Members of the WASP protein family are direct activators of the arp2/3 complex, thereby regulating the nucleation of branched actin assemblies within the cell. Each sub-class possesses a unique N-terminal domain architecture allowing a division of labour between its members, each coupling different signal transduction pathways to the nucleation of specific actin structures. WASH (WASP and SCAR homologue) is a newly identified member of the WASP protein family. Due to its disruption in \(Drosophila\) proving lethal (Linardopopoulou et al., 2007) little is know as to the functional role of WASH at the cellular level. Other than it is important in the development of multicellular organisms. Here we successfully disrupt WASH in the single celled amoebae \(Dictyostelium\) \(discoideum\) and discover a role for WASH in the endocytic pathway. WASH was shown to be essential for the trafficking of indigestible material through the endocytic pathway, with its disruption causing a complete bock in cellular defecation. This was shown to be due to a defect in lysosomal maturation into neutral post-lysosomes. Using fluorescently tagged fusion proteins we show that WASH recruitment coincides with removal of the Vacuolar H+ ATPase from lysosomal membranes, and suggests a possible role for WASH and actin in regulating the luminal pH of intracellular compartments.

\(Nesidiocoris\) \(tenuis\) Reuter (Hemiptera: Miridae) and \(Lysiphlebus\) \(testaceipes\) (Cresson) (Hymenoptera: Braconidae, Aphidiinae) are candidate biological control agents known to play an important role in the management of agricultural and horticultural pests in southern Europe. Through a series of laboratory and field assessments, this study investigates the establishment potential of these two species in cool temperate climates typical of northern Europe. Laboratory results demonstrated a low level of cold tolerance in \(N.\) \(tenuis\) with a developmental threshold of 12.9°C and no indication of ability to diapause. Field trials supported these findings with 100% mortality occurring after less than 4 weeks of winter field exposure. Collectively, these data suggest that \(N.\) \(tenuis\) is unlikely to establish outdoors in northern Europe and would therefore have little or no non-target effects on native species in such regions, thereby constituting a ‘safe’ candidate for release. Additionally, investigations into temperature-related thresholds indicated that \(N.\)\(tenuis\) would be an effective control agent against species with a similar activity profile to the two-spotted spider mite \(Tetranychus\) \(urticae\) Koch (Acari: Tetranychidae). \(Lysiphlebus\) \(testaceipes\)demonstrated a greater ability to tolerate cold than \(N.\) \(tenuis\) but there was no indication of ability to diapause. With a developmental threshold of 5.8°C, parasitoid larvae and pupae continued to develop during the 70 d of winter field trials yielding reproductively viable adults. With this level of cold tolerance and a host range in excess of 100 aphid species, including some known to overwinter in the UK and other temperate regions, it seems reasonable to predict that \(L.\) \(testaceipes\) would be able to establish in northern Europe. Thermal activity threshold investigations also indicated that \(L.\) \(testaceipes\) would constitute an effective control agent for pest species with similar activity profiles to \(Aphis\) \(fabae\) Scop. (Hemiptera: Aphididae) under a range of climatic conditions. These data are discussed in relation to current debate on the environmental risk assessment and regulatory system in Europe for the release of non-native biological control agents.

Insects are ectotherms and have limited ability to regulate body temperature above or below ambient and are consequently greatly affected by temperature. The aphid \(Myzus\) \(persicae\) has an extensive distribution throughout Europe from Scandinavia to Southern Spain, representing three distinct climatic regions: sub-Arctic, temperate and Mediterranean. The aphid also has genetically distinct clones within its holocyclic (sexual) and anholocyclic (asexual) life cycles. This raises the possibility that aphids are regionally-adapted to distinct climatic zones along the latitudinal cline of its European distribution. Genetically distinct clones of \(M. persicae\) were collected from Sweden, UK and Spain. Indices of temperature tolerance (upper and lower lethal temperature50, coma temperatures and mobility thresholds) were determined for each aphid clone at different rearing temperatures. Acclimation at 10°C for one generation increased cold tolerance by depressing lower lethal, chill movement and chill coma temperatures when compared to 20°C and 25°C and further enabled mobility to be maintained to lower temperatures. Acclimation at 25°C for one generation increased heat tolerance by raising upper lethal, heat movement and heat coma temperatures when compared to 10°C and 20°C. Acclimation at 10°C also acted to raise upper lethal temperatures, indicating that the physiological processes conferring heat tolerance are induced at both high and low temperatures. Data did not support intergenerational acclimation to higher or lower temperatures. Lower thermal limits were more plastic than upper limits, enabling tolerance ranges to be increased following acclimation at 10°C, but reduced on acclimation at 25°C. Rates of change varied between clones, suggesting that certain clones could be more affected by climate change. A relationship between thermal tolerance range and latitude was not supported by data on thermal traits investigated with the exception of heat coma temperature. This suggests that clonal mixing across Europe is extensive and prevents local adaptation, although long term populations could persist in the Mediterranean allowing increased heat tolerance. Clonal type, as identified by microsatellite analysis, did show a relationship with thermal tolerance, suggesting that clonal types could respond independently to climate change, affecting relative proportions of clones within populations.

Cryptococcus neoformans is an opportunistic fungal pathogen and a leading cause of fungal infection related fatalities in immunocompromised hosts. Compared to well-studied; Cryptococcus neoformans virulence factors like the polysaccharide capsule and melanin synthesis, very little is known about phospholipase B1 (Plb1). Plb1 is a phospholipid modifying enzyme that is implicated in multiple stages of cryptococcal pathogenesis. Herein I demonstrate that a Plb 1 deficient strain of C.neoformans has a profound defect in intracellular growth within macrophages. In addition, I show that the Δplb1 strain undergoes a novel morphological change during in vitro and in vivo infection, resulting in a sub-population of very large 'titan cells' that may arise as a result of the mutant's inability to cope within the macrophage. I go on to test whether these phenotypes are due to a reduction in eicosanoid production caused by Plb 1 deficiency. Finally, I present an addition project where I optimise a C. neoforman's intracellular proliferation assay for high throughput analysis via flow cytometry. This work provides a new insight into the function of this unappreciated virulence factor and helps to lay the foundation for new treatment strategies to combat cryptococcosis.

Brominated flame retardants (BFRs) and alkylphenols (APs) are pollutants commonly found within the environment and have human health concerns due to their endocrine disrupting and cytotoxic effects. BFRs are used to reduce the flammability of a variety of consumer products such as foam furnishings, whereas APs are found in plastic products used by the food industry. This study investigated the neurotoxicity of the most commonly used groups of BFRs and APs on SH-SY5Y human neuroblastoma cells. The results presented in this thesis showed (using cell viability assays) that these pollutants are toxic at low concentrations. Some compounds such as hexabromocyclododecane (HBCD) and 4-nonylphenol (4-NP) induce cell death (apoptosis) by caspases activation (Casp-8, Casp-9 and Casp-3) and cytochrome c release at low micromolar concentrations (IC50 ~ 4μM and 6μM, respectively). Consequently this study also showed that these compounds increased intracellular [Ca2+] levels and the production of reactive oxygen species (ROS) within SH-SY5Y cells by causing Ca2+-dependent depolarization of the mitochondria. In support of a Ca2+-mediated mechanism, the data presented here shows that some BFRs and APs inhibit Sarcoplasmic/ Endoplasmic Ca2+-ATPase (SERCA) and to corroborate this over-expressing SERCA1 improved cell viability especially in cells exposed to certain cytotoxic chemicals such as HBCD; this study is the first experiment of this type to be performed. This study also showed that some of these chemicals, at low concentrations had amyloidgenic effects causing the cleavage amyloid precursor protein (APP) into Beta-amyloid (Aβ) and could therefore be implicated in Alzheimer‟s disease (AD).

There is good evidence that high dietary calcium intake reduces the incidence of colorectal cancer. The mechanism by which calcium acts is unknown. It was hypothesised that parathyroid hormone (PTH) acts on colorectal epithelium and that calcium acts by suppressing serum PTH. Real-time PCR demonstrated expression of the PTH receptor PTHR1 in normal and neoplastic colon, indicating that the colon was sensitive to PTH. Gene expression profiling using oligo-nucleotide micro-arrays was performed to identify genes; a) Differentially expressed in response to PTH in an in-vitro model of the colorectal epithelium. b) For which expression in normal rectal mucosa correlated with serum PTH level. c) Differentially expressed in normal rectal mucosa in response to modulating PTH by dietary calcium supplementation. A common finding of all three studies was regulation of the wnt signalling pathway by PTH. In the in-vivo studies, expression of genetic markers of differentiation was negatively correlated with PTH. Calcium supplementation resulted in increased expression of genetic markers of differentiation. This suggests PTH inhibits differentiation and that calcium by suppressing PTH promotes differentiation. There was also significant overlap between the genes changed by calcium, those negatively correlated with PTH and those changed by PTH in-vitro. These findings provide support for the stated hypotheses.

Meiosis is a specialised cell division essential for sexual reproduction. During meiosis the chromosomes are highly organised and correct chromosome architecture is required for meiotic recombination and faithful segregation of chromosomes at anaphase. Condensin is involved in chromosome organisation during meiotic and mitotic cell divisions. Three condensin subunits, including condensin I and II specific subunits AtCAPD2 and AtCAPD3 respectively, have been studied for their role in meiosis using T-DNA insertion plants and an RNAi approach. The condensin subunit AtSMC4 localised to meiotic chromosomes at metaphase I, where it coated the entire chromosome and remained until telophase. Some evidence of centromere localisation of AtSMC4 was also seen during prophase I and metaphase I. AtSMC4RNAi lines were able to condense their chromosomes into metaphase bivalents which were essentially normal. However at anaphase the chromosomes appeared to lose structural integrity such that a thin curtain of lagging chromatin was seen to trail behind the segregating chromosomes. As in AtSMC4RNAi lines, both condensin I and II specific subunits were also required for chromosome segregation at anaphase I and II. However their roles in this process differed. AtCAPD2RNAi resembled AtSMC4RNAi lines but Atcapd3 plants had a unique anaphase phenotype. It is possible that these two phenotypes represent different manifestations of the same role of maintaining chromosome organisation. Condensin I, but not condensin II was also shown to be required for the organisation of the rDNA and to maintain the structure of the centromeres at metaphase.

Infection with human cytomegalovirus (HCMV), usually asymptomatic in healthy individuals, can cause severe or fatal disease in infants and immunocompromised patients. The generation of a potent protective vaccine is necessary to protect vulnerable people. Because of host restriction, murine cytomegalovirus (MCMV) is used as a model for HCMV. Previously, we have generated a temperature-sensitive mutant, tsm5, which failed to replicate in mice yet protected them against virus challenge. Several mutations have been identified in this mutant by Comparative Genome Sequencing (GCS) (Roche NimbleGen); 10 synonymous and 15 non-synonymous single nucleotide polymorphisms (SNPs). Among these are m139 (Y565X) and m141 (V195M), shown to be essential for replication in macrophages but not in fibroblasts, m143 (M232I), shown to play an important role in the inhibition of the PKR-mediated host antiviral response, M27 (A658S ), involved in interference with interferon- signalling, and M36 (V54I), an anti-apoptotic protein. In the present study, the above mentioned mutations were introduced individually into the MCMV K181 (Perth) variant bacterial artificial chromosome (BAC) using RecE/T homologous recombination. An in vitro phenotypical analysis revealed that only the double (Mt[M27\(^A\)\(^6\)\(^5\)\(^8\)\(^S\)M36\(^V\)\(^5\)\(^4\)\(^I\)]) and the m139 (Mt[m139\(^Y\)\(^5\)\(^6\)\(^5\)\(^X\)]) mutants showed a temperature sensitive phenotype in MEF and/or Raw 264.7 macrophages cells

The opportunistic fungal pathogen Cryptococcus neoformans is the major etiological agent of the life threatening disease cryptococcosis, which is responsible for over half a million human deaths per annum. Professional phagocytes, such as alveolar macrophages, phagocytose inhaled spores and attempt to destroy the pathogen. However, this process is inefficient in immunocompromised hosts, such as those suffering from HIV/AIDS. In such hosts the macrophage is thought to behave like a “Trojan Horse”, acting as both a cryptococcal dissemination vector and as a protective niche against antifungal agents/cells present in the circulation. Vomocytosis, first discovered in C. neoformans, is a non-lytic expulsive mechanism whereby C. neoformans or C. gattii exit the macrophage leaving both pathogen and the host macrophage with a morphologically normal phenotype. The clinical implications of vomocytosis are poorly understood however; data from this research suggests that the induction of a pro-inflammatory response increases vomocytosis rates, suggestive of a pathogen escape mechanism from a harsh antimicrobial environment i.e. the pro-inflammatory primed macrophage. Regulating the rates of vomocytosis in vivo may have dramatic consequences on pathogen dissemination and also patient prognosis. For instance, enhancing the rate of vomocytosis within circulation could allow other antifungal cells and compounds access to destroy the freshly released cryptococci, hence reducing pathogen burden and improving patient prognosis.

Many microbial pathogens are able to evade killing by phagocytes of the innate immune system. This thesis focuses on two pathogens: the fungal pathogen \(Cryptococcus\) \(neoformans\) and the bacterial pathogen \(Streptococcus\) \(agalactiae\). \(C\). \(neoformans\) causes severe cryptococcal meningitis in mostly immunocompromised hosts, such as those with HIV infection. In contrast, \(S\). \(agalactiae\) is the leading cause of neonatal sepsis and meningitis. The interaction between macrophages and these pathogens is likely to be critical in determining dissemination and outcome of disease in both instances. A collection of \(S\). \(agalactiae\) clinical isolates, ranging in origin from colonisation cases to severe infection cases, were tested for their ability to persist with a macrophage cell line. Surprisingly, persistence within macrophages was a characteristic shared by all of the isolates tested. Furthermore, by investigating the \(Streptococcus\)-containing phagosome, it was revealed that streptococci are able to manipulate the acidification of macrophage phagosomes. Similarly, the maturation of phagosomes containing the fungal pathogen \(C\). \(neoformans\) was explored. Cryptococci are shown to be able to manipulate the phagosome they reside within. This is driven by modified acquisition of Rab GTPases to the phagosome, as well as altered acidification and cathepsin activity within \(Cryptococcus\)-containing phagosomes.