Cancer cells have been noted to have an altered metabolic phenotype for over ninety years. In the presence of oxygen, differentiated cells predominately utilise the tricarboxylic acid (TCA) cycle and oxidative phosphorylation to efficiently produce energy and the metabolites necessary for protein and lipid synthesis. However, in hypoxia, this process is altered and cells switch to a higher rate of glycolysis and lactate production to maintain their energy and metabolic needs. In cancer cells, glycolysis is maintained at a high rate, even in the presence of oxygen; a term described as “aerobic glycolysis”. Tumour cells are rapidly dividing and have a much greater need for anabolism compared to normal differentiated cells. Rapid glucose metabolism enables faster ATP production as well as a greater redistribution of carbons to nucleotide, protein, and fatty acid synthesis, thus maximising cell growth. Recently, other metabolic changes, driven by mutations in genes related to the TCA cycle, indicate an alternative role for metabolism in cancer, the “oncometabolite”. This is where a particular metabolite builds up within the cell and contributes to the tumorigenic process. One of these genes is isocitrate dehydrogenase (IDH) IDH is an enzyme that forms part of the tricarboxylic acid (TCA) cycle and converts isocitrate to α-ketoglutarate (α-KG). It exists in three isoforms; IDH1, IDH2 and IDH3 with the former present in the cytoplasm and the latter two in the mitochondria. Point mutations have been identified in the IDH1 and IDH2 genes in glioma which result in a gain of function by converting α-KG to 2-hydroxyglutarate (2HG), an oncometabolite. 2HG acts as a competitive inhibitor of the α-KG dependent dioxygenases, a superfamily of enzymes that are involved in numerous cellular processes such as DNA and histone demethylation. It was hypothesised that the IDH1 mutation would result in other metabolic changes in the cell other than 2HG production, and could potentially identify pathways which could be targeted for therapeutic treatment. In addition, 2HG can act as a potential competitive inhibitor of α-KG dependent dioxygenases, so it was hypothesised that there would be an effect on histone methylation. This may alter gene expression and provide a mechanism for tumourogenesis and potentially identify further therapeutic targets. Metabolic analysis of clinical tumour samples identified changes associated with the IDH1 mutation, which included a reduction in α-KG and an increase in GABA, in addition to the increase in 2HG. This was replicated in several cell models, where 13C labelled metabolomics was also used to identify a possible increase in metabolic flux from glutamate to GABA, as well as from α-KG to 2HG. This may provide a mechanism whereby the cell can bypass the IDH1 mutation as GABA can be metabolised to succinate in the mitochondria by GABA transaminase via the GABA shunt. JMJ histone demethylases are a subset of the α-KG dependent dioxygenases, and are involved in removing methyl groups from histone tails. Changes in histone methylation are associated with changes in gene expression depending on the site and extent of chemical modification. To identify whether the increase in 2HG and fall in α-KG was associated with inhibition of histone demethylases a histone methylation screen was used. The IDH1 mutation was associated with an increase in methylation of H3K4, which is associated with gene activation. ChiP and RNA sequencing identified an increase in H3K4me3 at the transcription start site of the GABRB3 subunit, resulting in an increase in gene expression. The GABRB3 subunit forms part of the GABA-A receptor, a chloride channel, which on activation can reduce cell proliferation. The IDH1 mutation was associated with an increase in GABA and GABRB3 subunit of the GABA-A receptor. This raises the possibility of GABA transaminase as a potential therapeutic target. Inhibition of this enzyme could reduce GABA metabolism, potentially reducing any beneficial effect of the GABA shunt in IDH1 mutant tumours, and increasing activation of the GABA-A receptor by increasing the concentration of GABA in the brain. This in turn may reduce cell proliferation, and could be achieved by using Vigabatrin, a GABA transaminase inhibitor licensed for use in epilepsy.

No description available.

‘Cutting out’ HIV-1 proviral DNA could potentially cure a person of the infection. Genome editing approaches have been proffered for eradicating the provirus in infected persons by activating all latent viral reservoirs for further antiretroviral therapy or for the excision of the proviral DNA from memory T- cells. Previous approaches to do this have used nuclease-based tools or reprogrammed tyrosine recombinases; the former presenting unpredictable therapeutic outcomes and the latter, lengthy design time for newer tool variants if viral mutability erodes their effectiveness. Unlike nuclease-based tools that only cut DNA and rely on host-mediated repair mechanisms, chimaeric recombinases (CRs) cut DNA and carry the inherent ability to re-ligate cut ends at the cleavage site. The modular domain architecture of small serine recombinases can be redesigned to mediate site-specific recombination on non-cognate sites, by replacing the C-terminal DNA binding domains (DBDs) of serine recombinases with programmable DBDs such as Zinc Finger (ZF) proteins, TAL effector proteins and CRISPR-dCas9. For HIV-1 proviral DNA excision, CR requirement for the interaction of two recombinase-bound sites, and the lack of necessity for host cell-encoded factors should maximize the fidelity and efficiency of provirus removal. In this work, the engineering and characterization of CRs with the specificity to recognize and promote site-specific recombination at highly conserved regions within the HIV-1 proviral DNA is explored. This research provides a solid proof-of-concept for the use of CRs to target divergent novel target sequences, expanding their applicability for applied genome editing and wider biotechnological applications.

Mitophagy allows for the removal of damaged and dysfunctional mitochondria from the cell thereby attenuating any deleterious, potentially tumorigenic effects malfunctioning mitochondria may cause. Mitophagy is a specific from of macro-autophagy whereby mitochondria are selectively degraded. What controls this specificity is an area of active research. The translocation of various proteins such as PINK1 and PARKIN, to the mitochondria prior to mitophagy is thought to act as signals for recruitment of the autophagosome to the mitochondria. However what is the initiating signal for mitophagy that causes these proteins to act remains unclear. Damaged and dysfunctional mitochondria generate increased levels of reactive oxygen species and we hypothesized that these cause the oxidation of the mitochondrial membrane poly-unsaturated lipid, cardiolipin (CL), which acts as an indicator of mitochondrial health and as an initiating signal to the mitophagic machinery. Using human fibroblasts (derived from Barth’s syndrome patients) deficient in functional tafazzin (Taz), the enzyme responsible for CL maturation (poly-unsaturation), and control fibroblasts created by re-introducing a fully functional Taz gene into the parental Barth’s syndrome cells. The frequency at which mitophagy occurs in these deficient and revertant cell lines was analysed under different oxidative stress conditions, in conjunction with other factors known to affect the occurrence of mitophagy; such as mitochondrial morphology, dynamics, mass, membrane potential and function. We observed that not only were mitochondrial morphology, dynamics and function affected by the levels of polyunsaturated CL, but that indeed mitophagy is abrogated in cells lacking expression of functional TAZ and therefore lacking mature polyunsaturated CL. Further to this initial experiments have confirmed reduced levels of oxidized CL in the Barth’s syndrome cells, which combined with the evidence of reduced mitophagy suggests this could indeed be the initiating signal for mitophagy. Thus the data presented within this thesis provides evidence of the role of polyunsaturated CL, in mitophagy and suggests that through its oxidation it provides the initiating signal for mitophagy.

This project examined whether it was possible to create functional hybrid serine integrases – proteins responsible for recombining DNA in a site-specific manner. Creating hybrid recognition sites, specifically engineered to be recognised by the new integrases, was examined concurrently. Ultimately, new serine integrases and recognition sites were created with the intention of increasing the repertoire of serine integrases available for use as independently functioning modules in synthetic biology assemblies. Experiments were carried out primarily on two groups of hybrid integrases – BxbI integrase and ΦC31 integrase, and the smaller recombinase Tn3 resolvase and ΦC31 integrase. It was determined that either the BxbI integrase/ΦC31 integrase hybrids were not active on hybrid or parental recognition sites, or that the proteins themselves were not expressed at a high enough level to exhibit any activity. However, one ΦC31 integrase/BxbI integrase hybrid did exhibit activity on ΦC31 integrase recognition sites in vivo, though not on hybrid sites. However, Tn3 resolvase/ΦC31 integrase hybrid proteins proved far more promising. The two hybrids exhibited recombination on sites created for them, whilst exhibiting no activity on any parental recognition sites. When both Tn3 resolvase and either hybrid integrase were present in vitro, recombination on combination substrate plasmids containing one copy of the Tn3 resolvase recognition site res site I and one copy of a hybrid recognition site was much higher than for either hybrid against hybrid sites on its own. Additionally, throughout this investigation, it was discovered that ΦC31 integrase cleaved and recombined several sites very dissimilar to its natural attP and attB sites.

VEZF1 is a DNA-binding transcription factor that is highly conserved in vertebrates. Human VEZF1 has recently been found to interact with transcriptionally active gene promoters and erythroid specific enhancers. It is unclear how the broadly expressed VEZF1 interacts with enhancers in a cell type-specific manner or what functions it plays at these elements. This work begins to address the cell-type specific roles of VEZF1 by creating loss of function models in a human erythroid cell line.

The harvesting of light is an important, primary event in photosynthesis, allowing for the transfer of energy to the reaction centre. Purple photosynthetic bacteria use two types of antenna complexes (LH1, LH2) to enhance the efficiency of light harvesting and funnelling energy into the photosynthetic reaction centre. Marichromatium (Mcr). purpuratum is an example of a species that can synthesise antenna complexes that have an unusual absorption spectrum. This LH2 complex has a single strong absorption band at approximately 830nm, with a shoulder at 800nm, also known as the B800-B830 LH2 complex This thesis investigates Mcr. purpuratum and the structural basis for the unusual spectrum of the LH2 complex. The LH2 complex was isolated, purified and, after optimising the purification for stability, crystallised. A low-resolution crystal structure of the LH2 complex is presented, which suggests that this LH2 complex is an octamer, similar to Phaeospirillum (Phs.) molischianum. During this thesis the genome sequence of Mcr. purpuratum became available and I was able to show it contained three pucBA operons, which expressed three β and three α polypeptides, respectively. These were all identified in the purified LH2 complex, confirming that it is heterogeneous. Mcr. purpuratum is not able to produce an LH2 complex in the absence of carotenoid biosynthesis. This was shown by chemical inhibition of phytoene desaturase with DPA, however this checmical is also toxic to the cell. In order to overcome this genetic manipulation by knock-out of the CrtI gene was attempted but due to time constraints was not completed.

Throughout this body of work low amplitude high frequency (500 Hz – 5000 Hz) mechanical stimulation and its effect to induce osteogenesis on bone marrow derived MSCs has been investigated. Due to the nanolevel amplitudes of these high frequency vertical vibrations the term nanokicking appeared to be appropriate and was subsequently used throughout this thesis to refer to these high frequency sinusoidal stimulations provided by the bioreactor. In the first instance this work was performed in 2D and biological analyses to determine osteogenesis were carried out at a transcript (mRNA), protein and mineralisation level. Affirmative results for osteogensis were observed from genes and proteins (RUNX2, osteocalin, osteopontin) related to the osteoblast phenotype by qRT PCR, in cell western, and immunostaining. To determine the prescence of inorganic osseous minerals, more specific techniques such as Raman spectroscopy, micro computed tomography and histological stainings (Von Kossa/Alizarin Red) were further employed. The results observed remained in line with previously published material (Gentleman et al., 2009) drawing the conclusion that calcium phosphate (Ca10(PO4)6, through nanokicking,was formed in vitro. The natural progession of this research meant that a novel vibrational bioreactor was conceived and designed, through the use of Lean and Six Sigma principles (Andrew Thomas, 2004; Caldwell, 2006), in order to assess the potential of nanokicking in 3D. Here collagen was employed as a biomimetic scaffold and affirmative results for osteogenesis were observed. The bioreactor was unique in that long term (up to 46 days) sterile culture was achieved, it was easy to use and there was no requirement for osteogenic media, growth factors or complex chemistries (e.g. dexamethasone, rhBMP2) in order to induce osteogenesis. The cost of use and maintence was relatively cheap compared to available commercial bioreactors (Rauh et al., 2011b). It is envisaged that this technology may one day have real world use for ossesous tissue regeneration and care in a GMP and clinical setting, or for the preparation of autologous tissue for medical testing in the burgeoning field of personalised medicine.

Trypanosoma brucei is the protozoan parasite causing African trypanosomiasis, a neurological disease that affects humans and farm-stock in the impoverished sub-Saharan areas where tsetse fly transmission vector is endemic. Although it has great impact on public health and local economies, it has been neglected in drug discovery for almost a century. Current treatments are either toxic or of difficult administration, besides having serious risks of inducing resistance. Protein kinases are the primary set of signaling proteins in eukaryotes, including Trypanosoma brucei. Their druggability has been widely exploited in cancer research, and has been established in the parasite too. A recent kinome-wide RNAi screen with 176 individual cell lines of mammalian infective bloodstream forms of Trypanosoma brucei identified protein kinases required for proliferation in vitro. In order to investigate which protein kinases are also essential virulence factors in vivo, lines were pooled, inoculated into mice and screened for loss of fitness after 48 hours RNAi compared to uninduced controls. The presence of trypanosomes in the bloodstream was assessed using RNAi target sequencing (RITseq) and compared to an in vitro control. This revealed 49 protein kinases with a significant loss of fitness in vivo in two independent experiments, and a strong correlation between in vitro and in vivo loss of fitness for the majority. However, depletion of nine protein kinases affected more pronouncedly the growth in vivo than in vitro. Amongst these protein kinases were several with putative functions related with stress responses mediated through the PI3K/TOR or MAPK signaling cascades including CK2A2, a promiscuous protein kinase whose activity can be stress-induced; two MAP3Ks, involved in cell integrity upon osmotic shock; VPS15, component of the PI3K complex with roles in autophagosome formation and vesicular trafficking; BUD32, transducer of the PI3K/TOR pathway involved in translational regulation; and FAZ20, a parasite-specific pseudo-kinase localizing to the flagellum attachment zone. The other three have been implicated in repair of alkylation-induced cellular damage: SRPK1, a stress response RNA splicing regulator; AUK2, which acts during mitosis; and CAMKL, an AMPK with calcium-binding domains putatively involved in metabolic regulation. Identification of these virulence-associated protein kinases provides new insights in T. brucei-host interaction and reveals novel potential drug targets for protein kinase inhibitors. This RNAi screen revealed that the evolutionary divergent NEK kinase Repressor of Differentiation Kinase 2 (RDK2) has severe loss of fitness both in vitro and in vivo. Depletion of RDK2 had been shown previously to promote differentiation from bloodstream to procyclic-like forms causing the parasite’s death. Further investigation showed RDK2 to be an active protein kinase capable both of phosphorylating a substrate and to autophosphorylate. Protein kinase activity could be ablated by mutation of lysine 70 to methionine. Mutation of both serine residues (195 and 197), identified as sites of phosphorylation by phosphoproteomics, to alanine or glutamic acid, preventing and mimicking phosphorylation respectively, had no effect on protein kinase activity, suggesting they do not have a direct regulatory role on protein kinase activity. Introducing in the RNAi line a recoded RDK2 whose transcript eluded interference, permitted to some extent the rescue of the induced phenotype, while introducing a recoded inactive mutant did not. This may suggest that the lack of kinase activity was responsible for the RNAi phenotype and not depletion of the protein alone. RDK2 RNAi-differentiated cells could be maintained in conditioned procyclic form media for more than a week. However, they were unable to proliferate. Overexpression of RDK2 blocked the differentiation mediated by sequential treatment with 8-pCPT-cAMP and citrate/cis-aconitate (CCA). RNAi experiments in combination with known differentiation cues, suggested that when differentiation is triggered by the CCA signalling pathway, RDK2 inactivation happens downstream of the phosphatase TbPTP1. Differentiation caused by RDK2 inactivation could be traced in flow cytometry by the detection of EP procyclin expression. This was exploited in a cell-based mechanism-directed phenotypic screen for RDK2 inhibitors. A preliminary run with 518 drug-like molecules that had shown protein kinase inhibition, trypanocidal activity and/or activation of the EP procyclin promoter, unveiled 6 compounds triggering EP procyclin expression and parasite death in the low micromolar range. These compounds can be investigated further to assess whether RDK2 is their in vivo target.