Blood is one of the most common biological fluids encountered at crime scenes and is therefore constantly being tested for in the laboratory. Confirming the presence of blood can illuminate essential elements of a case as well as allow for identification via downstream DNA analysis. This significant investigative value is why it is crucial to use robust forensic testing techniques for blood detection. In the forensic laboratory, blood is identified using serological techniques. A presumptive test, such as a colorimetric test, is performed first. A confirmatory test, such as an immunochromatographic assay, is often performed following a presumptive positive result. While both types of tests have numerous advantages, they have several limitations as well. These limitations have served as the basis for exploring alternative techniques for forensic blood detection, such as FTIR. FTIR spectroscopy is a qualitative, non-destructive, confirmatory analytical technique. This technique uses infrared light to characterize organic compounds based on molecular structure. There are also several different FTIR techniques, such as ATR and DRIFTS. ATR-FTIR analysis has been widely researched for the detection of blood and other biological fluids, across several applications. ATR-FTIR may be preferable to serological blood detection because it can be quicker than combined serological blood testing, it requires minimal sample preparation, it does not damage DNA downstream, and it can detect multiple biological fluids at once. Despite all the advantages that ATR-FTIR analysis has over traditional forensic blood techniques, it has not yet been implemented in casework. This may be due to skepticism in using subjective and complex spectroscopic data that results from ATR-FTIR analysis of body fluids. The initial objective of this research was to develop an optimized protocol using ATR-FTIR and chemometric analysis to identify blood on cotton round substrates. Using these techniques together would allow for a rapid, nondestructive, confirmatory approach, that would be more objective than serological testing or FTIR analysis alone. However, due to complications throughout the research process, this objective was altered. The revised objective was to develop an optimized protocol using DRIFTS and chemometric analysis to identify blood on cotton round substrates. An optimized DRIFTS protocol for forensic blood identification was successfully developed. Blood samples from multiple donors were tested using this protocol, and all samples showed similar data. Human biological samples other than blood as well as non-human samples were also tested. These samples showed dissimilar data from the donors’ blood sample data. Chemometric analysis was then performed using AnalyzeIQ Lab software. After testing 93 pair-wise combinations of pre-processing methods and algorithms, a model was developed. Unfortunately, this model was not completely optimized. It had a 9.09% error rate, resulting from the misclassification of one sample. Future research is needed before implementation into casework. Alternative cotton substrates and data collection software should be considered. Additional time should be spent using AnalyzeIQ Lab software, to develop a model with a 0% error rate. If this cannot be achieved, an alternative chemometric analysis software should be considered.

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Inhibiting inflammatory processes or eliminating inflammation represents a logical role in the suppression and treatment strategy of cancer. Several studies have shown that anti-inflammatory drugs (NSAIDs) have promise as anticancer agents while reducing metastases and mortality. NSAIDs are seriously limited by side effects and their toxicity, which can become cumulative with their long-term administration for chemoprevention. The huge development in nanotechnology allows the drugs to exhibit novel and significantly improved properties compared to the large particles of the respective bulk compound, leading to more targeted therapy and reduced dosage. The overall aim of this thesis is to add to our understanding of cancer prevention and treatment through studying the genotoxicity mechanisms of NSAIDs agents in lymphocytes. In this study, the genotoxicity mechanisms of NSAID in bulk and nanoparticles forms a strategy to prevent and minimise the damage in human lymphocytes. Aspirin nano (ASP N) caused a significant decrease in deoxyribonucleic acid (DNA) damage compared to aspirin bulk (ASP B). Also, ibuprofen nano (IBU N) showed a significant reduction in DNA damage compared to ibuprofen bulk (IBU B). Micronuclei (MNi) decreased after ASP N, ASP B and IBU N in prostate cancer patients and healthy individuals, and the ibuprofen bulk showed a significant increase of MNi formation in lymphocytes from healthy and prostate cancer patients when compared to untreated lymphocytes from prostate cancer patients. In order to study the geno-protective properties of these drugs, the protective effect of NSAIDs and the quantification of the DNA repair capacity in lymphocytes was studied. ASP N was found to increase the DNA repair capacity and reduced the reactive oxygen species (ROS) formation significantly more than ASP B. Finally, the role of NSAIDs on some key regulatory signal transduction pathways in isolated lymphocyte cells was investigated by studying their effect on ataxia-telangiectasia-mutated kinase (ATM) and ataxia-telangiectasia and Rad3-related kinase (ATR) mRNA. ATM mRNA significantly increased after treatment with ASP B, ASP N and IBU N. ATR expression also increased after treatment with IBU B and IBU N, but was only significant with IBU N. These findings indicate that a reduction in particle size had an impact on the reactivity of the drug, further emphasising the potential of nanoparticles as improvement to current treatment options.

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DNA damage response (DDR) maintains genome integrity through checkpoint activation and lesion repair. While ATM and ATR are essential in DDR, mechanisms regulating their activation remain unclear. BMI1 is a component of the polycomb repressive complex 1 (PRC1), and contributes to PRC1’s E3 ubiquitin (E3-Ub) ligase activity though binding the catalytic subunit RING2. BMI1 binds RING2 through its ring finger (RF) domain. The E3-Ub ligase activity contributes to BMI1-deirved facilitation of the homologous recombination-based repair of DNA double-stranded breaks (DSBs). My research demonstrates that BMI1 reduces ATM and ATR activation during DDR. DSBs and single-strand DNA (ssDNA) lesions respectively activate ATM and ATR. ATM subsequently phosphorylates CHK2 at threonine 68 (CHK2pT68) and induces G2/M arrest. ATR produces CHK1pS345 and S-phase arrest. Both kinases phosphorylate histone H2AX at serine 139 (γH2AX) to prepare for lesion repair. Hydroxyurea initiates DDR via producing ssDNA lesions, and increases ATR activation (phosphorylation of T1989/ATR pT1989), CHK1pS345, γH2AX, and S-phase arrest. These events were significantly reduced and enhanced following the respective BMI1 overexpression and BMI1 knockdown in MCF7 and DU145 cells. BMI1 also displays similar effects towards ATM during DDR induced by etoposide-caused DSBs. Activation of ATM and ATR requires the formation of the ATM-NBS1 and ATR-TOPBP1 complexes. We observed that BMI1 interacted with NBS1 or TOPBP1. Deletion of the RF domain from BMI1 did not affect the associations and also had no effects on BMI1’s activity in reducing ATM activation and ATR-mediated CHK1 pS345. Collectively, our research suggests that BMI1 attenuates ATM and ATR signaling independently of the E3-Ub ligase activity. Genotoxic treatments elicit DDR in cells that are directly exposed and also in cells that are not exposed, a phenomenon known as bystander effect (BE). However, it remains unclear what mediates the BE. Microvesicles are small membrane-enclosed sacks that are shed from donor cells and communicate specific messages to recipient cells. We demonstrated that microvesicles isolated from cells treated with etoposide and ultraviolet induced BE in recipient cells. Neutralization of microvesicles through annexin V reduced the microvesicles-associated BE. / Thesis / Doctor of Philosophy (PhD)

Yes / Targeted therapy remains the future of anti-cancer drug development, owing to the lack of specificity of current treatments which lead to damage in healthy normal tissues. ATR inhibitors have in recent times demonstrated promising clinical potential, and are currently being evaluated in the clinic. However, despite the considerable optimism for clinical success of these inhibitors, reports of associated normal tissues toxicities remain a concern and can compromise their utility. Here, ICT10336 is reported, a newly developed hypoxia-responsive prodrug of ATR inhibitor, AZD6738, which is hypoxia-activated and specifically releases AZD6738 only in hypoxic conditions, in vitro. This hypoxia-selective release of AZD6738 inhibited ATR activation (T1989 and S428 phosphorylation) and subsequently abrogated HIF1a-mediated adaptation of hypoxic cancers cells, thus selectively inducing cell death in 2D and 3D cancer models. Importantly, in normal tissues, ICT10336 is demonstrated to be metabolically stable and less toxic to normal cells than its active parent agent, AZD6738. In addition, ICT10336 exhibited a superior and efficient multicellular penetration ability in 3D tumor models, and selectively eradicated cells at the hypoxic core compared to AZD6738. In summary, the preclinical data demonstrate a new strategy of tumor-targeted delivery of ATR inhibitors with significant potential of enhancing the therapeutic index. / UoB STARTER Fellowship Award