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Vaccinia virus (VV) is a large DNA virus belonging to the Orthopoxvirus family. The viral replicative life cycle takes place solely within the cytoplasm of a mammalian host cell. The VV genome contains 196 open reading frames which are expressed in a highly regulated and temporal fashion in order to bring about the production of a mature virion. In the process of viral replication many VV proteins are synthesized that require posttranslational modifications to become functional. A few of these modifications include, glycosylation, ADP-ribosylation, phosphorylation, fatty acid acylation, and proteolytic processing. This last modification is especially important with regard to the structural proteins of the virus in that they undergo prysis for an infectious virus particle to be formed, a common theme in viral systems. In order to understand these events in more detail, three abundant virion protein constituents 4a, 4b, and 25K were chosen as models for study. The three main questions we wanted to answer were: Is there a cleavage consensus site within the precursors, what protease(s) and/or factors are necessary for the process, and how are the events regulated in vivo? Our approach included development of specific immunological reagents to identify cleavage products as well as to show where these core proteins are located during virion assembly. We have subsequently identified cleavage products by N-terminal microsequence from each of the three structural proteins and this information has elucidated a putative cleavage consensus site of Ala-Gly- X, where cleavage is proposed to take place between the Gly and X and X is usually an aliphatic residue. The immunological reagents were used in conjunction with immunofluorescent and immunogold labeling analyses to identify the location of these core proteins during virion assembly. Core proteins were localized to the virosomes in VV infected cells, to the viroplasm of immature virus particles, and to the center of mature virions. Precursor specific antiserum indicated that the larger molecular weight precursors of core proteins are within immature virions as well. From these results the following conclusions can be made. Identification of a putative cleavage consensus site suggests that proteolytic processing is an endoproteolytic event. The observation that precursor structural proteins were found within immature particles indicates that the proteinase responsible for cleavage is also present. The fact that assembly has to occur before proteolytic processing of VV structural proteins suggests that the cleavage events are dependent upon a specific core protein conformation. However the nature of this conformational requirement is not known. Further research is underway to develop a full understanding of the proteolytic events during virion morphogensis. / Graduation date: 1993

This work presents an investigation of the potential development of phenoxodiol, a novel isoflavone, for the treatment of prostate cancer. The literature concerning isoflavone epidemiology, pharmacology, clinical use and their effect on prostate cancer is reviewed. Isoflavone impact on signal transduction pathways is also reviewed with a particular focus on the Akt / mTOR signal transduction pathway, a key signaling pathway in prostate cancer cells. In vitro experiments and xenograft nude mice studies show that phenoxodiol inhibits the growth of DU145 and PC3 prostate cancer cells, when used alone and in combination with cisplatin or carboplatin. Phenoxodiol in combination with cisplatin is highly synergistic when inhibiting the growth of DU145 cells in particular. We show for the first time, that phenoxodiol and cisplatin or carboplatin, inhibits phosphorylation of Akt and mTOR in DU145 and PC3 cells. A first-in-human study of single dose phenoxodiol investigating its pharmacokinetic properties in cancer patient volunteers was performed. Extensive conjugation of phenoxodiol and a short half-life was noted in this study. A Phase I study of intravenous phenoxodiol administered weekly to patients with advanced cancer was then performed to assess toxicity of a repeated dose schedule, as well as the maximum tolerated dose. This schedule was well tolerated in patients, with lymphocytopenia noted as the main toxicity. Given the short half-life of phenoxodiol and our preclinical studies suggesting that combination with cisplatin and carboplatin synergistically inhibited prostate cancer cell growth, we also undertook a Phase I dose escalation study of oral phenoxodiol administered in combination with cisplatin or carboplatin to patients with advanced cancer. The main adverse events include hyperglycemia, hypocalcemia, mild transaminase rises, as well as nausea, constipation, infusion site reactions and lethargy. Three patients with ovarian cancer responded to treatment according to CA125 criteria, and there was a 45% reduction in prostate specific antigen level in one man with androgen independent prostate cancer. The bioavailability of the oral formulation of phenoxodiol was calculated to be 17.5%. Phenoxodiol shows promise for further development in the potential treatment of prostate cancer.

This work presents an investigation of the potential development of phenoxodiol, a novel isoflavone, for the treatment of prostate cancer. The literature concerning isoflavone epidemiology, pharmacology, clinical use and their effect on prostate cancer is reviewed. Isoflavone impact on signal transduction pathways is also reviewed with a particular focus on the Akt / mTOR signal transduction pathway, a key signaling pathway in prostate cancer cells. In vitro experiments and xenograft nude mice studies show that phenoxodiol inhibits the growth of DU145 and PC3 prostate cancer cells, when used alone and in combination with cisplatin or carboplatin. Phenoxodiol in combination with cisplatin is highly synergistic when inhibiting the growth of DU145 cells in particular. We show for the first time, that phenoxodiol and cisplatin or carboplatin, inhibits phosphorylation of Akt and mTOR in DU145 and PC3 cells. A first-in-human study of single dose phenoxodiol investigating its pharmacokinetic properties in cancer patient volunteers was performed. Extensive conjugation of phenoxodiol and a short half-life was noted in this study. A Phase I study of intravenous phenoxodiol administered weekly to patients with advanced cancer was then performed to assess toxicity of a repeated dose schedule, as well as the maximum tolerated dose. This schedule was well tolerated in patients, with lymphocytopenia noted as the main toxicity. Given the short half-life of phenoxodiol and our preclinical studies suggesting that combination with cisplatin and carboplatin synergistically inhibited prostate cancer cell growth, we also undertook a Phase I dose escalation study of oral phenoxodiol administered in combination with cisplatin or carboplatin to patients with advanced cancer. The main adverse events include hyperglycemia, hypocalcemia, mild transaminase rises, as well as nausea, constipation, infusion site reactions and lethargy. Three patients with ovarian cancer responded to treatment according to CA125 criteria, and there was a 45% reduction in prostate specific antigen level in one man with androgen independent prostate cancer. The bioavailability of the oral formulation of phenoxodiol was calculated to be 17.5%. Phenoxodiol shows promise for further development in the potential treatment of prostate cancer.

The subcellular localisation of mRNA is a widespread mechanism to determine the fate of mRNAs in eukaryotes. Translationally repressed mRNAs localise to P-bodies and stress granules where their decay and storage, respectively, are directed. In a study from the Ashe lab, specific mRNAs were identified to localise, in actively growing S. cerevisiae, to cytoplasmic granules that do not seem to be related to P-bodies or stress granules but appear to be associated with active translation (Lui et al., 2014).It is possible that this might represent a strategy to co-regulate the expression of proteins from the same pathway. In the work of this thesis, microscopy techniques to visualise RNAs in live cells were used to extend the localisation analysis to several mRNAs encoding translation factors. The investigated transcripts were all found to localise to mostly one or two cytoplasmic granules per cell and would sometimes overlap with other transcripts, suggesting that each granule contains a mixture of mRNAs. Granules tend to migrate to the bud tip and may provide the daughter cell with a "start-up kit" of transcripts essential for rapid growth. A similar pattern can be observed in yeast cells growing undergoing filamentous growth, with granules harbouring translation factor transcripts often found in the apical quarter of the elongated cell. Although the mechanism by which the granules form and their protein composition are not yet known, high-throughput genetic screens performed as part of this work offer some insight into factors that might be involved in granule assembly and proteins that partially overlap with the granules. We propose that granules containing translation factor mRNAs might be functioning as a specialised factory for the translation machinery and are possibly being directed to the point in the cell where the rhythm of protein production is highest.

Collaboration between academia and industry has been the focus of numerous government reports and initiatives over the past 15 years, and is increasingly recognized as an effective way to capitalize on the UK's world-class research base. However, there is a need to further understand the role of such collaborations in the field of translational medicine, where the path to market is particularly lengthy, expensive, and risky, due to complexities associated with the clinical trial process. This research uses a mixed methods approach to investigate collaboration in translational medicine at the University of Oxford. The project comprises three principal stages. First, a broad understanding of the current landscape of academia-industry collaboration in translational medicine was obtained by administering a questionnaire to academics who had received industry funding. Next, a deeper understanding of the barriers to collaboration was sought through semi-structured interviews with 27 academics. Finally, potential strategies to reduce practical barriers to the collaboration process were investigated through interviews with members of three groups within the university who interact directly with industry: Research Services, Oxford University Innovation, and Business Development. This research constitutes the first empirical study on university-industry collaboration in translational research in the United Kingdom. It contributes to existing theory through the development of a new theoretical framework for the evaluation of barriers in terms of a) the practicalities of the collaboration process, b) the institutional environment and c) presiding cultures. Through these analyses, differences in experiences of barriers to collaboration emerged for clinical and non-clinical researchers. Furthermore, industry was seen as playing a crucial role in the translation of new therapeutics, especially in the funding of research that was perceived as being ‘too risky’ for Research Councils. Thus, reducing barriers to university-industry collaboration was seen as important to the realisation of public benefit from university research. Barriers were seen as being overcome, or avoided, via the formation of relationships between academics and companies at several different levels; while systems exist within the university to facilitate this, awareness and uptake of these systems was poor amongst the study population. Finally, if universities are to deliver impact as a key metric of performance, incentives within the university need to reward academics for commercialisation activities, in addition to publication. Through the suggestion of long and short-term strategies and a detailed analysis of industrial collaboration in this setting, this research has implications for both university and government policy.

Abstract Lysyl hydroxylase (EC1.14.11.4, LH) catalyzes post-translationally the hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. Hydroxylysyl residues may also be glycosylated by hydroxylysyl galactosyltransferase (EC 2.4.1.50, GT) or galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66, GGT) to form galactosylhydroxylysyl or glucosylgalactosylhydroxylysyl residues, structures unique to collagen. Three LH isoenzymes (LH1, LH2a/2b, LH3) have been characterized so far. We analyzed mRNA levels of these isoforms, as well as the mRNAs of the main collagen types (I, III, IV, V) and the α subunit of PH-4 in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. The mRNA levels of LH1, LH2, and the α subunit of PH-4 showed significant correlation with each other whereas LH3 correlated with none. No correlation was observed between the LH isoforms and individual collagen types. Three human LH isoforms were expressed in different expression systems. The purified recombinant protein produced by LH3 cDNA was found to be the only one possessing LH, GT and GGT activities. The molecular weight of the partially purified LH3 expressed in Sf9 or Cos-7 cells corresponded to about 85 kDa whereas that in E.coli cells was about 81 kDa probably due to a deficiency of glycosylation in bacterial cells. The recombinant protein of C. elegans LH cDNA was expressed in a cell-free translation system and in E.coli cells. The data indicated that the glycosyltransferase activities, GT and GGT, were also associated with this gene product. The sequence alignment of LH isoforms from different species revealed that there are 29 amino acids conserved between human LH3, mouse LH3 and C. elegans LH sequences and scattered evenly in the molecule, but differing from those of LH1 and LH2. In vitro mutagenesis data showed that the amino acids important for the glycosyltransferase activities were located at the amino-terminal part of the molecule, being separate from the LH active site. Mutation of a conserved LH3 specific, non-disulfide linked cysteine to isoleucine caused a dramatic reduction in GT and GGT activity but had no effect on LH activity. Mutations of the amino-terminal DxD motif (D187-191) characteristic of many glycosyltransferases eliminated both GT and GGT activities, showing the importance of this motif for collagen glycosyltransferases and suggesting that it might serve as the Mn2+ binding site in the molecule.

Lysine acetylation is a conserved post-translational modification (PTM) which was traditionally believed to be limited to histones and the regulation of gene expression. However, recent proteomic studies have identified lysine acetylation on proteins implicated in virtually all cellular processes indicating that this PTM plays a global regulatory role. Indeed, in humans, aberrance of lysine acetyltransferase (KAT) activity is associated with various pathogenesis. To date, over 2500 human proteins are known to be acetylated in vivo, but very few acetylations have been linked to specific KATs. Hence, to understand the biological relevance of KATs and acetylation in human pathology, it is important to learn about the mechanism regulating KAT activity and the identity of their in vivo targets. This is a complex task and will require the use of model organisms and system biology approaches. The work presented here explores the significance of self-acetylation in regulating KAT function by focusing on the highly NuA4 lysine acetyltransferase in the model organism Saccharomyces cerevisiae or budding yeast. Using genetics and biochemical assays I have identified NuA4 subunit Epl1 as a novel in vivo NuA4 substrate. I have also shown that Epl1 acetylation regulates NuA4 function at elevated temperatures. In an attempt to identify new biological processes regulated by yeast KATs and putative novel substrates, I have also performed a genome-wide synthetic dosage lethality screen with six non-essential yeast KATs; Hat1, Rtt109, Hpa2, Sas3, Sas2, and Elp3. My screen identified largely distinct sets of genetic interactions for each KAT suggesting that each KAT has specific cellular functions. Together, this study demonstrates the importance of auto-acetylation in regulating KAT function and the diversity of cellular processes impacted by KAT activity in vivo.

Amyotrophic lateral sclerosis (ALS) is a devastating adult onset neurodegenerative disorder characterized by the selective degeneration of upper and lower motor neurons. Patients typically die from respiratory failures within 2-5 years after diagnosis. One of the milestones in ALS research is the discovery Fused in Sarcoma (FUS), an ALS causative gene. FUS is an RNA/DNA-binding protein and predominantly resides in the nucleus. Majority of the FUS mutations are located in the C-terminus and causing aberrant misdistribution to the cytoplasm. Currently, only a few binding partners of FUS are known, which makes it difficult to speculate on the function and interaction of the protein. In this study, we conducted a kinome-wide RNAi screen to identify kinases that affect the localization of FUS. A dual specificity protein kinase named CDC2-like kinase (CLK1) from the screen was found to be responsible for in post-translational modification of FUS and affects the localization of FUS in the nucleus. The identification of CLK1 as FUSmodifying kinase is consistent with roles ascribed to both in the binding and regulation of RNA.

Dissipative spots are found in physical experiments of many branches of natural science. In this thesis we use three-component reaction-diffusion systems on two-dimensional domains in order to generate these patterns. Using a dynamical system approach we proceed with a Fourier analysis on a linearized reaction-diffusion system in order to provide the bifurcation conditions for a given homogeneous state. We validate our results and establish it's limitations through numerical experiments. We report very interesting behavior during these simulations, notably hysteresis and multi-stability. We will then turn our attention to the relatively unexplored phenomenon of rotating spots. Based on previous work done for spiral waves, we investigate the effect of translational symmetry-breaking on a rotating spot mainly through careful numerical analysis.