Thrombopoietin, the Mp1 ligand, is essential for full megakaryocyte development.

The development of megakaryocytes (MKs) from their marrow precursors is one of the least understood aspects of hematopoiesis. Current models suggest that early-acting MK colony-stimulating factors, such as interleukin (IL) 3 or c-kit ligand, are required for expansion of hematopoietic progenitors into cells capable of responding to late-acting MK potentiators, including IL-6 and IL-11. Recently, the Mp1 ligand, or thrombopoietin (Tpo), has been shown to display both MK colony-stimulating factor and potentiator activities, at potencies far greater than that of other cytokines. In light of these findings, we tested the hypothesis that Tpo is absolutely necessary for MK development. In this report we demonstrate that neutralizing the biological activity of Tpo eliminates MK formation in response to c-kit ligand, IL-6, and IL-11, alone and in combination, but that these reagents only partially reduce MK formation in the presence of combinations of cytokines including IL-3. However, despite the capacity of IL-3 to support the proliferation and initial stages of MK differentiation, elimination of Tpo prevents the full maturation of IL-3-induced MK. These data indicate that two populations of MK progenitors can be identified: one that is responsive to IL-3 but can fully develop only in the presence of Tpo and a second that is dependent on Tpo for both proliferation and differentiation. Thus, our results strongly suggest that Tpo is the primary regulator of MK development and platelet production.

Xce haplotypes show modified methylation in a region of the active X chromosome lying 3' to Xist.

During early mammalian embryogenesis, one of the two X chromosomes in somatic cells of the female becomes inactivated through a process that is thought to depend on a unique initiator region, the X-chromosome inactivation center (Xic). The recently characterized Xist sequence (X-inactive-specific transcript) is thought to be a possible candidate for Xic. In mice a further genetic element, the X chromosome-controlling element (Xce), is also known to influence the choice of which of the two X chromosomes is inactivated. We report that a region of the mouse X chromosome lying 15 kb distal to Xist contains several sites that show hypermethylation specifically associated with the active X chromosome. Analysis of this region in various Xce strains has revealed a correlation between the strength of the Xce allele carried and the methylation status of this region. We propose that such a region could be involved in the initial stages of the inactivation process and in particular in the choice of which of the two X chromosomes present in a female cell will be inactivated.

(Table 1) Composition of organic matter in bottom sediments from the Kuril-Kamchatka Trench

From results of analyses of sediment samples collected on a profile crossing the Kuril-Kamchatka Trench distribution of organic D, N. carbohydrates, lipids and humic substances was established, as well as nature of their relationship with amorphous silica and clay fraction. Sum of the main biochemical groups of organic matter in the surface layer of sediments (0-1 cm) from the Kuril-Kamchatka Trench amounts to about 15%; neogenetic forms not encountered in living organisms make up 85% of organic matter. Among such forms 26% comprise humic substances formed during initial stages of polymerization of decomposition products of biochemical macromolecules.

Characterisation of three ACC synthase gene family members during post-harvest-induced senescence in broccoli (Brassica oleracea L. var. italica)

We investigated the gene expression profiles of different members of the 1-aminocyclopropane-1-carboxilic acid (ACC) synthase (EC 4.4.1.14) gene family in broccoli (Brassica oleracea L. var. italica) during the post-harvest-induced senescence process. Using RT-PCR, three different cDNAs coding for ACC synthase (BROCACS1, BROCACS2 and BROCACS3) were amplified from floret tissue at the start of the senescence process. The three genes share relatively little homology, but have highly homologous sequences in Arabidopsis thaliana, and could be functionally related to these counterparts. Southern analyses suggest that BROCACS1 and BROCACS3 are present as single copy genes, while there are probably two copies of BROCACS2. All three genes showed different expression patterns: BROCACS1 is likely to be either wound - or mechanical stress-induced showing high transcript levels after harvesting, but no detectable expression afterwards. BROCACS2 shows steady expression throughout senescence, increasing at the latest stages, and BROCACS3 is almost undetectable until the final stages. Our results suggest that BROCACS1 could be required to initiate the senescence process, while BROCACS2 would be the main ACC synthase gene involved throughout the post-harvest-induced senescence. BROCACS3's expression pattern indicates that it is not directly involved in the initial stages of senescence, but in the final remobilization of cellular resources.

Corrosion resistance of aged die cast magnesium alloy AZ91D

The corrosion behaviour of die cast magnesium alloy AZ91D aged at 160degreesC was investigated. The corrosion rate of the alloy decreases with ageing time in the initial stages and then increases again at ageing times greater than 45 h. The dependence of the corrosion rate on ageing time can be related to the changes in microstructure and local composition during ageing. Precipitation of the beta phase (Mg17Al12) occurs exclusively along the grain boundaries during ageing. The beta phase acts as a barrier, resulting in a decreasing corrosion rate in the initial stages of ageing. In the later stages, the decreasing aluminium content of alpha grains makes the alpha matrix more active, causing an increase in the corrosion rate. Electrochemical testing results also confirm the combined effects of the changes in alpha and beta phases on the corrosion resistance of the aged die cast AZ91D alloy. (C) 2003 Elsevier B.V. All rights reserved.

Improving the efficiency of wildlife monitoring by estimating detectability: a case study of foxes (Vulpes vulpes) on the Eyre Peninsula, South Australia

Demonstrating the existence of trends in monitoring data is of increasing practical importance to conservation managers wishing to preserve threatened species or reduce the impact of pest species. However, the ability to do so can be compromised if the species in question has low detectability and the true occupancy level or abundance of the species is thus obscured. Zero-inflated models that explicitly model detectability improve the ability to make sound ecological inference in such situations. In this paper we apply an occupancy model including detectability to data from the initial stages of a fox-monitoring program on the Eyre Peninsula, South Australia. We find that detectability is extremely low (< 18%) and varies according to season and the presence or absence of roadside vegetation. We show that simple methods of using monitoring data to inform management, such as plotting the raw data or performing logistic regression, fail to accurately diagnose either the status of the fox population or its trajectory over time. We use the results of the detectability model to consider how future monitoring could be redesigned to achieve efficiency gains. A wide range of monitoring programs could benefit from similar analyses, as part of an active adaptive approach to improving monitoring and management.

Nervous and muscle system development in Phascolion strombus (Sipuncula)

Recent interpretations of developmental gene expression patterns propose that the last common metazoan ancestor was segmented, although most animal phyla show no obvious signs of segmentation. Developmental studies of non-model system trochozoan taxa may shed light on this hypothesis by assessing possible cryptic segmentation patterns. In this paper, we present the first immunocytochemical data on the ontogeny of the nervous system and the musculature in the sipunculan Phascolion strombus. Myogenesis of the first anlagen of the body wall ring muscles occurs synchronously and not subsequently from anterior to posterior as in segmented spiralian taxa (i.e. annelids). The number of ring muscles remains constant during the initial stages of body axis elongation. In the anterior-posteriorly elongated larva, newly formed ring muscles originate along the entire body axis between existing myocytes, indicating that repeated muscle bands do not form from a posterior growth zone. During neurogenesis, the Phascolion larva expresses a non-metameric, paired, ventral nerve cord that fuses in the mid-body region in the late-stage elongated larva. Contrary to other trochozoans, Phascolion lacks any larval serotonergic structures. However, two to three FMRFamide-positive cells are found in the apical organ. In addition, late larvae show commissure-like neurones interconnecting the two ventral nerve cords, while early juveniles exhibit a third, medially placed FMRFamidergic ventral nerve. Although we did not find any indications for cryptic segmentation, certain neuro-developmental traits in Phascolion resemble the conditions found in polychaetes (including echiurans) and myzostomids and support a close relationship of Sipuncula and Annelida.

Predator-specific changes in the morphology and swimming performance of larval Rana lessonae

1. We investigated the morphological responses of larval Rana lessonae to the presence of two predators with substantially different prey-detection and capture techniques; larval dragonflies (Aeshna cyanea) and the Pumpkinseed Sunfish (Lepomis gibossus). 2. We also examined the functional implications of any predator-induced morphological variation on their swimming ability by assessing performance during the initial stages of a startle response. 3. We found the morphological responses of larval R. lessonae were dependent on the specific predator present. Tadpoles raised in the presence of dragonfly larvae preying upon conspecific tadpoles developed total tail heights 5.4% deeper and tail muscles 4.7% shallower than tadpoles raised in a non-predator environment, while tadpoles raised with sunfish possessed tails 2% shallower and tail muscles 2.5% higher than non-predator-exposed tadpoles. 4. Predator-induced morphological variation also significantly influenced swimming performance. Tadpoles raised with sunfish possessed swimming speeds 9.5 and 14.6% higher than non- and dragonfly predator groups, respectively. 5. Thus, the expression of these alternative predator-morphs leads to a functional trade-off in performance between the different environments.

Arousal and re-feeding rapidly restores digestive tract morphology following aestivation in green-striped burrowing frogs

During aestivation, the gut of the green-striped burrowing frog, Cyclorana alboguttata undergoes significant morphological down-regulation. Despite the potential impact such changes might have on the re-feeding efficiency of these animals following aestivation, they appear to be as efficient at digesting their first meals as active, non-aestivating animals. Such efficiency might come about by the rapid restoration of intestinal morphology with both arousal from aestivation and the initial stages of re-feeding. Consequently, this study sought to determine what morphological changes to the intestine accompany arousal and re-feeding following 3 months of aestivation. Arousal from aestivation alone had a marked impact on many morphological parameters, including small and large intestine masses, small intestinal length, LF heights, enterocyte cross-sectional area and microvilli height and density. In addition, the onset of re-feeding was correlated with an immediate reversal of many morphological parameters affected by 3 months of aestivation. Those parameters that had not returned to control levels within 36 h of feeding generally had returned to control values by the completion of digestion (i.e. defecation of the meal). Re-feeding was also associated with several changes in enterocyte morphology including the incorporation in intracytoplasmic lipid droplets and the return of enterocyte nuclear material to the 'active' euchromatin state: In conclusion, morphological changes to the gut of aestivating frogs which occur during aestivation are transitory and rapidly reversible with both arousal from aestivation and re-feeding. The proximate causes behind these transitions and their functional significance are discussed. (C) 2005 Elsevier Inc. All rights reserved.

User satisfaction with allied health services delivered to residential facilities via videoconferencing

We have investigated the role of videoconferencing in allied health service provision to high-care clients in rural residential facilities. Videoconferencing equipment was set up at a rural aged-care facility and a metropolitan allied health centre; ISDN transmission at 384 kbit/s was used to link the equipment. Twelve residents were assessed by both videoconference and face to face across five allied health disciplines (a total of 120 assessments). User satisfaction was measured using questionnaires and focus groups. Face-to-face assessment took significantly longer than videoconferencing assessment. However, the mean satisfaction ratings for face-to-face assessments were higher than for videoconferencing and the majority of the staff preferred the face-to-face format. Videoconferencing was particularly useful for consultations and the initial stages of the assessment process. A number of issues relating to the videoconferencing equipment, to the environment in which assessments were performed and to the clients themselves need to be addressed in order for this form of service delivery to be effective.

Practicing social e-atmospherics:French and Turkish web designers' perspectives

E-atmospherics have motivated an emerging body of research which reports that both virtual layouts and atmospherics encourage consumers to modify their shopping habits. While the literature has analyzed mainly the functional aspect of e-atmospherics, little has been done in terms of linking its characteristics’ to social (co-) creation. This paper focuses on the anatomy of social dimension in relation to e-atmospherics, which includes factors such as the aesthetic design of space, the influence of visual cues, interpretation of shopping as a social activity and meaning of appropriate interactivity. We argue that web designers are social agents who interact within intangible social reference sets, restricted by social standards, value, beliefs, status and duties embedded within their local geographies. We aim to review the current understanding of the importance and voluntary integration of social cues displayed by web designers from a mature market and an emerging market, and provides an analysis based recommendation towards the development of an integrated e-social atmospheric framework. Results report the findings from telephone interviews with an exploratory set of 10 web designers in each country. This allows us to re-interpret the web designers’ reality regarding social E-atmospherics. We contend that by comprehending (before any consumer input) social capital, daily micro practices, habits and routine, deeper understanding of social e-atmospherics preparatory, initial stages and expected functions will be acquired.

Cellular interaction with novel biomaterials

The objective of this thesis is to report the behaviour of mammalian cells with biocompatible synthetic polymers with potential for applications to the human body. Composite hydrogel materials were tested as possible keratoprosthetic devices. It was found that surface topography is an important consideration, pores, channels and fibres exposed on the surface of the hydrogels tested can have significant effects on the extent of cell adheson and proliferation. It is recommended that the core component is fabricated out of one of the following to provide a non cell adhesive base; A8, A11, A13, A22, A23. The haptic periphery fabricated out of one of the following would provide a cell adhesive composite; A16, A30, A33, A37, A38, A42, A43, A44. The presence of vitronectin in the ocular tissue appears to lead to higher cell adhesion to the posterior surface of a contact lens when compared to the anterior surface. Group IV contact lenses adhere more cells than Group II contact lenses - this may indicate that more protein (including vitronectin) is able to adhere to the contact lens due to the Group IV contact lenses high water content and ionic hydrogel matrix. Artificial lung surfactant analogues were found to be non cytotoxic but also decreased cell proliferation when tested at higher concentrations. Poly(lysine ethyl ester adipamide) [PLETESA] had the most favourable response on cell proliferation and commercial styrene/maleic anhydride (pMA/STY sp2) the most pronounced inhibitory response. The mode of action that decreases cell proliferation appears to be through membrane destabilization. Tissue culture well plates coated with PLETESA allowed cells to adhere in a concentration dependent manner, multilaminar liposomes possibly of PLETESA were observed in solution in PLETESA coated wells. Polyhydroxybutryate (PHB) and polyhydroxyvalerate (PHV) blends that contained hydroxyapatite were found to be the most cell adhesive material of those materials tested. The blends that were most susceptible to degradation adhered the most cells in initial stages of degradation. The initial slight increase in cell adhesion may be due to the increased rugosity of the material. As the degradation continued the number of cells adhering to the samples decreased, this may indicate that the polarity was inhibitory to cell adhesion during the later stages of degradation.

Characterisation of a harvesting step for monoclonal antibodies from mammalian cell culture

The focus of this research was defined by a poorly characterised filtration train employed to clarify culture broth containing monoclonal antibodies secreted by GS-NSO cells: the filtration train blinded unpredictably and the ability of the positively charged filters to adsorb DNA from process material was unknown. To direct the development of an assay to quantify the ability of depth filters to adsorb DNA, the molecular weight of DNA from a large-scale, fed-batch, mammalian cell culture vessel was evaluated as process material passed through the initial stages of the purification scheme. High molecular weight DNA was substantially cleared from the broth after passage through a disc stack centrifuge and the remaining low molecular weight DNA was largely unaffected by passage through a series of depth filters and a sterilising grade membrane. Removal of high molecular weight DNA was shown to be coupled with clarification of the process stream. The DNA from cell culture supernatant showed a pattern of internucleosomal cleavage of chromatin when fractionated by electrophoresis but the presence of both necrotic and apoptotic cells throughout the fermentation meant that the origin of the fragmented DNA could not be unequivocally determined. An intercalating fluorochrome, PicoGreen, was elected for development of a suitable DNA assay because of its ability to respond to low molecular weight DNA. It was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing pertinent monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 89.0 % of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples. Application of the fluorescence based assay resulted in characterisation of the physical parameters governing adsorption of DNA by various positively charged depth filters and membranes in test solutions and the DNA adsorption profile of the manufacturing scale filtration train. Buffers that reduced or neutralised the depth filter or membrane charge, and those that impeded hydrophobic interactions were shown to affect their operational capacity, demonstrating that DNA was adsorbed by a combination of electrostatic and hydrophobic interactions. Production-scale centrifugation of harvest broth containing therapeutic protein resulted in the reduction of total DNA in the process stream from 79.8 μg m1-1 to 9.3 μg m1-1 whereas the concentration of DNA in the supernatant of pre-and post-filtration samples had only marginally reduced DNA content: from 6.3 to 6.0 μg m1-1 respectively. Hence the filtration train was shown to ineffective in DNA removal. Historically, blinding of the depth filters had been unpredictable with data such as numbers of viable cells, non-viable cells, product titre, or process shape (batch, fed-batch, or draw and fill) failing to inform on the durability of depth filters in the harvest step. To investigate this, key fouling contaminants were identified by challenging depth filters with the same mass of one of the following: viable healthy cells, cells that had died by the process of apoptosis, and cells that had died through the process of necrosis. The pressure increase across a Cuno Zeta Plus 10SP depth filter was 2.8 and 16.5 times more sensitive to debris from apoptotic and necrotic cells respectively, when compared to viable cells. The condition of DNA released into the culture broth was assessed. Necrotic cells released predominantly high molecular weight DNA in contrast to apoptotic cells which released chiefly low molecular weight DNA. The blinding of the filters was found to be largely unaffected by variations in the particle size distribution of material in, and viscosity of, solutions with which they were challenged. The exceptional response of the depth filters to necrotic cells may suggest the cause of previously noted unpredictable filter blinding whereby a number of necrotic cells have a more significant impact on the life of a depth filter than a similar number of viable or apoptotic cells. In a final set of experiments the pressure drop caused by non-viable necrotic culture broths which had been treated with DNase I or benzonase was found to be smaller when compared to untreated broths: the abilities of the enzyme treated cultures to foul the depth filter were reduced by 70.4% and 75.4% respectively indicating the importance of DNA in the blinding of the depth filter studied.

Marketing in indigenous and Asian small firms in the West Midlands.

This study is concerned with examining the application of marketing during the start-up, development and growth of small firms in the West Midlands. As an exploratory study, it provides evidence to support the central hypothesis of the thesis that whilst many small firms have the potential to progress through to the successful growth stage of development, they fail to do so because of their owner-managers' orientation towards production and selling and because they do not apply formal marketing during the initial stages of business development. A comparative approach to studying marketing in indigenous and Asian firms is adopted in an attempt to fill a gap in the literature on the characteristics and differences in the formation and development processes of these two groups of enterprises. The study has three main objectives and is based on qualitative research techniques of in-depth interviews, case studies and longitudinal studies among sixty-six firms representing the key activities of the small firms sector of the local economy. Firstly, it investigates owner-managers' orientation in developing and managing new and established businesses and explores the sources of, and changes in their orientation during the various stages of development. Secondly, it assesses the owner-manager's awareness and understanding of what constitutes the marketing function and investigates what aspects of marketing are applied during the different stages of business development. Finally, the study monitors and evaluates the outcomes and implications of applying formal marketing techniques in a small sample of firms over a period of two years. The thesis concludes by using the findings of the study to contribute additions to existing models of growth and by proposing new models of evolution and application of marketing in small firms.

The controlled release of macromolecules from macroporous hydrophyllic polymer matrices

This work has used novel polymer design and fabrication technology to generate bead form polymer based systems, with variable, yet controlled release properties, specifically for the delivery of macromolecules, essentially peptides of therapeutic interest. The work involved investigation of the potential interaction between matrix ultrastructural morphology, in vitro release kinetics, bioactivity and immunoreactivity of selected macromolecules with limited hydrolytic stability, delivered from controlled release vehicles. The underlying principle involved photo-polymerisation of the monomer, hydroxyethyl methacrylate, around frozen ice crystals, leading to the production of a macroporous hydrophilic matrix. Bead form matrices were fabricated in controllable size ranges in the region of 100µm - 3mm in diameter. The initial stages of the project involved the study of how variables, delivery speed of the monomer and stirring speed of the non solvent, affectedthe formation of macroporous bead form matrices. From this an optimal bench system for bead production was developed. Careful selection of monomer, solvents, crosslinking agent and polymerisation conditions led to a variable but controllable distribution of pore sizes (0.5 - 4µm). Release of surrogate macromolecules, bovine serum albumin and FITC-linked dextrans, enabled factors relating to the size and solubility of the macromolecule on the rate of release to be studied. Incorporation of bioactive macromolecules allowed retained bioactivity to be determined (glucose oxidase and interleukin-2), whilst the release of insulin enabled determination of both bioactivity (using rat epididymal fat pad) and immunoreactivity (RIA). The work carried out has led to the generation of macroporous bead form matrices, fabricated from a tissue biocompatible hydrogel, capable of the sustained, controlled release of biologically active peptides, with potential use in the pharmaceutical and agrochemical industries.