225 research outputs found
Data deposition of 'carbon nanotubes allow capture of krypton, barium, and lead for multichannel biological x-ray fluorescence imaging'
No full textThe authors declare that all the data supporting the findings of this study (post-processing) are available within the article and its supplementary information files. Unprocessed data relating to this manuscript are available from the corresponding author upon request and XRF, TEM, Raman, XPS, TGA, NMR, and microscopy data have been deposited on the Oxford database
Synthesis and self-assembly of DNA-β turn Conjugates
No full textThis thesis discusses the synthesis and self-assembly of several DNA-peptide conjugates, through different bi-orthogonal conjugating methods. Chapter 1 explores the current literature and perspective for which this work is based. Chapter 2 discusses the synthesis of DNA by solid phase synthesis, before being modified with a maleimide based phosphoramidite. The chapter goes on to discuss the development of these methods. Commercially bought DNA was modified with two bi-functional crosslinkers, before being further modified with different peptides. The chapter discusses the full scale of method development to obtain DNA-peptide conjugates, including the first known example of DNA β turn conjugates in the literature.
Chapters 3-5 discusses the self-assembly of DNA-peptide conjugates, with peptide directed, DNA and peptide directed, and DNA directed self-assembly respectively. Chapter 3, peptide driven assembly is explored through the doping of the parent peptide ILVAGK with varying amounts of DNA-ILVAGK. These experiments were observed over 28 days, and the resultant structures analysed by atomic force microscopy. In Chapter 4, both peptide and DNA driven self-assembly of DNA-ILVAGK is studied through DNA hybridisation with complementary sequences of 1, 2, and 3 lengths. As in Chapter 3, these studies were supported by atomic force microscopy, dynamic light scattering and circular dichroism. The structures observed in Chapter 4 differed in timeline and type to those seen in Chapter 3. Chapter 5 investigates DNA driven self-assembly with the peptide assembly disabled using sodium dodecyl sulphate. The structures observed during 28 days of aging showed little change in morphology over the period. This is indicative of the presence of hierarchy within this system.
Chapter 6 consists of an overall conclusion, to draw together the individual conclusions drawn in each chapter
Synthesis and Characterisation of Peptide-Oligonucleotide Conjugates Derived from β-sheet Forming or Collagen Mimetic Peptides
No full textGold Nanoparticles as Adjuncts in Electrophoretic Analysis of Biopolymers
No full textThis thesis discusses two projects which investigate the analysis and application of biomolecules. Chapter 1 provides the background and context of this work. Chapter 2 discusses the synthesis and characterisation of three types of gold nanoparticles, sodium citrate, cetyltrimethyl ammonium bromide (CTAB) and 4-dimethylamino pyridine (DMAP) protected gold nanoparticles, as well as two types of polyacrylamide gel, TBE and tris-HCl. Successful characterisation of these gold nanoparticles was carried out by dynamic light scattering, ultra-violet visible spectroscopy and transmission emission microscopy. The characterisation of polyacrylamide gels was achieved by investigation by rheology, optical coherence tomography and scanning electron microscopy. Rheological analysis demonstrated that the addition of gold nanoparticles to polyacrylamide reduces the elasticity of the gel but stabilises the linear viscoelastic range, while not changing the core properties of the material. These materials were then used to separate sulfur containing biomolecules by polyacrylamide gel electrophoresis.
Chapter 3 discusses the efficacy of the three types of gold nanoparticles to separate sulfur containing biomolecules when added to polyacrylamide gel electrophoresis. Citrate and CTAB gold nanoparticles where unsuccessful when used to analyse phosphorothioated DNA, however, DMAP gold nanoparticles showed promising results. DMAP gold nanoparticles were further tested with the proteins lysozyme, bovine serum albumin (BSA), glutathione-S-transferase (GST) and RNAse A (RNAse). The retention of BSA and GST was successful however there was continued problems with visualising lysozyme and RNAse. This led to the development of ATP polyacrylamide gel electrophoresis.
Chapter 4 discusses the theoretical application of the development of catalytic DNA aptamers using in vitro selection. We synthesised a DNA aptamer library, two primers and two biotinylated strands of DNA, to be used in the selection process. However due to poor purity and yields, the project remains in its infancy. We discuss the methods by which we would conduct this research and our justifications for these processes
Synthesis and selection of novel phosphoester oligomers to inhibit the interaction between KRASG12D and RAF1
No full textProtein-protein interactions (PPIs) have been a challenging area for drug discovery, due in part to the lack of small molecule binding sites and large flat surface areas. Whilst there have been some small molecule PPI inhibitors that have successfully passed through clinical trials in recent years, other alternative approaches are being examined that could overcome these issues, including the use of peptide-based inhibition and modified oligonucleotides that can bind across the larger surface area.
Oncogenic RAS proteins are responsible for almost one-third of human cancers, with KRAS being the most prevalent isoform. Oncogenic RAS is trapped in the GTP-bound active state and PPIs with proteins such as RAF and PI3K allows for continuous signaling, leading to tumour formation. Currently, only KRASG12C has clinically approved small molecule inhibitors, which covalently modify the cysteine at position 12, but there is no way to directly inhibit other more prevalent mutations (for example KRASG12D), and a new approach is needed to target these PPIs.
The main objective of this research is to develop and synthesise oligomers incorporating non-nucleosidic monomers that can disrupt the interactions between KRASG12D and RAF1. Seven monomers that offer different chemical reactvities were synthesised and combined in an oligomer library of over 800,000 unique oligomers using the split-and-mix method. Flow cytometry was then used to select oligomers that only bind to KRAS-GMPPnP (a non-hydrolysable analogue of GTP), do not bind to the inactive KRASG12D-GDP, and disrupt interactions between KRASG12D and the RAS binding domain of RAF1, resulting in 200 top binding oligomers. Tandem mass spectrometry was used to determine the sequences of these top binding oligomers, of which six oligomers were resynthesised. Validation assays using an ELISA based RAS-RAF interaction assay showed five of the six top binding oligomers disrupted the interaction between KRASG12D GMPPnP and fluorescently tagged RAF1 and three of these five did not inhibit the interactions of wild type KRAS (KRASWT).
This work demonstrates the first example of novel oligomers synthesised and selected that act as inhibitors of the KRASG12D/RAF1 PPI and has the potential to impact both the field of RAS drug discovery as well as PPI inhibitor drug discovery overall
Nanocarriers for the Targeted Delivery of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) to Tumours
No full textA growing body of evidence suggests that the regular use of non-steroidal anti-inflammatory drugs (NSAIDs), especially aspirin, is associated with a reduced risk of many types of cancer including colorectal, breast, and prostate malignancies. It has also been shown that they might be beneficial in reducing metastasis and lengthening remission periods. However, the prolonged exposure to this class of drugs does not come without a cost as it carries with it the risk of augmenting the serious side effects of NSAIDs. These include haemorrhages, increased bleeding time, peptic ulcers, and hypersensitivity, to name a few. This work aimed at employing the principles of nanotherapy to maximise the benefits of NSAIDs and minimise the risks of their harmful side effects. By exploiting the enhanced permeability and retention effect (EPR) unique to inflammatory tissues, NSAID-loaded nanocarriers could deliver high concentrations of the drugs to cancer tissue, compared to conventional therapy, while reducing the systemic exposure to their effects
Identifying cellular targets of 3',4',5',5,7-pentamethoxyflavone in colorectal cancer
No full textDespite increasing positive prognosis for those living in high income countries (HICs), the incidence of colorectal cancer (CRC) in low- and middle-income countries (LMICs) such as Malaysia, is rising and increasingly becoming a burden for people in these regions. Natural products with promising bioactive properties have been widely investigated for clinical usage and often show positive properties such as reduced toxicity. Flavonoids are polyphenol phytochemicals that often possess bioactive properties. Flavones, a sub-class of the flavonoid family, have shown promise as potential drug compounds in areas such as anti-inflammatory, anti-obesity, antioxidant, and anticancer medicine. 3',4',5',5,7-Pentamethoxyflavone (PMF) has been shown in previous research to possess anticancer and chemopreventive properties in CRC, this research aims to identify potential cellular targets of PMF to give insight into the mechanisms of action of the compound which are currently unknown. A structure-activity relationship study identified the 3'-position as a suitable modification point on the compound structure that could be changed without compromising biological activity. After synthesis of a photoaffinity labelling probe, protein cellular targets were then investigated using pull down experiments and mass spectrometry proteomics. RNA sequencing was also used to investigate changes in gene expression in CRC cells after treatment with the active compound to further aid in identifying a mechanism of action. Members of the RAB subfamily of small GTPases were isolated using the proteomics analysis. Pathway analysis showed that the key interacting proteins were involved in the cellular response to stress and protein folding. This suggests that the flavone could be involved in the unfolded protein response (UPR). RNA sequencing showed that exposure of the cells to the flavone compound caused changes in expression of genes associated with the cell cycle and the UPR. Future studies of PMF should aim to validate the interaction between the protein targets identified and the compound. Synthetic modification of PMF can improve both the binding properties to the target proteins and aqueous solubility of the compound and enable progression as a potential anticancer treatment
Crystal Engineering of Spin-Crossover Materials
No full textSpin-crossover (SCO) materials are transition metal complexes that can switch their spin state in response to external stimuli such as changes in temperature and pressure. They have potential applications in sensing, actuation and cooling technologies. Crystal engineering concepts have been applied to develop a huge range of new SCO materials based on the [Fe(3-bpp)2] 2+ SCO complex (where 3-bpp = 2,6-di(pyrazol-3-yl)pyridine). Chapter 2 focuses on the initial development of this approach by synthesising a series of eleven spin-crossover cocrystals using ditopic coformers, where the supramolecular architectures are dictated by the structure of the coformers. Eight of these new SCO materials were found to be SCO-active, displaying interesting switching properties including hysteresis, stepped SCO and irreversible transitions, and structure-property relationships were developed based on the findings. Chapter 3 investigates the effects of solvates and anions on SCO materials, showing that the choice of anion affects the resulting supramolecular structure of SCO cocrystals. A series of four SCO-active isostructural cocrystal solvates were synthesised and analysed by variable temperature single-crystal X ray diffraction, showing that stronger hydrogen bond acceptors stabilise the low-spin state. Chapter 4 builds on the work from Chapter 2 and Chapter 3 by developing the cocrystallisation strategy further and looking into the effects of using more complex coformers. This resulted in supramolecular structures that deviate from those seen in Chapter 2. Cocrystals were also synthesised using monotopic coformers, which do not form extended supramolecular architectures but were found to be SCO-active and showed interesting domain formation. Mixed cocrystallisation was used to synthesise new materials, demonstrating that it is another strategy that can be implemented to modify SCO V materials. Chapter 5 demonstrates that mechanochemistry using a pestle and mortar or a ball mill can be used to synthesise SCO materials and SCO cocrystals. The SO4 2- salt of the [Fe(3-bpp)2] 2+ SCO complex was synthesised using mechanochemistry and a crystal structure of the material has been reported for the first time. Liquid-assisted grinding in the ball mill was investigated, showing that specific SCO cocrystal polymorphs can be obtained selectively by modifying the milling condition
A Novel Strategy for the Synthetic Selection of Enhanced Therapeutic Aptamers
No full textThe epidermal growth factor receptor protein (EGFR) promotes tumorigenesis in many cancers, including breast, lung, colon and glioblastoma. Deregulation of EGFR activity in cancer is mainly due to point mutation, kinase domain deletion or gene amplification. It is essential to have EGFR-targeted therapies because it is among the most commonly altered genes in cancer. Aptamers are single-stranded oligonucleotide sequences that bind with high affinity and specificity to diverse targets. The recognition capacity of aptamers can be harnessed for therapeutic agents. The chemistry of aptamers is largely limited to that of nucleic acids, and although non-natural modifications of nucleic acids are known to enhance aptamer affinity, there is not yet a technology for selecting the right modifications amongst billions of possibilities.
This project aims to develop the first general method for the discovery of nucleoside modifications that increase aptamer binding efficacy. A library will be created of over 65,000 different chemical modifications on a known aptamer sequence (MinE07), which binds to EGFR protein. The modifications will use different types of chemistry to see how they will affect the binding and folding of the aptamer MinE07, aiming to improve the binding between MinE07 and EGFR. These will be attached on beads such that each bead displays millions of copies of a single aptamer sequence. The selection will then be carried out using a flow cytometer (FACS) to separate out the aptamer sequences with the highest affinity for EGFR from the one-bead-one-sequence aptamer library. These will then be identified using mass spectrometry. These top selected aptamer sequences will be subjected to biophysical and biological testing. This research provides a new method for synthesising and screening in a short space of time large drug candidate libraries producing new drug candidates and impacting drug discovery processes
- …
