Liver X receptor (LXR) is a member of the nuclear hormone receptor superfamily that is activated by hydroxylated cholesterol derivatives referred to as oxysterols. It has also been shown to play a crucial role in regulating cholesterol trafficking and lipid metabolism in liver and macrophages. Furthermore, LXR. has also been directly implicated in the reduction of atherosclerosis in several murine models of the disease by virtue of its ability to promote reverse cholesterol efllux from intima-resident lipid-loaded macrophages. While roles for LXR in monocyte biology have focused primarily on cholesterol trafficking, evidence for other functions for the receptor outside of its traditional role as a mediator of cholesterol homeostasis is lacking. Presented herein is evidence that LXR also serves as a mediator of cytokine expression. This work has shown that treatment of human peripheral blood monocytes or monocytic THP-1 cells with the LXR ligand 22(R)-hydroxycholesterol (22R-HC), in combination with 9-cis-retinoic acid (9cRA), a ligand for the LXR. heterodimerization partner retinoid X receptor (RXR), results in the specific induction of the potent pro-apoptotic and pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α.). Promoter analysis, inhibitor studies, and order-of-addition experiments demonstrated that TNF-α. induction by 22R-HC and 9cRA occurs by a novel two-step process. The initial step involves 22R-HC-dependent induction of TNF-α. mRNA, and intracellular accumulation of TNF-alpha protein, mediated by binding of LXRα/RXRα to an LXR response element at position -879 of the TNF-α promoter. Subsequent cell release of TNF-alpha protein occurs via a separable RXR-dependent step that requires de novo transcription and protein synthesis. Furthermore, the RXR-dependent secretory event can be mimicked by agents that induce monocytic differentiation like phorbol esters that culminate in RXR activation by a pathway that does not require exogenous ligand. In this context, RXR was also shown to be a down stream target of the protein kinase C (PKC) signal transduction cascade, that results in the activation of RXR and the induction of secretory factor(s) which facilitate secretion of LXR-derived TNF-α. These studies have provided evidence that should help to expand the currently known role for LXR in monocyte biology and have furthermore identified a new role for RXRs in promoting the secretion of soluble factors like cytokines. Furthermore, in light of reports that show LXR activity promotes a reduction in atherosclerosis, it stands to reason that this regulatory circuit of LXR-dependent production of TNF -α from monocytes would similarly contribute to the attenuation of atherosclerosis 𝘪𝘯 𝘷𝘪𝘷𝘰. / Thesis / Master of Science (MSc)

Ultra-wideband microwave imaging, with its advantages of absence of breast compression, nonionizing and noninvasive properties, is a complementary method to X-ray mammography for breast cancer detection that is safe and reasonably inexpensive. The motivation for employing microwave imaging techniques for detecting early-stage breast cancer stems from published research results showing the strong contrast in the dielectric properties at microwave frequencies between normal breast tissue and malignant lesion. This thesis contributes to development of novel techniques for the detection of early-stage breast cancer tumors well below a centimeter with specificity and high degree of accuracy, i.e., with minimum false negatives/positives. In our proposed approach, a modified Shannon entropy criterion (SEC) is formulated for determining when the time-reversed wave focuses back to the source target in the presence of an inhomogeneous lossy medium. It is demonstrated through two examples, the time-reversal mirror and cavity, that the SEC is found to be more robust than the inverse varimax norm. TR has been shown to be superior to other simple delay-based focusing techniques and here we have extended the TR algorithm by making it more robust in localizing small tumors. The importance of this finding becomes evident as the SEC allows for the detection of tumors that are sub-wavelength in size. Our novel sub-wavelength ultra-wide band (UWB) microwave radar imaging technique exploits the principle of phase-shifting mask (PSM) from optical lithography and is implemented using a time-reversal (TR) algorithm based on the transmission-line matrix (TLM) method. We incorporate the SEC in a TR algorithm to achieve a robust imaging algorithm exploiting the measurements acquired by our phase-shifting mask (PSM) experimental set-up. Unlike the FDTD TR algorithm by Kosmas et al., which excites one of the 23 antenna elements, we propose a different system where all antennas are stimulated simultaneously with their excitation based on the PSM principle. A 0.5-mm diameter tumor was detected and located using a 200-ps UWB pulse in a realistic inhomogeneous two dimensional breast model. The breast model was derived from magnetic resonance imaging data and simulated using the TLM method. The effect of the dielectric contrast and proximity of tumor to the antenna receivers are examined. A TLM-based TR algorithm employing two types of time reversal mirrors (TRMs) is proposed to improve the accuracy of localizing the sub-wavelength tumors. The final part of the thesis examines the feasibility and the design of a narrowbeam UWB antenna for microwave breast cancer detection focusing on the antenna feed structure and the printed TEM horn antenna. A feed structure of an UWB antenna for microwave radar imaging is designed. The UWB antennas are fundamental components of the UWB radar imaging hardware. The performance of the antenna is crucial for the resolution and the reliability of the whole imaging system. A TEM horn antenna is studied and suggestions are made regarding the hardware implementations of the experimental setup. We conclude by suggesting future work toward hardware and practical implementation of UWB microwave radar imaging with high resolution. / Thesis / Master of Applied Science (MASc)

No / The plant derived indoloquinoline alkaloid cryptolepine was investigated for its cytotoxic properties in 12 human tumour cell lines and in primary cultures of tumour cells from patients. The fluorometric microculture cytotoxicity assay was used to assess cytotoxicity and DNA mocro-array analysis to evaluate gene expression. Cryptolepine mean IC50 in the cell line panel was 0.9 microM compared with 1.0 and 2.8 microM in haemaotological and solid tumour malignancies respectively. Among patient solid tumour samples, those from breast cancer were the most sensitive and essentially as sensitive as haematological malignancies, respectively. Among patient solid tumour samples, those from breast cancer were the most sensitive and essentially as senstive as haematological malignancies. Cryptolepine activity showed highest correlations to topoisomerase II and microtubule targetting drugs. In the cell lines cryptolepine activity was essentially unaffected by established mechanisms of drug resistance. A number of genes were identified as associated with cryptolepine activity. In conclusion, cryptolepine shows interesting in vitro cytotoxic properties and its further evaluation as an anticancer drug seems warranted.

Yes / This data article is related to the research article entitled Proteomics of Tissue Factor silencing in cardiomyocytic cells reveals a new role for this coagulation factor in splicing machinery control by Lento et al [1]. Tissue Factor (TF) is the key player in the coagulation cascade, but it has additional functions ranging from angiogenesis, tumor invasion and, in the heart, the maintenance of the integrity of cardiac cells. This article reports the nano-LC-MSE analysis of the cardiomyocytic HL-1 cell line proteome and describes the results obtained from a Gene Ontology analysis of those proteins affected by TF-gene silencing.

Yes / Circulating tumour cells (CTCs) have significant diagnostic potential as they can reflect both the presence and recurrence of a wide range of cancers. However, this potential continues to be limited by the lack of robust and accessible isolation technologies. An alternative to isolation might be their direct detection amongst other peripheral blood cells, although this would require markers that allow them to be distinguished from an exceptionally high background signal. This review assesses the potential role of HOX genes, a family of homeodomain containing transcription factors with key roles in both embryonic development and oncogenesis, as unique and possibly disease specific markers of CTCs.

Alterations to the tumour microenvironment is a common feature of many cancers, including breast cancer, and there is increasing evidence that alterations to the microenvironment, including; increased integrin expression, ECM deposition and protease activity, promote cancer progression. Most invasive breast cancers arise from a preinvasive stage, ductal carcinoma in situ (DCIS). Previous work in our laboratory has shown the microenvironment of DCIS is altered, such that myoepithelial cells (MECs) switch to a tumour-promoting phenotype, associated with upregulation of integrin αvβ6 and fibronectin (FN) expression. Mechanisms by which integrin αvβ6 and FN expression are regulated is unclear. We show DCIS progression into invasion is accompanied by an increase in MEC expression of integrin αvβ6 and periductal FN deposition, and their expression were associated in DCIS. These findings were modelled in isolated primary DCIS-MECs, primary normal MECs and MEC lines, with and without integrin αvβ6 expression, where integrin αvβ6-positive MECs upregulating FN expression. We identified integrin αvβ6-positive DCIS ducts were larger than integrin αvβ6-negative DCIS ducts, and mechanical stretching of primary normal MECs and a normal MEC line led to upregulation of integrin αvβ6 expression and FN deposition in a TGFβ-dependent manner. We further show upregulation of integrin αvβ6 and FN by MECs mediate TGFβ-dependent upregulation of MMP13 which promotes breast cancer cell invasion in vitro. These data show altered tissue mechanics in DCIS and MEC expression of integrin αvβ6 and FN deposition are linked, and implicate TGFβ in their activation. These findings suggest integrin αvβ6 and FN may be used as markers to stratify DCIS patients.

Background: Breast and endometrial cancers are the most common gynaecological cancers in women in the UK. Early detection of tumours is crucial for improving patient survival rates. In breast cancer, mammography is the most reliable screening method for asymptomatic patients; however, its sensitivity is limited by breast density. Currently, there are no early screening assays for endometrial cancer. Thus, there is an urgent need to identify clinical biomarkers for improved non-invasive diagnosis of breast and endometrial cancer. Macrophages are abundant in the tumour microenvironment and their density has been associated with poor prognosis in breast cancer and decreased survival in endometrial cancer. Monocytes are precursors of macrophages and recent studies have shown an association with pro-tumoral functions. The aim of this study has been to examine the transcriptional profiles of human circulating monocytes and tumour associated macrophages (TAMs) in order to investigate their biological relevance and potential as biomarkers for cancer detection and prognosis. Methods: RNA-sequencing was performed on purified monocytes (22 healthy individuals, 21 breast cancer, 16 endometrial cancer samples), as well as purified normal macrophages, TAMs from breast tissue (4 breast cancer, 4 healthy breast) and endometrium tissue (5 endometrial cancer, 9 healthy endometrium). Results: A shift in the transcriptional profile of monocytes in cancer compared to controls was observed. Given these cancer-associated alterations circulating monocytes from cancer patients were called “Tumour Educated Monocytes” (TEMo). A TEMo-derived 13-gene signature was extracted that detected cancer, yielding an accuracy of 94%, a positive predictive value (PPV) of 92% and a negative predictive value (NPV) of 97%. External validation confirmed the ability of the signature to accurately identify cancer patients with perfect accuracy. Transcriptome profiling of TAMs revealed a significantly altered gene expression profile when compared to normal tissue resident macrophages. Furthermore, comparison of TAMs between breast and endometrial cancer also revealed differences suggesting that different tumour microenvironments induce different gene expression profiles in TAMs. Functional analysis of significant genes in breast cancer revealed similar biological pathways to those of murine studies suggesting that TAMs in humans and mice may have similar functions. A gene list of transmembrane receptors has been extracted by comparing breast cancer TAMs with publicly available datasets that could serve as markers for their identification. Finally, exploratory analysis identified a subset of 49 genes associated with recurrence-free and overall survival in publicly available datasets. Conclusion: To my knowledge this is the first genome-wide profiling study of human circulating monocytes and TAMs in breast and endometrial cancer. It provides evidence that monocytes and TAMs can alter their expression profile in the presence of cancer and, using bioinformatics tools I was able to identify biomarkers for diagnosis and prognosis of breast and endometrial cancer.

Three novel, synthetic biarylheterocycles bearing imidazole terminal groups had previously been discovered with high cytotoxicity (IC50 16¿640 nM) against a number of human tumour cell lines. Notably, this biological activity was independent of duplex DNA binding affinity. The compounds were tested in the NCI 60-cell line panel and COMPARE analysis suggests they have a novel mechanism of action, targeting the product of a ¿gene-like sequence¿ of unidentified function. The identity of likely protein targets was explored using a chemical proteomic strategy. Bespoke affinity matrices for chromatography were prepared in which test compounds were attached to a solid support through a biotin tag. A synthetic route to hit compounds containing a biotin moiety in place of one of the imidazole sidechains was developed. Chemosensitivity studies confirmed that the biotinylated compounds retained their activity showing IC50 = 6.25 ¿M in a susceptible cell line, compared with > 100 ¿M for an insensitive cell line. The biotinylated ligands were complexed to a streptavidin-activated affinity column and exposed to cell lysates from the susceptible cell lines. Bound proteins were eluted from the column and separated using SDS-PAGE. Proteins were characterised by MALDI MS and MS/MS and identified using Mascot database searches. Heterogeneous nuclear ribonuclear protein A2/B1 was found to selectively bind to the affinity probes. / Yorkshire Cancer Research, BMSS, School of Life Sciences and the Frank Hudson Memorial Fund

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer and urgently requires new therapies. Oncolytic viruses (OV) are a strong contender. OVs interact with immune components of the TME, which can be altered due to specific genetic mutations. The present study evaluates the impact of specific tumour mutations on the response to carboplatin, the current standard of care, and VSV∆M51, a promising OV candidate. After a study of genetically diverse models, constitutive KRas activation enhanced VSV∆M51 replication in-vitro and sensitivity in syngeneic in-vivo models. VSV∆M51 prolonged survival in syngeneic tumour- bearing mice with KRas, Trp53 and Pten mutations, including one tumour model that did not respond to carboplatin. Response to VSV∆M51 in-vivo was associated with activation of CD4+ and CD8+ T lymphocytes in the peritoneal TME. In summary, VSV∆M51-based immunotherapy has shown promise in diverse murine models of EOC bearing clinically relevant mutations.

The full text will be available at the end of the embargo period: 3rd April 2025 / The author's name as given on this thesis is Francis MPRAH BARNIEH. His publications use the name format Francis M. Barnieh.