite, Leishmania major, was investigated. Far fewer changes were seen in the L.major infected cell proteome, proving that the host response to infection is unique to the pathogen. Phosphorylation is one the most common post translational modifications therefore the host cell phosphoproteome was investigated alongside the steady state proteome of infected cells using three techniques and proteins specifically phosphorylated due to the presence of the parasite were identified. Functional analysis of the modulated proteins revealed extensive modulation of many pathways and functions within the infected host cell including glycolytic, mitotic and structural proteins. Over one third of all the modulated proteins identified were mitochondrial. Overall the findings detailed in this thesis indicate that the host cell has a highly specific response to Toxoplasma gondii infection and that many host cell functions are modulated due to the presence of the parasite and reveal the intimate molecular relationship between host and parasite.

The diagnosis and management of patients requires professional know-how or medical craft knowledge. To explain how this knowledge is acquired, this research asked 'How do medical experts pass on their craft?' Other questions arose through successive data collections and progressive focusing on what medical experts did well in their work and teaching. The programme comprised: pilot interviews with three expert physicians; a case study in a hospital medical unit; and paired consultant/SHO interviews. Participant observation, interviews, and expert-novice comparisons explored clinical work, teaching, and learning in apprenticeship. Data analysis of participants' responses and ward round discussions allowed identified categories to cluster within three inter-related constructs instrumental to the acquisition of medical knowledge: gaining experience in the experiential process of clinical practice (1); and the products of experience which manifest as experts' clinical expertise (2) and teaching/learning expertise (3). These constructs can be located within a model of apprenticeship based on Spady's (1973) analysis of authority in effective teaching containing two frames of reference: the social, 'traditional-legal'; and the individual, 'expert-charismatic'. The medical apprenticeship is associated with similar perspectives: the 'traditional-experiential' represents the professional process of learning through patient care with its infrastructure of clinical methods in presentation, discourse, and commentary; and the 'expert-charismatic' represents clinical and teaching expertise coupled with vocational enthusiasm. Experienced experts synthesised two repertoires of knowledge and skills derived from the craft knowledge of medicine and pedagogy, respectively. Both crafts are required for effective clinical education. While apprenticeship accommodates a range of teaching/learning experiences, in postgraduate education experts pass on knowledge through the deliberate engagement of junior doctors in diagnosis and management. The skills involved in this process were largely unrecognised by most senior and junior doctors and were not perceived as 'clinical teaching' although learning was structured through service-based work.

The acid hydrolase N-acetyl-Beta-D-glucosaminadase (NAGase) la a high activity lysosomal enzyme in trichomonads. Tritririchomonas foetus (strains F2, KV1 and CA84-2), Trichomonas vaginalis and Trichomonas augusta all contained and released NAGase. Analysis of NAGase using electrophoretic techniques demonstrated in all the Tritririchomonas foetus strains the same four major forms, named NAGase 1-4, whose mobility suggested apparent M,s of 54 000, 89 000, 100 000 and 158 000 respectively. Trichomonas vaginalis and Trichomonas augusta, however, each contained a single form of this enzyme of apparant M,s 138 000 and 107 000 respectively. In all cases the intracellular and extracellular forms of NAGase appeared to be identical. Growth of Tritririchomonas foetus, F2, in the presence of the glycosylation inhibitor, tunicamycin, resulted in an extra NAGase band with higher mobility assumed to be a non-glycosylated but active enzyme form. NAGase 3 mobility was simultaneously diminished. Extra NAGase forms wars also observed after incubating this parasite In serum free media containing the two proteinase inhibitors, leupeptin and Z-Phe-Phe-CHN2, indicating that proteolytic processing may be involved in the production of NAGase forms. NAGase 1, the enzyme with an apparent M, of 54 000, was purified from Tritririchomonas foetus, F2. Preparative Isoelectric focusing was used to separate the four NAGase forms and as the first step in its purification. Total denuturation of NAGase 1 before electrophoresis produced a decrease in mobility from an apparent M, of 54 000 to a doublet of apparent M,s of 68 000 and 70 000. This transformation was found to be complete within 2 min at 50°C but was dependent at this temperature on the presence of both SDS and Beta-mercaptoethanol. This transformation also occured without sample buffer at 100°C and may relate to the breaking of disulphide bridges. Treatment of NAGase 1 with endoglycosidase reduced its apparent M, by 2000, indicating the enzyme to be an N-linked glycoprotein. Trichomonas vaginalis and Tritririchomonas foetus were also found to contain and release acid phosphatase. SDS-PAQE analysis of this hydrolase found one form with an apparent M, of 126 000 In Tritririchomonas foetus, strains F2 and KV1, Trichomonas augusta also had one form of apparent M, 135 000 while Trichomonas vaginalis contained and released two forms with apparent M,s of 155 000 and 160 000. Despite several immunisation attempts purified NAGase 1 was found to be non-antigenic in rabbits and so It was not possible to analyse the release processes In the detail originally proposed. However, although the results have not allowed details of the release mechanisms and physiology, they have confirmed release and provided more Information on the properties of the hydrolases, especially NAGase.

This thesis set out to investigate the role of Prostaglandin F-2alpha (PGF-2alpha) in luteolysis in the mare and to determine any alterations in PGF-2alpha production between normal cycling mares and mares in prolonged dioestrus.