Satisfactory development of preantral follicles from humans and domestic ruminants <i>in vitro</i> remains elusive. The aims of this thesis were to use a serum-free culture system to identify regulators of early follicle and oocyte development. Preliminary experiments determined that bovine preantral follicles grow and produce increasing amounts of oestradiol throughout a six day culture period. Neither FSH nor IGF-1 significantly increased follicle diameter. However, FSH did promote follicular oestradiol secretion. The dissociation of follicular growth from steroidogenenic function indicated that measurement of follicular diameter may not be a reliable marker of physiological follicular development <i>in vitro</i>. In addition, stimulation of granulosa cells by FSH may result in inappropriate differentiation of these cells during the early stages of folliculogenesis. During follicular development, turnover and reconstruction of the basement membrane is facilitated and regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). When MMP-9 was secreted by prenatal follicles <i>in vitro, </i>the probability of follicles having healthy granulosa or theca cells at the end of culture was 0.85 and 0.60, respectively. If TMP-1 was released, there was a probability of 0.79 that the follicles would have healthy somatic cells. When TIMP-2 was detected, the probability of granulosa and theca cell health was 0.78 and 0.67, respectively. These results indicate that MMP-9 and TIMPs are related to follicular health, and can therefore be used as markers of follicular development. Ascorbic acid has been implicated in several processes associated with follicular development, including collagen biosynthesis, steroidogenesis and apoptosis. The effect of this vitamin on the development of bovine preantral follicles was investigated during a twelve day culture period. Ascorbic acid had no effect of follicular growth or oestradiol secretion. Serum addition from Day 0 stimulated follicular growth but compromised follicular integrity. By Day 12 of culture, a higher proportion of follicles remained intact in the presence of ascorbic acid in serum-free conditions, with significantly less granulosa and theca cell death than control follicles.

Mouse embryonic stem (mES) cells can be maintained <i>in vitro </i>without loss of pluripotency in the presence of leukaemia inhibitory factor (LIF). Germline competent mES cells can be genetically modified <i>in vitro</i> and used to make transgenic mice via chimaeras. Germline competent chicken ES (chES) cells would be a powerful tool for the production of transgenic chickens. ES cells have been isolated from primates and mice but attempts from other species have been unsuccessful. Novel ES cell isolation strategies have been tested using inbred mouse strains. Using standard techniques, mES cells cannot be isolated from CBA strain embryos whereas they can be isolated from 129Sv strain embryos. A vital function of LIF in mES cells is activation of signal transducer and activator of transcription 3 (STAT3). LIF also activates the mitgoen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathway that appears to promote differentiation. LIF is a member of the interleukin-6 (IL-6) family of cytokines that includes ciliary neurotropic factor (CNTF). Manipulation of IL-6 family signals is a new approach to mES cell isolation that may be applied to development of methods of chES cell isolation. Prior to investigation of new approaches, standard mES cell isolation was performed. chES cells were not isolated in conditions adapted from standard mES cell isolation. A new approach involving the manipulation of LIF-mediated signals were evaluated in mES cell isolation experiments. The drug PD98059 inhibits the MEK/ERK pathway by preventing phosphorylation of MEK I. ES cell isolation frequency from strain 129Sv embryos increased significantly in the presence of 25μM PD89059 and 500 U/ml LIF (p<0.1) however, CBA ES cells could be isolated in the same conditions. The drug U0126 blocks and MEK/ERK pathway by directly inhibiting both phosphorylated MEK I and MEK II. CBA ES cells were isolated at a frequency of 22.3% in medium containing 2μM U0126 and 2x10³ U/ml LIF.

The development of a culture system to sustain preantral follicle growth to a stage where fertilisable oocytes can be obtained remains elusive in the domestic species. The objectives of this thesis were to develop a culture system capable of supporting porcine preantral follicles, and identify possible markers of oocyte and follicle development. Follicles were cultured in two systems; individually in McCoys medium to identify the effects of ascorbic acid, FSH, and serum, and in NCSU medium individually (96-well plates) or in-groups (24-well plates) to identify the effects of follicle interactions. Results from the McCoys experiment revealed that somatic cell death was significantly reduced in follicles cultured in the presence of ascorbic acid in comparison with other treatment groups, but the vitamin was found to have no effect on follicle growth. Follicle growth was significantly enhanced by the addition of serum and FSH to serum-free medium, but FSH had no effect as a survival factor on granulosa cell death in follicles. Culture in NCSU medium revealed that follicles grew best when cultured individually in the presence of serum in 96-well plates in comparison to follicles cultured in-groups in 24-well plates. All other parameters of follicle health and development were found to be no different between follicles cultured in serum in-groups of individually. The identification of markers of development for follicles and oocytes could also aid the development of a culture system for preantral follicles. GDF-9 is a possible indicator of follicle and oocyte developmental stage. It has been identified in several species, including human, rodents, and domestic species, but not in the pig. In this study it was isolated using human and mouse primers, and sequenced. It was found to display 88% homology to the human sequence. RT-PCR revealed that it appears to be expressed strongly in the porcine oocyte, but not in granulosa, skin or intestinal cells. BMP-15 was also found to be oocyte-specific in the pig, using sheep primers to identify its location.

The use of hollow fibre bioreactors in mammalian cell culture has provided a means by which large populations of viable cells can be grown continuously, for extended periods, in a relatively small volume of vessel. Bioreactors in which one set of fibres are used for nutrient and metabolite exchange from the cell mass are limited by their reliance on an axial flow regime. This pressure mediated flow pattern leads to the formation of nutrient gradients within the cell mass, which occur due to the large diffusional distances present in the growth space, resulting in cell death within the bioreactor. The Edinburgh Dual Hollow Fibre Bioreactor solves this diffusional problem by using two sets of fibres to mimic the arterio-venous flow found in the body. In this instance the diffusional distances are reduced due to the close proximity of the two sets of fibres. Using this design, and a murine hybridoma (ES4) which produces an IgM blood typing antibody, cells were grown to a maximum density of 1.2x10<SUP>7</SUP> viable cells ml<SUP>-1</SUP>, with a maximum antibody production rate of 0.16 mgh<SUP>-1</SUP>. This was a 10 fold improvement in the viable cell number obtained using an airlift fermenter. The successful operation of this design was found to be dependent upon the pressure profiles developed within the bioreactor, with a number of extended culture experiments having been carried out. The influence of bioreactor design, fibre selection and the methods by which these bioreactors should be operated are discussed in this work.

Techniques for the isolation and genetic manipulation of embryonic stem cells (ES cells) in the mouse are well established. However, little is understood regarding the mechanisms which enable pluripotent cells from the early mouse embryo to be diverted experimentally from their normal fate of differentiation <i>in vivo</i> and maintained instead in culture, as stable ES cell lines. Such knowledge might aid the establishment of ES cells from other mammalian species; particularly from farm animals, with the potential applications of genetically manipulating the stem cells to modify production traits. The objective of this thesis was to investigate various factors which may influence the potential of murine and ovine embryos to yield ES cells. Mouse genotype influences the capacity of embryos to give rise to ES cells. A significantly greater proportion of day 3.5 <i>p.c.</i> mouse embryos from the inbred 129/Sv-<i>CP</i> strain yielded ES cell lines compared to crossbred F<SUB>2</SUB> (C57BL/6 X CBA/Ca) embroys. The effect of mouse genotype was first observed in the significantly greater proportion of 129/Sv-<i>CP</i> embryos that gave rise to ES-like colonies at the first passage, following the disaggregation of inner cell mass (ICM) outgrowths. The efficiency of ES cell isolation was significantly increased by culturing murine blastocyst-stage embryos which had been induced experimentally, to enter a period of implantational delay for five days. Typically, murine ES cells have been derived from the day 3.5 <i>p.c.</i> ICM, although day 2.5 <i>p.c.</i> morulae have also been used. Here, a study describes the isolation of pluripotent ES cell lines from the primitive ectoderm of day 5.5 <i>p.c.</i> egg cylinder-stage mouse embroys. Although this study has shown that, in the mouse, primitive ectodermal cells influenced by endoderm can be established as ES cells in culture, the frequency is significantly lower than from the ICM. ES cells isolated from the three different embryonic stages equivalent to three days of development, have the same morphological characteristics. The brief exposure of day 3.5 <i>p.c.</i> embryos from the 129/Sv-<i>CP</i> mouse strain to treatments expected to perturb gene expression, heat shock or puromycin-containing medium, significantly increased the frequency of ES cell isolation. These effects resulted from increases in the proportion of embryos giving rise to ES-like colonies at the first passage which, in the case of heat shocked embryos, were also less likely to differentiate in subsequent passages. ES cell lines derived from embryos exposed to heat shock or puromycin have retained the capacity for <i>in vivo</i> development, as the stem cells colonised the germline in some chimaeric mice. These results suggest that the isolation of ES cells depends upon reversible, epigenetic changes in the pattern of gene expression and that the heat shock and puromycin treatments utilised here, may have helped to promote these changes <i>in vitro</i>. It is suggested that heat shock and implantational delay may act through a similar mechanism; whereby the treatments may have interfered with the transcriptional activation of genes responsible for differentiation of the ICM.

No description available.

This thesis describes the development of a new variety of lumophores, in particular luminescent rhenium(I) complexes of the type Re(bisim)(CO)3L n+, where bisim represents a bisimine derivative, e.g. bipyridine, and Lisa pyridine derivative (n = 1) or a chloride (n = 0), suitable for specific cell imaging and whose modified photophysical properties overcome some of the problems associated with traditional imaging agents (small Stokes shift, short lifetimes, etc). Specifically the successful synthesis and photophysical studies of a chemical model to validate the concept of a membrane permeable cell-imaging device, fac-Re0)ipyXCO)3(PyCH2COO(CH2)3OTBD) +, are presented. Further investigations towards and improved cell imaging agent, considering different issues like water solubility, lipophilicity and cell encapsulation rate, are then undertaken. Consequently, a new range of water soluble rhenium complexes, ykc- Re(bisimXCO)3(L) n" where bisim is a water soluble bathophenanthroline and L is a pyridine derivative (n = 2) or a chloride (n = 1), as well as a variety of novel yac-Re(bisimXCO)3Cl complexes, where bisim is a terpyridine derivative bearing a self destruction device which can be triggered by nitroreductase, are also presented. Additionally, the synthesis and structural description of a trinuclear rhenium complex is reported, along with a discussion of its use in inclusion experiments and attempts at analogue formation. The design and synthesis of a thiol selective luminescent rhenium complex, fac-Re(bipyXCO)3(PyCH2Cl) +, is described, along with the demonstration of its thiol selectivity both in vitro and in vivo as demonstrated by specific mitochondrial localization. Finally, studies of membrane permeability in liposome models, cellular uptake and localization in different cells such as Spironucleous vortens, yeast cells and human breast cancer cells of a variety of rhenium complexes above cited are also reported. These results combine to demonstrate the promising future of this new class of imaging probes as specific cell imaging agents and therefore, this work represents the initial step in the growing area of the development of transition metal based imaging agents.

Luminescent complexes of europium and terbium, incorporating new sensitizing chromophore moieties containing carboxylic acid functional groups, have been synthesised. It has been shown that the new tetraazatriphenylene chromophore leads to highly emissive complexes. A thiaxanthone chromophore, despite sensitizing lanthanide emission, results in complexes with lower quantum yields and is only practically useful for europium. Modification of the azaxanthone chromophore by formation of its N-oxide was investigated as a possibility of extending its longest wavelength absorption maximum beyond 340 nm. Phosphinate pendant arms were introduced into complexes containing the azaxanthone chromophore and resulted in highly emissive complexes Complexes of the new chromophores were investigated, together with numerous further examples, in terms of their susceptibility to quenching by electron rich species (iodide, ascorbate, urate). These experiments enhanced the mechanistic understanding of this process. Complexes using an azaxanthone chromophore with a carboxylic function and phenyl amide arms were used in coupling reactions. The coupling reactions involved isolation of the NHS ester of the complex. Cellular uptake and cytotoxicity experiments were carried out with several of these conjugate complexes. The possibility to observe these complexes using two photon excitation fluorescence microscopy was demonstrated.

No description available.

Cytochemical studies have been performed to investigate several enzymes which are involved in the metabolism of drugs, toxins, carcinogens and a variety of endogenous substances. Three groups of enzymes were studied: cytochrome P-450, gamma-glutamyl transpeptidase and NADP linked dehydrogenases. Also studied was glutathione, a peptide closely associated with these enzymes. The distribution of cytochrome P-450 in human tissues was determined by immunocytochemistry with monoclonal antibodies to a major constitutive form of human hepatic cytochrome P-450. Within the liver, cytochrome P-450 was present predominantly in hepatocytes of zone 3 of the liver acinus. Cytochrome P-450 was also demonstrated in the columnar absorptive cells of small intestinal villi, polymorphonuclear leucocytes and their precursors and mast cells. A novel cytochemical technique for demonstrating glutathione was developed. Glutathione was identified using o-phthaldialdehyde to produce a fluorophore which was visualised with fluorescence microscopy. This method was used to study the localisation and distribution of glutathione in breast lesions, particularly invasive carcinomas, where a variable level of glutathione in different carcinomas was demonstrated. A method was devised for the enzyme histochemical demonstration of gamma-glutamyl transpeptidase in fixed resin embedded tissue. Enzyme activity was optimally maintained and accurately localised in tissue displaying excellent morphology by fixing tissue in cold methanol free formaldehyde and then embeddding the tissue at low temperature in glycol methacrylate resin. Several NADP linked dehydrogenases and NADPH-cytochrome P-450 reductase were localised by enzyme histochemistry in sections of freeze-dried resin embedded tissue. This novel method of tissue preparation was evolved to allow the demonstration of the activity of dehydrogenases in resin embedded tissue.