No description available.

Freshwater planarian Schmidtea mediterranea is an emerging model for studying in vivo gene functions and regulation in native cell niches. The obligate asexual strain of this species reproduces by fission, in which succession of soma occurs without passing through the germline. To achieve this somatic immortality the somatic stem cells need to overcome the end replication problem. Therefore it can be hypothesised that somatic telomere maintenance in asexual S. mediterranea must possess a germ-like property, with which age-related erosions can be adequately repaired. In this PhD project, the telomere repeat unit in S. mediterranea was confirmed to be the vertebrate-like TTAGGG. Attrition of whole body telomere length was found in ageing sexual worms and also in asexual worms which had not gone through recent fission events. Opposite telomere length dynamics were observed in regenerated samples of the two strains, with erosion in the sexuals and reset in the asexuals. The telomere maintenance was found to increase during regeneration in both strains, with a higher level of increase in asexual worms. A homolog of the telomerase reverse transcriptase subunit, Smed_Tert, was identified and characterised in this organism. High level of Smed_Tert expression was seen in germ cells in mature sexual worms and adult stem cells in asexual worms. Knockdown of Smed_Tert expression by RNA interference caused progressive telomere erosion, however effects on cell proliferation and viability have not been observed in knockdown samples. Four alternate splice isoforms of Smed_Tert were identified. The enhanced telomerase activity during regeneration correlates with a proportional increase in the full-length isoform and a decrease in isoforms with a truncated TRBD domain, suggesting a dominant negative regulation of telomerase by alternative splicing. Significant increase in the expression of the full-length isoform was seen in regenerating asexual samples but not in sexual strains, which correlates with their telomere length dynamics. It is hoped that the comparative studies between the sexual and asexual strains can improve our understanding of how soma can evolve to become an effective inheritable unit.

During development, polarity is a common feature of many cell types. One example is the polarisation of whole fields of epithelial cells within the plane of the epithelium, a phenomenon called planar cell polarity (PCP). It is widespread in nature and plays important roles in development and physiology. Prominent examples include the epithelial cells of external structures of insects like the fruit fly Drosophila melanogaster, polarised tissue morphogenesis in vertebrates and sensory hair cells in the vertebrate ear. In this work we focus on the wing and the abdomen of Drosophila, where PCP becomes obvious in the alignment of hairs and bristles. The underlying dynamics are not fully understood yet, but two distinct protein networks centred around the transmembrane proteins Frizzled and Dachsous, respectively, have been shown to play essential roles. We will present and analyse five models for different aspects of the process of planar cell polarisation. The first two models assess the nature of PCP in a generic setting, ensuring that the results are valid for whole classes of PCP models. Models three and four are existing more complex models that include detailed assumptions about the underlying protein interactions of the Frizzled system in the Drosophila wing. Model five considers the Dachsous system in the Drosophila abdomen. We describe the features of the different types of mechanisms and determine the conditions under which they can yield polarity. All five models can establish wild-type polarity for a wide range of parameter values. We find, however, that for model one, three and four an inhomogeneous pattern exists for the same parameter values as the polarised state. Therefore, in these cases either specific initial conditions, which are unlikely in nature, or a global bias are necessary to ensure correct polarisation. Furthermore, we present the effects of clonal clusters of cells on the polarity of the surrounding cells in our models and relate them to the phenotypes observed in experiments. Model one and five show the largest discrepance between the numerical and the experimental results. We discuss the biological relevance of these findings and indicate outstanding questions.

Acanthamoeba castellanii is an amoeboid protozoan which causes opportunistic infections, including granulomatous encephalitis in immune-compromised patients. Haematogenous dissemination follows initial infection and the pathogen exhibits an ability to cross the blood-brain barrier (BBB). In the bloodstream and at the site of BBB penetration in the brain microvasculature A. castellanii is exposed to host humoral immunity. Here, we have provided insights into A. castellanii pathogenesis and the identity of amoeba antigens participating in immune control. We have investigated the role circulating immunoglobulin plays in preventing penetration of the BBB, and whether trophozoites can alter the efficacy of the immune response. Furthermore we have extended previously published data, demonstrating that amoeba proteases can degrade all antibody classes including physiologically-derived antibody. Nonspecific binding of polyclonal antibody was also observed, and attributed to Fc-binding activity by trophozoites. Additionally, we have examined the binding dynamics of A. castellanii under physiological conditions. BBB disruption was shown to be not directly linked to binding, instead it is reliant on secreted proteases. This study provides insights into mechanisms by which A. castellanii evades host immunity and crosses the BBB. This has the potential to enhance therapeutic strategies aimed at restoring essential disease prevention processes. In addition we have identified a number of amoeba antigens that are targets for the immune system and which may therefore be exploited through vaccination or immunotherapy.

No description available.

Silver nanoparticles (AgNPs) have gained much popularity in consumer products due to their strong antimicrobial ability. The majority of research concerning the biological effects of AgNPs has been limited to humans, mammals and freshwater organisms. Marine organisms, especially invertebrates, have been studied to a lesser extent. The objective of this thesis was to understand the effects of AgNPs on the marine benthic invertebrates. Specifically, we focused on the acute and sub-lethal toxicity of two AgNPs (Oleic acid coated AgNP (OAgNP) and Polyvinylpyrrolidone coated AgNP (PAgNP)) on marine invertebrate larvae across three phyla (i.e. the barnacle Balanus amphitrite, the limpet Crepidula onyx and the polychaete Hydroides elegans) in terms of mortality, growth, development and metamorphosis. Bioaccumulation and biodistribution of silver, as well as apoptosis induction were also investigated. To distinguish the toxic effects derived from nano-silver and aqueous form of silver, larvae were also exposed to silver nitrate (SN) in parallel. In the acute toxicity test, larvae were exposed to OAgNPs and PAgNPs for 48 hours, and the concentration leading to 10 % mortality (〖LC〗_10) were determined and compared. The results indicated that B. amphitrite and H. elegans were more sensitive to OAgNPs (〖LC〗_10: 0.138 and 2.63 × 〖10〗^(-4) μμg L-1, respectively) than PAgNPs (〖LC〗_10: 0.502 and 0.317 μμg L^(-1), respectively). In contrast, C. onyx was more susceptible to PAgNPs (LC10: 38.5 μμg L^(-1)) than OAgNPs (〖LC〗_10: 467 μμg L^(-1)). Among the three taxonomic groups, C. onyx was most tolerant of AgNPs, following by B. amphitrite and H. elegans. The sub-lethal effect of AgNPs resulted in a significant retardation in growth and development, and the reduction of settlement rate of all three species tested. In particular, the settlement rate of H. elegans was significantly lower in AgNPs treatment than in SN treatment, suggesting that toxicity of AgNPs might not be solely evoked by the release of silver ion (Ag+) into the test solution. The three species took up and accumulated silver efficiently from all forms. Importantly, AgNP aggregates were found along the digestive tract of C. onyx and the TEM images further confirmed that AgNPs were able to move across the plasma membrane. In addition, TUNEL assay indicated that AgNPs could induce apoptosis in B. amphitrite and C. onyx. In view of the very low number of detected apoptotic cells and the random occurrence of cell death found, AgNP-induced apoptosis does not appear to be the major toxicity mechanism in causing delayed growth and settlement failure. Unlike the results revealed from acute toxicity test, surface coatings did not affect the sub-lethal toxicity of AgNPs. This research clearly demonstrated that AgNPs exerted toxic effects in a speciesspecific manner, and long-term exposure of AgNPs might allow bioaccumulation of silver, induce apoptosis, and affect growth, development and recruitment of marine invertebrates. This study also highlighted the possibility that toxicity of AgNPs might be mediated through toxic Ag+ as well as the novel modalities of AgNPs. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

No description available.

Investigations have been made into the genetic control of immunity to the nematode Trichuris muris. Both background genes and genes within the mouse major histocompatibility complex (MHC), H-2, were shown to influence the expulsion of T. muris with the former having the stronger influence. At least two genes within the H-2 complex determined response phenotypes, the effects of "resistance" or "susceptibility" alleles at I-A being modulated by resistance or susceptibility alleles at aD end locus/loci. Differential responsiveness within slowly responding mouse strains suggested that parasite-dependent effects were also important. The primary antibody response to T. muris excretory/secretory (E/S) antigen, predominantly an IgG response, was also shown to be controlled by background and H-2-linked genes. In general, mouse strains less resistant to infection developed higher levels of IgG than- more resistant strains of mice. However strains of mice possessing the H-2q haplotype, irrespective of their genetic background, rapidly developed higher levels of IgG1 antibodies than strains of other haplotypes, H-2q haplotype mice tending to be more resistant to infection. Recognition of two high molecular weight (MW) E/S antigens by IgG as revealed by immunoprecipitation was also found to be almost exclusively H-2q restricted. This restriction may be partly quantitative but as such would operate in vivo due to the restriction on the ability to produce high levels of specific IgG. Both H-2q restricted phenomena may be part of, but not absolute requirements for, protective immunity. Parasite-induced effects on host immunity were also studied. Later larval and adult stages of T. muris were shown to be immunosuppressive, immunosuppression being long lasting and preventing the expulsion of subsequent infections. Vaccination with E/S antigen was shown to protect strains of mice which are slow to expel worms (poor-responder) or totally unable to expel worms (non-responder) from a primary infection with T. muris. However protection was slow to be expressed. Antigen recognition profiles of vaccinated strains of mice differed from their primary infection recognition profiles and included the recognition of the two high MW antigens shown to be H-2q restricted in a primary infection. Thus altering the mode or route of E/S antigen presentation may lead to shifts in responsiveness of H-2 genotypes to specific determinants and/or boost specific antibody levels sufficiently to reveal recognition of these antigens. Prior experience of a patent primary infection prevented vaccination protecting non-responder mice against subsequent infections. This inability was correlated with suppressed IgG1 antibody levels and failure to recognise three high MW antigens including the IL-2q restricted antigens. Using a panel of monoclonal antibodies raised against E/S antigen it was shown that E/S antigens, apparently including both immunogenic and immunosuppressive molecules, were localised to granules within the stichocyte cytoplasm of the adult T. muris stichosome.

The hoverflies (Diptera:Syrphidae) represent an apparently paradoxical visual Batesian mimicry complex, with what appear to be "poor" Mimics outnumbering their more accomplished counterparts. The purpose of this thesis is to determine how far conventional mimicry theory is capable of explaining the apparent paradoxes of mimicry in the hoverflies. It becomes obvious that determining the mimetic status of the supposedly poor Mimics is not a trivial task. Conventional experimental tests of mimicry, using captive predators, seem incapable of predicting the degree of protection enjoyed by a Mimic in the field. The research therefore concentrates on developing some novel empirical approaches to the study of mimicry. This includes developing a method of image analysis which yields an objective, single-value measure of the similarity between Model and Mimic patterns. This index of similarity is used to produce unique descriptions of the structure of mimetic communities in terms of Mimic frequency and similarity to the supposed Model. These profiles indicate that there is an objective basis to the perceived paradox, and suggest that there is not a simple relationship between the actual and perceived similarity of two patterns. The perceived similarity of Model and Mimic will be a key determinant of mimetic success. The index of similarity is also used as a basis for direct comparison of the supposedly mimetic hoverflies with a more established example of mimicry in the butterflies. This exercise demonstrates that an index of pattern similarity enables a unique comparative analysis of mimicry. It is proposed that an index of similarity also provides a unique opportunity to test our theoretical understanding of mimicry, if it is used in conjunction with a mathematical model that possesses some specific attributes. A suitable prototype model is developed and demonstrated. The thesis concludes with an indication that the novel empirical approaches developed here, have been adopted elsewhere. This latter work indicates that those hoverfly species which are apparently "poor" Mimics, may be exploiting some constraint in predator perceptual and cognitive systems to achieve mimetic protection, despite a relatively low degree of actual similarity to the Model species.

The development of the gastrointestinal nematode Heligmosomoides polygyrus (syn. Nematospiroides dubius) in the mouse was studied. The stage specific production of acetylcholinesterase was measured in both excretory/ secretory products and in worm homogenates and found to be maximal between days 4-6 post infection, corresponding to the fourth larval stage of the parasite's life cycle. Analysis of the proteolytic enzymes found in the same preparations of the parasite again revealed a stage specific release. Quantitative examination showed a maximum concentration of proteolytic enzymes in the early third larval stage, whilst qualitative analysis revealed a number of molecules at 96, 76, 42, 33, 18, 16, and 13 kDa in the early stages, which gradually disappeared as the parasite aged until only those at 76, 18, 16, 13 kDa remained by day 120. The molecules present on the surface of the various stages of the parasite were extracted using a number of procedures. Various stage specific surface molecules were identified as were two possible sex specific molecules at 76 and 145 kDa. The immune response to a primary infection of the parasite was characterised in three strains of mice with different degrees of susceptibility to infection (SJL, BALB/c and CBA). It was noted that the better the strain was at expelling the parasite, the greater and swifter was the response as assessed through the use of a number of criteria. These included white blood cell counts, differential cell counts, the Band T cellularity of the secondary lymphoid organs, the response of these cells to mitogens, the mucosal mast cell response, quantitative antibody response (Mancini and ELISA) and qualitative antibody response to parasite antigens (immunoblot). In each case SJL responded better than BALB/c which, in turn responded to higher degree than CBA. Functional host protective immunity was stimulated in the same three strains of mice using a challenge infection following a 9-day anthelmintic abbreviated infection. The same criteria were used to measure the immune response to the parasite as for the primary infection and, as for the primary infection, it was found that the high responder strains gave a more rapid and more intense reaction to the parasite than the low responder strain. Immunisation prevented the establishment of a proportion of the challenge infection and also resulted in the premature expulsion of parasites. The parasite surface molecules which were recognised by mice undergoing either a primary infection or an immunising infection were identified. It was revealed that molecules at 208, 145, 92, 76 and 62 kDa on adult parasites were recognised by mice which had expelled a primary infection. Mice which were immune to a challenge infection recognised molecules at 62 and 20-15 kDa on larval parasites. A molecule at 30.5 kDa was also recognised by immune mice and corresponded to the molecular weight of acetylcholinesterase in the ES.