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Supported by grants from North West Cancer Research (Grant number: CR945), The Wellcome Trust (Grant number: 074287/Z/03/Z) and a research studentship (HG) from the Libyan Government. Matrix metalloproteinase (MMP)‐7, unlike many MMPs, is typically expressed in epithelial cells. It has been linked to epithelial responses to infection, injury, and tissue remodeling including the progression of a number of cancers. We have now examined how MMP‐7 expression changes in the progression to esophageal adenocarcinoma (EAC), and have studied mechanisms regulating its expression and its functional significance. Immunohistochemistry revealed that MMP‐7 was weakly expressed in normal squamous epithelium adjacent to EAC but was abundant in epithelial cells in both preneoplastic lesions of Barrett's esophagus and EAC particularly at the invasive front. In the stroma, putative myofibroblasts expressing MMP‐7 were abundant at the invasive front but were scarce or absent in adjacent tissue. Western blot and ELISA revealed high constitutive secretion of proMMP‐7 in an EAC cell line (OE33) that was inhibited by the phosphatidylinositol (PI) 3‐kinase inhibitor LY294002 but not by inhibitors of protein kinase C, or MAP kinase activation. There was detectable proMMP‐7 in cultured esophageal myofibroblasts but it was undetectable in media. Possible metabolism of MMP‐7 by myofibroblasts studied by proteomic analysis indicated degradation via extensive endopeptidase, followed by amino‐ and carboxpeptidase, cleavages. Myofibroblasts exhibited increased migration and invasion in response to conditioned media from OE33 cells that was reduced by MMP‐7 knockdown and immunoneutralization. Thus, MMP‑7 expression increases at the invasive front in EAC which may be partly attributable to activation of PI 3‐kinase. Secreted MMP‐7 may modify the tumor microenvironment by stimulating stromal cell migration and invasion. Publisher PDF

Matrix metalloproteinase (MMP)-7, unlike many MMPs, is typically expressed in epithelial cells. It has been linked to epithelial responses to infection, injury, and tissue remodeling including the progression of a number of cancers. We have now examined how MMP-7 expression changes in the progression to esophageal adenocarcinoma (EAC), and have studied mechanisms regulating its expression and its functional significance. Immunohistochemistry revealed that MMP-7 was weakly expressed in normal squamous epithelium adjacent to EAC but was abundant in epithelial cells in both preneoplastic lesions of Barrett's esophagus and EAC particularly at the invasive front. In the stroma, putative myofibroblasts expressing MMP-7 were abundant at the invasive front but were scarce or absent in adjacent tissue. Western blot and ELISA revealed high constitutive secretion of proMMP-7 in an EAC cell line (OE33) that was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitor LY294002 but not by inhibitors of protein kinase C, or MAP kinase activation. There was detectable proMMP-7 in cultured esophageal myofibroblasts but it was undetectable in media. Possible metabolism of MMP-7 by myofibroblasts studied by proteomic analysis indicated degradation via extensive endopeptidase, followed by amino- and carboxpeptidase, cleavages. Myofibroblasts exhibited increased migration and invasion in response to conditioned media from OE33 cells that was reduced by MMP-7 knockdown and immunoneutralization. Thus, MMP-7 expression increases at the invasive front in EAC which may be partly attributable to activation of PI 3-kinase. Secreted MMP-7 may modify the tumor microenvironment by stimulating stromal cell migration and invasion.

Background Ejaculates contain a diverse mixture of sperm and seminal fluid proteins, the combination of which is crucial to male reproductive success under competitive conditions. Males should therefore tailor the production of different ejaculate components according to their social environment, with particular sensitivity to cues of sperm competition risk (i.e. how likely it is that females will mate promiscuously). Here we test this hypothesis using an established vertebrate model system, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our study tests for the first time how both sperm and seminal fluid components of the ejaculate are tailored to the social environment. Results Our quantitative proteomics analysis reveals that the relative production of different proteins found in seminal fluid – i.e. seminal fluid proteome composition – differs significantly according to cues of sperm competition risk. Using a conservative analytical approach to identify differential expression of individual seminal fluid components, at least seven of 31 secreted seminal fluid proteins examined showed consistent differences in relative abundance under high versus low sperm competition conditions. Notably three important proteins with potential roles in sperm competition – SVS 6, SVS 5 and CEACAM 10 – were more abundant in the high competition treatment groups. Total investment in both sperm and seminal fluid production also increased with cues of heightened sperm competition risk in the social environment. By contrast, relative investment in different ejaculate components was unaffected by cues of mating opportunities. Conclusions Our study reveals significant plasticity in different ejaculate components, with the production of both sperm and non-sperm fractions of the ejaculate strongly influenced by the social environment. Sperm competition risk is thus shown to be a key factor in male ejaculate production decisions, including driving plasticity in seminal fluid composition.

The fungal pathogen Candida albicans survives in humans most commonly as a commensal organism in the flora of the gastrointestinal and urogenital tracts, occasionally causing opportunistic infections, such as thrush. However, in people who are immunocompromised, C.albicans can cause severe and life-threatening infections. Stress adaptation is critical for the pathogenicity of C.albicans, and hence has been studied in detail at the genomic level to try to assess the mechanisms for host-defence implemented as adaptations to the environments encountered.

Insect identification and monitoring are essential to a number of diverse fields and settings, seeking to identify and study insect populations to learn more about their place in ecosystems as well as their impact on the environment and other species. The long-established approach to identifying insects is by morphological taxonomy, which utilises taxonomic keys and requires or at least greatly benefits from experience. However, identification based on morphological characteristics can be difficult when facing morphologically indistinguishable species, immature life stages or damaged specimens. Additionally, there is the challenge of processing large sample numbers that are being collected for analysis. New, easy-to-use high-throughput tools, capable of handling a variety of samples in vast amounts and with minimal sample preparation, are still needed and could provide much needed support in the wide array of fields requiring rapid insect identification. This PhD project explored the capabilities of Rapid Evaporative Ionisation Mass Spectrometry (REIMS) and its potential as a new tool for insect identification and characterisation. REIMS utilises an ambient ionisation source, specifically designed to analyse aerosols resulting from thermal disintegration caused by the passage of electricity through the sample of interest. The electric current is applied through diathermy tools and the resulting aerosol evacuated through a tube to the source and subsequently the mass spectrometer. Instead of focusing on the identification of single molecules, pattern recognition is applied to identify unique mass patterns that facilitate classification and consequently sample identification. After the first test using a mixture of wild-trapped arthropod species, successfully generating informative mass spectra, a larger proof-of principle study was conducted, based on 800 adult specimens of different Drosophila species. By analysing the REIMS data using random forest analysis, in addition to principal component and linear discriminant analysis (PCA-LDA), high classification rates were achieved when using test data sets. The results demonstrated the ability of REIMS to distinguish species, even closely related ones, as well as discriminate males and females. Further, the same approach correctly discriminated Drosophila species at the larval stage, where specimens are morphologically highly similar or identical. The next stages of the project focussed on mosquitoes and the use of REIMS to help with population characterisation – using both laboratory reared and semi-wild/trapped specimens. Laboratory reared Anopheles mosquitoes from three sibling species, usually requiring DNA analysis to be distinguished, extended the species separation challenge. Furthermore, the ability of REIMS to separate sample groups according to their age was investigated. Establishing the age profile of a mosquito population is challenging, but potentially useful as it allows prediction of disease transmission intensities and evaluation of disease vector control actions. The resulting models allowed for clear distinction between age groups separated by only 24 h and high classification rates when leaving more distinct gaps between age groups. Further, REIMS analyses of local mosquito specimens were completed, testing the system using wild-caught mosquitoes as well as semi-wild specimens, which had been collected as larvae in the field and raised under changing conditions. While the focus remained on species and age, these data sets possessed far more variability and confounding factors than those based on specimens reared under controlled laboratory conditions. The increased variance provided a powerful way to gauge REIMS suitability as identification device and helped underpin results and findings obtained with laboratory reared insects. The species of over 180 unknown specimens, part of a blinded sample set, were correctly identified at a rate of 94 % using a pre-built model and recognition software. The exploration of the potential of REIMS concluded with preliminary proof-of-principle experiments that focussed not on the insects themselves, but the frass (droppings) they produce. Successful separation of different cricket species using only their faecal matter proved that REIMS could have the potential for insect identification and population monitoring on various levels, whether its adult specimens, immature forms or ‘calling cards’ left behind. Without the need for sample preparation, entomological expertise or perfectly preserved specimens, REIMS offers a novel approach to insect typing and analysis and has considerable potential as a new tool for the field biologist.

As relatives share genes that are identical by descent, organisms can gain additional fitness benefits by improving the reproductive success of known kin. There are a number of costs associated with close inbreeding, including an increased likelihood of the expression of recessive deleterious alleles and reduced survivorship. The ability to recognise kin therefore enables individuals to improve their inclusive fitness and avoid problems associated with close inbreeding. Female house mice (Mus musculus domesticus) will nest and nurse offspring communally. Choosing an appropriate nest partner is therefore important and competition between nesting females can result in reproductive inhibition and infanticide. Kin selection theory suggests that females could gain inclusive fitness benefits from nesting with relatives. This thesis explores the mechanisms of kin recognition in female house mice in the contexts of social partner choice and inbreeding avoidance. Female house mice recognised unfamiliar relatives, suggesting a phenotype matching mechanism for kin recognition. Females were presented with maternal and paternal half-siblings to investigate recognition template formation. Females nested with maternal but not paternal half-sisters, suggesting that female house mice may use a recognition template learnt from their mother for social partner choice decisions. However females avoided both maternal and paternal half-brothers suggesting that females may use a match-to-self mechanism for inbreeding avoidance. Female house mice were able to identify relatives from urine, suggesting that genetic markers are present in urine. To investigate the molecular markers of kin recognition mice were bred to control for the major histocompatibility complex (MHC) and major urinary proteins (MUPs). Females nested with females that matched for MHC or MUP type, suggesting that both gene families may be used for kin recognition between females. A non-significant trend was observed for females to avoid males that matched themselves for MUP type, but females showed no avoidance of males that matched for MHC type. A small pilot experiment investigated the physiological effects of female social environment. Housing with unfamiliar females resulted in short-term decreases in female body mass and urinary protein concentration but an increase in urinary creatinine concentration. Competition between unfamiliar females may have resulted in a decreased water uptake and an increase in scent marking which could explain the physiological changes observed. Together these results suggest that female house mice may use two mechanisms of kin recognition. For social partner choice females may use a match-to-maternal MHC and MUP type mechanism, whilst for inbreeding avoidance females may use a match-to-own MUP type mechanism. The possibility of a single species using two separate mechanisms suggests that kin recognition may be considerably more complex than previously thought.