The 5-year survival rate of head and neck squamous cell carcinoma (HNSCC) has remained at ~50% for over 50 years. HNSCC is categorised by multiple anatomical sites, but oral (oral SCC) and oropharyngeal squamous cell carcinoma (OPSCC) account for approximately 90% of all cases. At the time of writing, only one targeted agent, cetuximab (a monoclonal antibody targeting the epithelial growth factor receptor), has been approved for the treatment of recurrent/metastatic HNSCC. However, despite the high expression of EGFR in oral SCC tumour samples, the clinical benefit of cetuximab has been modest thus far. Using a phenotypic screening approach, I sought to identify putative therapeutic targets. A whole genome siRNA screen carried out using an aggressive patient-derived cell line (‘Liv7k’) in normoxic and hypoxic conditions provided the foundation for this project. In addition, a drug-repurposing screen tested the efficacy of 1,351 compounds, approved for cancer and non-cancer indications. A number of approaches were used to identify potential targets, including a whole genome siRNA screen in normoxic and hypoxic conditions, a drug-repurposing screen, and a data multiplexing approach combining the two screens with pathway analysis and datasets from The Cancer Genome Atlas and the International Cancer Genome Consortium. Genomic characterisation of oral cancer cell lines confirmed the importance of a previously identified frequently amplified region of chromosome three, which contains a number of driver genes in HNSCC. In addition, a differential susceptibility of oral SCC cells in hypoxia formed the basis of a line of inquiry centred on triglyceride and ether lipid metabolism. Finally, compound screening identified a dependence of oral SCCs on cysteinyl leukotriene signalling, which is involved in inflammatory conditions such as asthma.

No description available.

This study investigated the beneficial potential effects of grape pomaces obtained after winemaking of different Mediterranean grape varieties from crude materials to their in vivo effectiveness. Grapes and their respective grape pomaces from six different V. vinifera L. cultivar were studied namely Grenache (from two different locations [GRE1 and GRE2]), Syrah (from two different locations [SYR1 and SYR2]), Carignan (CAR), Mourvèdre (MOU), Counoise (COU) and Alicante (ALI) grape varieties from the Rhône Valley. The comparison of several wine industry by-products with their respective grapes provided evidence that pomace remaining at the end of the winemaking process can be very rich sources of antioxidants. The quantitative and qualitative distribution of polyphenols by HPLC-PDA-Fluo-MS in grape pomaces showed significant differences through varieties and vintages varying from 15% to 70% of polyphenols extracted. Seeds from Grenache (GRE1), Syrah (SYR1) and skins from Syrah (SYR1), Carignan and Alicante were of particular interest because of their higher polyphenol contents in terms of flavan-3-ols (monomers, dimers and trimers) up to 8.7 mg/g DW and anthocyanins (glycosides, acetylated and coumaroylated derivatives up to 17.40, 1.57 and 2.38 mg/g DW, respectively). The investigation of aqueous and hydro-alcoholic 70% extracts of seeds from Carignan and Syrah (SYR1) and skins from Carignan and Alicante was carried out as they contained high levels of total phenols and antioxidant activity. Several extracts, were tested in order to evaluate their in vivo biological effects on hypertension using a spontaneously hypertensive rat (SHR) model. A series of different grape pomace extracts were tested in association with verapamil. All in vivo experiments demonstrated that some grape pomace extracts administrated with or without co-ingestion with verapamil possessed an anti-hypertensive activity. This was evident with GRE1 (EA70) seed pomace extract, SYR1 (EA70) seed pomace extract, ALI (EA70) skin pomace extract administrated alone and with GRE1 (EA70) seed pomace extract, SYR1 (EAQ) seed pomace extract, ALI (EA70) skin pomace extract and SYR2 (EAQ) skin pomace extract administrated in association with verapamil. Grape pomace extracts with or without co-ingestion with verapamil were absorb as phase II metabolites mainly including glucuronide, O-methyl glucuronide, sulfate, and O-methyl sulfate derivatives of (epi)catechin which arise from the metabolism of monomeric flavan-3-ols. The detection by HPLC-PDA-Fluo-MSn and GC-MS of microbial-derived metabolites of flavan-3-ols, hydroxyphenyl-γ-valerolactones in their glucuronide and sulfate forms confirmed the absorption of metabolites derived from both monomeric and polymeric flavan-3-ols from grape pomace extracts and subsequent post-absorption conjugation. Numerous metabolites derived from further microbial degradation of hydroxyvalerolactones were also detected. The urinary excretion of these metabolites accounted for a larger proportion of the total polyphenol ingested than phase II metabolites of monomeric flavan-3-ols, indicating the important role of intestinal bacteria in the metabolism of polymerized procyanidins. All these metabolites may have exerted biological effects during the period in which they circulated in the bloodstream. This study constitutes the first step of assessing grape pomace as an enhancer of the verapamil, an anti-hypertensive drug. Substantial levels of polyphenols, especially flavan-3-ols, procyanidins and anthocyanins, remain in pomace after the winemaking process in quantities sufficient to exert anti-hypertensive effects. In addition, according to the extract used and its composition, it is feasible to modulate anti-hypertensive effects by amplifying or decreasing polyphenols and/or verapamil absorption.

Candida biofilms have become an increasingly important clinical problem. The widespread use of antibiotics, frequent use of indwelling medical devices, and a trend towards increased patient immuno-suppression has resulted in a creation of opportunity for clinically important yeasts to form biofilms. Whilst there is growing evidence of the importance of Candida biofilms in clinical medicine, not all clinical isolates are able to form biofilms. There is therefore a fundamental gap in understanding exactly what drives biofilm formation and its clinical implications. These structures have become increasingly recognised as a significant clinical problem. One of the major reasons behind this is the impact that these have upon treatment, as antifungal therapy often fails and surgical intervention is required. This places a large financial burden on health care providers. Therefore, the discovery of alternative antifungal agents to be used in the treatment of fungal biofilms is in great demand for the management of these infections. A panel of Candida albicans bloodstream isolates were assessed for their biofilm forming ability by using the crystal violet assay and measuring cellular surface hydrophobicity. Scanning electron microscopy was used to visualise differences in the clinical biofilms. The impact of amphotericin B (AMB) treatment was determined next by broth microdilution method to assess differences in susceptibility profiles of the clinical isolates. The virulence of these clinical isolates was evaluated in vivo using a Galleria mellonella model and transcriptional analysis used to assess the expression of various genes associated with C. albicans biofilm formation within clinical isolates. Extracellular DNA (eDNA) in clinical biofilms was quantified using a microplate fluorescence assay and chitinase activity measured using a biochemical assay. Moreover, the potential of a novel antimicrobial agent Carbohydrate-derived fulvic acid (CHD-FA) was assessed against a panel of fungal and bacterial species. The mechanism of action of CHD-FA was determined using membrane assays include ATP release, and propidium iodide fluorescence, with various inhibitors used to determine whether CHD-FA activity is affected by known resistance mechanisms. Finally, the immunomodulatory properties of CHD-FA were investigated using ELISA and PCR arrays. The results from this study have shown C. albicans biofilm formation is differential within clinical isolates, where those with high biofilm formation (HBF) predominately consisted of hyphal cells, were more virulent in vivo and had decreased susceptibility to AMB, when compared to those with low biofilm formation (LBF). Furthermore, transcriptional analysis identified a number of genes that positively correlated with C. albicans biofilm formation. The novel agent carbohydrate-derived fulvic acid (CHD-FA) was shown to not only be highly active against C. albicans biofilms, but also against a range or orally relevant bacteria through non-specific membrane activity. Furthermore, CHD-FA was shown to down-regulate a number of pro-inflammatory mediators in an oral epithelial cell line. In conclusion, this study has characterised C. albicans clinical isolates based on their biological characteristics, where clear difference in virulence and antifungal treatment have been shown. It may be possible to develop a panel of genetic markers that could be used as a diagnostic tool for detecting biofilm formation in clinical isolates. CHD-FA is a microbiocidal compound that may serve as a potential novel antiseptic agent for the treatment of oral candidiasis and other candidal biofilm infections, whereby the immunomodulatory properties of CHD-FA could be exploited for controlling inflammation in a number of diseases.

Being one of the most rapidly advancing cancer imaging techniques in recent years, Positron Emission Tomography (PET) represents a standard of excellence with respect to sensitivity and resolution for the non-invasive in vivo molecular imaging of solid tumours via the detection of radiotracer molecules. Amongst these radiotracers, radiolabelled fluorinated nucleosides such as 3’-Deoxy-3’-[18F]-fluorothymidine (18F-FLT) has been widely recognized as a key, specific biomarker for tumour cellular proliferation. Current methods for the commercial production of [18F]FLT are characterized by low overall yields and time-consuming high-performance liquid chromatography (HPLC) purification. These disadvantages could be rectified by the development of a fast, efficient synthetic route to FLT that could enhance the productivity and reduce the reaction time of the fluoridation step necessary for the synthesis of short-lived radioisotope, 18F (t1/2=110 min) installed nucleoside. This project focussed mainly on the development of a new efficient chemical synthetic route to FLT. The synthesis and in vitro evaluation of a series of pro-nucleotide (ProTide) analogues of FLT as new potential therapeutic agents was also carried out. Various studies regarding FLT synthesis have been carried out on the cold (non-radioactive) 19F and radioactive 18F isomer by varying and optimizing conditions for the incorporation of different protecting groups and also different fluoridation reactions by nucleophilic displacement. Further optimization studies were made for the fluoridation step and the synthesis of [18F]FLTProTide analogues as new diagnostic PET imaging agents by variation of different chemical parameters on the phosphoramidate group was attempted. The synthesis of 19F- FLT based ProTides as new therapeutic agents were initiated by the introduction of the phosphoramidate group at the 5’-position of the furanosyl group of thymidine under basic conditions followed by fluoridation to generate the desired analogues. In addition to that, biological evaluation of the newly developed 19F-FLT ProTide analogues for anti-HIV 1 and anti-HIV 2 activities was undertaken on CEM cells. The results in those models indicated that the synthesized compounds were less potent than the parent nucleoside FLT. However in TK- (HIV-2) cells, the analogues retained biological activity in contrast to FLT. This suggested that the FLT ProTides bypassed the first phosphorylation step. However, the therapeutic in vitro evaluation for anti-tumour activity on L1210, CEM and HeLa cells showed no significant activity.

In this PhD project we show for the first time that novel dideoxy bicyclic pyrimidine nucleoside analogues (ddBCNAs) with L-chirality represent promising antiviral candidates for use against pox and measles viruses. We suggest a mechanism of action based on a cellular target. Our lead compound (Cf2642, with side chain C9H18–O–C5H11) is active against vaccinia virus (a surrogate poxvirus for smallpox) and measles virus, with IC50 concentrations of 0.19 and 7.5 µM, respectively. This is a 60-fold enhancement over cidofovir (viral DNA polymerase inhibitor; IC50 of 11.5 µM against VACV). A structure activity relationship was established, which was similar for both viruses, indicating a common and specific mechanism of action. Cf2642 does not inhibit HSV-1/2, influenza, adeno or yellow fever viruses. The mechanism of action for the ddBCNAs has been investigated and, though not defined, has been narrowed down. Based on our observations of drug activity in cell lines derived from various sources, we have suggested a cellular target for the ddBCNAs, most likely cellular membrane compartments or the proteins located therein. Though inhibition of vaccinia is observed within two hours of infection, we have shown that the ddBCNAs are unlikely to be entry inhibitors. Acidification of the extracellular medium was observed but, whilst it may be linked to the mechanism of action, this is not the cause of the antiviral effects. With a possible cellular target, toxicity was carefully evaluated. We have not observed significant cytotoxicity in any of our cell models. Antivirals active against cellular targets are less subject to viral resistance, which may develop rapidly with virus-targeting drugs. This could be critical since, there are currently no effective measles antiviral drugs available on the market, and resistance to measles RNA polymerase inhibitors and the potential antipoxviral drug cidofovir has already been described.

Consideration of broader outcomes of disease, especially those exclusively experienced and reported by the patient, such as HRQOL, is not only consistent with the ‘whole person’ view of health contained in the 1948 WHO definition, but is also a prerequisite to building health-care systems that are responsive to the needs of the patients. For chronic skin diseases, such as hyperhidrosis, these provide a useful indicator of how a patient feels and functions disease for both practical and methodological reasons. The aims of this study therefore were to investigate the impact of hyperhidrosis on patients’ HRQoL using a mix of qualitative and quantitative methods. In addition, a further aim was to develop and validate a disease-specific instrument for assessing HRQoL in hyperhidrosis. In pursuing the above aims, the feasibility of applying online social networking sites for outcomes research in dermatology was assessed. Patients were recruited through online social networking communities related to hyperhidrosis for all stages of the study. Interviews, focus groups and surveys were used for collecting qualitative data from patients (n = 71) to understand quality of life issues of patients, and to provide the content of the new instrument. Dermatologists (n= 5) and patients (n=7) took part in the content validation of the HidroQoL©. Item reduction and the development of the scale’s structure was carried out through several field-testing studies (n: USA, 559; UK, 115), using the item response theory (IRT) Rasch model and factor analyses. Further psychometric testing was performed in a separate study (n = 241). Distribution-based methods were applied in establishing minimum clinically important difference (MCID). A thematic analysis of the qualitative data collected produced 29 quality of life themes and 102 sub-themes, forming the content for the initial 49-item HidroQoL©. The two expert panels judged the instrument as content valid, with a few suggestions. The Rasch analysis modelling led to the collapsing of response categories (from five to three) and the reduction in number of items (from 49 to 18), to ensure a perfect model fit. Factor analyses supported both a single- and a two-factor structure. In subsequent construct validation study the HidroQoL correlated with the DLQI (rs = 0.572, p < 0.01) and the Skindex-17 (rs = 0.551, p < 0.01). Reliability was high (Cronbach alpha = 0.9; test-retest ICC = 0.93). The scores were sensitive to change in patients’ disease severity (standard response mean = 0.8, 95% C.I: 0.34-1.27). The scale banding proposed for the HidroQoL score is as follows: 0 – 1, no effect at all; 2 – 11, small effect; 12 – 22, moderate effect; 23 – 32, large effect; 33 – 36, very large effect. The MCID values were 1.94 – 3.07, for generalised v hyperhidrosis, 2.16 – 4.36, for axillary hyperhidrosis, 2.15 – 3.39, for palmo-plantar hyperhidrosis. An MCID of three is currently being proposed for all types of hyperhidrosis. This study has provided the initial evidence supporting the appropriateness of the content of the HidroQoL and validity of inferences from its scores for assessing HRQoL in hyperhidrosis. In addition, the availability of MCID estimates for the HidroQoL will facilitate its clinical interpretation in both research and routine clinical practice. This study has also demonstrated how CTT and IRT can be integrated in the development and validation of a new generation of HRQoL instruments, using social network for patient recruitment.

RNA viruses are a major cause of disease that in the last fifteen years counted for frequent outbreaks, infecting both humans and animals. Examples of emerging or ri-emerging viral pathogens are the Foot-and- Mouth disease virus (FMDV) for animals, Chikungunya virus (CHIKV), Coxsackie virus B3 (CVB3) and Respiratory Syncytial virus (RSV) for humans, all responsible for infections associated with mild to severe complications. Although both vaccines and small-molecule compounds are at different stages of development, no selective antiviral drugs have been approved so far, therefore for all four these viruses improved treatment strategies are required. Promising targets are the viral non-structural proteins, which are commonly evaluated for the identification of new antivirals. Starting from the study of different viral proteins, several computer-aided techniques were applied, aiming to identify hit molecules first, and secondly to synthesise new series of potential antiviral compounds. The available crystal structures of some of the proteins that play a role in viral replication were used for structure- and ligand-based virtual screenings of commercially available compounds against CVB3, FMDV and RSV. New families of potential anti-CHIKV compounds were rationally designed and synthesized, in order to establish a structureactivity relationship study on a lead structure previously found in our group. Finally, a de-novo drug design approach was performed to find a suitable scaffold for the synthesis of a series of zinc-ejecting compounds against RSV. Inhibition of virus replication was evaluated for all the new compounds, of which different showed antiviral potential.

Bacterial drug resistance is an increasingly serious problem that threatens public health and researchers need to develop new drugs. The biosynthetic pathway of the bacterial peptidoglycan is a known and good target for the development of novel antibacterial agents. This research project focused on the first lipid-linked step of peptidoglycan biosynthesis. The enzyme required for this step is Phospho-MurNAc-pentapeptide Translocase MraY. Our aim was to screen for novel inhibitors of MraY. A continuous fluorescence MraY assay was developed and optimised to test known and potential inhibitors such as nucleoside natural products, antimicrobial peptides and structurally new small molecule potential inhibitors. The fluorescence assay was adapted to a high-throughput fluorescence assay in microtitre plate format and around 2,000 compounds were screened from the diversity set of the National Cancer Institute (NCI) against MraY in order to identify novel inhibitors. However, around 22 % of the test compounds from the diversity set interfered with fluorescence. Therefore, a radiochemical MraY assay was developed as an independent method. We eventually identified one potential MraY inhibitor from the diversity set, the naphthylisoquinoline alkaloid, michellamine B, with IC50 values of 400 and 340 μg/ml against E. coli and B. subtilis MraY respectively. The compound showed antibacterial activity against B. subtilis with an MIC value of 16 μg/ml. It was established that MraY inhibition from the pacidamycin producer, S. coeruleorubidus, was detectable directly from culture supernatants by the fluorescence and by the radiochemical MraY assays. Therefore, we tested culture supernatants and cell extracts from various Streptomyces strains. MraY inhibition was observed using cell extracts from Streptomyces venezuelae, and higher levels of inhibition were observed from a gbnB/gbnR S. venezuelae mutant, though it was not possible to identify the active species present. Following an earlier report of halogenated fluoresceins identified from a combined MraY/MurG screen, we also tested several halogenated fluoresceins, and found that phloxine B, a tetra-bromo fluorescein analogue, was an inhibitor of E. coli MraY with an IC50 value of 32 μM, and also inhibited MraY from P. aeruginosa, B. subtilis, M. flavus and S. aureus with IC50 values ranging between 100 and 210 μg/ml.

The pharmaceutical industry has been criticised for a lack of innovation associated with the drug discovery and development process, for example when compared with the computer or music industries. In fact, bringing a new medicine to the market requires, on average, the screening of up to 10 000 molecules, an expense in the range of $500 million-$2 billion and roughly 10-15 years of research. Such a situation not only has a direct impact on the health and life expectancy of every single human being on the planet, but also indicates that alternative strategies for drug development should be investigated. In this thesis, studies of direct formulation-membrane interactions, both in a high throughput (HT) manner and at a nanometre scale, were initially identified as an important approach that could offer advantages for in vitro-in vivo correlations of in-man drug behaviours. Subsequently, supported lipid bilayers (SLBs) of physiologically-relevant lipid compositions were indicated as experimental models of preference for pre-clinical drug development. For that reason, the characterisation and assessment of physicochemical and behavioural properties of the model SLBs at a nanometre scale, as well as development of an SLB microarray for HT applications were the focus of this research. Here, the optimisation and characterisation of model lipid films was performed using atomic force microscopy (AFM), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). Additionally, the AFM-investigated assessment of the interactions between model SLBs and formulation components (e.g. Pluronics®, siRNA, DNA polyplexes) enabled both the correlation of in vitro observations with literature-reported in vivo performances of the components of interest and the development of hypotheses with regards a number of phenomena in biology. Furthermore, the development of a SLB microarray prototype suitable for HT applications is reported. Directly, this research improves: the understanding of SLB behaviours and experimental investigation at a nanometre scale of the mechanisms of interactions between membranes and: Pluronics®, nucleic acids and their complexes, as well as the technology of SLB microarray development. Indirectly, this research contributes towards the progress in a number of research areas within pharmaceutical sciences, potentially resulting in new scientific disciplines, such as immunolipidomics or nanopharmacology.