http://www.era.lib.ed.ac.uk:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. Fri, 11 May 2012 14:05:05 GMT 2012-05-11T14:05:05Z http://hdl.handle.net/1842/5924 Title: Investigating the role of eEF1A2 in motor neuron degeneration Authors: Griffiths, Lowri Ann Abstract: Abnormal expression of the eukaryotic translation elongation factor 1A (eEF1A) has been implicated in disease states such as motor neuron degeneration and cancer. Two variants of eEF1A are found in mammals, named eEF1A1 and eEF1A2. These two variants are encoded by different genes, produce proteins which are 92% identical but have very different patterns of expression. eEF1A1 is almost ubiquitously expressed while eEF1A2 is expressed only in specialised cell types such as motor neurons and muscle. A spontaneous mutation in eEF1A2 results in the wasted mouse phenotype which shows similar characteristics in the mouse to those seen in human motor neuron degeneration. This mutation has been shown to be a 15.8kb deletion resulting in the complete loss of the promoter region and first non coding exon of eEF1A2 which completely abolishes protein expression. The main aim of this project was to further investigate the role of eEF1A2 in motor neuron degeneration. Firstly, although the wasted phenotype is considered to be caused by a recessive mutation, I established a cohort of aged heterozygote mice to evaluate whether any changes are seen later in life that might model late onset motor neuron degeneration. A combination of behavioural tests and pathology was used to compare wild type and heterozygous mice up to 21 months of age. Whilst results indicate that there is no significant difference between ageing heterozygotes and wildtype controls, there is an indication that female heterozygote mice perform slightly worse that wildtype controls on the rotarod (a behavioural test for motor function). Secondly, I aimed to investigate the primary cause of the wasted pathology by generating transgenic wasted mice expressing neuronal eEF1A2 only. This would complement previous experiments in the lab which studied transgenic wasted mice expressing eEF1A2 in muscle only. Unfortunately the expression of eEF1A2 in the transgenic animals was not neuronal specific. However a transgenic line with expression of eEF1A2 in neurons and skeletal muscle but not cardiac muscle has been generated which clearly warrants further investigation. Thirdly, I wished to assess whether eEF1A2 has any role in human motor neuron degeneration. To achieve this, eEF1A2 expression was investigated in spinal cords from human motor neuron disease (MND) patients. Preliminary data suggests that motor neurons from some MND patients express significantly less eEF1A2 than motor neurons of control samples. Further work is required to confirm these findings. Finally, I investigated the individual roles of eEF1A1 and eEF1A2 in the heat shock response. I used RNAi to ablate each variant separately in cells and subsequently measured the ability of each variant individually to mount a heat shock response. Results indicate a clear role for eEF1A1 but not eEF1A2 in the induction of heat shock. This may explain in part why motor neurons exhibit a poor heat shock response as they express eEF1A2 and not eEF1A1. These experiments shed light on our understanding of the role of eEF1A2 in motor neuron degeneration and uncover many new avenues of future investigation. Fri, 25 Nov 2011 00:00:00 GMT http://hdl.handle.net/1842/5924 2011-11-25T00:00:00Z http://hdl.handle.net/1842/5923 Title: Determining the role of follicular dendritic cells in TSE agent neuroinvasion Authors: McCulloch, Laura Abstract: Transmissible spongiform encephalopathies (TSEs), such as scrapie and variant Creutzfeldt-Jakob disease are infectious, fatal, neurodegenerative diseases. Following peripheral infection TSE agents usually accumulate in lymhoid tissues before spreading to the central nervous system. In mice, follicular dendritic cells (FDCs) expressing the host prion protein (PrPC) are essential for scrapie agent accumulation in lymphoid tissues. The accumulation of the scrapie agent on FDCs is critical for the efficient spread of infection to the brain. However, it is unknown whether FDCs themselves actively replicate the scrapie agent, or simply accumulate it following production by other cells types such as neurones, lymphocytes or other stromal cell populations. To definitively address this issue a transgenic mouse model was created in which PrPC is switched on or off exclusively on FDCs. Expression of cre-recombinase (Cre) under the action of cell-specific gene promoters can be used to induce or delete the expression of a target gene in specific cell populations. In this model, Cre expression is driven by the complement receptor type 2 gene (Cr2/CD21) which is expressed by FDCs and mature B lymphocytes. Characterisation of the CD21-cre mouse line was achieved by crossing with a ROSA26 reporter strain. The CD21-cre mouse line was subsequently crossed with floxed-PrP mouse lines to produce compound transgenic mouse lines in which PrPC expression was switched on or off, only in FDCs. Cre expression by B lymphocytes was eliminated by γ-irradiation and grafting recipient mice with Cre-deficient bone marrow. Immunohistochemical analysis confirmed the expression PrPC had been switched on or off exclusively on FDCs. Subsequently, the mice were challenged with scrapie by intra-peritoneal injection to determine the precise role of FDCs in the accumulation of scrapie in lymphoid tissues. Switching off PrPC expression exclusively on FDCs prevented the accumulation of TSE agent specific disease-associated PrPSc in the spleen after i.p inoculation. Conversely, in mice in which PrPC was expressed only on FDC, successful replication of the agent occurred on the FDC network in the spleen. Taken together, these data show PrPC-expressing FDCs alone are sufficient to support the accumulation of the scrapie agent within lymphoid tissues. Furthermore, these data suggest FDC replicate the TSE agent and do not simply accumulate it following synthesis by other cell types. Fri, 25 Nov 2011 00:00:00 GMT http://hdl.handle.net/1842/5923 2011-11-25T00:00:00Z http://hdl.handle.net/1842/5922 Title: Cyclin-dependent kinase inhibitor drugs drive neutrophil granulocyte apoptosis by transcriptional inhibition of the key survival protein MCL-1 Authors: Leitch, Andrew Edward Abstract: The normal physiological response to bacterial infection or wounding with threat of infection, termed inflammation, has been shown to be dysregulated in certain human diseases including (but not limited to): idiopathic pulmonary fibrosis, acute lung injury, arthritis and glomerulonephritis. The earliest arriving and most abundant cell responding to an inflammatory stimulus is the neutrophil granulocyte. It has been shown that under inflammatory conditions neutrophil granulocytes have extended longevity, enhanced responsiveness and upregulated activation parameters. In the setting of non-infective, or prolonged, ineffectuallycleared infective disease where resolution of inflammation does not occur then neutrophil granulocytes may cause tissue damage which is mediated by excessive, misdirected exocytosis of toxic granule contents or by spillage of the same products from necrotic or netotic cell carcasses that have lost membrane integrity. A key process in the resolution of inflammation is the induction of apoptosis in recruited neutrophils following a successful response to an inflammatory stimulus. Cellular signalling from apoptotic cells and from professional phagocytes that have ingested apoptotic cells has been shown to favour resolution of inflammation and restoration of tissue homeostasis. Additionally, the removal of key inflammatory cells in a highly regulated, non-phlogistic fashion robustly assists the resolution process. Cyclin-dependent kinase (CDK) inhibitor drugs are being developed as anti-cancer agents as it is hypothesized that they should interfere with the enhanced cellcycling ability (increased proliferative capacity and extended longevity) which is such a key feature of cancer cell biology. The CDKs that drive the cell cycle are CDKs 1, 2, 4 and 6 and consequently agents were designed to have enhanced specificity for these targets. CDK inhibitor drugs target the ATP-binding domain of CDKs and as a result usually have activity against more than one CDK. The CDK inhibitor drug, R-roscovitine which targets CDKs 2, 5, 7 and 9 was shown to promote neutrophil apoptosis and consequently resolution of inflammation. This thesis aims to investigate the mechanism by which apoptosis is induced in neutrophil granulocytes by CDK inhibitor drugs. The first experimental chapter of this thesis explores in detail the time-course and active concentration range of CDK inhibitor drugs in comparison to known promoters and inhibitors of neutrophil apoptosis. It then dissects the apoptotic machinery which is responsible for the effects of CDK inhibitor drugs before investigating their capacity to promote apoptosis even in the presence of survival mediators relevant to the context of inflammatory disease. Flow-cytometry, light and confocal microscopy as well as western blotting for caspases, mitochondrial dissipation assay, fluorometric caspase assay and the detection of DNA laddering demonstrate that CDK inhibitor drugs promote classical neutrophil apoptosis by the intrinsic pathway and show similar kinetics of apoptosis induction to drugs that inhibit transcription. The second experimental chapter investigates the key neutrophil survival protein and bcl-2 homologue Mcl-1. By flow cytometry, western blotting and RT-PCR it is demonstrated that Mcl-1 is down-regulated at the level of transcription and that this occurs even in the presence of inflammatory mediators that would normally promote neutrophil survival. Additionally, it is shown that pro-apoptotic bcl-2 homologues are affected to a lesser degree suggesting an imbalance of bcl-2 proteins is caused by effects at a transcriptional level mediated by CDK inhibitor drugs. The third experimental chapter identifies CDKs and their binding partner cyclins in neutrophil granulocytes and investigates the impact of CDK inhibitor drugs on CDK protein levels and cellular distribution by differential lysis and western blotting as well as by confocal microscopy. The key transcriptional enzyme RNA polymerase II is also identified and the effect of CDK inhibitor drugs on phosphorylation of this enzyme is documented. Western blotting and confocal microscopy demonstrate the presence of key CDKs 2, 5, 7, 9 and cyclin binding partners of CDKs 7 and 9. It is shown that the phosphorylation of RNA polymerase II mediated by CDKs 7 and 9 is inhibited by CDK inhibitor drugs. This suggests that a key mechanism by which neutrophil apoptosis is induced by CDK inhibitor drugs is the inhibition of transcription of key proteins and suggests that neutrophils require survival proteins for functional longevity. The fourth experimental chapter addresses the production and use of HIV-tat dominant negative CDK 7 and 9 proteins to knockdown CDKs 7 and 9 in neutrophil granulocytes in vitro to provide a molecular biology surrogate for the pharmacological data already presented. The cloning, production, purification and use of HIV-tat dominant negative CDK proteins are described. The final chapter describes the use of a more specific pharmacological inhibitor of CDKs 7 and 9, DRB, in the mouse bleomycin lung injury model. Resolution of inflammation by a compound specifically targeting CDKs 7 and 9 is described. This thesis identifies CDKs 7 and 9 as key targets of CDK inhibitor drugs in neutrophilic inflammation. It shows these drugs acting at the level of transcription to drive neutrophil apoptosis by exploiting the unique dependency of neutrophils on the short-lived survival protein Mcl-1. In so doing the presence of functional and essential transcriptional machinery is identified in neutrophils and the transcriptional profile of resting, stimulated and inhibited neutrophils is delineated. These findings suggest novel approaches to the pharmacological promotion of resolution of inflammation and indicate key new targets for rational drug design. In future, it will be important to further characterize the effects of CDK inhibitor drugs on other cell-types including epithelial cells, fibroblasts and mononuclear cells. This information should prove important to the continued investigation of CDK inhibitor drugs in resolution of inflammation and also to the ongoing experimental trial of these drugs in idiopathic pulmonary fibrosis. Fri, 25 Nov 2011 00:00:00 GMT http://hdl.handle.net/1842/5922 2011-11-25T00:00:00Z http://hdl.handle.net/1842/5921 Title: Theoretical modelling of ultrasound contrast agents Authors: Looney, Padraig Abstract: This thesis compares theoretical models of ultrasound contrast agents to the acoustic response from single Microbubbles(MBs). The acoustic response was compared using a range of driving parameters. A rigid shelled contrast agent and a lipid shelled contrast agent were used in the comparison. While attempts to model the behaviour of some contrast agents at low mechanical index (MI) have been successful at higher MI the behaviour of MBs is still not well understood. Understanding and predicting the response ofMBs to medical ultrasound can lead to improvements in the clinical use of MBs through improved contrast agent design or improved signal processing. Numerical models were developed to compare to three specific cases; 1) Rigid shelled contrast agents 2) Lipid shelled contrast agents 3) Responses from lipid shelled contrast agents that are hit by subsequent driving pulses. Three models were used to compare to the responses from single rigid shelled contrast agents. Two of these models have been used before and the third was developed based on the optical observations of the responses of these rigid shelled agents at these MI. Two shelled models were used to compare to the response of single lipid shelled MBs. Using statistical methods the parameters defining the shell properties were found. The parameters that gave best agreement with the lipid shelled data was then used with a model to account for the molecular diffusion of gas from a MB and a new model to account for the optically observed shedding of the shell from a MB to compare to the multiple response from single MBs. While the theoretical prediction of an acoustic response of a suspension of MBs or the radial oscillation of single MBs has been compared before to experimental data, the successful comparison of the acoustic response of single MBs to the theoretical prediction is the first of it’s kind known to the author. The new theoretical model of the rigid shelled MB that was developed in this thesis gave better agreement with the experimental data than the other previously used models. The shell parameters of the lipid shelled MB were determined for the lowest driving amplitude and were in agreement with those measured previously from optical observations. Finally, the model for the shedding of the shell was shown to give quantitative agreement with the multiple acoustic responses from single MBs. When shedding of the shell was included the choice of constitutive equation for the shell was shown to strongly affect subsequent responses from the MB. Fri, 25 Nov 2011 00:00:00 GMT http://hdl.handle.net/1842/5921 2011-11-25T00:00:00Z http://hdl.handle.net/1842/5920 Title: Vascular lesion development: influence of endogenous and exogenous glucocorticoids Authors: Low, Lucinda Abstract: Atherosclerotic and restenotic lesions develop as a result of an excess inflammatory response to vascular injury. Glucocorticoid hormones have widely-recognised anti-inflammatory and anti-proliferative properties which appear to make them ideal candidates for inhibition of vascular lesion development. Indeed, administration of glucocorticoids to experimental animals does inhibit the growth of vascular lesions in some models. In addition, glucocorticoids are currently being trialled clinically as anti-restenotic agents. However, glucocorticoid excess in patients, either as a result of Cushing’s syndrome or chronic steroid therapy, is associated with enhanced CVD risk. Therefore, the effects of glucocorticoids on vascular lesion development remain imperfectly understood. The overall objective of these studies was to explore the influence of endogenous and exogenous glucocorticoids on vascular lesion development using murine models of atherosclerosis (ApoE-/- mice fed a “western” diet) and neointimal hyperplasia (wireinduced femoral artery injury). The work described in this thesis addresses the hypothesis that glucocorticoids are pro-atherogenic, yet anti-restenotic. Mice were bilaterally adrenalectomised to investigate the role of endogenous glucocorticoids on vascular lesion development. Removal of the adrenal glands had no influence on atherogenesis or neointima development. The influence of exogenous glucocorticoids on lesion development was assessed by orally administering dexamethasone (0.1 or 0.8mg/kg/day). Atherosclerotic lesion burden was augmented by dexamethasone administration. Conversely, fibro-proliferative neointimal proliferation was inhibited by dexamethasone. However, this effect was obscured by thrombotic lesion development. It was proposed that this thrombotic effect is attributable to increased plasminogen activator inhibitor-1 (PAI-1), which was tested using PAI-1 deficient mice. Although PAI-1 was found to mediate the systemic pro-thrombotic effect of dexamethasone, it is not required for the enhanced development of thrombotic lesions at the site of intra-luminal injury. These results suggest that physiological levels of endogenous glucocorticoids play a limited role in vascular lesion development. Conversely, although exogenous glucocorticoids inhibit fibro-proliferative intimal hyperplasia, they appear to have significant detrimental influences on lesion development, augmenting atherosclerosis and inducing thrombotic neointimal lesion formation following vascular injury. Further research is therefore required to improve the cardiovascular outcome of patients requiring glucocorticoid therapy and for the use of glucocorticoids as antirestenotic agents. Fri, 25 Nov 2011 00:00:00 GMT http://hdl.handle.net/1842/5920 2011-11-25T00:00:00Z http://hdl.handle.net/1842/5919 Title: Determinants and correlates of intra-individual variability in reaction time Authors: Dykiert, Dominika Abstract: Traditionally, reaction time (RT) was conceived of as an average speed of a number of responses made by an individual, or mean RT. Increasingly, however, intraindividual variability in reaction time (RT IIV) – the consistency of responses by a single person across trials – is used as an additional or even alternative measure. RT IIV is often found to be elevated in a number of conditions that affect the central nervous system functioning, such as traumatic brain injury or neurodegenerative diseases. It can predict change in cognitive performance in ageing, progression from normal ageing to mild cognitive impairment, and even death. Therefore, RT IIV may be of great practical importance. However, RT IIV and mean RT are correlated; therefore it is often problematic to draw conclusions about unique associations between these and other variables. One objective of the work presented in this thesis was to investigate determinants and correlates of simple and choice RT IIV and to test which associations may be accounted for by the individual differences in mean RT. The first investigation was concerned with age differences in RT IIV. Following a systematic review of literature, a series of meta-analyses demonstrated that older individuals (aged 60 years and above) have greater RT IIV than young or middle-aged adults in simple and choice RT tasks. The effects were reduced but still significant when RT IIV was adjusted for mean RT. The next study was a cross-sectional investigation of the associations between age and RT IIV, as well as of sex differences in RT IIV, across the lifespan in participants ranging in age from 4 to 75. Non-linear effects of age were found for RT IIV measures, such that variability decreased with age in children and increased with age in older adults. A novel finding from this study was that sex differences in RT IIV were present among adults but not children, suggesting that there might be an age threshold at which sexes diverge in their RT IIV trajectories. The results also indicated that findings regarding RT IIV may differ depending on the variability measure used (that is, whether and how mean RT is controlled). The second study on the same sample investigated variability on a trial-by-trial basis. Specifically, it tested the hypothesis that sex differences in variability are due to females being disproportionately slower at the first trial which inflates their overall RT IIV. This hypothesis was not supported. Another investigation used longitudinal data from the West of Scotland Twenty-07 study. Three cohorts of individuals aged approximately 15, 35 and 55, were followed up for 20 years and had RT data collected at four occasions. Analyses confirmed non-linear effects of age on RT IIV found in the earlier cross-sectional investigation. The final study investigated the effect of high altitude on RT IIV. It found that altitude-related increase in RT IIV is fully accounted for by general slowing of RT at high altitude. The overall pattern of results obtained from the investigations suggests that RT IIV increases with age in adults and that not all of the increase is due to general slowing. Moreover, the results show that sex differences in RT IIV are not uniform across the lifespan. Finally, whereas associations of RT IIV with some variables, for example age, are relatively robust to controlling for mean RT, others are fully attenuated by such practice. Fri, 25 Nov 2011 00:00:00 GMT http://hdl.handle.net/1842/5919 2011-11-25T00:00:00Z http://hdl.handle.net/1842/5918 Title: Analysis of the expression and function of mammalian CSP isoforms Authors: Gorleku, Oforiwa Afi Abstract: Exocytosis, the fusion of intracellular vesicles with the plasma membrane, is fundamental to intercellular communication in multicellular organisms. This pathway facilitates the release or secretion of molecules from the cell. In addition, exocytosis is essential for delivery of resident proteins to the plasma membrane. There are two different pathways of exocytosis, constitutive and regulated exocytosis. Constitutive exocytosis occurs without regulation, e.g. pathways regulating the delivery of lipids and ‘house-keeping’ proteins to the plasma membrane or the secretion of antibodies and extra-cellular matrix components from the cell. In contrast, regulated exocytosis facilitates the controlled release of extra-cellular molecules or insertion of new membrane components only in response to a physiological signal. The most common signal for regulated exocytosis is an increase in intracellular Ca2+ concentration. Several proteins function in exocytosis, and the membrane fusion step is widely believed to result from an interaction between SNARE (SNAP receptor) proteins on the vesicle membrane and plasma membrane. In neuroendocrine cells, these SNARE proteins are VAMP2, which is bound to vesicle membranes and syntaxin1A and SNAP25, which are associated with the plasma membrane. Several proteins have been implicated as SNARE regulators, such as NSF (N-ethylmaleimide-sensitive factor) and its cofactor α-SNAP, Munc18 and synaptotagmin. Another possible SNARE regulator is the cysteine string protein (CSP). CSPα was first identified in Drosophila melanogaster and was later identified in Torpedo as a possible Ca2+-channel regulator. Inactivation of the CSPα gene in Drosophila is lethal at an embryonic stage and in embryos synaptic vesicle exocytosis was decreased by ~50% at 22°C and was abolished at higher temperatures. These results provided strong evidence that CSPα has an important role in presynaptic neurotransmission. However, more recent work on CSPα null mice uncovered an important neuroprotective function for CSPα in brain, but also challenged the proposed function of CSPα in neuronal exocytosis, as no defect in this pathway was evident, at least in young animals. The only reported developmental abnormality of CSPα null mice was bilateral cryptorchidism, a failure of testicular descent during development. Interestingly, two additional CSP isoforms were recently identified in mouse and human testis, CSPβ and CSPγ. One consequence of the identification of CSPβ and CSPγ is that they may complicate analysis of CSPα knockout mice. Here, we have used a combination of techniques, cell systems and human brain samples to examine the function of CSPα in exocytosis, the expression of novel CSPα isoforms in testis, and expression changes of CSPα and its partner proteins in neurological disorders. Furthermore, we have initiated studies to examine how CSPα function is linked to cryptorchidism at the molecular level. My results show that CSPα depletion perturbs regulated exocytosis in neuroendocrine cells, but has no consistent effect on constitutive exocytosis. CSPα has been reported to have an important neuroprotective function; however, no significant changes in CSPα expression were detected in brain samples for schizophrenia, depression and bipolar disorder. Nevertheless the expression of specific CSPα binding partners was found to be significantly changed in some of these disorders. In addition to these studies focussing on CSPα function and expression in neuronal and neuroendocrine cells, studies were undertaken to analyse expression profiles of CSP isoforms in testis. This analysis found that CSPβ and CSPγ are exclusively expressed in testis, and that mRNA transcription of both isoforms is initiated with sexual maturation. Furthermore expression of both isoforms is restricted to germ cells, whereas CSPα is expressed throughout testes. Previous work has shown that the secretory hormone INSL3, which is exclusively expressed in testicular Leydig cells, is involved in the development of cryptorchidism. Confocal microscopic analysis revealed that CSPα and INSL3 colocalise on vesicles in Leydig cells, suggesting the intriguing possibility that CSPα inactivation might cause cryptorchidism due to a loss of INSL3 secretion. Fri, 25 Nov 2011 00:00:00 GMT http://hdl.handle.net/1842/5918 2011-11-25T00:00:00Z http://hdl.handle.net/1842/5917 Title: Role of SUMO modification in hepatocyte differentiation Authors: Hannoun, Zara Abstract: Primary human hepatocytes are a scarce resource with variable function, which diminishes with time in culture. As a consequence their use in tissue modelling and therapy is restricted. Human embryonic stem cells (hESCs) could provide a stable source of human tissue due to their properties of self-renewal and their ability to give rise to all three germ layers. hESCs have the potential to provide an unlimited supply of hepatic endoderm (HE) which could offer efficient tools for drug discovery, disease modelling and therapeutic applications. In order to create a suitable environment to enhance HE formation, hESC culture needed to be standardised. As such, a media trail was carried out to define serum free media capable of maintaining hESC in a pluripotent undifferentiated state. We also ensured hESC cultured in the various media could be directly differentiated to HE in a reproducible and efficient manner. The project then focused on the effect of post-translational modifications (PTMs), specifically SUMOylation, in hepatocyte differentiation and its subsequent manipulation to enhance HE viability. SUMOylation is a PTM known to modify a large number of proteins that play a role in various cellular processes including: cell cycle regulation, gene transcription, differentiation and cellular localisation. We hypothesised that SUMO modification may not only regulate hESC self renewal, but also maybe required for efficient hESC differentiation. We therefore interrogated the role of SUMOylation in hESC differentiation to hepatic endoderm (HE). hESC were differentiated and the cellular lysates were analysed by Western blotting for key proteins which modulate the conjugation and de conjugation of SUMO. We demonstrate that peak levels of SUMOylation were detectable in hESC populations and during cellular differentiation to definitive endoderm (DE), day 5. Following commitment to DE we observed a decrease in the level of SUMO modified proteins during cellular specialisation to a hepatic fate, corresponding with an increase in SENP 1, a SUMO deconjugation enzyme. We also detected reduced levels of hepatocyte nuclear factor 4 α (HNF4α), a critical regulator of hepatic status and metabolic function, as SUMOylation decreased. As a result, we investigated if HNF4α was SUMOylated and if this process was involved in modulating HNF4α’s critical role in HE. HNF4α is an important transcription factor involved in liver organogenesis during development and is a key regulator for efficient adult liver metabolic functions. We observed a decreasing pattern of HNF4α expression at day 17 of our differentiation protocol in conjunction with a decrease in SUMO modified proteins. In order to further investigate and validate a role of SUMOylation on HNF4α stability Immunoprecipitation (IP) was employed. HNF4α protein was pulled down and probed for SUMO 2. Results show an increase in the levels of SUMO2 modification as the levels of HNF4α decrease. Through deletion and mutation analysis we demonstrated that SUMO modification of HNF4α was restricted to the C-terminus on lysine 365. Protein degradation via the proteasome was responsible for the decrease in HNF4α, demonstrated by the use of a proteasome 26S inhibitor MG132. Additionally, a group at the University of Dundee has shown that polySUMOylation of promyelocytic leukaemia protein (PML) leads to its subsequent ubiquitination via RNF4, an ubiquitin E3 ligase, driving its degradation. Using an in vitro ubiquitination assay, we show that polySUMOylated HNF4α is preferentially ubiquitinated in the presence of RNF4. Overall polySUMOylation of HNF4α may reduce its stability by driving its degradation, hence regulating protein activity. In conclusion, polySUMOylation of HNF4α is associated with its stability. HNF4α is subsequently important for HE differentiation both driving the formation of the hepatocytes and in maintaining a mature phenotype, in agreement with a number of different laboratories. Creating the ideal environment for sustaining mature functional hepatocytes, primary and those derived from hESCs and iPSCs, is essential for further use in applications such as drug screening, disease modelling and extracorporeal devices. Fri, 25 Nov 2011 00:00:00 GMT http://hdl.handle.net/1842/5917 2011-11-25T00:00:00Z http://hdl.handle.net/1842/5916 Title: Novel approaches to the development and assessment of an ovine model of polycystic ovary syndrome Authors: Hogg, Kirsten Abstract: Polycystic ovary syndrome (PCOS) is a common reproductive, endocrine and metabolic disorder present in women of reproductive age. Despite the widespread prevalence and heritability of PCOS, the heterogeneous and polygenic traits have made the successful identification of candidate genes difficult. Animal models have been developed on the premise that early exposure to sex steroids can programme epigenetic changes that predispose the fetus to the adult features of PCOS. Past research has modelled ovarian dysfunction, endocrine abnormalities and metabolic perturbances in rodent, non-human primate and sheep PCOS models, through the enhanced neonatal or prenatal exposure to the male sex hormone, testosterone. The modelling of PCOS in a large domestic species such as the sheep is advantageous due to similar biological reproductive function as the human. In this regard the sheep has been extensively used to model PCOS by the treatment of pregnant ewes from early to midgestation with androgens such as testosterone propionate (TP). These experiments have demonstrated the fetal programming effects of androgens on offspring that go on to develop PCOS-like characteristics in adulthood. One of the caveats of assessing steroid effects in this way is the effect of the placenta in mediating the transfer of these hormones. TP is an aromatisable androgen and thus some of its effects in the fetus may be attributable to placental by-products such as estrogens. This thesis describes the development and assessment of a novel model of prenatal androgenisation. Two models were compared: the indirect maternal exposure to TP (the current model) and the direct fetal injection of TP. In directly treating the fetus this allowed control over the dose of TP administered and avoidance of secondary effects that androgens may exert in the mother that could be transferred to the fetus. For the maternal model, pregnant Scottish Greyface ewes were administered TP twice weekly from day (d)62-102 of a 147 day gestation. For the fetal model, fetuses were injected twice while the ewe was anaesthetised with graded doses of TP during the same period of treatment as the maternal model. The effects of prenatal androgenisation were assessed in the female fetus shortly after treatment and also in young adult sheep. Fetal ovarian and adrenal steroidogenic gene expression was monitored and found to be altered in response to elevated levels of sex steroids. At d90 the morphology of the developing ovary was not changed by prenatal androgens. In the adult a detailed ovarian and endocrine assessment was undertaken, by examination of ovarian morphology, hormone levels, ovulatory cycles, hypothalamic pituitary ovarian function and follicle steroidogenesis, during the first breeding season. In addition, the metabolic effects of prenatal androgens were monitored by measuring body fat, insulin and glucose homeostasis and liver function. Neither maternal nor fetal prenatal androgenisation during mid-gestation resulted in a perturbed hormonal milieu or polycystic ovaries in young adults. These treatments did however programme a clear ovarian phenotype demonstrated by the increased capacity of follicles to secrete androgens, independently of an abnormal endocrine environment and disordered folliculogenesis. Furthermore, animals that were exposed maternally to TP developed fatty liver and had increased insulin secretion in response to glucose load. A major outcome of this study was the finding that the fetally injected control animals were phenotypically different than the maternal control animals. In fact, some of the reproductive and metabolic features of maternal TP exposure were found in the fetal control group. This unexpected finding has raised the possibility that it is the fetal exposure to stress, that is secondary to elevated maternal androgens, rather than androgens per se that is responsible for at least some of the multitude of anomalies encountered in PCOS. Fri, 25 Nov 2011 00:00:00 GMT http://hdl.handle.net/1842/5916 2011-11-25T00:00:00Z http://hdl.handle.net/1842/5915 Title: Investigation into taxane resistant breast cancer Authors: Kenicer, Juliet Elisabeth Margaret Abstract: One group of chemotherapeutics that are used successfully to treat breast cancer, alone or in combination with other agents, are the taxanes; paclitaxel and docetaxel. They act by interfering with the spindle microtubule dynamics of the cell causing cell cycle arrest. However, the complexities underlying the mechanism of action are yet to be fully elucidated. Arguably, one of the most significant problems with taxanes is chemoresistance. Unfortunately, some patients are intrinsically resistant to taxanes and others acquire resistance to taxanes as treatment advances. This problem is exacerbated by a lack of understanding of the mechanisms underlying taxane resistance. Isogenic breast cancer cell lines that were taxane resistant were generated to use as an experimental model. Paclitaxel resistant (PACR) MDA-MB-231, paclitaxel resistant ZR75-1 and docetaxel resistant (DOCR) ZR75-1 cell lines were successfully generated by incrementally increasing taxane dose in respective native cell lines in vitro. An extensive characterisation of each of the resistant cell lines was conducted, focussing primarily on the 25nM resistant cells which were determined to be the most clinically relevant dose of taxane. A suboptimal dose of 5nM, a “superoptimal” dose of 50nM and the native, taxane sensitive cells was included. Dose response cell count experiments were performed that confirmed taxane resistant cells had been generated. It was shown that MDA-MB-231 native cells were more sensitive to paclitaxel than the ZR75-1 native cells, suggesting that ZR75-1 cells may already have low level inherent resistance. The MDA-MB-231 25nM PACR cells were tested to determine whether they retained PACR when maintained in media containing no paclitaxel. MDA-MB-231 25nM PACR cells were maintained in a taxane free environment for six months and then rechallenged with taxane. When rechallenged, the PACR cells previously maintained in the absence of paclitaxel mirrored the pattern of growth of corresponding PACR cells that had been maintained in the presence of paclitaxel. This proved that in the absence of paclitaxel, PACR cells did not revert to parent phenotype. This meant that experiments could be designed to grow cell lines as xenografts in mice, (in the absence of paclitaxel) & bring in vitro experiments into an in vivo setting. Effects of taxane treatment on both native and resistant cells were analysed using flow cytometry. Paclitaxel treatment exerted G2/M block in native MDA-MB-231 cells but when PACR cells were treated with the same dose of paclitaxel no G2/M block was observed, suggesting that PACR cells had developed a mechanism for escaping G2/M block. ZR75-1 native lines were also investigated and we established that treatment with paclitaxel also exerted a G2/M block in these lines. In future studies this process will be repeated to investigate the effect of taxane treatment on the ZR75-1 PACR and DOCR lines. CD 1 nude mice were injected with cells from all five cell lines to grow xenografts, unfortunately MDA-MB-231 PACR cells failed to grow so they could not be used for further xenograft experiments. PACR, DOCR and Native ZR75-1 cells did successfully grow as xenografts in mice and confirmed that all 3 groups showed very similar growth patterns. A cross resistance experiment was conducted and it was determined that the DOCR xenografts maintained a taxane resistant phenotype to docetaxel, and not paclitaxel and the PACR xenografts may be perpetuate the paclitaxel resistant phenotype in xenografts and that there may be cross resistance to docetaxel in the paclitaxel resistant xenografts. This is the first time that taxane resistant cell lines grown in this way have been established as xenografts in mice. These cross resistance experiments represent novel findings and merit further investigation. Extensive genomic and transcriptomic analyses were carried out on the cell lines to help identify potential taxane resistance markers. aCGH experiments were carried out to compliment the illumina experiments. The first set of experiments used DNA from pooled whole female blood as ref sample and DNA from each of the native and taxane resistant cell lines as test samples. The second set of experiments used DNA from native cells as a ref sample and DNA from their respective taxane resistant cells as a test, which allowed areas of loss or gain to be tracked in the genome as resistance increased. In the MDA-MB-231 cell lines the following areas of loss extended with increasing resistance: 1p36.13-q44, 6p25.3-q12, 8p, 10p, 19q, X Chr and the following areas of gain 2p25.3-23.3, 3p24.3-q13.3, 4p16.1-q12, 5q14.3-q31.1, 8q21.13-24.3, 11q15.1-q25, centromeric 12, and centromeric 14. In the ZR75-1 PACR and DOCR cell lines the areas of loss extended with increasing resistance in the following regions: 7q, 12p and 16q. For gene expression analysis RNA was extracted from the MDA-MB-231 cell lines, labelled and hybridised them to illumina human ref 8 vs. 2 chips. Data showed a progressive increase in mRNA dysregulation as paclitaxel resistance increased. Eleven genes were dysregulated across all resistance levels in the PACR MDA-MB-231 cells when compared to the relative cell lines; RGS16, CLDN1, IL7R, P&PP1R14C, COBL, TRPV4, TSPAN8, CD33, NLRP2, P13, and PAGE5. The experiment was repeated using MDA-MB-231 PACR, ZR75-1 PACR and DOCR cells and resulting data was analysed to determine genes commonly dysregulated across resistance levels, between MDA-MB-231 PACR and ZR75-1 PACR and between ZR75-1 PACR and DOCR cell lines. An extensive literature search was conducted and established four genes of interest in the context of our genomic and transcriptomic experiments including AURKA, Mdr-1, Stathmin and YY1. The novel biomarkers identified in the illumina experiments were validated with complimentary qPCR gene expression experiments looking at expression levels of the eleven commonly dysregulated genes identified and a panel of 19 other genes with significantly increased or decreased expression as resistance increased including AURKA, Mdr-1, Stathmin and YY1. Western blots were performed with lysates from the cell lines using a standard panel of predictive breast cancer markers and AURKA, Mdr-1, Stathmin and YY1. Combining the data from the genomic study, the gene expression profile, qPCR and Western blotting it was established that Mdr-1 had increased expression in the taxane resistant ZR75-1 lines and YY1 had increased expression in the MDA-MB-231 PACR line. Material from the LAPATAX trial was used to observe any transcriptomic changes occurring in tumours following treatment with docetaxel and to compare them to changes identified in our in vitro and xenograft models, this allowed the final step to be taken into a translational environment. LAPATAX (EORTC 10054) is a phase I-II study of Lapatanib and Docetaxel as neoadjuvant treatment for HER-2 +ve locally advanced/inflammatory or large operable breast cancer. Tumour material from eighteen core biopsies pre and post treatment was obtained, the mRNA was extracted, labelled and hybridised to the illumina array. This allowed the changes in gene expression pre and post docetaxel treatment to be tracked. The gene expression data from the LAPATAX trial was combined with gene expression data from our cell line panel and identified two novel putative markers of taxane resistance DUSP1 and FOS. Although sample size is small this has provided extremely valuable evidence directly from the clinic. These two novel putative biomarkers are extremely intriguing and certainly merit further investigation, ideally using additional taxane treated breast tumour tissue. Ultimately, an isogenic in vitro model of taxane resistance was developed in two different cell lines and with two different taxanes within one cell line. The cell lines were characterised and the effect of the taxanes on the cell cycle was determined in the native and taxane resistant lines. Selected cell lines were grown as xenografts in mice and performed successful cross resistance studies upon them. A large transcriptomic and genomic analysis was conducted and has identified a panel of potential taxane resistance markers and areas of loss and gain in the genome perpetuated by increasing taxane resistance. This analysis was validated using qPCR and Western blotting. This allowed a panel of novel taxane resistance markers to be identified. In future studies it is hoped that these targets will be knocked down with shRNA to observe if the taxane resistant cell lines revert to the parental phenotype. In vitro studies will be conducted to find agents that may be used to reduce expression of these markers and restore sensitivity to taxanes and consequently restore the efficacy of these drugs in a clinical setting. As far as the author is aware this is the first time that isogenic taxane resistant cell lines have been generated and investigated in this way. Fri, 25 Nov 2011 00:00:00 GMT http://hdl.handle.net/1842/5915 2011-11-25T00:00:00Z