This thesis proposes to show that Hemingway's novels reveal a change of attitude which culminates in an increased faith in the ultimate goodness and dignity of man.

When a cell divides into two, it is imperative that each resultant daughter receives a full complement of chromosomes; DNA is ultimately responsible for all cellular processes. Cohesion between sister chromatids from the moment of their generation in S phase is central to ensuring the fidelity of chromosome segregation. Smc1 and Smc3 proteins interact with each other via their hinges and with a bridging kleisin subunit via their heads to form the cohesin ring. It is cohesin, through entrapment of sister chromatid within its ring, that confers sister chromatid cohesion. The process of cohesin’s loading onto DNA is poorly understood. While it is thought to depend on ATP hydrolysis, opening of the ring at one of its three interfaces, and the as yet undefined action of the kollerin complex, comprising Scc2 and Scc4 proteins, the sequence of events as they occur are yet to be defined. A recent screen for suppressors of a thermosensitive scc4 allele in budding yeast revealed a mutation within Smc1’s hinge that could bypass the kollerin subunit. Here, the Smc1 suppressor mutation is investigated. Through targeted mutagenesis, the Smc1D588Y mutant identified in the screen and two additional point mutants, Smc1D588F and Smc1D588W, are herein proven able to bypass Scc4 function completely. Thus we provide the strongest evidence to date to suggest that cohesin’s hinge is a critical factor in its loading. Biochemical evidence shows that isolated Smc1 hinge mutants are defective in their binding to Smc3 hinges. This, together with the genetic link made between the hinge and loading complex, suggests that hinge opening might be a requisite for loading. Through mutagenesis of Scc2 and Scc4 we show that the N-terminus of each protein is responsible for their dimerisation. Furthermore, the N- terminus of Scc2 confers no function other than in its binding to Scc4. Finally, we show that Scc4 is required for the enrichment of both Scc2 and cohesin at centromeres, but not at arm loci. Our results are therefore indicative of there being two different pathways of cohesin loading.

Subcellular sequestration of proteins within membrane bound compartments is widely acknowledged to be an important mode of enzymatic regulation. Recently a novel paradigm for metabolic enzyme compartmentalisation has become apparent with the identification of several proteins which are able to form filamentous structures in vivo. Multiple studies independently identified the essential de novo pyrimidine biosynthesis enzyme CTP synthetase as a major constituent of a novel filamentous structure which has been termed “the cytoophidium”. Cytoophidia have been observed to form in multiple organisms including bacteria (C. crescentus), yeast (S. cerevisiae) and fruit fly (D. melanogaster) as well as in human cultured cells. In this thesis I describe the development and results of a high throughput genomescale screen to identify factors involved in cytoophidia biogenesis. Observations of tissue specific CTPS distribution lead to the identification of the well-conserved growth regulator dm/dMyc as an essential factor for CTPS regulation in vivo. These results provide new insights into the coordination of cellular growth and metabolic regulation during normal development and indicate the potential of CTPS/cytoophidia as a future therapeutic target.

Endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded proteins of the secretory pathway is crucial for ER homeostasis and the physiological importance of this mechanism is reflected by more than 60 diseases that have been linked to ERAD to date. The best characterised mammalian ERAD complex is centred on the ubiquitin ligase Hrd1, and for a complete understanding of the dynamics of the ERAD network it is important to thoroughly characterise the interactions within the Hrd1 complex and to decipher the functions of the individual accessory factors. SEL1L is a well characterised interaction partner of Hrd1 and here we identify a highly hydrophobic region in the lumenal part of SEL1L as necessary, but not sufficient, to interact with Hrd1; as a consequence, the topology of SEL1L may need to be re-evaluated. Furthermore, we investigate the roles of the novel Hrd1 interaction partners AUP1 and FAM8A1 in ERAD. We establish here that AUP1 adds to the complexity of the ERAD network by making the cognate E2 of gp78, Ube2g2, accessible to Hrd1 and by regulating the access of polyubiquitinated proteins to the Hrd1 complex. Moreover, we demonstrate that Hrd1 is the regulator of FAM8A1 half-life and that the interaction between these two proteins is necessary for degradation of the lumenal ERAD model substrate TTR_D18G.

Membrane-bound vesicle secretion provides a novel intercellular communication mechanism, whose roles and regulation remain poorly characterised, particularly in vivo. I have identified two classes of lipid-containing, vesicle-like structures secreted into seminal fluid by epithelial cells of the Drosophila male accessory gland (AG). Exosomes, one class of membrane-bound vesicle formed inside late endosomal multivesicular bodies, are specifically secreted by secondary cells (SCs). The unusual cell biology of SCs allowed me to develop a powerful new high resolution in vivo system to characterise the mechanisms underlying intracellular membrane trafficking events underlying exosome biogenesis using real-time live imaging. I characterise how specific ESCRTs (endosomal sorting complexes required for transport) control SC exosome biogenesis, and identify a novel role for BMP signalling in regulating endolysosomal trafficking events necessary for exosome secretion. I also identify roles for epidermal growth factor receptor (EGFR) and phosphatidylinositide 3-kinase (PI3K) signalling in exosome biogenesis. Importantly, SC exosomes are transferred to females during mating. Here, they fuse with sperm, mirroring in vitro interactions between human prostate exosomes and sperm, and interact with the female reproductive tract epithelium. Blocking SC exosome production specifically suppresses post-mating effects on female receptivity to remating, demonstrating that exosomes have an important reproductive signalling function in vivo, directly or indirectly reprogramming female cells. Finally, I show that main cells, the major epithelial AG cell type, shed lipid-containing microvesicle-like structures from their apical surface. Remarkably, these vesicles carry the seminal peptide, sex peptide, into females during mating and also contribute to the anterior mating plug. In summary, my data reveal previously unsuspected roles for exosomes and microvesicles in Drosophila reproduction that may be evolutionarily conserved. Since these vesicles mediate physiological processes previously thought to involve soluble peptides, my work suggests that current models explaining male reprogramming of female behaviours in flies and higher organisms need substantial revision.

Evidence in different systems suggests that local translation is involved in synaptic plasticity in both neuron and muscle, but the mechanism by which this occurs is still poorly understood. The mRNA-binding protein Syncrip is conserved from fly to mammals and is thought to be involved in localized translation in both oocytes and neurons. Previous work has shown that Syncrip associates with mRNAs encoding key synaptic proteins at the Drosophila larval neuromuscular junction. Here I show that Syncrip is necessary for the structure and function of the neuromuscular junction. First, the loss of Syncrip leads to overgrowth of the neuromuscular junction. Second, Syncrip is required for proper expression of the Ca²⁺-sensor Synaptotagmin1 at the presynapse, and loss of Syncrip causes a decrease in vesicle release probability. Third, while it was not possible to measure mRNA distribution in neurons, Syncrip mutants, like other perturbations in synaptic plasticity, correlate with changes in mRNA localization in muscle. Fourth, the overexpression and loss of Syncrip function suggest that the nuclear and nucleolar trafficking of the eukaryotic translation initiation factor eIF4E may be important to regulating synaptic morphology. These data suggest that Syncrip is involved in mRNA localization and translation in synaptic plasticity.

The period following World War I can be characterized in literature by the trauma and changes that promoted crises of masculinity. These crises, however, are not discussed between the men that suffer similar feelings of insecurity and anxiety; not approached as a tension in need of resolution. Exploring the narrative voices of Nick, Jake, Darl and Anse in The Great Gatsby, The Sun Also Rises, and As I Lay Dying, this thesis addresses the ways in which this unspoken phenomenon is essential to the modernist male narrative. I propose that, despite the widespread nature of this phenomenon, it is the voice of the individual – the preoccupations of his consciousness – that is the most appropriate point through which to examine these crises of masculinity.

The function of the endometrium is to mediate implantation of the embryo. During the early stages of implantation, the endometrial stroma undergoes differentiation known as decidualization, a process critical for successful embryo implantation. The precise mechanisms involved are not clearly understood but extracellular matrix (ECM) remodelling is a key feature. Fibronectins (FNs) are large glycoproteins abundant in the ECM of the human endometrium. Up to twenty isoforms of FNs are generated from alternative splicing, including the EDIIIA+ and EDIIIB+ variants. This thesis investigated changes in endometrial stromal ECM levels, in particular FN and its splice variants, during decidualization and in response to the endometrial cytokines and growth factors that drive the implantation process. Furthermore, the influence of these splice variants on the functional properties of FN was explored, including cell attachment, spreading and proliferation, integrin binding and focal adhesion kinase activation. Structural studies including crystallization trials were carried out to investigate how the insertion of EDIIIA modulates the conformation of FN and accessibility of its integrin binding sites. These combined studies allow us to test the hypothesis that the regulation of alternative splicing provides a biological mechanism for modulating function of FN in the endometrium. The main findings from this study can be summarized as follows: Immunocytochemistry and immunoblotting demonstrated reduced endometrial stromal levels of EDIIIA+FN, total FN and tenascin during in vitro decidualization. Substrate-associated FN production by endometrial stromal cells was reduced in response to the endometrial cytokine TNFalpha as detected by ELISA. Recombinant FIII7-12A±B± fragments were expressed, purified and mediated endometrial stromal cell adhesion. Inclusion of EDIIIA in the recombinant FIII7-12 fragment decreased binding affinities to integrin alpha5beta1. These findings suggest that production of FNs in the endometrial stroma is modified during in vitro decidualization and in response to endometrial TNFalpha. This modification in ECM composition is likely to result in modulation of cellular processes, perhaps to allow for cellular differentiation and migration that is required for invasion of the implanting embryo.

Current practice for sample size computations in clinical trials is largely based on frequentist or classical methods. These methods have the drawback of requiring a point estimate of the variance of treatment effect and are based on arbitrary settings of type I and II errors. They also do not directly address the question of achieving the best balance between the costs of the trial and the possible benefits by using a new medical treatment, and fail to consider the important fact that the number of users depends on evidence for improvement compared with the current treatment. A novel Bayesian approach, Behavioral Bayes (or BeBay for short) (Gittins and Pezeshk, 2000a,b, 2002a,b; Pezeshk, 2003), assumes that the number of patients switching to the new treatment depends on the strength of the evidence which is provided by clinical trials, and takes a value between zero and the number of potential patients in the country. The better a new treatment, the more patients switch to it and the more the resulting benefit. The model defines the optimal sample size to be the sample size that maximises the expected net benefit resulting from a clinical trial. Gittins and Pezeshk use a simple form of benefit function for paired comparisons between two medical treatments and assume that the variance of the efficacy is known. The research in this thesis generalises these original conditions by introducing a logistic benefit function to take account of differences in efficacy and safety between two drugs. The model is also extended to the more general cases of unpaired comparisons and unknown variance. The expected net benefit defined by Gittins and Pezeshk is based on the efficacy of the new drug only. It does not consider the incidence of adverse reactions and their effect on patients’ preferences. Here we include the costs of treating adverse reactions and calculate the total benefit in terms of how much the new drug can reduce societal expenditure. We describe how our model may be used for the design of phase III clinical trials, cluster randomised clinical trials and bridging studies. This is done in some detail and using illustrative examples based on published studies. For phase III trials we allow the possibility of unequal treatment group sizes, which often occur in practice. Bridging studies are those carried out to extend the range of applicability of an established drug, for example to new ethnic groups. Throughout the objective of our procedures is to optimise the costbenefit in terms of national health-care. BeBay is the leading methodology for determining sample sizes on this basis. It explicitly takes account of the roles of three decision makers, namely patients and doctors, pharmaceutical companies and the health authority.

‘Gender and Development’ (GAD) is currently seen as the dominant theoretical model within international development for promoting social justice and equality for women. As a consequence, many development organisations are undertaking gender mainstreaming. The most interesting fact about the vast number of analyses about gender mainstreaming is the consistency with which they tell of GAD influenced policies failing to implement GAD approaches in practice. This should raise suspicion rather than simple condemnation. It is time to ask: ‘How are, often very progressive, gender policies and strategies consistently silenced across the range of organisational contexts?’ This thesis focuses upon the contemporary process of gender mainstreaming in development organisations – a term that specifically refers to a ‘process of organisational change’ that aims to explicitly develop the ‘use of GAD approaches within all projects and programmes’ of development institutions in order to achieve ‘a vision of development that creates gender equitable social change’ in society. Moreover, it takes an approach that specifically details the ‘organisational process’ element of change inferred in the term. As such, this thesis uses the literature of organisational culture as a lens to make previously unnoticed and submerged sites of conflict and acts of resistance visible, allowing an understanding to be gained of how gender mainstreaming has so consistently faced a policy-practice impasse. It develops this analysis using an in-depth case study of Oxfam GB and demonstrates that the process of gender mainstreaming in the organisation has resulted in the removal of ‘responsibility for’ implementing GAD approaches among staff in the organisation. It goes on to highlight that the unwillingness of development organisations and practitioners to recognise gender mainstreaming as an explicitly feminist and political process of change directly couched at the level of the organisation and not just at the level of the actual development project (or society more widely) has resulted in the ‘process of organisational change’ becoming rationalised and technical rather than personal and politically charged. In reaching this understanding of gender mainstreaming, the thesis develops an awareness of organisational change processes and highlights that ‘norms’ and ‘values’ in organisations are often confused. This confusion has led to an ineffective process of change in institutions as well as a poor conceptualisation and practice of gender mainstreaming in international development.