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Biotechnology enables the design and assembly of customized biological systems to deliver solutions to challenges in medicine, agriculture, sustainable energy production and industrial processes. One current objective within biotechnology, as well as synthetic biology, is to create tools that can be used to manipulate biological systems in a reliable and predictable manner. While many transcription-based and translation-based control devices have been reported, they are based on a limited repertoire of biological component types, and there is a need for new systems that can be used to implement more elaborate devices. This work explored the potential of reprogramming translation for engineering new gene-regulatory tools. Chapter 1 reviews the standard process of protein synthesis and the numerous translation reprogramming mechanisms that exist in nature, focusing on the role of the RNA message and the nascent translated peptide in these processes. Previous efforts to reprogram translation are also covered, including an examination of potential opportunities for reprogramming translation that have not yet been explored. In Chapter 2, the materials and methods used throughout this work are presented. In Chapter 3, a reporter system is developed for examining elements of bacteriophage T4 DNA topoisomerase gene 60 mRNA that reprogram protein synthesis by translational bypassing. It is demonstrated that the reporter system is a reliable tool for identifying and verifying previously characterized gene 60 translational bypassing elements and proposes the use of the sequences for reprogramming translation through RNA. In Chapter 4, an experimental framework is developed for engineering de novo translational bypassing devices (based on T4 gene 60 bypassing elements) and their application to regulate protein synthesis in bacteria. Evidence is presented that demonstrates that these engineered devices can regulate gene expression and control the relative stoichiometry of two major distinct protein outputs from one gene. The utility of these devices in expanding our synthetic capabilities to manipulate biological systems is explored, as well as revealing unforeseen details of the bypassing mechanism in T4 gene 60. A recent focus of synthetic biology has been the control of protein function in cells. The achievement of this goal has focused on split protein complementation approaches, in particular when the N- and C- terminal fragments are fused to inducible protein-protein interaction systems, that enable spatiotemporal control of gene expression. In Chapter 5, a previously predicted protein-protein interaction system between two T4 DNA topoisomerase subunit fragments is demonstrated and its application for generating functional split proteins is explored. Lastly, the concluding remarks on this work are presented in Chapter 6.

RNA polymerase II (Pol II) catalyses the transcription of many RNA classes including protein-coding, small nuclear RNA (snRNAs) and some non-coding RNA classes within eukaryotes. Its journey is ended by the cessation of transcription and the dissolution of the DNA-bound complex in a process known as transcriptional termination. Given the diverse array of transcript classes, several termination mechanisms can induce Pol II termination. One of the most studied is polyadenylation signal (PAS)-dependent termination and occurs at the ends of most protein-coding as well as some other transcript classes. Two long-standing models have been used to explain the role of the PAS in termination and their relevance has been a topic of much debate. The allosteric model suggests Pol II undergoes conformational changes after the PAS to instigate termination, while the torpedo model suggests degradation of the downstream RNA product of PAS-cleavage is important to instigate termination. Here, rapid depletion cell lines are employed to describe the widespread dependence of protein-coding transcript termination on the XRN2 torpedo and the CPSF73 PAS-cleaving endonuclease. CPSF73 depletion leads to profound "run-away" transcription, whereas XRN2 depletion results in a more limited read-through. XRN2 targets Pol II complexes that have undergone slowing or pausing in a protein phosphatase 1 (PP1)-mediated process occurring downstream of the PAS. Additionally, XRN2 can degrade RNA and cause torpedo termination from some other PAS-independent cleavage events. However, this is not a universal process with XRN2 dispensable at snRNA and histone transcripts. Together these results suggest a unified allosteric/torpedo mechanism at protein-coding transcripts, where PAS cleavage precedes a PP1-dependent slowing of Pol II. This facilitates the degradation of downstream RNA by XRN2 and thereby instigates transcriptional termination.

Delivering undamaged genetic material to the next generation is the primary aim of every living organism. Given the continual threat of DNA damage caused by endogenous and exogenous factors, a number of systems that continually detect, alert, and repair DNA damages, have evolved naturally in the whole spectrum of living organisms. The protozoan parasites are excellent examples of eukaryotes that show extreme adaptation to the environment they are exposed to. However, there is much to uncover about the DNA repair in these human parasites. This work presents here the overall picture emerging from my studies of DNA repair mechanisms in eukaryotic organisms with an emphasis on the proteins found in human-pathogenic parasites with a particular interest in G. intestinalis, T. vaginalis, T. brucei, and Leishmania species. In the first chapter of results, I demonstrate that a purified recombinant family 1-related uracil-DNA glycosylase from G. intestinalis (GiUDG) that lacks various residues that were thought to be essential for the activity of the previously characterised Family 1 UDGs is indeed an active uracil-DNA glycosylase. GiUDG remained active in the presence of Ugi. The site-directed mutagenesis studies revealed that many of the remaining amino acid residues that are conserved between GiUDG and other Family 1 enzymes could be mutated without affecting the enzyme's activity. In the second chapter of results, I demonstrate that T. vaginalis possesses two active 3-alkyladenine DNA glycosylases: AAG1 that showed a robust activity against deamination lesions and AAG2 that was not active on the deamination lesions; nevertheless, the preferable substrate was identified as one of the exocyclic DNA adducts, 1,N6-ethenoadenine. Neither did TvAAG1 nor TvAAG2 compensate for the lack of OGG1 in T. vaginalis. In this chapter I also present studies of NTH1 from G. intestinalis. I have shown that GiNTH1 uses the same catalytic residues as its previously characterised homologs, the protein with deleted two extra amino acids within the iron-sulphur cluster retained majority of the activity. The wild type protein was found to possess a very weak ability to remove 8-oxoguanine from double-stranded oligonucleotide and even less activity was detected for the GiNTH1_Del. mutant. The preliminary experiments presented in this chapter provide the first evidence that human NTH1 iii containing a [2Fe-2S] iron-sulphur cluster not only is an active protein, but in particular, it is able to remove 8-oxoG from double-stranded oligonucleotide containing an 8-oxoG:C pair. The third chapter of results focuses on a polynucleotide 5´-kinase 3´-phosphatase (PNKP), an important enzyme of single-strand DNA break repair that typically works in response to oxidative damage within trypanosomatid parasites. PNKP is absent in microaerophilic organisms such as G. intestinalis or T. vaginalis, however curiously, PNKP was also found to be unexpectedly absent in certain obligately aerobic Leishmania parasites. I show that PNKP from T. brucei, appears to be the only one DNA repair enzyme with 5´-kinase activity and it contributes to the majority of 3'phosphatase activity. Here, I present that both PNKP -/- and PNKP -/-APE -/- cells are more sensitive to neocarzinostatin and camptothecin than the wild-type cells with only minor difference in cell viability between PNKP -/- and PNKP -/-APE -/- cell lines. In the fourth chapter of results, I demonstrat that apurinic/apyrimidinic endonuclease (APE1) from G. intestinalis possessed predicted AP endonuclease activity and additional phosphatase and non-specific exonuclease activities. Further site-directed mutagenesis studies demonstrated that mutation of aspartic acid at position 213 to alanine abolished the GiAPE1 activity. The phosphatase activity of GiAPE1 might compensate in G. intestinalis, at least to some extent, for the phosphatase activity of the absent PNKP protein.

The overarching aim of this thesis was to investigate how proton-sensitive members of the C. elegans DEG/ENaC family regulate cellular processes and drive behaviour. In the initial screen, I identified five C. elegans DEG/ENaCs subunits that can form proton-sensitive homomeric channels when expressed in vitro in Xenopus oocytes. These could be further clustered into two groups based on their response to neutral and low pH in vitro: One group that is inhibited by increasing concentrations of protons (ACD-5 and DEL-4, as well as the previously identified ACD-1 (Wang et al., 2008)) and the other one that can be activated by increasing concentrations of protons (ASIC-1, ACD-2 and DEL-9). Exploration of cellular expression pattern and amino acid sequence similarity revealed that C. elegans DEG/ENaCs can form clusters that reflect expression in neuronal or non-neuronal tissue but these clusters do not correspond to the electrophysiological properties presented here. Further electrophysiological characterisation of candidate subunits showed that increasing concentrations of protons inhibit or activate the homomeric channels in a dose-dependent manner. The ACD-5 and DEL-4, which are inhibited by low pH, show specific pH ranges in which the channel is open or closed, which might reflect their physiological environment in vivo. A similar observation has been made for the human ENaC currents, they are regulated by a pH range that is comparable to the one in epithelia where ENaCs are expressed (Collier and Snyder, 2009). The electrophysiological characterisation confirmed previous results that the C. elegans DEG/ENaCs are highly diverse in their ion selectivity (Fechner et al., 2020). This again, is likely to reflect their diverse physiological function in vivo. Based on expression pattern and in vitro electrophysiological evidence, I further characterised one candidates of each group in more detail, DEL-9 which belongs to the group that is activated by low pH and ACD-5 which is inhibited by low pH. Interestingly, both can be expressed in neuronal and non-neuronal tissue, suggesting that their proton-sensing properties are important for various physiological processes and are not restricted to neuronal functioning, and mutants of either gene show defects in rhythmic behaviours. This fits well with evidence across species that DEG/ENaCs and ASICs are involved in regulating cellular excitability and communication to modulate behaviour (Du et al., 2014, Wemmie et al., 2003, Wemmie et al., 2002, Voglis and Tavernarakis, 2008). Finally, in the last chapter, I identified daf-7/TGFβ-like signalling as the regulatory genetic pathway for neuronal and global upregulation of ACD-5 in dauers and post-dauers. I have further explored the genetic relationship between daf-7/TGFβ and acd-5 and explored potential behaviours relating to expression in ASK sensory neurons. Taking together the evidence presented suggests that at some level ACD-5 regulation depends on food and that ACD-5 is implicated in food-sensing: It is expressed in the chemosensory ASK neuron and in the intestine, it is implicated in food-sensing behaviours, and it is regulated by TGFβ-like signalling which again is a developmental pathway linked to food abundance.

Mutation is a fundamental driver of evolution. It occurs non-randomly throughout the genome, influenced by factors such as chromatin architecture, DNA replication, transcription and repair. In human populations an important, but poorly understood, subset of mutations are short insertions and deletions (indels), the formation of which has traditionally been ascribed to deficiencies in mismatch repair to correct polymerase slippage events. However in S. cerevisiae, the formation of short 2 to 5 base pair deletions has been shown to arise as a consequence of the activity of Topoisomerase 1 (Top1) on DNA-embedded ribonucleotides. In normal cells, such ribonucleotides are the most common aberrant non-canonical nucleotides. They occur stochastically throughout the genome, mainly as a result of polymerase misincorporation. In this thesis I detail the development of a novel, nanopore based methodology to detect ribonucleotides embedded in DNA at single nucleotide resolution. I also describe the design and implementation of a highly sensitive reporter construct in S.cerevisiae to detect the Top1 dependent deletion signature. Transferring this reporter to HeLa cells, I show that the same deletion signature is also present in human cells, and confirm this through the orthogonal analysis of a whole genome sequencing mutation accumulation experiment in RPE1 cells. I identify the same mutation signature in de novo mutations from human populations, showing similarities between this mutational process and a newly described COSMIC (Catalogue of Somatic Mutations in Cancer) indel signature. This work demonstrates the presence of a novel mutational process in human genomes, distinct to that caused by polymerase slippage and deficient mismatch repair activity. This process may be an important cause of short deletions in human cells, and has implications for our understanding of the generation of genetic diversity, the formation of de novo mutations, and the development of cancer.

During meiosis in diploid organisms, Mendel's laws suggest that homologous chromosomes should recombine and be randomly distributed to gametes. This is true for meiosis in most organisms, however, in some cases, chromosome transmission is not random, and can be biased depending on a number of factors including the parent of origin of a chromosome or selfish genes located on the chromosome. Why and how non-Mendelian inheritance evolves is not well understood in many cases, but having a good understanding of the evolution of these systems is important for establishing under what scenarios non-Mendelian inheritance might evolve and how meiosis is manipulated by certain chromosomes. This thesis focuses on two insect clades that exhibit non-Mendelian inheritance: scale insects in the family Eriococcidae and fungus gnats in the superfamily Sciaroidea. Both of these families exhibit a non-Mendelian inheritance system known as paternal genome elimination (PGE), where males only transmit chromosomes inherited from their mother to future generations. In Sciaroidea, I focus on one species with an unusual genetic system: Bradysia coprophila, which in addition to PGE has an unusual reproduction system in several other respects. I examine content and evolution of germline restricted chromosomes, which are present in the germ cells in this species but eliminated from somatic cells early in development. I characterise how many genes are on these chromosomes, and explore using genomic methods how these chromosomes evolved. I also conduct a mating preference experiment on B. coprophila to determine whether the chromosome inheritance system in this species affects male mating preferences, as this is one case among animals where we may expect males to evolve mating preferences for certain types of females. In Eriococcidae, how PGE occurs is labile, and transitions between different types of PGE are not well understood. I conduct of survey of species to establish how male meiosis differs in species across this clade, how the type of PGE affects paternal chromosomes in somatic cells, and whether there is any evidence for transitions to new genetic systems (i.e., loss of PGE) in any species within this clade. Studying these two insect systems can provide information about what happens in lineages when non-Mendelian chromosome inheritance evolves. We can determine how this affects a species behaviour, the mechanisms of chromosome inheritance, and genome evolution.

Accurate repair of DNA double-strand breaks (DSBs) is crucial for both cell survival and genome integrity. In Escherichia coli, these breaks are repaired by the homologous recombination (HR) pathway, normally using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include Holliday junctions (HJs), D-loops and replication forks. Due to the transient nature of these intermediates and the overlapping action of the pathway proteins, it is difficult to determine the mode and extent of action of the proteins in vivo. Towards that purpose, the study of the structure and distribution of branched repair intermediates that can be detected in recombination mutants is vital. For this, I have optimised the stabilisation and isolation of HR intermediates, using a specific DSB system (palindrome/SbcCD). This system allows the introduction of a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, leaving the second sister chromosome intact for repair by HR. This study has shown that in cells where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and replication forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, located on both sides of the engineered DSB. Limited movement of these HJs is observed in the presence of branch migration by RuvAB but in absence of RuvC. Surprisingly, these branched DNA structures can even be detected in the central region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of branch migration by RuvAB and RadA and of the helicase activities of RecG, RecQ and PriA. The detection of DNA fragments, of expected electrophoretic mobility, containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. HJs and replication forks in this central region display a constrained distribution, suggesting that these structures are confined in some way by the DSB site itself. Exonuclease I seems to play a role in converting D-loops into repair forks and RecJ seems to promote strand invasion further away from the recombination initiation sites. Using a strategy based on a synthetic region of chromosomal DNA devoid of sites for 4-base cutting restriction enzymes, I have also optimised a protocol for purifying DSB repair intermediates from a single chromosomal location for analysis by transmission electron microscopy (TEM). This promises to enable the direct visualisation of recombination intermediates and help infer the mechanism of their formation. All in all, my investigation into the structure and distribution of DNA repair intermediates, formed across a DSB region, in the absence of the HJ branch migration and resolution complex RuvABC, sheds light on how far initial events like resection and strand invasion following a site-specific DSB can extend in vivo, and how these initial events can lead to the accumulation of branched structures in a central region of DNA not engaged in these initial events. The impact, or lack of impact, of additional mutations in genes implicated in recombination (e.g. recG, recQ, radA, priA, recJ, xonA) place constraints on models of DSB repair by homologous recombination in E. coli.

Eukaryotes maintain their genomes in the form of linear chromosomes. Because of the inability of the replication machinery to copy linear DNA molecules to the very end, the chromosomes were predicted to become shorter with every round of replication. This phenomenon was called "the end replication problem" by Watson and Olovnikov. Later, it has been discovered that eukaryotes have evolved telomeres, non-coding DNA repeats at the chromosomal ends, and telomerase, a ribonucleoprotein that extends the telomeres to overcome "the end replication problem". Telomerase is known to be downregulated in humans through the development resulting in progressive telomere shortening and replicative senescence with age. This is believed to be one of the major tumour suppressor mechanisms. However, most cancer cells reactivate telomerase to become immortal. Thus, understating telomerase regulation is one of the central questions in the field of cancer biology. Telomerase complex formation involves several component maturation and assembly steps. In Saccharomyces cerevisiae, the steady-state levels of the catalytic subunit Est2 are significantly reduced when its interaction with the telomerase RNA component TLC1 is impaired. I have found that Est2 not bound to TLC1 undergoes degradation in a proteasome-dependent manner. Loss of Tom1, an E3-ubiquitin ligase, leads to an increase in the Est2 levels accompanied by a decrease in the Est2 degradation rate. Consistent with these findings, tom1 mutants have longer telomeres. Furthermore, Tom1 physically interacts with Est2, specifically through recognizing its RNA binding domain. Disruption of TOM1 does inhibit proteolysis of Est2 but does not stop it completely, suggesting the existence of an additional, Tom1-independent degradation pathway of Est2. Interestingly, the Est2 levels are reduced in cells grown at elevated temperatures. This decrease is caused by the temperature-triggered degradation of Est2. This degradation resembles proteolysis through the protein quality control system, however, the exact regulator of this mechanism is yet unknown. I propose that regulation of the telomerase through the proteolysis contributes to telomere length homeostasis indirectly through controlling the levels of Est2. The human Est2 homologue hTERT is also degraded in a proteasome-dependent manner. Thus, the proteolysis-dependent regulation of the telomerase might be evolutionarily conserved. RNA-free hTERT is believed to have extra-telomeric roles and this regulation might balance its telomeric and extra-telomeric functions.

An important challenge in understanding gene behavior is deciphering how the genome is organized in space and how this organization influences its function. Existing experimental and computational methods lack the ability to provide close up views of the structure of biomolecules inside nano-scale chromatin. In this thesis, we develop a multiscale coarse-grained chromatin model, which integrates all-atom representations of proteins, DNA, and nucleosomes; a chemically specific coarse-grained model of kb scale chromatin; and a minimal model of sub-Mb scale chromatin. A key feature of this model is its capacity to link the molecular details of nucleosomes to the collective behavior of mesoscale (up to sub-Mb scale) chromatin. Our chemically-specific model describes DNA at base-pair resolution and proteins at amino-acid level resolution. We have used this model to investigate how sub-nucleosome level physicochemical and structural properties, such as the spontaneous thermal breathing and sliding motion of DNA, affect larger scale chromatin self-assembly. Nucleosome breathing refers to the observation that nucleosomes, rather than being static particles, exhibit spontaneous structural fluctuations where the DNA binds and unbinds dynamically. We find that such plasticity of nucleosomes destabilizes the highly regular zig-zag fiber chromatin folding configurations, and promotes instead an irregular and dynamical organization of nucleosomes termed `liquid-like'. Our model can also be used to investigate the effects of DNA sequence, salt conditions, and binding of additional proteins on the behavior of chromatin. Our minimal model describes nucleosomes with just a few particles, while still explicitly representing the DNA. We have used our minimal model to investigate the phase behavior of systems of multiple interacting chromatin fibers. We find that chromatin undergoes salt-mediated liquid-liquid phase separation, and that nucleosome plasticity plays an important role in increasing the range of stability of the coexistence region. Additionally, the model is able to investigate the size scaling properties of chromatin fibers and the effect of nucleosome repeat length on chromatin compaction and inter-chromatin interactions. Together, our multiscale methodology provides a useful technique to extrapolate atomistic properties of nucleosomes to the modulation of large-scale chromatin organization.

microRNAs (miRNAs) are small, non-protein coding RNAs which post-transcriptionally downregulate gene expression. Circulating miRNAs are promising clinical biomarkers, and circulate either within extracellular vesicles (ECVs) or bound to proteins, both of which protect miRNAs against degradation. Recently, small RNA sequencing has demonstrated the existence of isomiRs, of which vary in length and/or sequence when compared to the parent (canonical) miRNA. microRNA-122 (miR-122) is a liver-specific, circulating biomarker of drug-induced liver injury (DILI), notably that caused by paracetamol (APAP; acetaminophen). It has been previously demonstrated by our group that miR-122 is present in circulating exosomes, a type of ECV. IsomiRs of miR-122 were identified in serum samples obtained from patients with paracetamol-DILI by small RNA sequencing. We observed that certain isomiRs are DILI-specific. Clinically relevant isomiRs of miR-122 underwent reverse transcription quantitative PCR (RT-qPCR) using two commercially available assays for miR-122. We demonstrated that these systems are not specific for canonical miR-122, and additionally that the presence of different isomiRs interferes with the accurate quantification of miR-122. A previously published PCR-based assay termed 3'-Dumbbell-PCR (3'-Db-PCR) was adapted and optimised for the selective detection of a DILI-specific miR-122 isomiR. The 3'-Db-PCR assay was demonstrated to be more selective for the DILI-specific isomiR than standard RT-qPCR, and was applied in the subsequent in vivo study. Using a wild-type mouse model of paracetamol-DILI, we demonstrated that liver miR-122 decreased and, concurrently, circulating miR-122 increased. Over time, miR-122 increased in the renal cortex, renal medulla, and spleen. Fluorescence-activated cell sorting (FACS) of the kidney revealed that levels of miR-122 increased specifically in renal tubular cells. 3'-Db-PCR produced results which correlated to standard RT-qPCR of blood plasma, but these correlations were found to be less significant or completely insignificant in the tissues. Validation of PCR products by Sanger sequencing was inconclusive. Following this study, we utilised a genetically-modified mouse model containing a double-fluorescent Cre reporter allele in order to investigate the release of miR-122 in ECVs from the liver. These mice were administered a liver specific adeno-associated viral (AAV) vector expressing Cre recombinase, with the aim of causing the liver to produce enhanced green fluorescent protein (eGFP) tagged ECVs which could be identified in the blood, kidney, and spleen. Additionally, some of these mice were also administered paracetamol to increase ECV release. After paracetamol administration, miR-122 decreased in the liver and increased in the blood, kidney and spleen, as previously observed. Western blotting and confocal microscopy confirmed the expression of eGFP in the liver, however the expression of eGFP in the blood, kidney and spleen, was not detected. These results therefore confirm that certain isomiRs of miR-122 are DILI-specific, but current 'gold standard' RT-qPCR assays are unable to discriminate between canonical miR-122 and its isomiRs. Therefore, miR-122 cannot be quantified by RT-qPCR due to isomiR interference, which may also translate to other miRNAs under clinical investigation. 3'-Db-PCR is more selective than RT-qPCR assays for miR-122, but further investigations are required to validate its PCR products and therefore what the assay actually detects. miR-122 is released from the liver in paracetamol-DILI into the circulation, and is then taken up by both the spleen and renal tubular cells. The confirmation of ECV-dependent or independent miR-122 release and uptake into these organs in liver-derived ECVs must be investigated further by refining our current model, using a different model, and/or by use of different techniques.

Over the course of 10 years and 14 publications, 7 of which are described in this thesis, I made significant contributions to the field of RNA processing, particularly RNA splicing in budding yeast. These contributions have been both in knowledge accrued and techniques developed and optimised. For the Ribo1 reporter (chapter 2), I developed the RT-qPCR assays, RNA copy number/cell estimation and 3' end cleavage assay. These show that the minimum time taken for signal induction, recruitment, assembly of transcription factors and ultimately transcription, is in the order of 4 minutes, with transcription rates of 60 to 90 seconds per kb. The 3' end cleavage assay reveals that the initial pulse of transcripts may be spliced only partially co-transcriptionally; presumably splicing factors are recruited to the site of transcription during this period. After this initial phase, in the reporter at least, splicing is almost exclusively co-transcriptional. Mutant transcripts on which spliceosomes assemble but are unable to complete splicing, are targeted for very rapid degradation. I have optimised the thio-labelling technique to the point where I can detect thiolated RNAs just 15 seconds after addition of the 4-thiouracil nucleotide analogue to cell cultures (chapter 3). From my data and transcriptome sequencing (chapter 4), I have constructed models of transcription and splicing, which indicate that co-transcriptional splicing is a general feature of most yeast pre-mRNA transcripts and almost all ribosomal protein gene transcripts. Intronic features that act against co-transcriptional splicing include runs of uracils and secondary structure, especially over the branch point adenosine (chapter 4). I have provided a highly detailed protocol and video for thio-labelling of RNA in vivo and its purification (chapter 3). Similarly, I have developed and prepared a comprehensive protocol for auxin-induced protein depletion, to the point where this is the most flexible and least metabolically perturbing technique for doing this in yeast (chapter 6). Applying this method, whilst thio-labelling, I revealed that depleting splicing factors sequesters spliceosome components, rapidly resulting in the cessation of splicing (chapter 7). My microarray results indicate that this is a common consequence of splicing factor deactivation for many or all transcripts. In this thesis I examine RNA metabolism, noting that the processes of transcription, splicing, 3' end formation and degradation are coordinated and harmonised to optimise fidelity, flexibility and efficiency.

Nuclear organisation shapes regulation of genes. However, the principles by which three-dimensional genome architecture influences gene transcription are incompletely understood. Condensin is a key architectural chromatin constituent, best known for its role in mitotic chromosome condensation. Yet, at least a subset of condensin is bound to DNA throughout the cell cycle. Studies in various organisms have reported diverse roles for condensin in transcriptional regulation, but no unifying mechanism has emerged. In this thesis, I use rapid conditional condensin depletion in cell cycle-staged budding yeast S. cerevisiae cells to study condensin's role in transcriptional regulation. I observed a large number of small changes in gene expression, enriched at genes located close to condensin binding sites. Nascent RNA sequencing revealed that transcription is overall slightly elevated following condensin depletion. More strikingly, transcriptional changes in response to environmental stimuli were subdued in the absence of condensin. Most notably, condensin contributes to the global transcriptional downregulation in response to heat shock. My results suggest that condensin facilitates transcriptional reprogramming as part of adaptation to environmental stimuli.

Nucleic acid detection, referred herein as 'DNA detection', is defined as the process of identifying the presence of a specific sequence of bases, and has a vital, far-reaching impact on healthcare applications such as pathogen detection and drug resistance screening. In particular, DNA amplification methods, such as those used in popular COVID-19 tests, present a rapid and affordable means of DNA detection. When performing DNA amplification, there are 3 main goals which are desired: detection, quantification, and multiplexing. In an ideal world, we would like: (i) detection to be rapid and robust; (ii) quantification to be accurate and precise; and (iii) the ability to multiplex several targets in the same chemical reaction, without increasing the complexity of the chemistry or instrument. The aim of this thesis is to develop intelligent algorithms which can be used to improve DNA detection, quantification and multiplexing. In an effort to achieve the aforementioned goals, several detection platforms have been developed which can be broadly classed as: quantitative, digital, and electronic instruments, corresponding to the 3 Parts in this thesis. This work exhibits a common philosophy throughout: increasing the value of data through algorithms that operate in higher dimensions, compared with existing methods which significantly reduce the dimensionality of data. In Part I, the use of multiple features from quantitative instruments are investigated by analysing each signal in multidimensions, compared to the traditional unidimensional perspective, where each signal is reduced to a single value. To this end, a new framework is proposed that formalises the process of quantification and leverages the benefits of multiple features using a novel concept called the multidimensional standard curve (MSC). This new method is shown to guarantee enhanced quantification and provides a natural method of outlier detection, solely through analysing existing data from a new perspective - incurring little to no cost. Subsequently, this new framework is extended to perform multiplexing, demonstrating the first data-driven method for simultaneous quantification and multiplexing of nucleic acids in a single fluorescent channel. In particular, this method showed an accuracy of 100.0% (using 11 clinical isolates) when multiplexing 4 of the most prominent carbapenem-resistant genes, which account for 97.1% of confirmed cases in the UK. In Part II, the use of machine learning methods for multiplexing is explored by considering the vast volume of data from digital instruments. To this end, a supervised machine learning classifier is trained to automatically perform a new form of data-driven multiplexing, referred to as Amplification Curve Analysis (ACA). This demonstrates the first application of machine learning for multiplexing in digital instruments based on amplification curves, leading to breaking the barrier of multiplexing one target per fluorescent channel and final intensity value. Moreover, a mathematical formula based on Multivariate Poisson statistics is derived to theoretically demonstrate the trade-off between multiplexing and quantification, and support evaluating quantification precision in practical settings. Following this work, a novel 3-step machine learning system called Amplification and Melting Curve Analysis (AMCA) is proposed, which combines the kinetic information from amplification curves with thermodynamic information from melting curves - leading to the largest digital multiplex (using cheap intercalating dyes) that has been reported in the literature. In particular, over 100,000 amplification events were analysed, where this method showed an accuracy of 99.3% (for positive events) when multiplexing 9 variants of mobilised colistin resistance, representing a 10.0% increase over existing methods. In Part III, the use of intelligent algorithms for increasing the robustness of electronic devices based on sensor arrays in highly noisy environments is explored. In particular, recent devices based on Ion-Sensitive Field-Effect Transistor (ISFET) sensors present a new form of data which is correlated in both space and time (similar to a video), and can produce a vast volume of rich data. To this end, a new algorithm is proposed based on modelling sensor drift using adaptive signal processing and clustering sensors based on their behaviour using unsupervised machine learning. This robust algorithm is shown to enable a high precision application such as distinguishing bacterial from viral infections using host signature-based classification. In particular, this new method results in robust values from 100.0% of experiments (N=22), compared with 63.6% from an existing method. In summary, as inspired by the field of Signal Processing and Machine Learning, this thesis develops new intelligent algorithms for a wide range of platforms, by analysing data in higher dimensions. Through exploring the data from instruments that represent past, present and future diagnostic systems, this thesis lays the foundation for maximising the value of amplification data, which will ultimately lead to better informed decisions across all healthcare applications.

RNA transcript abundance is largely decided by a careful balance between the transcription and degradation of any given gene's transcripts. RNA stability plays a critical role in the availability of an RNA species, and the time period over which it is able to elicit its functions and roles. The majority of the literature on regulation of RNA activity by degradation focuses on mRNAs, with the assumption that their role is to be translated into a functional protein, as described by the central dogma. Increasingly though, non-coding RNAs have been recognised as crucial to the normal function of biological organisms. The roles of RNA species such as miRNAs in controlling gene expression are now relatively well understood, as are the molecular mechanisms by which they bind to and regulate RNAs. In contrast, long non-coding RNAs (lncRNAs) are a very poorly understood (and arguably, defined) category of RNAs. However, they clearly have their own crucial roles to play, as this relatively recently discovered RNA species regulates gene expression in diverse ways, encodes small biologically relevant peptides, and has been involved in a large variety of important biological functions. Importantly, an increasing number of lncRNAs have also been associated with a range of human diseases, including neurodegenerative pathologies and cancer. The degradation of lncRNAs requires significant study, in order to bring understanding of this key regulatory step of a crucial class of transcripts up to the level of that of the rest of the transcriptome. The aim of this thesis is to investigate the degradation of lncRNAs by the exoribonucleases Pacman and Dis3L2, in Drosophila melanogaster. Within this overarching aim, several smaller goals arise. Firstly, this thesis investigates whether certain lncRNAs are specifically and significantly degraded by Pacman and Dis3L2, as is seen with canonical RNAs. By examining previous RNA sequencing data from experiments carried out on exoribonuclease deficient Drosophila (both in vivo, and in Drosophila derived cell lines,) it was possible to identify promising candidates for lncRNAs with significantly altered abundance in the absence of either Pacman or Dis3L2. This existing work was validated with qPCR, proving the principle of specific regulation of lncRNAs by Pacman and Dis3L2. Following this, an experiment was designed and carried out to examine the role of the translating ribosome in this degradation. Existing work has shown the ribosome to be associated with XRN1 and Pacman in humans and Drosophila respectively, and Dis3L2 has also been shown to associate with the ribosome in humans, although whether this occurs in Drosophila is unclear. By using the powerful technique polyribosome sequencing (poly-ribo-seq), on exoribonuclease deficient Drosophila samples, this work has identified a preliminary set of lncRNAs that appear not only to be specifically regulated by Pacman and Dis3L2, but also undergoing translation, indicating the presence of small open reading frames (smORFs) within the lncRNA genes. Ongoing work will validate not only the upregulation of these transcripts in the absence of the relevant exoribonuclease, but also the putative smORF from which a peptide is likely produced. Following this, it will investigate whether a block in transcription eliminates the differential abundance of these transcripts in the absence of Pacman or Dis3L2. This work then, identifies an initial subset of lncRNAs regulated by Pacman and Dis3L2, and shows several of them to be actively translated, identifying novel peptides, potentially of biological significance (given their active translation and specific degradation). With the completion of ongoing work, this project will also elucidate whether their translation is important to the degradation of these transcripts.

Acute kidney injury (AKI) is a common medical condition characterised by an abrupt decrease in kidney function. AKI can lead to chronic kidney disease (CKD), which has a global prevalence of approximately 10% and is linked to high morbidity and mortality. Despite the high prevalence of kidney injury, the pathophysiological mechanisms that elicit either tissue scarring or function recovery are poorly understood. Due to this lack of understanding, there is a shortage of options for effective treatments and depending on the severity of the disease the only available resources are dialysis and kidney transplantation. Hence, it is of utmost importance that new strategies are developed for the treatment of kidney injury. Macrophage polarisation has a crucial role in the context of kidney injury, as there is evidence that a shift in macrophage phenotype aids in tissue repair and function recovery. The means by which this shift takes place is still not completely elucidated, but mesenchymal stromal cells (MSCs) appear to display immunomodulatory properties that would prime macrophages towards an anti-inflammatory phenotype. MSCs have been also investigated as a means for treatment of renal diseases and recently there is a reckoning that the mechanism of action is not through engraftment and differentiation, but instead through paracrine factors, including extracellular vesicles (EVs). Therefore, the aim of this study was to assess the effects of MSC-derived EVs in activated human monocyte-derived macrophages (hMDMs), so as to verify whether these EVs would elicit a change in macrophage phenotype. For this purpose, MSCs were characterised and an isolation protocol was optimised. hMDMs were co-incubated with EVs and surface marker expression was evaluated through microscopy, cytometry and cytokine secretion. MSCs displayed required characteristics, including expression of CD73, CD90 and CD105. The method used for EV isolation was serum starvation followed by ultracentrifugation of medium supernatant. EVs had an average diameter ranging from 150 to 200 nm and expressed CD9, CD63 and CD81. MSC-derived EVs had an effect on hMDM morphology, but did not change surface marker expression or cytokine secretion. When hMDMs were incubated with human umbilical cord MSCs it was possible to notice changes in expression of CD14, CD80 and CD163, suggesting that soluble factors, including cytokines, are necessary to elicit a shift in phenotype. Angiogenic assays were put in place to confirm EV potency, but EVs did not promote vessel formation. Taken together, even though there was no significant change in surface marker expression, these results contributed to a better understanding of the role of MSC-derived EV in hMDM polarisation. Among the reasons for the lack of effect of MSC-derived EVs could be the low number of samples analysed and the high inter-individual variability regarding hMDMs, indicating the need for further investigations.

Nucleic acids are at the heart of extant biology and the structure of life's original genetic polymer is still a question of debate. The RNA world theory proposes that RNA was the first nucleic acid employed as life's genetic polymer due to its dual ability as informational storage (genotype) and primordial catalyst (phenotype). However, ribonucleotides are complex chemical structures, and simpler or more stable nucleic acids, such as threose nucleic acid (TNA) or DNA, can also carry genetic information. In principle, nucleic acids like TNA could have played a vital role in the origins of life but the advent of any genetic polymer requires synthesis of its monomers. This work demonstrates a high-yielding, stereo-, regio- and furanosyl-selective prebiotic synthesis of threo-cytidine, an essential component of TNA. This work uses key intermediates (aminooxazolines) and reactions previously exploited in the prebiotic synthesis of the canonical pyrimidine ribonucleoside cytidine. It avoids the low yielding glycosylations that have previously been demonstrated for constructing nucleic acids and utilises and efficient photochemical anomerization that is enabled by selective anhydronucleoside thiolysis. This work also demonstrates that erythro-specific 2',3'-cyclic phosphate synthesis provides a mechanism to photochemically select TNA cytidine and suggests that TNA may have coexisted with RNA during the emergence of life. This thesis also investigates whether DNA be delivered simultaneously with RNA, the co-emergence of both would further probe the place and importance of DNA at the origin of life. This work expands on previous work towards DNA and examines the role of irreversible thiol addition to anhydronucleosides as a route towards DNA precursors. Finally, the question of why the nucleobases present in extant biology (A, G, C, U/T) were chosen is addressed. This work explores the paradigm of UV stability as a selection pressure and the results contrast with current thinking that the purine nucleobases were chosen for their ability to resist degradation by UV-light.

Integration of viral DNA into a host chromosome, by action of the viral enzyme integrase (IN), is an essential step of the retroviral lifecycle. To fulfil its function, IN assembles into a multimer on the viral DNA ends, forming a highly stable nucleoprotein complex known as the intasome. The intasome architecture varies between the retroviral genera, and the maedi-visna virus (MVV) intasome contains a homo-hexadecamer (tetramer-of-tetramers) of IN. The conserved intasome core, observed in all structurally characterized retroviral intasomes, is formed between a pair of MVV IN tetramers, each providing one active site, and is completed by the insertions of the synaptic C-terminal domains (CTDs) donated by a pair of flanking IN tetramers. It was argued that this configuration is necessitated by the propensity of lentiviral INs to form tetramers in solution and the α-helical structure of the linkers connecting the catalytic core domain (CCD) and the CTD. Within the MVV IN hexadecamer, a pair of CTD tetrads bridge the IN tetramers by forming intra- and inter-tetramer interactions. Using site directed mutagenesis, the importance of these distinctive structural features was probed. The mutations disrupting the CTD-CTD interfaces or destabilizing the α-helical configuration of the CCD-CTD linkers perturb the ability of MVV IN to form multimers, assemble into stable intasomes and strongly affect its strand transfer activity in vitro. Moreover, these mutations strongly compromised infectivity of single-cycle MVV vector in cells. Lentiviral integration distinctively favours actively transcribing genes, which is facilitated by the interaction of IN with LEDGF/p75, a chromatin-bound adaptor protein. The presence of LEDGF/p75 was essential to observe MVV IN strand transfer activity or intasome assembly in vitro. To determine the importance of LEDGF/p75 for integration in the context of viral integration in cells, infectivity of MVV-derived vector in LEDGF-knockout cells was tested. Although ablation of the host factor in human and sheep cells did not lead to a reproducible reduction of infectivity, it led to a notable shift in integration pattern, away from the usual gene bodies and transcription units. Collectively, these observations indicate that the hexadecameric architecture is critical for the MVV IN function and suggest that LEDGF/p75 is relevant in targeting its integration towards favored regions of host genome.

Exosomes are an emerging sub class of extracellular vesicle which are rapidly gaining momentum as a novel therapeutic platform. Their regenerative and therapeutic potential is reflective of the plethora of cell types from which they can be derived. However, as the technology is in its nascent stage, relatively little exists in terms of defined manufacturing processes. As exosome technologies progress towards pre-clinical and clinical stages, the constraints in manufacturing processes, specifically in downstream processing, will need to be overcome. The current "gold standard" for exosome recovery from conditioned culture medium is ultracentrifugation. This step is time consuming, prone to operator error and difficult to scale and translate. The work presented here shows that monolith chromatography is a scalable, and reproducible purification option which can be used to successfully recover functionally active, and highly pure exosomes. Exosomes derived from the clinically relevant stem cell line, CTX0E03, were shown to present the biomarkers CD 9, CD 63 and CD 81, commonly conserved amongst exosome species throughout the literature. The vesicles were characterised as having a size distribution between 20 to 150 nm, and a flotation density between 1.136 - 1.185 g mL-1, as expected based on literature values. Furthermore, exosomes recovered by tangential flow filtration (TFF), were shown to promote fibroblast migration and wound closure (98% ± 1.5%) in an in vitro potency model, in a dose dependant fashion. In contrast exosomes purified by ultracentrifugation could not achieve wound closure, with no significant difference observed over the 72 hour period. TFF recovered exosomes were purified by the processes developed in this thesis. In the first instance they were purified by use of an anion exchange monolith using the quaternary amine ligand. Exosomes were shown to elute broadly over the elution gradient and overlap with DNA and albumin co-present within the feed material. Samples obtained post purification had purity ratios of 1.5 x 10^9 particles per µg of protein and 9.3 x 10^11 particles per µg of DNA impurity. Based on a hypothetical dose size of 10^9 particles per mL this result indicated purities within the WHO guidelines for injectable therapeutics (100 µg of protein, 10 ng DNA per dose) and benchmarked a potential method for purification of exosomes. A second monolith was also tested, using an orthogonal chemistry: hydrophobic interaction with an OH ligand. The results of this column surpassed those of the AEX process and showed a binding affinity beyond the hypothesized values. Unlike the AEX column, the HIC operation did not co-bind impurities in the form of albumin, DNA or even cell-0derived organelle matter. Resultantly, purities were even higher than those of the AEX column at 3.97 x 10^9 particles per µg of protein, and 3.12 x 10^12 particles per µg of DNA. Finally, combination of the processes showed the potential application of the chromatographic options within a larger process for exosome purification and high performance capillary electrophoresis analysis showed substantial removal of cell culture derived proteins from the recovered material, without substantial loss in particle number. The processes were assessed for potency, both individually and in sequence. No adverse effect in wound closure was noticed with all samples achieving wound closure over 90%. This showed improvement on the current gold-standard method, which could not retain product functionality.

Transmembrane pores are highly specialised nano-devices, with intrinsic specificity and gate-keeping properties that can be exploited in the field of nanobiotechnology. Recently, DNA-origami inspired transmembrane pores with tailorable surface chemistry and programmable dimensions have been rationally designed in an effort to overcome the limitations of protein-based membrane pores such as their fixed lumen size and limited structural repertoire. Ongoing experimental research into the potential applications of triethylene glycol-cholesterol DNA nanopores (DNPs) has been fruitful, with a particular emphasis on drug delivery and biosensing. In this thesis, I describe an ensemble-based coarse-grained MD protocol devised to probe the interactions between bilayer lipids and DNPs, and to determine the effect of membrane encapsulation and salt concentration on the dynamics, structure and conductance of these nanopores. Furthermore, I aim to elucidate the mechanisms by which DNPs mediate translocation of small molecules across lipid bilayers, and the energetics associated with these mechanisms with constant-velocity steered MD and umbrella sampling simulations. I have found that the DNP has no distinct lumen in bulk solution, where it adopts a bloated, amorphous structure with strained and constricted termini regardless of the salt conditions, with significant kinking and fraying of helices. However, salt conditions have a profound effect on the structure of a DNP as it spans a planar lipid bilayer, where it assumes a barrel-like structure with a defined lumen. Sites of constriction in the lumen of the membrane-spanning DNP present a significant barrier to translocation of fluorophores bearing dense negative charges.

The serum response factor (SRF) is a transcription factor involved in the regulation of cell proliferation and migration. At cytoskeletal genes, SRF acts cooperatively with the actin-regulated myocardin-related transcription factors MRTF-A and MRTF-B. In addition to regulating MRTFs' subcellular localization, G-actin also controls their nuclear activities. Previous work showed that nuclear accumulation of MRTF-A in the absence of an activating signal is sufficient for its association with genomic loci but not for target gene activation, demonstrating a repressive effect of nuclear actin on MRTF activity. However, the exact mechanism is unclear. The data presented below demonstrates that G-actin interferes with ternary complex formation between SRF and MRTF on DNA. In response to G-actin depletion, MRTF is recruited to target gene promoters and activates gene expression, whereas increasing the concentration of G-actin inhibits MRTF recruitment to target promoters. We used inhibition of the Crm1 nuclear export receptor and reconstitution of MRTF-A/MRTF-B null cells with a constitutively nuclear MRTF- NLS to induce MRTF nuclear accumulation in the absence of an activating signal. Under resting G-actin levels, nuclear MRTF is recruited to target promoters, albeit at reduced levels, and induces non-productive transcription. TTseq and RNAseq experiments demonstrate that while RNA Polymerase II is engaged in elongation, no pre-mRNA accumulates. This inhibited transcriptional state correlates with aberrant Pol II CTD phosphorylation, Mtr4 recruitment and degradation of the nascent transcripts by the nuclear exosome. Inhibition of phosphorylation at Ser7 of the Pol II CTD is sufficient to induce Mtr4-mediated degradation of MRTF- dependent transcripts under induced conditions.

Understanding the degradation behaviour of extracellular matrix (ECM) scaffolds is essential for predicting and advancing wound healing. Spider-silk based proteins are one type of biomaterial with the potential to be used as a matrix to improve would healing. In addition to good biocompatibility and low-pyrogenicity, silk-based biomaterials have displayed the capacity for controlled degradation, a characteristic that is investigated in this study. In silico studies took target sequences of human matrix metalloproteinase 2 and 9 (MMP2 and MMP9) and compared them to sequences of silk major ampullate spidroin 1 (MaSp1) termini of spider genera: Araneus, Argiope, Cyrtophora, Dolomedes, Euprosthenops and Nephila and the recombinant synthetic mini-spidroin 4RepCT to identify locations for potential mutations to influence the protein's degradation. Proteolytic degradation has been carried out in vitro with dragline silk fibres of a range of species from distantly related spider families namely Cyrtophora citricola, Dolomedes fimbriatus, Pisaura mirabilis, Pholcus phalangioides and Nephila madagascarensis to confirm the predicted degradation seen from in silico studies. Based on the MaSp1 of Euprosthenops australis, 4RepCT was recombinantly expressed and degraded by human neutrophil elastase (ELNE), MMP2 and MMP9. From the MMP profiles of 4RepCT, 14 mutation sites were identified, with a final seven being carried forward to experimentation due to location within the structured spidroin. Of these seven, two mutations located near the thrombin cleavage site and within the structured C-terminus were successfully expressed in DL41 and BL21 E. coli, respectively. Successfully expressed mutant spidroins were subjected to MMP2 (>1000 pmol/min/μg protein) and MMP9 (>1300 pmol/min/μg protein) concentrations that were approximately 10% of that typically seen in chronic wounds. Spidroins with a mutation in the amorphous region of the spidroin gene increased the degradation rate, degrading 1 mg/mL protein in 30 minutes with both proteases, whereas mutations within the structured C-terminus did not degrade in the same way. This suggests that while introducing target sites can influence the rate of degradation, the sites must be accessible to the protease in question.

Model systems are commonly used to investigate biological processes and understand molecular mechanisms. The choice of a model cell or organism is informed by the scope of the question being asked. Decades of research using predominantly animal and fungal models form the bulk of our knowledge of eukaryotic processes. The diversity of eukaryotic organisms, however, indicates that this may not be representative of what exists in more diverged groups, and key processes like cell division vary across the larger group. In the past, non-traditional models including Tetrahymena have been applied to answer biological questions because of peculiar traits that make them suitable for investigation. African trypanosomes offer an excellent opportunity as model organisms because of their divergence from common models and importance to human health and livelihood. Particularly Trypanosoma brucei, with several genetic tools available to study them, has become an interesting model to study processes like cell division, cytoskeleton and antigenic variation. Here, I present work exploring the application of two species of African trypanosomes as models to investigate common biology of eukaryotic kinesins and specific biology of animal African trypanosomiasis. The genetic tractability of T. brucei and its cytoskeletal structure, which is predominantly made up of tubulin, makes it a good model to study functional differentiation between kinesin families. I show cellular localisation and characterise the effect of RNAi depletion of representative kinesin families. The data suggest that Kinesin-2, -3 and 13-1 perform similar roles in T. brucei as seen in other systems, and can therefore be candidates for investigation. On the contrary, loss of both alleles of T. brucei Kinesin-1 does not affect in vitro growth, suggesting possible functional redundancy in trypanosomes. Trypanosoma congolense is the most significant cause of animal African trypanosomiasis, a wasting disease of livestock that results in huge economic losses to agriculture particularly in Africa. Disease presentation differs from T. brucei, and the lack of genetic tools makes it difficult to study the biology of T. congolense, specifically its pathogenesis in animal trypanosomiasis. I show the progressive development and application of genetic tools for endogenous locus tagging, RNA interference and high-complexity mutant library production. I demonstrate that loci on the minichromosome can be targeted for transgene integration, give good expression and are regulatable. Genes targeted by RNAi could only be depleted to about 50% of original levels, with similar proportion of cells expressing mutant phenotype, suggesting that T. congolense has lower RNAi penetrance than T. brucei. The development and optimization of a system that uses a homing endonuclease to increase DNA breaks at integration sites consistently increased the number of positive transformants to ~ 50,000 per transfection. This T. congolense line maintained stable transfection efficiency even after cycles of freeze-thawing. Finally, I demonstrate for the first time, a genome-wide technology in T. congolense. Using all the tools developed, I create two RNAi libraries containing > 5x105 independent transformants. The method of library generation by direct RNAi fragment sequencing (DRiF-Seq) allowed detection of very small changes in gene fitness cost due to RNAi. This enabled fitness screen of > 9000 genes in T. congolense even at a low RNAi penetrance. Analysis of differential biology between T. congolense and T. brucei gives some glimpse into similarities and differences in processes like endocytosis and quorum sensing. I also demonstrate application of the T. congolense RNAi libraries in a drug screen to investigate isometamidium mode of action and resistance.

Maintaining genome stability is essential to support DNA replication for all life to continue. Multiple systems are in place to ensure this occurs by scrutinising replication as it happens, detecting and repairing changes to DNA caused by damaging agents and removing physical blocks. Repair during replication is essential to ensure errors are not replicated, homologous recombination (HR) is one example; repairing DNA errors by using the homologous template from the sister chromosome. While these pathways exist to maintain genome integrity it is essential that they are tightly regulated to prevent unnecessary activation or downstream impacts on the genome. Therefore, a network of proteins are involved in controlling genome maintenance. Metazoan HelQ DNA helicase is a single- stranded DNA ATPase with 3' to 5' translocase activity that unwinds forked DNA structures. It is hypothesised that HelQ activity is crucial in promoting DNA replication and repair through the regulation of HR. However, the mechanism is unknown. While HelQ has shown to co-elute with essential repair proteins including RPA, the Rad51 paralogues and ATR and has been linked to cancer and repair-related diseases, little is known about how HelQ behaves in nature. Here, I was able to generate yields of HelQ and HelQ fragments for biochemical analysis of the recombinant proteins. We show that HelQ forms active dimers that unwind DNA fork substrates but no other intermediate DNA structures. Functional analysis of a catalytically active and non-catalytically active region of HelQ aids in the dissection of HelQ structure and function. I also report that HelQ interacts with the single-stranded DNA binding protein RPA in vitro and the N-terminal ORFan domain is able to displace RPA from DNA through an unknown mechanism. This leads us to hypothesis a model of HelQ activity and translocation on DNA.

Held out wings (HOW) is an RNA-binding protein essential for spermatogenesis in Drosophila melanogaster. HOW is a signal transduction and activation of RNA (STAR) protein family member. Its orthologues include Quaking (mammals) and GLD-1 (Caenorhabditis elegans); both are important post-transcriptional regulators of RNAs in processes including gametogenesis and myelination. Loss of HOW in D. melanogaster testes results in male infertility. Similar to Quaking, multiple protein isoforms of HOW exist, including the longer HOW(L) and the shorter HOW(S). HOW(L) is nuclear and regulates bam mRNA to control mitotic divisions prior to meiosis (Monk et al., 2010). The HOW(S) isoform is cytoplasmic and its function is not well characterised. To determine which RNAs are bound by HOW(S) in germ cells, in vivo RIP-seq was performed. This identified 343 genes and 121 transcripts bound by HOW(S), which were enriched for the GO terms related to signal transduction, consistent with HOW being a STAR protein. Hipk, a signalling kinase, was identified in the HOW(S) RIP-seq and knockdown of Hipk in the testis revealed a potential role for it in spermatogenesis, as these flies displayed a range of defects in testis morphology and fertility. Motif analysis found the (A/U/G)CUAAC motif enriched in 3'-UTRs of the HOW(S) bound mRNAs. This sequence is similar to the consensus sequences of other STAR proteins and is found in the 3'-UTR of Hipk transcripts. Fluorescence anisotropy assays revealed that HOW's STAR domain has strong nanomolar affinity for RNA oligos containing this motif. A novel motif was identified within 5'-UTRs, GCG(A/U)G, which HOW's STAR domain bound with micromolar affinity. Together, this work has identified many RNAs bound by HOW(S) in the cytoplasm of male germ cells. These RNAs have helped to expanded our understanding of HOW(S)– RNA interactions and may contribute to understanding the importance of HOW(S) RNAbinding activity during spermatogenesis.

Telomeres are the terminal parts of linear chromosomes made of tandem repeats of the TTAGGG motif. Telomeric repeats are associated with a six-protein complex called shelterin, which protects chromosome ends from the DNA Damage Response and regulates their maintenance. Telomeres are hard to replicate and behave like replication fragile sites and telomere replication constitutively requires shelterin and specialized helicases involved in the replication stress response. The repetitive nature of the telomeric repeats and the tendency to form secondary structures like G4 DNA, are thought to challenge fork progression at telomeres, however the molecular bases of telomere replication problems are not fully understood, in part due to the lack of techniques that can monitor structural transitions at telomeric replication forks. To overcome this limitation, we introduced telomeric repeats with different lengths and orientations, in an SV40-based construct that has been previously used to study the replication of specific DNA sequences in mammalian cells. Consistent with previous studies, we show that the shelterin complex associates with the telomeric repeats in our constructs, mimicking the sequence context of natural telomeres. Introduction of an episomal construct does not interfere with the structure of natural telomeres. Two-dimensional agarose gel electrophoresis showed that as telomeric insertions increase in length, they accumulate X-shaped intermediates compared to non-telomeric DNA sequences, independently of the orientation of the telomeric repeats with respect to the SV40 replication origin. In order to define the exact molecular nature of the intermediates that accumulate at telomeric repeats, we isolated replication intermediates and analyzed them in Electron Microscopy. Consistent with the 2D-gel analysis, we found that fragments containing 115 telomeric repeats have a 2-fold increase in replication fork reversion compared to control DNA sequences. These results show that replication forks have a higher probability of undergoing reversion at telomeric repeats in vivo.

Telomeres are essential in preventing the recognition of chromosome ends by the double-strand break (DSB) response, thereby permitting the maintenance of linear chromosomes. Extrachromosomal telomeric circles are important players in telomere maintenance, but little is known about their origin. We developed a new procedure for the purification of mammalian telomeric repeats in order to study telomere structures by Electron Microscopy (EM). Using this approach, we detected the expected telomeric features (i.e. t-loops) but we also found accumulation of internal loops (i-loops) at telomeric repeats that occur in the proximity of nicks and single-stranded DNA gaps. We showed that i-loops are induced by single-stranded damage at normal telomeres and represent the majority of the telomeric structures that accumulate in ALT (Alternative Lengthening of Telomeres) tumour cells, which are characterized by DNA damage accumulation at telomeric repeats. We also demonstrated that telomeric molecules containing i-loops can become a source of extrachromosomal telomeric circles. We propose that i-loops can form through strand exchange events at sites of damage, which are facilitated by the high sequence homology at telomeric repeats. This leads to the formation of intramolecular Holliday junctions that can be resolved to generate circular molecules made of telomeric DNA and resulting in a net loss of telomeric repeats. We propose that damage-induced i-loops are common intermediates in telomere maintenance that link telomere damage to the accumulation of extrachromosomal telomeric circles and to telomere erosion.

Obesity has considerable consequences on global health. Yet, it is relatively unexplored if the causes and consequences of obesity and obesity-related diseases differ between men and women. The increased scope of genetic studies provides an opportunity to leverage genetic data to characterise sex-specific epidemiological relationships using methods less prone to reverse causation and confounding, such as Mendelian randomisation. Thus, I aimed to investigate the sex-specific causes and consequences of obesity using genetic epidemiology, as well as to identify potential disease-mediating genes in two common obesity-related female reproductive diseases: polycystic ovary syndrome and miscarriage. In my first research chapter, I use Mendelian randomisation to investigate the sex-specific effects of obesity on leading causes of death from non-communicable diseases, before comparing the men- and women-specific effect estimates. My results indicate that the disease risk arising from obesity differs between the sexes for several diseases, including type 2 diabetes, renal failure, and chronic obstructive pulmonary disease. I then proceed to perform sex-stratified genome-wide association studies of 2,623 unique proteins and compare the effect estimates of the genetic variants between the sexes. Using these data, I compare the effects of proteins on obesity and cardiometabolic diseases between men and women. My results in this chapter indicate that up to 15% of protein-associated genetic variants may have different effects in the two sexes. I further identify seven proteins with sexually heterogeneous effects on obesity in men versus women, including two proteins - kynureninase and N-terminal pro b-type natriuretic peptide - with seemingly opposite effects in men compared to women. However, sensitivity analyses indicated that my results may be affected by poor specificity in the original protein measurements. Finally, in my third research chapter, I use colocalisation to highlight potential disease-mediating genes in polycystic ovary syndrome and recurrent miscarriage. While I was not able to identify any potential disease-mediating genes for recurrent miscarriage, I identify eleven genes and proteins with evidence of colocalising with risk of polycystic ovary syndrome. My thesis demonstrates that causal relationships in cardiometabolic disease can differ between the sexes, and emphasises the importance of investigating sex-specific effects. Furthermore, my results highlight several genes and proteins that may contribute to obesity and obesity-related diseases. Although their clinical utility will need to be established in future studies, several of these genes and proteins hold promise for use as biomarkers or for targeting by drugs to improve the health of both men and women.

In the era of genome sequencing, it has become clear that interpreting sequence variation in the non-coding genome represents a critical bottleneck for translating genomic insights to benefits for human health. The non-coding genome harbours the complex sequence patterns that encode regulatory elements and their intricate interplay over large genomic distances, ensuring precise gene regulation throughout time and cell differentiation. This complex sequence grammar is challenging for humans to comprehend and the experiments required to probe it are expensive and time-consuming. Machine learning approaches offer a powerful tool for studying the non-coding genome in silico. In this thesis work, we explored the use of deep neural networks for the purpose of interpreting genomic variations in the non-coding genome. We re-implemented, optimised and evaluated proposed convolutional neural networks models for predicting chromatin features from DNA sequence and utilise them in an integrated pipeline for dissecting non-coding sequence variants identified by Genome-Wide Association Studies (GWAS). We applied this pipeline to variants associated with red blood cell traits, demonstrating how neural networks can facilitate the interpretation of loss and gain of function variants in regulatory elements. Although chromatin feature networks evaluate variation in larger sequence context than previously possible, they still only offer a highly local perspective ignoring chromatin architecture and the distal interplay of regulatory elements. To address this limitation, we developed a novel neural network framework (deepC) using dilated convolutions and transfer learning to predict chromatin interactions from megabase scale DNA sequence. We validated deepC predictions using chromosome conformation assays at high sensitivity. Finally, we demonstrated how deepC can be used to dissect the sequence determinants of chromatin folding and to predict the impact of structural and single nucleotide variation on genome architecture. Together this thesis evaluates, utilises and expands the deep learning toolbox for genomics and our capabilities of interpreting non-coding genomic variation.

The discovery of ten-eleven translocation (TET) enzymes-mediated 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) gave rise to a new area of epigenetic study. Initially thought to be simply an intermediate in an active DNA demethylation pathway, 5hmC was since demonstrated to play critical roles in mammalian development, cellular differentiation and cancer progression. The global 5hmC content is reduced in the vast majority of studied cancers and mutations in the three TET genes are present to a varying extent in some tumour types. The reactivation of the TET proteins was proposed as a therapeutic intervention in melanoma patients. However, the preliminary data from the Murrell lab suggests that 5hmC levels increase during colorectal cancer (CRC) metastasis to the liver and few TET mutations are reported in CRC patients. Given that 90% of cancer deaths occur due to metastasis, the role of TETs and 5hmC in CRC progression warrants closer investigation. The in-house ultrasensitive liquid chromatography-mass spectrometry method of global cytosine, 5mC and 5hmC levels detection in various DNA samples was successfully optimised and validated. It was found to robustly detect the 5hmC content in almost all DNA samples studied and offer a superior sensitivity to an external method. The primary SW480 and metastatic SW620 CRC cell lines were chosen to study the effects of 5hmC and TET levels manipulation. The SW480 cells were shown to have higher migration rate and TET2, 5mC and 5mhC content but lower TET1 expression and proliferation rate compared to the SW620 cells. The treatment of the two cell lines with vitamin C increased their 5hmC content, but its phenotypic effects were found to be 5hmC-independent. The CRISPR/Cas9-based targeting of the three TET genes in these cell lines caused an incomplete ablation of TET and 5hmC levels and resulted in lower proliferation rate in the SW480 cells and reduced migration in the SW620 cells. The changes in TET expression and 5hmC levels are also described during the differentiation of human embryonic stem cells (hESCs) to hepatocyte-like cells (HLCs). Despite the initial attempt to knockdown TET1 levels in hESC to improve differentiation efficiency failing, future experiments utilising this approach could produce HLCs closer resembling human hepatocytes, which is critical for drug testing purposes. Taken together, these results contribute to the growing body of evidence for the critical role of 5hmC and TETs in cancer progression and hESC differentiation.

DNA modifications are epigenetic marks deposited throughout the mammalian genome and play an important role in gene regulation in normal and malignant biological processes. Biological DNA modifications are introduced by DNA methyltransferases (DNMT) and dioxygenases of ten- eleven translocation (TET) family. DNMT proteins can convert cytosine into 5-methylcytosine. TET proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. These oxidized forms of cytosine stay in the DNA or result in DNA demethylation. TET2, a member of TET family proteins, is one of the most frequently mutated genes in hematological malignancies. While TET2 has roles in normal hematopoiesis, including stem cell self-renewal and specific lineage commitment, the exact molecular mechanism of its function still remains unknown. The aims of the project are to identify the role of TET2 in regulating hematopoietic gene expression and to identify the molecular partners of TET2 in the hematopoietic-associated context, thus to arrive at the mechanism of action of TET2 in normal hematopoiesis. To study the Tet2 function in a hematopoietic context, I chose the mouse hematopoietic progenitor cell line, HPC-7, as a model system. I constructed the Tet2-knock-out (KO) HPC-7 cell and characterized the cell differentiation phenotype caused by Tet2 depletion. To understand the role of Tet2 in gene regulation, I firstly identified genes affected by Tet2 depletion. Then I mapped the Tet2 binding profile in HPC-7 and discovered genes directly targeted by Tet2. Finally, I assessed the contribution of DNA demethylation in Tet2-mediated gene regulation. The results revealed that Tet2 deficiency caused a block of cell differentiation in the Colony-forming-unit assay. Genes involved in hematopoiesis were differentially expressed upon Tet2 deficiency. Surprisingly, Tet2 binding is associated with both gene activation and gene repression. Tet2 binds to the hematopoietic-enhancers together with many hematopoietic transcription factors, and leads to DNA demethylation at enhancers, thus activates the expression of essential hematopoietic genes, such as csf1R, Cebpa, Cebpe. The mechanism by which Tet2 negatively regulates its targeted gene expression needs further investigation. To identify Tet2 molecular partners in the hematopoietic-associated context, I mapped the TET2 interactome in HPC-7 cells by immunoprecipitation coupled to mass spectrometry. Our data suggests that polycomb repressive complex 2 (PRC2) physically interacts with Tet2 via the C- terminus of Tet2 protein. The functional importance of Tet2-PRC2 interaction in gene regulation and cell differentiation remain further explored. Taken together, this study systematically investigated the Tet2-mediated gene regulation and identified new Tet2 binding partner, PRC2 complex, suggesting a novel mechanism of Tet2 function in hematopoiesis.

At fertilisation, terminally differentiated gametes combine to form a totipotent zygote, from which all subsequent cell types of the developing embryo are derived. Control of gene expression via epigenetic regulation represents an important mechanism contributing to the attainment of totipotency, and subsequent specification of cell lineages. During embryonic development, there are dynamic changes to the epigenetic systems controlling gene expression. Mammalian embryos differ from the embryos of other vertebrates in specifying an additional cell lineage that will lead to the development of a placenta. In addition, the evolution of mammalian XY sex chromosomes has resulted in the special requirement for dosage compensation of X-linked genes. The evolution of both the XY sex chromosomes and the placenta occurred prior to the divergence of the metatherian and eutherian lineages. Metatherian mammals are therefore perfectly placed in this evolutionary lineage to answer key questions about the evolution of mammalian traits. In this thesis, I have examined the role of one key component of epigenetic regulation, DNA methylation, in the early embryos and adults of the metatherian Monodelphis domestica, with the aim of understanding how the roles for DNA methylation in Xchromosome inactivation and early development have evolved in the mammalian lineage. I profiled the DNA methylation landscape of the M. domestica genome, and revealed that the inactive X chromosome displays a unique hypomethylated state. Using allelespecific analyses, I identified several novel X chromosome inactivation (XCI) escape genes, and demonstrated that XCI-escape genes are discernible by a unique DNA methylation signature. Finally, I profiled DNA methylation of M. domestica gametes and preimplantation embryos. I show DNA methylation at the region around RSX, the long non-coding RNA implicated in controlling metatherian XCI, to be a putative imprint controlling paternal XCI. Lastly, I discovered that unlike eutherian mammals, metatherian embryos do not undergo a dramatic genome-wide reprogramming of DNA methylation.

Cancer and bacterial infection are major killers across the world. Personalised cancer therapies are needed that respond to individual mixes of DNA damage, and new antibiotics are needed to r espond to resistance brought about by genetic mutations in bacteria [1] [2]. In this thesis, I analyse the gene sequence patterns next to small mutations in cancer cells, identifying those associated with substitutions and indels. I show that in the exome there is an excess of in-frame indels compared to frameshift mutations; evidence of negative selection. Next I analyse the associations between the sequences of driver genes and mutational frequency in cancers. I find most driver genes are more frequently mutated in those cancers where the re is a good match between the mean mutational fingerprint for that cancer and the fingerprint formed from the mutations found in the driver gene in question. I then extend existing work on mutational signatures, to identify novel bacterial mutational signatures. By comparing the signatures with those of human cancers and environmental mutagens, I identify alkylation as a driver of bacterial mutagenesis. Next I review translational drug discovery, highlighting the use of bioinformatics to identify drug targets and biomarkers, assess protein druggability; and predict opportunities for drug repositioning. Finally, I identify therapeutically actionable mutually exclusive gene pairs within human cancers. I show that the Poisson binomial distribution is better for identifying mutual exclusivity. The predictions are available on the new MexDrugs website, and my python implementation of the Poisson binomial test can be installed via pip.

A major goal of synthetic biology remains to create a fully orthogonal chassis, co-existing with its environment and unable to interfere with it, which would lead to the realisation of real-world applications of this discipline. Current biocontainment strategies often rely on easily circumvented auxotrophy or suicide networks triggered outside of a controlled environment. Rather than establishing a new genetic network to induce lethality, the present work intends to develop a method called genome ablation to obtain a DNA-free chassis unable to self-replicate or transfer genetic material to other species while remaining biochemically functional. In order to achieve absolute DNA degradation in vivo, exonuclease III and T5 bacteriophage exonuclease were overexpressed in Escherichia coli CSR603, a strain deficient in DNA repair, leading to DNA degradation without filamentation. Antibiotics mitomycin C and ciprofloxacin induced lethality and enhanced DNA degradation. Expression of restriction enzymes PvuII or HpaII, DNA gyrase poison ccdB, or T4 bacteriophage endonuclease denA alongside exonucleases reduced DNA content and cell viability; the lowest DNA content was achieved with E. coli CSR603 expressing PvuII at 4 h post-induction. Nuclease induction was found to exert a high selective pressure, leading to loss of nuclease activity. UV irradiation for 15 min induced lethality and absolute DNA degradation. However, this also induced extensive photodamage and compromised membrane integrity. Despite a functional chassis was not attained and further analysis is required, this is the first demonstration of DNA degradation in vivo applied to chassis engineering.

Increasing evidences suggest that post-transcriptional RNA modifications regulate essential biomolecular functions and are related to the pathogenesis of various diseases. To date, the study of epitranscriptome layer gene regulation is mostly focused on the function of mediator proteins of RNA methylation limited by laborious experimental procedures, i.e., the readers, writers and erasers. However, there is limited investigation of the functional relevance of individual m6A RNA methylation sites. To address this, we annotated human m6A sites in large-scale based on the guilt-by-association principle from complex biological networks. In the first chapter, the network was constructed based on public human MeRIP-Seq datasets profiling the m6A epitranscriptome under independent experimental conditions. By systematically examining the network characteristics obtained from the RNA methylation profiles, a total of 339,158 putative gene ontology functions associated with 1446 human m6A sites were identified. These are biological functions that may be regulated at epitranscriptome layer via reversible m6A RNA methylation. The results were further validated on a soft benchmark by comparing to a random predictor. In the second chapter, another approach was applied to annotate the individual human m6A sites by integrating the methylation profile, gene expression profile and protein-protein interaction network with guilt-by-association principle. The consensus signals on sites were amplified by multiplying the co-methylation network and the methylation-expression network. The PPI network smoothed the correlation for a query site to gene expression for furthering GSEA functional annotation. In the third chapter, we functionally annotated 18,886 m6A sites that are conserved between human and mouse from a larger epitranscriptome datasets using method previously described. Besides, we also completed two side projects related to SARS-CoV-2 viral m6A site prediction and m6A site prediction from Nanopore sequencing technology.