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1
Lyzhanov, Iurii. „Architektonická studie sakrálního objektu a komunitního centra Salesiánského Brno - Líšeň / druhá etapa“. Master's thesis, Vysoké učení technické v Brně. Fakulta stavební, 2021. http://www.nusl.cz/ntk/nusl-443691.
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The main task of diploma is creating a sacral object and community centre of Salesians in Brno-Líšeň. A construction of the Salesian complex started in 1998. As a result, the whole complex of institution got to the district of Brno-Lisen with the help of the endowment fund for youth. The Congregation focuses on the methods of educating the youth of Don Bosco, the father and teacher of the youth, the first head of the Salesian. The creation of a church is a very desirable step for the Salesian Center, because Masses are currently being celebrated in the existing building in the gym, where not everything is needed to carry out of the event. The church should be developed in the vicinity of Salesian centre and has to be available for a wide range of societies. The architectural project includes the designing of church, parish building and enviroment. The form and view of the complex suit to this environmental area and react on its entire structure. The object consists of two forms, which are connected by the horizontal communication. In the middle of the Salesian complex, there is a semi-open area. The first object is a church with a capacity of approximately 200 hundred visitors, the second object is a parish building, which perform the functions of administration with multifunction space for the exhibitions, aparmtments and offices for its employees.
2
Šomlo, Ivan. „Explorativní editace zdrojových textů“. Master's thesis, Vysoké učení technické v Brně. Fakulta informačních technologií, 2011. http://www.nusl.cz/ntk/nusl-237088.
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The thesis discusses the tools commonly used by developers in exploratory environments of languages Smalltalk and Self. These languages free their users from using plain unstructured source code. Moreover, they contain tools with richer features. The thesis consists of an analysis, a design proposal, and a description of Object Viewer, a Self based tool designed for languages C and C++. The tool graphically displays fragments of a program (objects and pointers) and allows for manipulating them during debugging. The conclusion consists of a summary and a recommendation for additional new features.
3
Ouldridge, Thomas E. „Coarse-grained modelling of DNA and DNA self-assembly“. Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:b2415bb2-7975-4f59-b5e2-8c022b4a3719.
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In this thesis I present a novel coarse-grained model of deoxyribonucleic acid (DNA). The model represents single-stranded DNA as a chain of rigid nucleotides, and includes potentials to represent chain connectivity, excluded volume, hydrogen-bonding and base stacking interactions. The parameterization of these interactions is justified by comparing the model's representation of a range of physical phenomena to experimental data. In particular, the geometrical structure and elastic moduli of duplex DNA, and the flexibility of single-stranded DNA, are shown to be physically reasonable. Additionally, the thermodynamics of single-stranded stacking, duplex hybridization, hairpin formation and more complex motifs are shown to agree well with experimental data. The model is optimized for capturing the thermodynamic and mechanical changes associated with duplex formation from single strands. Considerable attention is therefore given to ensuring that single-stranded DNA behaves physically, an approach which differs from previous attempts to model DNA. As a result, the model is the first in which an explicit stacking transition is present in single strands, and also the only coarse-grained model to date to capture both hairpin formation within a single strand and duplex formation between strands. The scope of the model is demonstrated by simulating DNA tweezers, an iconic nanodevice -- the first time that coarse-grained modelling has been applied to dynamic DNA nanotechnology. The simulations suggest that branch migration during toehold-mediated strand displacement -- a central feature of many nanomachines -- does not have a flat free-energy profile, as is generally assumed. This finding may help to explain the observed dependence of displacement rate on toehold length. Finally, the operation of a two-footed DNA walker on a single-stranded DNA track is considered. The model suggests that several aspects of the walker will reduce its efficiency, including a tendency to bind to an undesired site on the track. Several design modifications are suggested to improve the operation of the walker.
4
Fahlman, Richard P. „DNA nanotech, expanding the repertoire of DNA for the assembly of nanoscale objects and electrical devices“. Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/NQ61642.pdf.
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5
Dunn, Katherine Elizabeth. „DNA origami assembly“. Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:dff1bafd-e355-4df5-968b-b0deb7e6f44f.
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This thesis describes my investigations into the principles underlying self-assembly of DNA origami nanostructures and discusses how these principles may be applied. To study the origami folding process I designed, synthesized and characterized a polymorphic tile, which could adopt various shapes. The distribution of tile shapes provided new insights into assembly. The origami tiles I studied were based on scaffolds derived from customized plasmids, which I prepared using recombinant DNA technology. I developed a technique to monitor incorporation of individual staples in real time using fluorescence, measuring small differences in staple binding temperatures (~0.5-5 °C). I examined the tiles using Atomic Force Microscopy and I found that a remarkably high proportion of polymorphic tiles folded well, which suggests that there are assembly pathways, arising from strong cooperation between staples. In order to analyse the tile shapes quantitatively, I developed a specialized image processing technique. For validation of the method, I generated and analysed simulated data, and the results confirmed that I could measure individual tile parameters with sub-pixel resolution. I studied eleven variants of the polymorphic tile, and I proved that minor staple modifications can be used to change the folding pathway dramatically. The strength of cooperation between staples affects their behaviour, which is also influenced by their length and base sequences. Paired staples are particularly significant in assembly, and there are clear parallels with protein folding. I describe in an Appendix how I applied origami assembly principles in the development of my concept for an autonomous rotary nanomotor utilizing the sequential opening of DNA hairpins (already used for linear motors). This device represents an advance over non-autonomous rotary motors and I have simulated its performance. In this thesis I have answered important questions about DNA origami assembly, and my findings could enable the development of more sophisticated DNA nanostructures for specific purposes.
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Harrison, Ryan M. „Molecular biophysics of strong DNA bending and the RecQ DNA helicase“. Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:f02fc167-b705-4275-a413-21d13b5d94c3.
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Molecular biophysics is a rapidly evolving field aimed at the physics-based investigation of the biomolecular processes that enable life. In this thesis, we explore two such processes: the thermodynamics of DNA bending, and the mechanism of the RecQ DNA helicase. A computational approach using a coarse-grained model of DNA is employed for the former; an experimental approach relying heavily on single-molecule fluorescence for the latter. There is much interest in understanding the physics of DNA bending, due to both its biological role in genome regulation and its relevance to nanotechnology. Small DNA bending fluctuations are well described by existing models; however, there is less consensus on what happens at larger bending fluctuations. A coarse-grained simulation is used to fully characterize the thermodynamics and mechanics of duplex DNA bending. We then use this newfound insight to harmonize experimental results between four distinct experimental systems: a 'molecular vise', DNA cyclization, DNA minicircles and a 'strained duplex'. We find that a specific structural defect present at large bending fluctuations, a 'kink', is responsible for the deviation from existing theory at lengths below about 80 base pairs. The RecQ DNA helicase is also of much biological and clinical interest, owing to its essential role in genome integrity via replication, recombination and repair. In humans, heritable defects in the RecQ helicases manifest clinically as premature aging and a greatly elevated cancer risk, in disorders such as Werner and Bloom syndromes. Unfortunately, the mechanism by which the RecQ helicase processes DNA remains poorly understood. Although several models have been proposed to describe the mechanics of helicases based on biochemical and structural data, ensemble experiments have been unable to address some of the more nuanced questions of helicase function. We prepare novel substrates to probe the mechanism of the RecQ helicase via single-molecule fluorescence, exploring DNA binding, translocation and unwinding. Using this insight, we propose a model for RecQ helicase activity.
7
Benn, Florence. „Functional DNA nanotechnology“. Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:1ed7a9d7-acf2-46ee-97d1-b28084b3d4cc.
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This thesis sets out to further the field of functional DNA nanotechnology through the design of novel functional DNA scaffolds, and investigates their applications and efficacy. The work presented here comprises two parts: The design of a chiral DNA nanotube that acts as a scaffold for motor motion and for an enzyme cascade; and the design of two different tetrahedral scaffolds for selection of a combination of three ligands, which together have a greater binding effect than the sum of the individual components. It begins by proposing the design of a DNA origami nanotube which distinguishes between the inside and outside face of the tube at the design stage, which most previous designs reported do not. The previous designs in the literature result in a distribution of 50:50, of one face forming the inside surface on one tube and the same face forming the outside surface of a different tube. In the design presented in this thesis, this distinction results from making the tube chiral, which forces it to roll up in a predetermined manner. Chirality is introduced by varying the positions of staple crossovers and this process is explained. The chiral tubes may stack end-to-end to form long polymers, or exist in monomeric form with stacking suppressed, by inclusion of different sets of staples at the ends of the tubes. We confirm tube formation and right-handed chirality with AFM and CD respectively. The efficacy of the tube as a scaffold for an enzyme cascade is tested and discussed in context of the wider field. No significant enhancement is observed when enzymes are tethered to the inside of the tubes, compared to when they are tethered to the outside or are free in solution, although the same slight trend is always observed. Suggestions are made to better this experiment and further understand the underlying physics of such systems. We propose using the tube as a scaffold for a DNA track, upon which a DNA motor may walk. DNA motors are introduced and we attempt to observe micron-scale, inter-tube motion within the confines of our origami tube. Initial experiments show the motor moving and we propose methods of fluorescent labeling via PAINT to better the experimental set-up for TIRF microscopy, which currently is limited by photobleaching. The second part of this thesis proposes systems for selection of a combination of three ligands, which together have a greater binding effect than the sum of the individual components. Here we design two tetrahedral systems where either three ligands or three aptamers are brought together at a vertex of the tetrahedron to form a binding domain. The aptameric system allows for selection, amplification and reassembly of the strongest binders, because the functional and structural sequences are on one strand of DNA, following ligation. This design betters the initial tetrahedral system, where the coding/record strands for amplification are separate from the functional binding domain strands the ligands are attached to. This means it is not possible to reassemble this particular structure after amplification of the record strand.
8
Lucas, Alexandra. „Dynamic DNA motors and structures“. Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:5f0b0773-a7af-4edb-a6a2-790a0086553d.
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DNA nanotechnology uses the Watson-Crick base-pairing of DNA to self-assemble structures at the nanoscale. DNA nanomachines are active structures that take energy from the system to drive a programmed motion. In this thesis, a new design for a reversible DNA motor and an automatically regenerating track is presented. Ensemble fluorescence measurements observe motors walking along the same 42nm track three times. A second new motor was designed to allow motors on intersecting tracks to block each other, which can be used to perform logical computation. Multiple design approaches are discussed. The chosen approach showed limited success during ensemble fluorescence measurements. The 'burnt bridges' motor originally introduced by Bath et al. 2005 was also sent down tracks placed along the inside of stacked origami tubes that are able to polymerise to micrometre lengths. Preliminary optical microscopy experiments show promise in using such a system for observing micrometre motor movement. Scaffold-based DNA origami is the technique of folding a long single-stranded DNA strand into a specific shape by adding small staple strands that hold it in place. Extended staple strands can be modified to functionalise the origami surface. In this thesis, the threading of staple extensions through a freely-floating origami tile was observed using single-molecule Förster resonance energy transfer (smFRET). Threading was reduced by bracing the bottom of the extension or by using a multilayered origami. smFRET was also used to investigate the process of staple repair, whereby a missing staple is added to a pre-formed origami missing the staple. This was found to be successful when the staple is single-stranded, and imperfect when partially double-stranded. Finally the idea for a new "DNA cage", a dynamic octahedron called the "Holliday Octahedron", is presented. The octahedron is made of eight strands, one running around each face. Mobile Holliday junctions at each face allow the stands to rotate causing a conformational change.
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Boemo, Michael Austin. „Computation by origami-templated DNA walkers“. Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:bdea667e-a9aa-484a-9db0-a816339e5594.
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Interactions between DNA molecules can be used to perform computation. These DNA computing systems often use DNA molecules as freely diusing reactants in a well-mixed solution. We demonstrate how DNA walkers tethered to an origami-templated track can perform computation. A DNA walker can block a track that intersects with its own, preventing another walker from stepping down this blocked track. These blockages are primitive operations that can be used to perform computation. This thesis demonstrates how blocking interactions between DNA walkers can evaluate formulae posed in propositional logic. When anchorages in the track are viewed as networked machines and the DNA walker is viewed as a coordinated message passed between them, DNA walker circuits can be modelled as a distributed system. Techniques from formal veri- cation can be used to check this system for errors, determining the probability with which the system will end up in a certain state. This forms the basis of a compiler that can automatically design a DNA walker circuit that evaluates a given propositional formula within a specied error tolerance. To show how DNA walker circuits can be simplied, we create a propositional logic system called blocking logic that is proven to be both sound and complete. DNA walker circuits can be implemented and measured experimentally by using fluorescence spectrophotometry to track the position of a walker on the track. To demonstrate proof of principle, circuits were built that implement NOT and NOR operators. To make these circuits operate with minimal error, dierent sources of possible error were investigated and quantied. Cumulatively, the novel contributions that this thesis makes to the eld are: ⢠the experimental design and implementation of a DNA computing system that uses DNA walkers, ⢠probabilistic model checking software that automatically designs these DNA walker circuits, ⢠a propositional logic system that can simplify a DNA walker circuit to an equivalent circuit that uses fewer tracks.
10
Dannenberg, Frits Gerrit Willem. „Modelling and verification for DNA nanotechnology“. Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:a0b5343b-dcee-44ff-964b-bdf5a6f8a819.
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DNA nanotechnology is a rapidly developing field that creates nanoscale devices from DNA, which enables novel interfaces with biological material. Their therapeutic use is envisioned and applications in other areas of basic science have already been found. These devices function at physiological conditions and, owing to their molecular scale, are subject to thermal fluctuations during both preparation and operation of the device. Troubleshooting a failed device is often difficult and we develop models to characterise two separate devices: DNA walkers and DNA origami. Our framework is that of continuous-time Markov chains, abstracting away much of the underlying physics. The resulting models are coarse but enable analysis of system-level performance, such as âthe molecular computation eventually returns the correct answer with high probabilityâ. We examine the applicability of probabilistic model checking to provide guarantees on the behaviour of nanoscale devices, and to this end we develop novel model checking methodology. We model a DNA walker that autonomously navigates a series of junctions, and we derive design principles that increase the probability of correct computational output. We also develop a novel parameter synthesis method for continuous-time Markov chains, for which the synthesised models guarantee a predetermined level of performance. Finally, we develop a novel discrete stochastic assembly model of DNA origami from first principles. DNA origami is a widespread method for creating nanoscale structures from DNA. Our model qualitatively reproduces experimentally observed behaviour and using the model we are able to rationally steer the folding pathway of a novel polymorphic DNA origami tile, controlling the eventual shape.
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French, Catherine A. „The role of RAD51-like genes in the repair of DNA damage in mammalian cells“. Thesis, University of Oxford, 2003. http://ora.ox.ac.uk/objects/uuid:3408a30d-8833-4649-8d72-7194c60f944c.
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12
Olcina, del Molino Mónica. „Hypoxia-induced chromatin changes and ATM signalling“. Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:dbef3c19-b7f6-42a4-a321-97d00c572ae3.
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The DNA damage response (DDR) is a complex signalling cascade triggered in response to stress, in an attempt to maintain genomic integrity. Components of this pathway, such as ATM-mediated signalling, have been proposed to act as a barrier in the early stages of tumourigenesis. Regions of low oxygen concentrations (hypoxia) occur in most solid tumours and are associated with a poor prognostic outcome. Here, we investigated the DDR induced following hypoxia-induced replication stress in an attempt to decipher the mechanism of ATM activation in response to physiological stresses that do not induce DNA damage. We hypothesized that hypoxia-mediated chromatin changes could impact on ATM signalling. We have characterised H3 methylation in response to hypoxia and found oxygen dependent changes in H3K9me3, including both global and replication fork associated increases in this histone modification. Importantly, we have found that decreases in H3K9me3 result in loss or attenuation of ATM activation. Notably, in a background of replication stress and increased H3K9me3, ATM inhibition or loss leads to accumulation of DNA damage and a significant decrease in replication rates in hypoxia. We propose that when replication stress occurs in the presence of hypoxia-induced chromatin changes, ATM activation is facilitated by the induction of H3K9me3. In this context, we propose a novel and stress specific role for ATM-mediated signalling in maintaining replication and preventing the generation of DNA breaks that may compromise genomic integrity. Moreover, the biological consequences of the hypoxia-induced chromatin context and in particular hypoxia-induced H3K9me3 include the repression of APAK, a negative regulator of p53. Activation of p53 is a key consequence of the hypoxia-induced DDR. Here we found that SETDB1, one of the H3 methyltransferases induced by hypoxia, mediates APAK repression. We propose that H3K9me3 plays a role in regulating APAK expression to allow optimal induction of p53 dependent apoptosis in hypoxic conditions suggesting a further role for H3K9me3 in facilitating DDR signalling in hypoxia. Together, these data suggest that the hypoxic chromatin context is critical for the role of the DDR as a barrier to tumourigenesis and predict that altering the chromatin landscape in combination with DNA damaging therapies would be efficacious in the treatment of hypoxic tumours.
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Machinek, Robert R. F. „Control and observation of DNA nanodevices“. Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:89e131dd-5179-4ebd-924b-2939f16681d5.
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The uniquely predictable and controllable binding mechanism of DNA strands has been exploited to construct a vast range of synthetic nanodevices, capable of autonomous motion and computation. This thesis proposes novel ideas for the control and observation of such devices. The first of these proposals hinges on introducing mismatched base pairs into toehold-mediated strand displacement – a fundamental primitive in most dynamic DNA devices and reaction networks. Previous findings that such mismatches can impede strand displacement are extended insofar as it is shown that this impediment is highly dependent on mismatch position. This discovery is examined in detail, both experimentally and through simulations created with a coarse-grained model of DNA. It is shown that this effect allows for kinetic control of strand displacement decoupled from reaction thermodynamics. The second proposal improves upon a previously presented strand displacement scheme, in which two strands perform displacement cooperatively. This scheme is extended to be cascadable, so that the output of one such reaction serves as input to the next. This scheme is implemented in reaction networks capable of performing fundamental calculations on directed graphs. The third proposal is exclusively concerned with a novel observation methodology. This method is based on single-molecule fluorescence microscopy, and uses quantum dots, a fluorescent type of semiconductor nanocrystal, as a label. These quantum dots display a set of characteristics particularly promising for single-molecule studies on the time- and length scales most commonly encountered in DNA nanotechnology. This method is shown to allow for highly precise measurements on static DNA devices. Preliminary data for the observation of a complex dynamic device is also presented.
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Budworth, Helen Louise. „Role of Base and nucleotide excision repair pathways in processing of clustered DNA lesions induced by ionising radiation“. Thesis, University of Oxford, 2003. http://ora.ox.ac.uk/objects/uuid:357d0c93-77bf-4097-85a7-05fa0dca740a.
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Ionising radiation (IR) induces a wide spectrum of lesions in DNA, including double- and single-strand breaks, abasic (AP) sites and a variety of base lesions. IR-induced damage to DNA can range from simple, isolated lesions to clustered DNA damage in which multiple lesions are formed, usually within a single helical turn of the DNA. Individual lesions within a cluster are recognised by repair enzymes of the base excision repair (BER) pathway, however, clustered DNA damage may be recognised as a bulky lesion and be processed by nucleotide excision repair (NER). Additionally, the presence of other closely spaced lesions may affect the rate and fidelity of DNA repair and, in doing so, may contribute to the harmful effects of ionising radiation. The aim of this study is to gain further understanding of the repairability of clustered DNA damage and the effects of multiple lesions on cellular repair systems. 7, 8-dihydro-8-oxoguanine (8-oxoG), thymine glycol (Tg), AP sites and single-strand breaks (SSB), some of the most frequently formed IR-induced DNA lesions, were employed in synthetic oligonucleotides to model various types of clustered lesions and their repairability was studied using purified base excision repair enzymes and cell extracts. It was revealed that BER is the major repair system involved in the processing of clustered DNA lesions, and that some clustered lesions are repaired with decreased efficiency. Both the composition of lesions in a cluster and the positioning of the various lesions determine their repairability by base excision repair enzymes.
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Entwistle, Ngai Mun Aiman. „Actuation of DNA cages and their potential biological applications“. Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:0983ed77-77f6-4d15-9187-52aff44299ec.
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DNA cages are polyhedra self-assembled from synthetic oligonucleotides in a one-pot process. The main system described in this thesis is a reconfigurable, wire-framed DNA tetrahedron in nanometre-scale. On one of its vertices this tetrahedron has an overhang that can hybridise with a specific sequence of nucleic acids and open the cage. We describe the design of a reconfigurable cage that remained closed under physiological conditions and only opened in the presence of an appropriate signal in solution. Fluorescence techniques were employed to distinguish the open and closed states of the cage. We used flow cytometry and confocal microscopy to successfully established the open and closed states of the cage inside live cultured mammalian cells. Further experiments revealed that the DNA cage could be opened by a separately transfected signalling strand. Hybridisation between two separately transfected systems was possible. The DNA cage was then simplified to a DNA duplex so that the intracellular interactions between the two nucleic acids systems could be studied more efficiently. Microscopy images showed that the interaction occurred in membrane-bound compartments. We describe an investigation into the use of various cellular RNAs, including full-length mRNA and tRNA-RNA fusion, to actuate the DNA cages. Choosing an appropriate cellular opening signal remains a challenge. Analysis showed that bulky cellular RNA experienced steric hindrance with the rigid DNA cage. Finally, other potential biological applications of DNA cages, such as using DNA nanostructures as the carriers for genetic therapeutic agents, were also presented.
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Haley, Natalie Emma Charnell. „Structures and mechanisms for synthetic DNA motors“. Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:7bcdd990-cb31-40f2-b85b-4a9a1630eafb.
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DNA provides an ideal substrate for nanoscale construction and programmable dynamic mechanisms. DNA mechanisms can be used to produce DNA motors which do mechanical work, e.g. transportation of a substrate along a track. I explore a method for control of a DNA mechanism ubiquitous in DNA motor designs, toehold-mediated strand displacement, by which one strand in a duplex can be swapped for another. My method uses a mismatch between a pair of nucleotides in the duplex, which is repaired by displacement. I find that displacement rate can be fine-tuned by adjusting the position of the mismatch in the duplex, enabling the design of complex kinetic behaviours. A bipedal motor [1, 2] is designed to walk along a single-stranded DNA track. Previously the motor has only taken a single step, due to a lack of designs to extend the single-stranded track. I present a novel design for track held under tension using a 3D DNA origami tightrope, and verify its assembly. The bipedal motor design is adapted and a method to specifically place motors on tightropes is demonstrated. Motor operation is investigated on truncated tracks and tightrope tracks by electrophoresis and spectrofluorometry. The motor does not accumulate appreciably at the track end; this is tentatively attributed to rearrangement of the motor between track sites without interaction with fuel. Tightrope origami can hold single-stranded DNA under pN tension. I use tightropes to study hybridization kinetics under tension and find dramatic, non-monotonic changes in hybridization rate constants and dissociation constants with tension in the range ∼0-15 pN. Extended tracks for a 'burnt-bridges' motor which destroys its track as it moves [3] are created on the inside of DNA nanotubes, which can be polymerised to create tracks up to a few mm in length, and on tiles which I attempt to join in a specific order. Crossing of the motor between tubes is verified, and microscopy experiments provide some evidence that track is being cleaved by the motor, a requirement for movement along the track. Tile based tracks are imaged by super-resolution DNA PAINT [4], providing proof-of-principle for track observation to infer motor movement.
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Nicolay, N. H. „The role of DNA polymerase eta in determining cellular responses to chemo-radiation treatment“. Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:73cef89c-319d-4a14-a3a6-93e8b8dd186a.
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DNA polymerase η (pol η), a crucial component of the cellular translesion synthesis pathway, allows cells to bypass and thereby temporarily tolerate DNA damage. Inherited deficiency of pol η, as reported in the variant form of xeroderma pigmentosum, predisposes to UV light-induced skin cancers. To date, pol η is the only DNA polymerase shown to exhibit a causal link to the formation of cancers in humans. However, the role of pol η in the cellular response to forms of DNA damage other than UV-induced lesions is largely unknown. In the first part of this thesis, it is shown that cells deficient in pol η are resistant to ionising radiation. Deficiency in the polymerase was associated with accumulation of cells in S phase of the cell cycle. Cells deficient in pol η demonstrated increased homologous recombination-directed repair of DNA double-strand breaks created by ionising radiation, and depletion of the homologous recombination protein X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells compared to pol η-complemented cells. These findings suggest that homologous recombination mediates S phase-dependent radioresistance associated with pol η-deficiency. In the second part of this thesis, it is shown that pol η-deficient cells have increased sensitivity to the chemotherapeutic compound, oxaliplatin, compared to pol η-deficient expressing cells, but not to the drug 5-fluorouracil that is usually administered in combination with oxaliplatin in the clinical setting. Despite the importance of pol η for cellular survival following exposure to oxaliplatin, the drug did not upregulate the enzyme after either short-term or long-term exposure. Inhibition of pol η activity by siRNA-mediated knockdown of the protein sensitised cells to oxaliplatin treatment, and partially reversed acquired resistance in oxaliplatin-resistant tumour cell lines. These data suggest that pol η is an interesting target whose function can potentially be interfered with to optimise oxaliplatin-based chemotherapy. In the third part of this thesis, clinical samples obtained from oesophageal cancer patients before and after treatment with oxaliplatin-containing chemotherapy were analysed for POLH mRNA levels encoding pol η protein. Malignant tissue specimens obtained before treatment demonstrated a significantly higher level of POLH mRNA than matched normal oesophageal tissue samples. Contrary to the preclinical data, high POLH mRNA expression before therapy was shown to correlate with increased overall and disease-free survival of the patient cohort in the clinical trial. Additionally, patients with high POLH mRNA-expressing cancers had better therapeutic responses (measured by PET-CT) to oxaliplatin-based treatment than those with low levels. These data suggest that POLH mRNA expression should be tested as a biomarker to predict survival and therapeutic responses in oesophageal cancer patients treated with oxaliplatin-containing chemotherapy.
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Wallace, Emma Victoria Bristowe. „Selective modification and detection of the DNA bases“. Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:e30564de-0a84-4665-b7af-66a8ea589801.
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α-Hemolysin (αHL) is a biological nanopore, which is currently under investigation for implementation into a new method for DNA sequencing. It has been established that αHL is capable of discriminating the canonical bases (adenine, cytosine, guanine and thymine) when they are immobilised within a pore by means of a biotin•streptavidin complex. Work in this thesis develops this procedure for the discrimination of these standard nucleobases from the the epigenetic modifications of cytosine – 5-methylcytosine (5mC) and 5 hydroxymethylcytosine (hmC). Strategies to selectively modify and detect the modified bases are also explored. Introduction. DNA sequencing strategies from the initial methods employed by Sanger up to current techniques utilised for the sequencing of entire human genomes, are reviewed. The initial discoveries of the epigenetic modifications 5-methylcytosine and 5 hydroxymethylcytosine are discussed. The proposed effects these bases have in vivo and a number of methods for their detection are also covered. Finally, methods for the selective chemical modification of the DNA bases are reviewed. Results and Discussion. Initially, the biotin•strepatvidin immobilisation strategy is implemented for the discrimination of the epigenetically modified analogues of cytosine (5mC and hmC), without prior chemical modification. It is subsequently observed that an α-hemolysin mutant is capable of discriminating all six bases – adenine, cytosine, guanine, thymine, 5 methylcytosine and 5-hydroxymethylcytosine. A number of different chemical reactions are then investigated on the bis-TBS protected deoxynucleoside model system, for their ability to selectively modify one of the DNA bases. Two selective oxidation reactions are then further optimised for use on ssDNA. Finally, electrical recording experiments were used to investigate two selective chemical reactions, as well as the modification of the bases, by the cancer drug temozolomide.
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Baddock, Hannah. „Understanding the role of the SNM1B and EXD2 in DNA damage repair“. Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:e120ab1f-d001-4b9b-9a03-c0852f4dfd21.
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Unrepaired, or misrepaired, DNA damage can be carcinogenic or mutagenic; thus functional DNA damage repair pathways are essential for the safeguarding of the genome. SNM1B is a 5' to 3' exonuclease implicated in the repair of damaged DNA, particularly the repair of interstrand crosslinks. Genetic studies have identified SNPs in the SNM1B gene as related to cancer risk. One of these (rs11552449) encodes a single amino acid change, H61Y. This study shows that WT and H61Y SNM1B have comparable in vitro biochemical and biophysical characteristics. The structures of both WT and H61Y C-terminally truncated SNM1B (Δ-SNM1B) were solved to 2.8 and 3.1 Å, respectively, and reveal similar structural properties. The structure of WT Δ-SNM1B was also reported (to 1.8 Å) with two 2'-deoxy-5'- adenosine monophosphate molecules in the active site. The structure of SNM1B shows an accessible extended active site, which may facilitate the binding of a variety of non-canonical DNA substrates. Accordingly, in vitro, WT and H61Y SNM1B are able to exonucleolytically process a wide range of structurally diverse DNA substrates. By utilising SNM1B depleted cell lines, this study also shows that SNM1B is required for DNA repair in response to treatment with DNA-crosslinking genotoxic agents (including cisplatin and SJG-136). This study also identifies the novel double strand break repair factor, EXD2, as having intrinsic 3' to 5' exonuclease activity. EXD2 was shown to have enzymatic activity on a variety of substrates in vitro, including replication fork intermediates, 'nicked' or 'gapped' DNA duplexes, and RNA based substrates. Together with the cellular data this suggests a role for EXD2 in nucleolytically processing RNA or DNA-based intermediates in damage repair pathways.
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Mendes, de Oliveira Martins Carlos Daniel. „Real-time studies of DNA repair kinetics following low-LET short-pulse electron radiation“. Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:63a874a6-9348-4504-b4cd-b1cfd129b75d.
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Radiation-induced damage to the genomic DNA of cells may lead to errors in transcription and replication and, if not repaired correctly, these may result in mutations, genomic instability and cell death. Laser microbeams have generally been used by many research groups to investigate the real-time dynamics of protein recruitment in response to DNA insults in mammalian cells; however, such irradiations induce a plethora of DNA damage (including UV base damage, base damage, SSBs and DSBs and complex damage). A novel experimental setup has been designed capable of following the dynamics of protein recruitment in response to DNA insults in mammalian cells shortly following submicrosecond- pulsed electron irradiation of living mammalian cells, not possible using conventional irradiation techniques. This arrangement was developed based on a 6 MeV electron pulse linear accelerator, to deliver sparsely ionising radiation, coupled to an automated, time-lapse inverted epifluorescence microscope imaging system. An integrated robotic system contained within a physiological environment of 37°C and 5% CO2 was used to transfer remotely and repetitively custom-designed cell dishes containing the mammalian cells between irradiation and imaging locations. Following the development of the linear accelerator and associated imaging devices, preliminary ‘proof-of-principle’ investigations were carried out using living HT1080 mammalian cells containing YFP-tagged 53BP1, an established biomarker of DSB, to follow the recruitment and loss of 53BP1 to sites of radiation-induced DNA damage in real-time. This novel experimental setup has allowed for the first time observations of the appearance and disappearance of radiation-induced foci in the same cell population at very early times. These single-foci studies have provided evidence for the formation of not only promptly formed DSBs but also late appearing DNA damage signalled by 53BP1. These data highlight different classes of DSBs formed in response radiation damage. Additionally, the role of cell cycle on the repair kinetics was undertaken using HT1080- 53BP1-YFP cells which also express Geminin-mCherry under appropriate selection. Geminin is increasingly expressed from early S-phase onwards, but is degraded following mitosis. Geminin-associated fluorescence can be used as a marker of progression through the cell cycle. 53BP1 repair kinetics were characterised in response to radiation damage in combination with ATM and PARP inhibitors. These studies provided supporting evidence for the existence of different classes of DSBs, potentially assigned to radiation-induced replication breaks and DSBs formed by enzymatic conversion of clustered damage. These preliminary ‘proof-of-principle’ findings using DNA damage repair as an example, emphasize the use of this novel technology to explore the dynamics of numerous other biochemical processes in living cells in real-time with the knowledge of being able to quantify the range of damage induced by IR coupled with accurate dosimetry. The knowledge obtained may be used to identify potential biological targets coupled with drug discovery for translation into adjuncts for radiotherapy.
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Haile, James Seymour. „Ancient DNA from sediments and associated remains“. Thesis, University of Oxford, 2008. http://ora.ox.ac.uk/objects/uuid:8012c839-6a9c-47f9-83a6-b4a0f1d23b93.
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This thesis explores the potential of new substrates for ancient DNA studies and addresses novel questions that can now be asked. It also highlights an additional use of ancient DNA extracted from a traditional source.
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Lempidaki, Styliani. „Study of DNA double strand break repair in Dictyostelium discoideum“. Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:3d0035a5-6f17-435d-990f-22ec24ec441e.
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The homologous recombination (HR) pathway contributes to genome integrity by mediating double strand break (DSB) repair using a homologous DNA sequence as a template. In mammals Rad51 and Brca2 are molecules central to this process. Little is known about HR repair in Dictyostelium. However, research previously conducted on DSB repair using this organism has shown that DSB repair pathways are highly conserved when compared to humans. This encouraged study of HR in this organism. In this study, through a bioinformatics search I have identified putative orthologues of most human HR proteins and most interestingly of BRCA2, which cannot be found in other lower eukaryotes used as models for DSB repair, such as the budding yeast S.cerevisiae. Brcp, the Dictyostelium BRCA2 ortholog, shows similar domain structure when compared to BRCA2-related proteins identified in other organisms. To verify the implication of HR proteins in DSB repair, I developed a method to monitor recruitment of DNA repair proteins on chromatin upon DSB induction. Findings of this study suggest that both Brcp and Rad51 get recruited to chromatin upon DSB induction and are therefore implicated in DSB repair in Dictyostelium. To further study Brcp function and based on findings suggesting that disruption of brcp might be lethal, I developed a novel system for specific and conditional depletion of endogenous Dictyostelium proteins. Utilizing this system, I conducted phenotypic studies in a strain depleted of Brcp to examine its role in DNA repair. Overall this study shows that the HR pathway in Dictyostelium shows great similarity to vertebrates, making Dictyostelium an appealing model for the study of DSB repair and specifically HR.
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Journot, Céline M. A. „On the interaction of DNA nanostructures with lipid bilayers“. Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:f4d0fd64-7916-473e-a8ea-4b36745f8254.
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Much of our knowledge of cellular biology arises from direct observation of active cellular functions. Tools and techniques have steadily developed over the past several hundreds of years to aid in our understanding and control of the nanoworld and are referred to as nanotechnologies. In the context of nanotechnology, DNA is not used as a carrier for genetic information (as it is in cell), but as a construction material. DNA offers unprecedented control over the construction of simplified biomimetic models for the study of biological processes. This thesis first introduces and defines the field of DNA nanotechnology, with particular emphasis on the interaction of snthetic DNA nanostructures with biological membranes. Inspired by the protein clathrin, three-fold symmetric DNA tile made of eight, short DNA strands and capable of polymerising is described and studied, with the aim to interact with and controllably bend a membrane bilayer. This structure presented challenges during construction so an enhanced three-armed DNA structure built with DNA origami was designed. The succesful assembly of a rigid and functionalisable nanostructure is described. This origami structure was polymerised into large constructs in solution and on a supported lipid membrane. The shape of the structure was modulated to vary its curvature and apply a bending force to a lipid vesicle when anchored to it. Following the conclusion of this study, we present the construction of a small, unique DNA structure for enhanced electron microscopy imaging in cell lysate. This project is part of a developing technique to couple the interaction specificity of dyes in super-resolution microscopy and the high-resolution output of electron microscopy. Finally, the optimisation procedures and recommendations for TEM imaging of samples of DNA origami and lipid structures are discussed.
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Foskolou, Iosifina Petrina. „The role of hypoxia-induced RRM2B in DNA replication“. Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:e552002e-9303-4968-babe-6092331c2540.
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Regions of low oxygen (hypoxia) occur in most solid tumours and correlate with poor patient prognosis due to their resistance to chemo- and radiotherapy and to their increased metastatic potential. Severe levels of hypoxia induce DNA replication stress characterised by an increased number of stalled replication forks and significantly reduced replication rates, which occurs in the absence of DNA damage. Ribonucleotide Reductase (RNR) is the only enzyme capable of de novo synthesis of dNTPs - the building blocks of DNA synthesis and repair. However, oxygen is essential for mammalian RNRs (RRM1/RRM2 and RRM1/RRM2B), leading us to question the source of dNTPs in hypoxia. Here we show that the RRM2B subunit of RNR is significantly induced in response to hypoxia in a universal manner. Interestingly, the hypoxic induction of RRM2B occurs both at transcriptional and translational levels and likely through two distinct mechanisms, one of which is p53-dependent. Most importantly, we demonstrate that RRM1/RRM2B enzyme is capable of retaining activity in hypoxia and therefore is favoured over RRM1/RRM2 in order to preserve on-going replication. We found two distinct mechanisms by which RRM2B maintains hypoxic activity and we identified specific RRM2B-residues (Y164 and K37/K151) responsible for this function. The importance of RRM2B in the response to tumour hypoxia is further illustrated by increased expression in the hypoxic regions of glioblastoma biopsies, roles in tumour growth and radioresistance, as well as prevention of DNA damage and apoptosis. In this study we present multi-disciplinary evidence, demonstrating the molecular rationale for the ability of RRM1/RRM2B to function in hypoxia. We propose that RRM2B has been evolutionary conserved so as to act as the hypoxic specific RNR subunit in order to be able to react promptly when this physiologically relevant stress occurs. Our data provide new insight into RNR biology, highlighting RRM2B as an important, hypoxic-specific, anti-cancer therapeutic target.
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Neo, Jacqueline Pei Shan. „The importance of DNA replication termination and the MHF complex to genome stability“. Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:d8f726f9-e18e-43e0-b1f7-96c4f9afb064.
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The final stages of replication fork termination requires the timely and orderly orchestration of catalytic and enzymatic activities. Given the complexity of this process, it is conceivable that the final stages of fork termination is susceptible to problems that could trigger recombination, which could lead to deleterious genomic rearrangements if ectopic homologous sequences are recombined. Using the site-specific RTS1 barrier in fission yeast, I demonstrated that fork termination is generally not a recombinogenic process, and that hyper-recombination-induced by fork blockage at RTS1 is largely a result of replication fork restart. To investigate the actual mechanisms and proteins, which drive and influence recombination at a replication barrier, I studied the MHF proteins, which assist Fml1 in limiting crossovers during double-strand break (DSB) repair and promoting Rad51-mediated recombination at impeded replication forks, and are also components of the constitutive centromere-associated network (CCAN). Intriguingly, structural studies revealed that the MHF can exist as an octamer in vitro. I examined the biological significance of octameric MHF by employing three mutations that disrupt the octamer configuration in vitro. In fission yeast, these mutations cause hypersensitivity to methyl methanesulfonate (MMS), suggesting that the MHF octamer may have a role in DNA repair. One of the “octamerisation” mutants, exhibits greater hypersensitivity to MMS than the other two, and biochemical experiments indicated that this is because it confers an additional defect in MHF’s interaction with Fml1. Further genetic experiments on this mutant suggest that the ability of Fml1 to unwind D-loops depends more critically on its interaction with MHF than fork reversal. Additionally, I showed a synergistic interaction between Dcr1 and MHF, and demonstrated that in the absence of Dcr1, there is a greater need for recombination to tolerate/repair DNA damage. Lastly, I uncovered a novel function for the MHF in controlling the initiation of septation.
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Latusek, Robert. „The role of LATS1 in DNA damage signalling“. Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:7f21ecaf-20a6-45a9-8403-c8255955920e.
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Genomic DNA is constantly exposed to assaults, which if not dealt with, can lead to genomic instability and carcinogenesis. In response to stress including either Ionising Radiation (IR) or replication stress, ATM and ATR promote the activation of cell cycle checkpoints and initiate repair of DNA damage. Recent studies have revealed that ATM signalling can activate LATS1 via a cascade through RASSF1A and MST2. LATS1 is a tumour suppressor, which forms a barrier to carcinogenesis restricting cell proliferation and promoting apoptosis by stabilising a YAP/p73 transcriptional complex, hence upregulating p73 responsive genes. LATS1 is inactivated through promoter hypermethylation in a number of cancer types including breast cancer and soft tissue sarcoma. This research project seeks to define the mechanism through which LATS1 is involved in IR-induced DNA damage signalling. The data presented in this thesis shows that LATS1 controls CDK2 and regulates phosphorylation of S3291 on BRCA2. Cells lacking LATS1 exhibited enhanced accumulation of damage-induced Rad51 foci leading to cell cycle arrest at the G2/M checkpoint. Furthermore, the data presented here suggests that LATS1 may not be required for homologous recombination. This work supports the hypothesis that LATS1 inhibits CDK2-dependent phosphorylation of BRCA2 at S3291, hence protecting stalled replication forks from nucleolytic degradation.
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Minall, Leanne. „Synthetic glyco-viruses for targeted DNA vaccination vectors“. Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:9a7d2e5a-9053-40c6-969c-40dd39f2d02a.
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Viruses are powerful vectors for the delivery of nucleic acids, with applications in gene therapy and vaccination. However, major challenges for this technology include mis-targeting of the vector and neutralization by host antibody responses. Here we show that chemical addition of synthetic glycans to adenovirus (Ad) increased resistance to neutralizing antibodies and other critical host clearance mechanisms. Carbohydrate ligands were synthesised and functionalised with a cyanol methyl thiol group, which was subsequently activated to the amine reactive 2-imino- 2-methoxyethyl (IME). Ads were coated with ligand whilst maintaining virus cogency, with up to 58% of total Ad lysine resides glycosylated. The viral tropism of glyco-Ads was switched to target macrophages and dendritic cells (DCs), with high selectivity for the complementary sugar receptors. In vaccination studies, DC targeted glyco-virus enhanced antigen-specific T cell responses. Thus, chemical glycosylation of the Ad capsid allows modulation of tropism and shielding from sequestration and neutralizing antibodies. Since manipulation of this process is facile, it provides a flexible and potentially universal solution to key obstacles facing the utilization of viral vectors in therapeutic and vaccination contexts.
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May, Peter F. J. „Tethered fluorophore motion studies of DNA segregation machinery“. Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:1d909673-82a4-4b03-bb33-46e2a1526f63.
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Tethered fluorophore motion (TFM) is a single-molecule fluorescence technique, used to monitor an effective length along a biopolymer, such as DNA. In this work, I start by characterising TFM, focussing on the theoretical basis and potential applications, and then use TFM to study the bacterial site specific recombination system, XerCD-dif. I present a description of the sources of noise in TFM and derive an analytical expression for the resolution, which is compared to simulations and experiments. The work demonstrates that length changes as low as 100 bp of double-stranded DNA are distinguishable using TFM. The optimum pixel size for TFM is derived and validated experimentally. XerCD-dif recombination is responsible for chromosome decatenation in most bacteria with circular chromosomes. It is activated by the DNA translocase FtsK. I describe the application of TFM, in combination with Förster resonance energy transfer (FRET) and protein induced fluorescence enhancement, to observe the formation and activation of XerCD-dif synaptic complexes. The work followed the reaction, as Holliday junctions formed by XerD-mediated strand exchange isomerised and were resolved to product by XerC, and measured the lifetimes of all the intermediates observed. To analyse the role of FtsK in the activation of recombination, and to monitor its assembly, translocation and behaviour when encountering XerCD-dif, TFM was extended with the use of two spectrally distinct fluorophores, allowing simultaneous monitoring of two effective lengths along the same DNA. FtsK was observed to assemble on DNA, stepwise, into a single hexamer, and begin translocation rapidly (~0.25 s). Single hexamers approached XerCD-dif, and resided for 0.5s to 1s before dissociating, irrespective of whether XerCD-dif was synapsed or recombination was activated. The final section of this thesis details the design, construction and evaluation of a three colour microscope. Alternating laser excitation was implemented in the green, red and near-infrared spectral channels with three laser sources, and an inexpensive automatic focus system was implemented using the back reflection of the excitation lasers and a complementary metal oxide semiconductor camera.
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Carlos, A. R. „DNA damage responses to loss of telomere integrity“. Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:27bcf3b6-edb9-47e2-af7c-c7ba9b431572.
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Linear genomes end in characteristic structures consisting of repetitive DNA and proteins: the telomeres. These play two critical roles: on one hand they avoid the of loss of genetic information due to the incomplete replication of the chromosome ends and on the other, they provide capping structures for chromosome termini, differentiating them from double strand breaks. Telomeres contain specialized proteins (the shelterin complex), as well as proteins present elsewhere on the chromosomes (chromatin remodelling, DNA damage repair and response factors). Interestingly, several DNA damage factors are required for proper telomere maintenance, drawing a thin line between telomere protection and their recognition as broken DNA ends. Loss of telomere integrity has severe consequences for the cell, namely it can induce replicative senescence and cellular aging, or it can contribute to tumorigenesis. How telomeres are capped and how they are perceived by the cell when they become dysfunctional is essential for our understanding of the contribution of loss of telomere integrity to aging and disease. In order to unravel new factors involved in telomere maintenance, siRNA screens were performed. The optimization process has confirmed both telomeric foci and telomere dysfunction-induced foci (TIFs) as suitable readouts and the screens performed generated a list of potential candidate genes involved in telomere biology. Although some of the candidate genes tested in this work failed the validation process, other genes deserve further analysis. In addition this work also studied the role of several DNA damage factors at uncapped telomeres. Furthermore, BRCA1, CtIP and EXO1 were found to be critical for the formation of end-to-end fusions generated after TRF2 inactivation. The requirement of this proteins in this process, suggests that not only that not only the classical non-homologous end joining (C-NHEJ) pathway is active at TRF2-depelted telomeres, but emphasises the multiplicity of mechanisms that act to repair dysfunctional telomeres.
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Roy, Noemi Bernadette Alice. „The study of DNA methylation anomalies in chronic lymphocytic leukaemia“. Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:af6091aa-3d00-4436-b2b7-5dfe439b5c05.
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Many haematological malignancies are associated with widespread alterations of the transcriptional and epigenetic programmes. Changes in DNA methylation provide the clearest example of epigenetic changes, but the mechanism(s) underlying such changes is unknown. To investigate this I studied DNA methylation across an ~80kb segment of the genome which is not known to be mutated in haematological malignancies. Methylation was perturbed in 35-100% of samples of DNA from individuals with a wide range of haematological malignancies but not in non-malignant haematological disorders. DNA methylation was comprehensively assessed by Southern blot analysis, classical bisulphite sequencing and using a newly developed capture bisulphite sequencing protocol. The results were also compared with analysis by MeDIP, an immunoprecipitation-based technique. These analyses provide methylation status at various levels including individual CpG resolution. This showed both gain and loss of methylation at CpG dinucleotides. Of interest, hypomethylation was most frequently seen in intergenic regions corresponding to transcription factor binding sites and areas of increased chromosome accessibility. These observations suggested that hypomethylation of the genome in haematological malignancies could arise from aberrantly expressed DNA binding proteins which, recruited to sequences in regions of open chromatin, would protect the underlying CpG dinucleotides from the methylation machinery. This, in turn, could lead to passive demethylation accumulating with increasing cell divisions. This hypothesis was tested with electrophoretic mobility shift assays using oligonucleotides representing the DNA underlying one such region. This showed that, compared to nuclear extracts from the lymphocytes of normal individuals, those from patients with CLL were enriched for a protein which binds to oligonucleotides containing the underlying sequence. Using a mass spectrometry approach, I identified a variety of proteins that may bind such regions and account for their passive demethylation in haematological malignancies.
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Stracy, Mathew. „Single-molecule studies of DNA-binding proteins in live bacteria“. Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:d2b6ad0a-0fd4-4742-9ca2-dc13ba95f267.
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Protein-DNA interactions are critical to many important biological functions, from transcription to DNA replication. To better understand these processes we need to look at molecular details, such as the stoichiometries and binding kinetics of these proteins. However, focusing on the molecular level can miss the bigger picture; we also need to understand how protein-DNA interactions shape the organisation of chromosomes and cause phenotypical changes over the whole cell. In this thesis I describe the construction of a super-resolution fluorescence microscope to image single molecules in live bacteria, and show how analysis with tools like single-particle tracking allow individual proteins specifically bound to DNA to be distinguished from mobile molecules, offering a new perspective on protein-DNA interactions, from the molecular level to the length scale of whole bacterial cells. I detail how I have applied these techniques to answer key questions in transcription, chromosome organisation, and DNA segregation in Escherichia coli. Firstly, I looked at RNA polymerase (RNAP) to study how transcription affects the organisation of the nucleoid. Discriminating specifically bound RNAPs showed that low levels of transcription can occur throughout the nucleoid, but clustering analysis and 3D Structured Illumination Microscopy (SIM) showed that dense clusters of transcribing RNAPs format the nucleoid periphery, indicating a movement of gene loci out of the bulk of DNA as levels of transcription increase. Furthermore, I developed an assay to characterise the search process and non-specific DNA interactions of RNAP, which I also apply to a diverse selection of other DNA-binding proteins. I also characterized the in vivo behaviour of the type II topoisomerase, TopoIV. Imaging both subunits of TopoIV, combined with over-expression of unlabelled subunits, allowed the fraction of functional enzymes to be determined. Measuring the duration of catalytic events indicated that the majority of active TopoIV molecules catalyse decatenation. Finally, I studied MukBEF, an SMC (Structural Maintenance of Chromosomes) complex that acts in chromosome segregation, to show that TopoIV and MukB interact directly in vivo and determine the dissociation constant and turnover of this TopoIV-MukB complex.
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Orlando, Giulia. „Molecular mechanisms of ARF regulation in response to DNA damage“. Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:fde8ab19-bc7f-4ad8-8396-6c5f5f385d34.
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DNA is a highly unstable molecule. Endogenous souces of DNA damage, such as reactive oxygen species (ROS), can cause DNA damage and it has been estimated that 20000 lesions occur in a cell per day. BER is the major pathway for the repair of these lesions and therefore maintains genome stability, thus preventing the development of human diseases such as neurodegenerative diseases and cancer. Therefore, if BER cannot accomplish the repair, accumulation of DNA damage occurs, triggering different cellular responses, such as cell cycle delay and senescence. The ARF tumour suppressor protein, the gene of which is frequently mutated in many human cancers, plays an important role in the cellular stress response by orchestrating upregulation of p53 protein. Moreover, ARF expression is upregulated in senescent cells, suggesting that ARF induction might be triggerred in response to persistent DNA damage. Although ARF has been reported to be important in the regulation of proteins involved in the DNA damage response, its role is still controversial. Here, it has been shown that ARF gene transcription is induced by DNA strand breaks (SBs) and that ARF protein accumulates in response to persistent DNA damage generated by disabling BER. These data suggest that PARP1-dependent poly(ADP-ribose) synthesis at the sites of SBs initiates DNA damage signal transduction by reducing the cellular concentration of NAD+, thus inhibiting SIRT1 activity and consequently activating E2F1-dependent ARF transcription. These findings suggest a vital role for ARF in DNA damage signalling, and furthermore explain the critical requirement for ARF inactivation in cancer cells, which are frequently deficient in DNA repair and accumulate DNA damage.
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Cerf, Aline. „Assemblage dirigé de nano-objets“. Phd thesis, INSA de Toulouse, 2010. http://tel.archives-ouvertes.fr/tel-00525067.
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Un échange vigoureux au travers des frontières de la biologie et de la physique se développe autour de nouvelles méthodes et outils, et autour de nouveaux phénomènes. Les objets d'étude au coeur de ce recouvrement multidisciplinaire sont très divers. De manière non exhaustive, il s'agit de nanoparticules, de cellules ou encore d'objets encore plus petits et élémentaires tels que les molécules. Aussi bien pour des applications dans le domaine de la microélectronique que pour l'étude de mécanismes biologiques fondamentaux, l'intégration des objets d'intérêt à l'échelle de l'objet unique est essentielle. Dans le cadre de cette thèse, l'objectif que nous nous sommes fixés est de développer un volet technologique qui permette l'assemblage d'objets micro- ou nanométriques uniques à des endroits bien définis d'une surface solide de façon simple, fiable, bas-coût et parallèle. Pour ce développement, nous nous sommes intéressés tout particulièrement aux nanoparticules d'Au de 100 nm de diamètre, aux bactéries, puis aux molécules d'ADN. Nous décrirons les stratégies développées reposant sur la lithographie douce puis leurs potentialités pour différentes applications dans les domaines de l'analyse médicale et de la détection.
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Beattie, Thomas R. „The molecular biology of DNA replication in the archaeon Sulfolobus solfataricus“. Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:99d668a5-2d7a-4c7f-a1f8-b514e699347e.
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DNA replication is essential for the propagation of all living organisms. The ability of a cell to accurately duplicate its entire genome is dependent upon the activity of numerous proteins. Identifying the molecular mechanisms by which these proteins act, and determining how they are physically and functionally coordinated at sites of active DNA replication, is central to understanding this essential cellular process. Archaea possess a DNA replication machinery which is ancestral to the one present in eukaryotes, and thus these organisms serve as simplified model systems for understanding the complexities of eukaryotic DNA replication. This thesis investigates the molecular mechanisms underlying Okazaki fragment maturation in the crenarchaeon Sulfolobus solfataricus, which is essential to the completion of lagging strand DNA replication. Reconstitution of Okazaki fragment maturation in vitro demonstrated that the activities of three enzymes – PolB1, Fen1, and Lig1 – are required for this process in S. solfataricus. Furthermore, it was shown that optimum coordination of their three distinct activities is dependent on the ability of PolB1, Fen1 and Lig1 to simultaneously interact with a single PCNA ring, providing evidence for a mechanism of multi-enzyme coordination which may be universally employed by DNA sliding clamp proteins. The importance of protein flexibility in the accommodation of multiple proteins around a single PCNA was also investigated. Finally, the physical coordination of one of these key maturation enzymes – PolB1 – with other replisome proteins was examined. It was demonstrated that PolB1 exists in a trimeric complex in vivo with two previously unidentified factors, raising the possibility of uncharacterised activities and interactions for this crucial enzyme. Taken together, these data provide new insights into functionally important protein-protein interactions within the archaeal replisome, and facilitate a greater understanding of the DNA replication machinery in both archaea and eukaryotes.
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Stoddart, David. „Progress towards ultra-rapid DNA sequencing with protein nanopores“. Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:597b217c-c995-401d-b772-4cb373d195af.
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The sequencing of individual DNA strands with nanopores is being developed as a rapid, low-cost platform in which bases are identified in order as a DNA strand is transported through a pore under an electrical potential. Several challenges remain and this thesis focuses on one major area, the base identification properties of the a-hemolysin (aHL) nanopore. Under the potentials required for threading, DNA translocates too fast for single bases to be identified. However, immobilization of the DNA within the pore increases the residence time and therefore improves the precision of the electrical current reading and allows for the small differences in current flow, associated with different sequences, to be observed. DNA molecules with a 3'-terminal biotin-tag were complexed with streptavidin. Streptavidin is too large to be transported through the aHL pore and therefore the DNA-btnestreptavidin complex is not fully translocated; thus, the DNA strand is immobilized within the pore. Using this approach the nucleobase recognition properties of the aHL pore were mapped. The data suggest that the transmembrane 13 barrel domain of the pore contains at least three nucleobase recognition sites, termed R1, R2 and R3. Additional sequence information can be gained when multiple recognition sites are employed within a single aHL pore, as compared to the simple case of a single recognition site. Recognition site R1, which is located near the central constriction, can be modified by site-directed mutagenesis of Met-113. It was observed that amino acids with related side chains produce similar patterns of nucleobase recognition. Amino acids that provide an energy barrier to ion flow (e.g. bulky or hydrophobic residues) strengthen base identification, while amino acids that lower the barrier, weaken identification. Deletion and site-directed mutagenesis were used to remove one recognition site and generate an αHL pore. With truncated β barrel domain that contains only two recognition sites.
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Hua, Hui. „Regulation of DNA replication during meiosis in fission yeast“. Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:5d4d66ab-5441-4e96-adb1-28f4b51a975b.
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The interval between meiotic nuclear divisions can be regarded as a modified mitotic cell cycle where DNA replication is blocked. Mechanisms regulating this critical aspect of meiosis that allows haploid cells to be generated from a diploid progenitor were investigated in this project. Licensing is restricted after meiosis I due to down-regulation of Cdc18 and Cdt1. Late meiotic expression of Cdc18 and Cdt1, which load the MCM helicase onto replication origins, can lead to partial DNA replication after meiosis I. This implies that block to initiation via licensing forms an important component of this regulation. As detecting any minor DNA re-replication after meiosis I requires a technique more sensitive than flow cytometry for detection of total cell DNA contents, I also investigated a procedure to allow incorporation and detection of 5-ethynyl-2'-deoxyuridine (EdU) in fission yeast. Additional inactivation of Spd1 or stabilization of Dfp1 after MI when Cdc18 and Cdt1 are also expressed does not enhance re-replication, but cyclin-dependent kinase Cdc2 plays a role in preventing re-replication during the MI-MII interval. Unexpectedly, when the licensing block is subverted, replication forks only move a short distance in the interval between meiosis I and II, implying that the elongation step of DNA replication is also inefficient. In addition, I show that the regulation of entry into meiosis II is not delayed by a partial round of DNA replication or DNA damage, indicating that replication and DNA damage checkpoints do not operate in late meiosis.
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Snodin, Benedict E. K. „Simulating large DNA nanostructures with a coarse-grained model“. Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:86b2d03e-3cad-423c-8db0-3d56c65ccc20.
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In this thesis we investigate the self-assembly and structural properties of DNA origamis, which are large-scale DNA nanostructures comprising thousands of DNA base pairs, using the coarse-grained model of DNA called oxDNA. We use simulations of oxDNA, with both a "brute-force" approach and with a rareevent method, to probe how the origamis self-assemble from single strands of DNA in solution. With the brute-force approach we are able to simulate the complete assembly of a small 384-base-pair origami at a high strand concentration, and identify kinetic traps under certain conditions, as well as cooperativity between staple strands and optimal assembly windows. These findings help to rationalise certain successful design strategies. Using the rare-event method we are able to use the lower strand concentrations typical of experimental conditions and make more quantitative measurements, identifying sequential staple binding for our origami design at these conditions, and probing how a staple binds each of its domains. We introduce oxDNA2, an enhanced version of oxDNA, which has improved predictive power for origami structure, as well as other improvements such as salt dependence. We verify that the model reproduces well the structural properties of Holliday junctions, which are a vital feature of DNA origamis. We then use the model to investigate the basic structural properties of 2D and 3D origamis, characterising their generic structural features such as, for 2D structures, the "weave pattern," where adjacent double helices push apart away from the junctions, and "corrugation," a systematic, out-of-plane bending of the double helices. We find a good agreement with experiment where data is available.
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Tomkova, Marketa. „The relationship between DNA modifications and mutations in cancer“. Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:013eed80-45e9-48b9-84a7-718c6111e407.
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Somatic mutations are the main triggers that initiate the formation of cancer. Large sequencing data sets in recent years revealed a substantial number of mutational processes, many of which are poorly understood or of completely unknown aetiology. These mutational processes leave characteristic sequence patterns, often called "signatures", in the DNA. Characterisation of the mutational patterns observed in cancer patients with respect to different genomic features and processes can help to unravel the aetiology and mechanisms of mutagenesis. Here, we explored the effects of DNA modifications and DNA replication on mutagenesis. The most common mutation type, C>T mutations in a CpG context, is thought to result from spontaneous deamination of 5-methylcytosine (5mC), the major DNA modification. Much less is known about the mutational properties of the second most frequent modification, 5-hydroxymethylcytosine (5hmC). Integrating multiple genomic data sets, we demonstrate a twofold lower mutagenicity of 5hmC compared to 5mC, present across multiple tissues. Subsequently, we show how DNA modifications may modulate various mutational processes. In addition to spontaneous deamination of 5mC, our analysis suggests a key role of replication in CpG > TpG mutagenesis in patients deficient in post-replicative proofreading or repair, and possibly also in other cancer patients. Together with an analysis of mutation patterns observed in cancers exposed to UV light, tobacco smoke, or editing by APOBEC enzymes, the results show that the role of DNA modifications goes beyond the well-known spontaneous deamination of 5mC. Finally, we explored which of the known mutational processes might be modulated by DNA replication. We developed a novel method to quantify the magnitude of strand asymmetry of different mutational signatures in individual patients followed by evaluation of these exposures in early and late replicating regions. More than 75 % of mutational signatures exhibited a significant replication strand asymmetry or correlation with replication timing. The analysis gives new insights into mechanisms of mutagenicity in multiple signatures, particularly the so far enigmatic signature 17, where we suggest an involvement of oxidative damage in its aetiology. In conclusion, our results suggest that DNA replication or replication-associated DNA repair interacts with most mutagenic processes.
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Jalan, Manisha. „Investigating the recombinational response to replication fork barriers in fission yeast“. Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:aed1673a-f967-41a5-9643-2e432052e174.
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Timely completion of DNA replication in each cell cycle is crucial for maintaining genomic integrity. This is often challenged by the presence of various replication fork barriers (RFBs). On collision with a RFB, the fate of the replication fork remains uncertain. In some cases, the integrity of the fork is maintained until the barrier is removed or the fork is rescued by merging with the incoming fork. However, fork stalling can cause dissociation of all of the associated replication proteins (fork collapse). If this occurs, the cell's recombination machinery can intervene to help restart replication in a process called recombination-dependent replication (RDR). Programmed protein-DNA barriers like the Replication Terminator Sequence-1 (RTS1) have been used to demonstrate that replication fork blockage can induce recombination. However, it remains unclear how efficiently this recombination gives rise to replication restart and whether the restarted replication fork exhibits the same fidelity as an origin-derived fork. It is also unknown whether accidental replication barriers induce recombination in the same manner as programmed barriers. In this study, I introduce recombination reporters at various sites downstream of RTS1 to obtain information on both the fidelity and efficiency of replication restart. I find that unlike break induced replication (BIR), the restarted fork gives rise to hyper-recombination at least 75 kb downstream of the barrier. Surprisingly, fork convergence, rather than inducing recombination, acts to prevent or curtail genetic instability associated with RDR. I also investigate a number of genetic factors that have a role in either preventing or promoting genome instability associated with the progression of the restarted fork. To compare RTS1 with an accidental protein-DNA barrier, a novel site-specific barrier system (called MarBl) was established based on the human mariner transposase, Hsmar1, binding to its transposon end. Replication fork blockage at MarBl strongly induces recombination, more so than at RTS1. This appears to be a general feature of accidental barriers as introduction of the E. coli TusB-TerB site-specific barrier in S. pombe gives rise to a similar effect. Here, I compare and contrast accidental barriers with programmed barriers. I observe that there is very little replication restart, if any, at MarBl measured by direct repeat recombination downstream. This points to the fact that accidental barriers do not trigger fork collapse in the same way as programmed RFBs and that the increased recombination that they cause may be a consequence of the inability of replication forks to terminate correctly, owing to the bi-directional nature of the barrier. Several genetic factors are assessed for their impact on MarBl-induced recombination, which further highlights both similarities and differences with RTS1-induced recombination.
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Lally, Parminder. „Construction of a synthetic ribosome using DNA as the building material“. Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:26573255-79bc-482d-9dd9-8c9f771ccbd8.
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This thesis forms part of an ongoing project in the DNA Group to build and operate a synthetic ribosome. We present two synthetic ribosome designs that can be combined with DNA-templated chemistry to generate libraries of functional synthetic small molecules. In Chapter 2 we use the DNA strand displacement technique to construct a mechanism that is capable of moving along a DNA track. We explore ways to control the speed and the driving force of the mechanism, and present a mathematical model of the system. We discuss the ability of the design to incorporate chemically-functionalised DNA strands. In Chapter 3 we use a 2D DNA origami tile as the basis of the synthetic ribosome mechanism. Functionalised DNA strands are arranged on the surface of the tile, and we demonstrate the ability to template reactions between the strands, and discuss the possibility of creating a library of distinct chemical products from a single origami tile.
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Fry, Andrew Mark. „Phosphorylation of the human topoisomerase II protein“. Thesis, University of Oxford, 1992. http://ora.ox.ac.uk/objects/uuid:70d2dbb9-d3fe-43ed-8206-44a95202eeff.
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DNA topoisomerase II is an essential enzyme in eukaryotes and is required for many aspects of DNA metabolism including DNA replication, recombination, chromosome segregation and chromosome condensation. It is also a major component of the nuclear scaffold. Topoisomerase II from lower eukaryotes has been shown to be phosphorylated in vivo and this phosphorylation leads to a modulation of activity. However, unlike these lower eukaryotes, human topoisomerase II exists as two closely related, but genetically distinct, isozymes which have markedly different expression and localization patterns. Topoisomerase IIα is a 170kDa protein and topoisomerase IIβ is 180kDa. This study set out to analyse the phosphorylation of these specific isozymes and understand how this leads to the regulation of their distinct biological functions. In order to undertake this study, two polyclonal anti-topoisomerase II antibodies were generated and a series of other polyclonal and monoclonal antibodies characterized. Furthermore, the α isozyme of human topoisomerase II was purified to near homogeneity from cultured HeLa cells. A kinase activity with the biochemical characteristics of casein kinase II co-purified with and could phosphorylate the purified topoisomerase Hot protein. The α and β isozymes of human topoisomerase II were both shown to be phosphoproteins in vivo. The α isozyme is phosphorylated predominantly on serine residues but with a minor proportion of phosphothreonine. Both the α isozyme and a stable ISOkDa fragment of the β isozyme are phosphorylated in vitro by casein kinase II and the catalytic subunit of PKA (cAMP-dependent protein kinase). The α isozyme can also be phosphorylated in vitro by Ca2+-dependent and -independent isozymes of protein kinase C and the cell cycle-regulated p34cdc2 kinase. Two-dimensional tryptic phosphopeptide mapping suggested that the pattern of phosphorylation of human topoisomerase Ha protein in vivo is complex with phosphorylation occurring on multiple residues. Comparison with in vitro maps suggested that casein kinase II and PKA could account for most of the phosphorylation seen in vivo. Using a one- dimensional phosphopeptide mapping approach, a major site of phosphorylation in vivo appeared to be within the C-terminal 20kDa, and that casein kinase II, PKA and PKC may all phosphorylate this region. Phosphorylation of human topoisomerase Hoc protein by casein kinase II, PKA and PKC all led to a stimulation of activity as measured by plasmid relaxation and decatenation. In contrast, dephosphorylation led to a marked decrease in activity of the enzyme. The dephosphorylated enzyme could be reactivated by casein kinase II but not PKA phosphorylation. These data suggest that phosphorylation plays a crucial role in the control of DNA tertiary structure in human cells via regulation of the activity of topoisomerase II proteins.
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Evans, Geraint Wyn. „Real-time single-molecule observations of conformational changes in DNA polymerase“. Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:fdf11b59-2e58-4174-9219-9d61e4528f65.
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Genetic information is encoded in the long sequence of bases which form DNA, which is replicated during cell division by enzymes known as DNA Polymerases. Polymerases replicate DNA extremely accurately to avoid errors which can cause cell death and diseases such as cancer, although the mechanisms behind these extraordinary fidelities are not well understood. A large conformational change in the protein, in which the “fingers" subdomain closes around an incoming nucleotide, is thought to be implicated in these fidelity mechanisms. Here we present an assay to monitor this conformational change in single polymerase molecules, in real-time. We achieve this using total-internal-reflection-fluorescence (TIRF) microscopy to monitor the fluorescence resonance energy transfer (FRET) of an intra-protein dye labelled DNA Polymerase I (KF) as it binds to surface-immobilised DNA. Initially, we investigated the polymerase fingers-conformations during the pre-chemistry polymerisation reaction, resolving forward and backward rates which would be challenging to observe using ensemble techniques. These observations confirmed that KF closes rapidly around complementary nucleotide, but we discovered that the reverse step, fingers-opening, is particularly slow relative to chemistry. These finger kinetics act to remove the influence of the reaction rate-limiting step on fidelity, surprising given decades of investigations have focused on the rate-limiting step as the key determinant of fidelity. We also use our kinetic measurements to quantify contributions of different reaction steps to the macroscopic error rate of the polymerase. Subsequently, we developed our assay to investigate the fingers-conformations across the entire DNA polymerisation reaction. We observed single-nucleotide incorporations, and processive DNA polymerisation at high and low nucleotide concentrations, which suggested heterogeneous nucleotide incorporation rates. The observations demonstrated that the post-chemistry slow step that limits processive polymerisation occurs before post-chemistry fingers-opening, or is accounted for by post-chemistry fingers-opening. We observe a correlation in turn-over kinetics and binary complex kinetics, suggesting that turn-over rates could be limited by the intrinsic dynamics of the binary complex, as seen in other protein systems, although more work is needed on this.
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Haugland, Marius Myreng. „A 2'-alkynylnucleotide strategy for site-directed spin labelling of DNA“. Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:c344cf05-813c-4cbe-85a5-b1131ad4c5ad.
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This thesis describes the synthesis of derivatives of 2'-ethynyl-2'-deoxynucleosides, and their conversion into phosphoramidite building blocks for DNA synthesis. Herein is also outlined the preparation of azide-bearing nitroxyl radicals. After site-selective incorporation of 2'-alkynylnucleosides into DNA using a standard solid-supported phosphoramidite protocol, the modified oligonucleotides were spin labelled with the azide-bearing nitroxyl spin labels via the Cu(I)-catalysed azide-alkyne [3+2] Huisgen cycloaddition (CuAAC or 'click' reaction). The destabilising effect of the spin labelling was determined by UV denaturation studies and circular dichroism, and the spin labelled DNA was investigated by EPR spectroscopy. It was found that this novel site-directed spin labelling strategy afforded conformationally restricted systems, and that the structure of the spin label exerts a significant influence on the results.
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Rakhimova, Alina. „Role of histones in DNA double-strand break repair in Dictyostelium“. Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:f6ae6f6a-9f83-4326-89e3-719ea9c43cc0.
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Correct repair of DNA double-strand breaks is crucial for maintenance of genome integrity. Despite data showing the importance of histones variants and histone post-translational modifications in the cellular response to DNA damage, there is still a lack of knowledge concerning the role of histone H3 and its variants as well as histone ADP-ribosylation in such processes. In this work Dictyostelium discoideum was employed as genetically tractable model organism to address the role of histone H3 variants and histone ADP-ribosylation in DNA double-strand break (DSB) repair. Vegetative cells lacking two out of three histone H3 variants - H3b and H3c, were shown not to be sensitive to DNA DSB. No evidence for altered DSB repair was found as phosphorylation of histone H2AX (a marker of DSB) and one of the pathways of DSB repair, non-homologous end joining, were not altered. Altogether, this work demonstrates that H3b and c variants are not required for overall DNA DSB repair in Dictyostelium. Among the core histones histone H2B was discovered to be the major acceptor of ADP-ribosylation by major ADP-ribosyl transferase involved in DSB repair, Adprt1a, in vitro. ADP-ribosylation in vitro was shown to occur on glutamate E18 with E19 being a potential regulator of this modification. Using an epitope-tagged overexpressed H2B, in vivo H2B ADP-ribosylation in response to DSBs was observed in Dictyostelium for the first time. Decreased ADP-ribosylation of epitope-tagged H2B mutated in both E18 and E19 residues was demonstrated. Overall, this work demonstrates the presence of the ADP-ribosylation of H2B in Dictyostelium in response to DSBs and identifies the major site of this modification.
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Chan, Katie Kin Ling. „Overexpression of DNA polymerase beta and its effect on genome stability“. Thesis, University of Oxford, 2006. https://ora.ox.ac.uk/objects/uuid:e7b8d575-6117-4112-a1f5-dd0907041698.
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DNA polymerases play key roles in DNA synthesis by catalysing the polymerisation of deoxynucleotides opposite a parental (template) DNA strand to generate a new or repaired complementary daughter strand. Faithful replication by DNA polymerases is essential in maintaining genome integrity during cell division, DNA repair and DNA recombination. DNA polymerase beta (Pol β) plays a pivotal role in the base excision repair (BER) pathway by performing repair synthesis to fill single nucleotide gaps which arise during DNA repair. However, overexpression of Pol β is found in many human cancers and has been shown to promote a mutator phenotype. The aim of this study was therefore to investigate the effect of Pol β overexpression on BER. In vitro repair assays using whole cell extracts and DNA substrates containing site-specific BER lesions were conducted to compare two cell lines, one of which was derived from a cancer patient overexpressing Pol β. I found that overexpression of Pol β results in a 5 to 10-fold increased frequency of one nucleotide frameshift mutations and based on biochemical studies a mechanism is proposed to explain this phenomenon. I therefore conclude that an excess of Pol β can have potentially mutagenic consequences.
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Shepherd, Marianne E. A. „The cell cycle and DNA damage-dependent regulation of Cdt1 in schizosaccharomyces pombe“. Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:7f2ebafb-7d0d-4c0d-b6a6-277025f71850.
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Cdt1 is a conserved and essential eukaryotic protein that is required for the licensing step of DNA replication. In order to control replication licensing and ensure a single round of DNA replication occurs per cell cycle, Cdt1 is subject to strict regulation. In Metazoa and S. pombe, Cdt1 is targeted for ubiquitylation and proteolysis in S phase and after DNA damage by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 is activated in Metazoa by an unusual mechanism that requires an interaction between the substrate and chromatin-loaded proliferating cell nuclear antigen (PCNA). This study addressed the involvement of PCNA in S. pombe Cdt1 proteolysis. A mutational analysis was undertaken to establish whether the Cdt1-PCNA interaction is conserved in S. pombe and the extent to which it regulates CRL4Cdt2-dependent turnover of the protein. S. pombe Cdt1 was shown to interact with PCNA in vivo and two variant PCNA-interacting peptide (PIP) motifs were identified in the protein. The two motifs function near-redundantly to promote both the Cdt1-PCNA interaction and the CRL4Cdt2-dependent proteolysis of Cdt1 in S phase and after DNA damage. The mutational analysis also resulted in the characterisation of two in-frame AUG codons in the cdt1+ reading frame. The second in-frame AUG codon was shown to be the principal initiator codon and was required to maintain wildtype Cdt1 protein levels and cell viability. CRL4Cdt2 is emerging as an important regulator of proteins that are involved in the control of cell cycle progression and the maintenance of genome stability. However, there are a number of outstanding questions regarding the mechanism and regulation of CRL4Cdt2. In order to address these questions, a genomics approach was taken to identify novel genes involved in Cdt1 regulation. A screen of non-essential S. pombe genes identified 17 candidate genes that, when inactivated, caused up-regulation of Cdt1. Unexpectedly, deletion of genes involved in homologous recombination resulted in a Rad3-dependent up-regulation of Cdt1. Further work is required to establish the biological significance of this finding.
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Blaikley, Elizabeth Jane. „Analysis of nucleotide synthesis and homologous recombination repair in Schizosaccharomyces pombe“. Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:2fcd2cab-e8bc-4164-8068-5c50af5f91e4.
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Nucleotide synthesis is a conserved and highly regulated response to DNA damage, required for the efficient repair of DNA double strand breaks (DSB) by homologous recombination (HR). This is essential to prevent loss of heterozygosity (LOH) and maintain genome stability. The aim of this study was to identify new genes important for HR through roles in damage-induced nucleotide synthesis. A screen was performed to identify S. pombe gene deletion strains whose DSB sensitivity was suppressed by deleting the ribonucleotide reductase (RNR) inhibitor spd1+ to promote nucleotide synthesis. The screen identified a number of genes including ddb1+, cdt2+, rad3+ and csn1+ which have known roles in nucleotide synthesis. Distinct roles were identified for the DNA damage checkpoint in suppressing LOH. rad3+, rad26+, rad17+ and the rad9+, rad1+ and hus1+ genes encoding the 9-1-1 complex were required for DNA damage-induced nucleotide synthesis through Cdt2 induction to promote Spd1 degradation. The HR repair defect of rad3+ and rad26+ deletion strains was partially suppressed by spd1+ deletion. However, the HR repair defect of rad17+, rad9+, rad1+ and hus1+ deletion strains was not suppressed. An additional role was confirmed for Rad17 and the 9-1-1 complex in preventing LOH by promoting DSB resection. A role was identified for the Gcn5 histone acetyl transferase (HAT) protein module, consisting of Gcn5, Ngg1, Ada2 and Sgf29, in suppressing DSB sensitivity by promoting nucleotide synthesis. This was independent of Cdt2 or RNR protein levels. The Gcn5 HAT module was also found to regulate DSB repair pathway choice consistent with previous observations. Deletion of gcn5+, ngg1+ or ada2+ decreased HR and increased non-homologous end joining. Surprisingly, deletion of spd1+ in a gcn5∆, ngg1∆ or ada2∆ background also promoted HR. This predicts a role for nucleotide pools in regulating DSB repair pathway choice. Eleven other candidates showed repeatable suppression of DSB sensitivity following spd1+ deletion. However many of these candidates did not show reduced nucleotide levels. This suggests deleting spd1+ may also suppress DSB sensitivity by a different mechanism.
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Chitnis, Meenali M. „Type 1 insulin-like growth factor receptor inhibition as treatment for urological cancer“. Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:21282ce9-ce6b-4d26-b262-a3fca6d9c9fc.
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The type 1 insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase that mediates diverse cellular functions including growth, differentiation, migration and apoptosis protection. IGF-1R signalling has been implicated in tumorigenesis in a variety of cancers, and IGF-1R inhibitory drugs are currently undergoing clinical evaluation. Previous work in our laboratory has shown IGF-1R over-expression in urological cancers at both the mRNA and protein level, thus making it a potential therapeutic target. The first aim of this project was to develop a protocol for IGF-1R immunohistochemistry, investigate the expression and cellular distribution of the IGF-1R receptor in clear cell renal cell carcinomas (ccRCC), and assess correlation with clinical parameters. In tissue microarray analysis, IGF-1R was detected in ~90% of 195 ccRCCs, with signal in the plasma membrane, cytoplasm and also in the nucleus. The presence of nuclear IGF-1R in up to 50% of ccRCCs and its association with adverse prognosis was a novel finding, and suggests that nuclear IGF-1R may influence ccRCC biology. Further investigations will clarify its role in the nucleus and its potential as a prognostic biomarker. The second aim was to investigate effects of IGF-1R inhibition on radiosensitivity and DNA repair, following previous work in our laboratory showing that IGF-1R depletion enhances chemo- and radio-sensitivity, delays double strand break (DSB) resolution, and may play a role in the homologous recombination (HR) pathway of DNA DSB repair. However, the repair defect seen in these early experiments was larger than could be entirely explained by a defect in HR. The current project used a small molecule IGF-1R tyrosine kinase inhibitor AZ12253801 (AstraZeneca), which blocked IGF-1 induced IGF-1R activation and inhibited cell survival. AZ12253801 enhanced the radiosensitivity of prostate cancer cells, which appeared to be independent of effects of IGF-1R inhibition on cell cycle distribution and apoptosis induction. IGF-1R inhibition delayed the resolution of γH2AX foci, supporting a potential role for the IGF-1R in DSB repair. This delay in focus resolution was apparent at early time-points (less than 4 hr), and was epistatic with DNA dependent protein kinase (DNAPK) inhibition in prostate cancer cells and DNAPK deficiency in glioblastoma cells. These results suggest a role for the IGF-1R in the non-homologous end-joining (NHEJ) pathway of DNA DSB repair. A cell-based reporter assay in HEK-293 cells confirmed that IGF-1R inhibition suppressed DSB repair by NHEJ, helping to explain the radiosensitization demonstrated upon IGF-1R inhibition. There was lack of support for a transcriptional effect, with no significant change observed in gene expression on microarray analysis. Although the mechanism of this effect remains unclear, the observed inhibition of NHEJ has implications for the use of IGF-1R inhibitors in combination with DNA damaging agents in cancer treatment.
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Long, Hannah Katherine. „Evolutionary usage and developmental roles of vertebrate non-methylated DNA“. Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:78b14c1d-1fa3-46f1-815f-a8ba55579c43.
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Vertebrate genomes exhibit global methylation of cytosine residues where they occur in a cytosine-guanine dinucleotide (CpG) context and this epigenetic mark is generally thought to be repressive to transcription. Punctuating this pervasive DNA methylation landscape are short, contiguous regions of non-methylated DNA which are found at two thirds of mammalian gene promoters. These non-methylated regions exhibit CpG content close to expected levels as they escape the depletion of CpGs observed across the methylated fraction of the genome. The unique nucleotide properties of these CpG island (CGI) regions enable their identification by computational prediction in mammalian genomes. Owing to a lack of high-resolution genome-wide DNA methylation profiles in non-mammalian species, these CGI predictions have often been used as a proxy for non-methylated DNA in these organisms. In contrast to mammals, CGI predictions in cold-blooded vertebrates rarely coincide with gene promoters, leading to the belief that CGls are significantly divergent between vertebrate species, and that unique promoter-associated features may have been acquired during warmblooded vertebrate evolution. This thesis is primarily concerned with the location, establishment and biological function of non-methylated islands of DNA in vertebrate genomes. To experimentally determine genome-wide profiles of non-methylated DNA, a novel biochemical technique was established called biotinylated ZF-CxxC affinity purification (Bio-CAP), and development of this method is discussed in Chapter 3. Experimental analysis of non-methylated DNA profiles in this thesis initially addresses two main questions: (1) 'How does the non-methylated DNA landscape compare genome-wide for seven vertebrates considering distinct tissue types and developmental stages?' (2) 'How are vertebrate non-methylated regions of DNA defined and interpreted in the nuclear environment?' To address the first question, non-methylated DNA was profiled by Bio-CAP sequencing across the genomes of seven diverse vertebrate species, representing all major branch points of vertebrate evolution, and the results are discussed in Chapters 4 and S. Contrary to previously held dogma, experimentally determined nonmethylated islands of DNA (NMls) constitute an ancient epigenetic feature of vertebrate gene regulatory elements. However, despite having numerous high-resolution maps of vertebrate non-methylated DNA, the means by which NMls are identified and maintained in the nuclear environment remains poorly understood. To address the second question and identify features which determine the methylation state of DNA, exogenous DNA sequences were introduced into mouse embryonic stem (ES) c~.II~. Non-methylated DNA was profiled by Bio-CAP sequencing to investigate how different features, such as sequence-specific binding motifs, chromatin architecture and nucleotide composition of a given DNA sequence impact local DNA methylation patterns. Interestingly, the majority of exogenous promoters were appropriately non-methylated in mouse ES cells, germline and somatic cells suggesting that gene promoters have retained strong signals for the nonmethylated state across millions of years of evolution (discussed in Chapter 6). During mouse embryogenesis, genome-scale DNA demethylation and remethylation events occur to remodel the epigenetic landscape and loss of DNA methylation during this time leads to embryonic lethality. To investigate the biological function of non-methylated DNA, the third question addressed in this thesis is (3) 'What is the developmental importance of non-methylated islands of DNA during vertebrate embryogenesis?' To investigate this, members of the ZF-CxxC domain-containing family of chromatin modifiers were ablated in zebrafish embryos to perturb the chromatin landscape at NMls, and therefore interfere with their function during early development (Chapter 7). Early embryonic development and patterning was disrupted in knockdown embryos, suggesting that interpretation of non-methylated DNA and placement of chromatin modifications at NMls is essential for normal zebrafish embryogenesis. Together this work sheds light on the evolutionary origins of NMls, the mechanisms involved in the recognition and establishment of nonmethylated loci and provides an insight into the function of non-methylated DNA during early embryonic development.
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Brazauskas, Pijus. „The biological role of DNA modifications deposited over gene bodies“. Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:807458f2-1c34-4474-a0c5-0f82abe7640e.
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5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the most common DNA modifications in vertebrate genomes. Promoter-residing 5mC is a well-established transcriptional repression mark. However, the role of gene body DNA modifications, especially those involving 5hmC, remains elusive and controversial. In this study, the role of genic 5mC and 5hmC in transcriptional regulation is investigated. By engineering a cell line with inducible 5hmC it is shown that deposition of 5hmC causes elevated expression of genes with the highest accumulation of this modification. The transcriptional elevating potential of 5hmC is also confirmed via expression analysis of integrated and transfected artificial reporters. Furthermore, it is demonstrated that promoter proximal gene body DNA modifications can modulate transcription initiation in cis. Finally, the ability of methyl-CpG-binding domain protein 1 (MBD1) to interact with genic DNA methylation and repress transcription is analysed. Upon oxidation of 5mC to 5hmC or demethylation, MBD1 binding is abolished and 5mC-mediated repression is relieved. Altogether, this study suggests that 5hmC plays a de-repressive role and proposes a mechanism by which this is achieved.