Head and neck squamous cell carcinoma (HNSCC) is the sixth commonest cancer in the UK. Despite recent therapeutic developments, survival rates remain poor, particularly in advanced cancer and Human Papilloma Virus (HPV) negative disease. Novel treatments approaches are therefore urgently required. The type-1 insulin-like growth factor receptor (IGF-1R) regulates cellular growth and survival and is over-expressed in a range of cancer types. Other groups reported that inhibition of IGF-1R reduces HNSCC cell survival and sensitises to ionising radiation, but a clinical trial of IGF-1R inhibition as monotherapy was inactive in unselected palliative patients with HNSCC. These data suggest that predictive biomarkers for response to IGF-1R inhibition are required. The aims of this project were threefold. The first objective was to define factors associated with morbidity and mortality in patients with oropharyngeal cancer (OPSCC) treated with primary surgery alone or with adjuvant chemoradiotherapy. The five-year overall and disease specific survival rates were 68% and 78% respectively. In line with previous data, HPV negative status, current smoking status, high tumour T stage and the presence of perineural spread of tumour or lymphovascular invasion were associated with adverse survival outcomes. In surviving patients, quality of life outcomes were evaluated using the University of Washington Quality of Life score and functional outcomes were assessed with the MD Anderson Dysphagia Inventory. Increasing age, higher tumour T stage, lip-splitting mandibulotomy and free flap reconstruction were associated with reduced quality of life outcome scores following multivariate analysis. The second aim was to assess the significance of IGF-1R expression in HNSCC and test for correlates with clinico-pathological variables. Immunostaining of cores from 346 primary HNSCCs showed that IGF-1R expression was higher in tumour tissue than matched benign epithelium. High IGF-1R was significantly associated with reduced overall and disease specific survival, HPV negative status and high tumour T stage, although was not an independent predictor of survival in multivariate analysis. The final aim was to test the utility of IGF-1R inhibition in HNSCC cell lines as monotherapy and in combination with established treatments, aiming to identify predictive biomarkers for resistance to IGF-1R inhibition. In a panel of 6 HNSCC cell lines, the IGF-1R inhibitor BMS-754807 reduced IGF-1R, AKT and ERK phosphorylation in a dose dependent manner. IGF-1R inhibition with BMS-754807 reduced cell survival and sensitised cells to ionising radiation in clonogenic assay, although the magnitude of this effect varied between cell lines. Combination of BMS-754807 with the EGFR inhibitor Gefitinib caused supra-additive reduction in cell survival. Correlation analysis showed a trend towards an association between high levels of phosphorylated AKT and resistance to BMS-754807 monotherapy. To test the hypothesis that RAS signalling conferred resistance to IGF-1R inhibition, cells were infected with retroviral constructs encoding wild-type or mutant activated HRAS. Cells expressing mutant HRAS were more resistant to BMS-754807 than empty vector or wild-type HRAS infected controls, suggesting that HRAS mutation status may represent a biomarker of resistance to IGF-1R inhibition in HSNCC. Taken together, the results from this project highlight the significance of IGF-1R biology in HNSCC, and form the basis for further in-vivo and clinical research.

Background and aims Pin site infection (PSI) is a common complication of external fixation which can cause significant morbidity. Additional treatment required as a result of PSI can be costly in terms of NHS time and resources, but also personal and financial losses for those undergoing additional treatment. A reduction in PSI would therefore reduce the complications and costs associated with infection. The aim of the study is to determine the feasibility of antimicrobial impregnated collars in preventing PSI in patients with external fixation. Methods Current pin site care practices were identified by using an electronic questionnaire. Assessment of bacteria present at clinically non-infected and infected pin sites was determined through evaluating swabs obtained from the out-patient setting. Bacterial isolates were tested for antimicrobial sensitivity and biofilm capacity. Pre-clinical testing of the antimicrobial collar used bacterial isolates identified from the PSI evaluation. Test strains included S aureus, S epidermidis, corynebacteria and E coli. Pre-clinical tests included SPTT, tK100, and an in-vitro pin site model. A single-centre randomised control feasibility study was conducted with participants allocated to standard care or antimicrobial collar. Primary endpoints included number of participants with PSI, number of infected pin sites and bacteria present at pin sites at 3 months. Main findings Pin site care remains varied across the UK despite the availability of the UK Consensus Guidelines (Timms et al 2011). Pin site care is also inconsistent internationally. The bacterial evaluation identified large number of CoNS (46.2%), corynebacteria (10.7%) and S aureus (4.1%) in clinically non infected pin sites, although the significant increase of S aureus to 17.9% in PSI (p<0.01) suggests that this is a key causative organism in PSI. Antimicrobial sensitivity testing identified that 91.9% CoNS were sensitive to at least two of the antimicrobials used in the collar indicating that the antimicrobial collar has the potential to prevent PSI. SPTT demonstrated the efficacy of the antimicrobial collar in maintaining a zone of bacterial inhibition in excess of three months. TK100 methods also demonstrated the ability of the antimicrobial collar to kill attached bacteria within 72 hours. The feasibility study identified poor participant compliance with the antimicrobial collar. Participants in the antimicrobial collar group had significantly reduced bacteria at pin sites when the antimicrobial collar was retained in place for the duration of treatment (p=0.01). No significant difference was detected with number of participants with pin site infection or number of infected pin sites. Conclusion Results from preliminary testing indicates that the antimicrobial collar may be effective in reducing PSI in participants with external fixation, however further product development is required prior to rigorous testing with an adequately powered randomised control trial and economic evaluation.

No description available.

Myotonic dystrophy (DM) is a progressive multisystemic genetic disorder which is inherited as an autosomal dominant trait. There are two subtypes of the disorder, DM type 1 and DM type 2. DM type1 is caused by an expansion of a CTG repeat located in the 3' untranslated region of the DMPK gene on chromosome 19q13.3, whereas DM type 2 is caused by a CCTG expansion in intron 1 of ZNF9 gene located on chromosome 3q. The mutant RNAs containing the expanded CTG/CCTG repeats alters the activity of various alternative splicing factors like Muscleblind-like (MBNL) proteins, which are sequestered in the ribonuclear foci in nucleus by the expanded mutant transcripts resulting in a number of splicing defects observed in DM patients. In first part of my thesis, I have assessed the nuclear and cytosolic distribution of MBNL proteins in both normal and DM cells. In both DM1 and DM2 cells the amount of nuclear MBNL1 was found to be at least 50% greater than seen in normal cells. In addition to this, I studied the distribution of MBNL1 protein in nuclear and cytosolic fractions of DM cells before and after treatment with compounds chromomycin A3, gemcitabine, IMOX, RO 31-8220 and hypericin which were highlighted in the primary screen. Treatment with the compounds produced a significant reduction in the proportion of nuclear MBNL1 compared to DMSO treated cells in DM fibroblast and myoblasts. In second part of this thesis I have examined the effect of MBNL1/2 down regulation on both RNA and MBNL1 foci in DM cells. MBNL1 and MBNL2 double knockdown resulted in a 40% increase of nuclear RNA foci than observed in scrambled siRNA treated cells, though a significant reduction was observed in case of MBNL (protein) foci. Also, MBNL 1 and MBNL2 down regulation did not result in the release of mutant transcript from the nucleus to the cytoplasm in KB-Telo MyoD (DM) cells as seen in BpmI restriction polymorphism assay. However, it had a degradative effect on the mutant DMPK transcript.

Aggregation of the abundant neural protein α-synuclein contributes to cellular toxicity in Parkinson‘s disease. We have created transgenic nematodes carrying fusion constructs encoding human α-synuclein (S) tagged with YFP (V) and/or CFP (C) as a fluorescent marker. Using the unc-54 myosin promoter, a synuclein-YFP (unc-54::SV (NI)) fusion construct was abundantly expressed in the body wall muscles of Caenorhabditis elegans. Permanent integrated lines were successfully generated for unc-54::V (NI), unc-54::S+V (I), unc-54::SC+SV (I), unc-54::C+V (I), and unc-54::CV (I) using gamma irradiation. The outcrossed transgenic synuclein strains were radiation sensitive and have shorter life span and lower pharyngeal pumping compared to wild type N2 and unc-54::V (I) worms. Fluorescence Resonance Energy Transfer (FRET) was measured for all the transgenic strains. The unc-54::SC+SV (I) worms showed FRET signals intermediate between the negative (unc-54::C+V (I)) and positive (unc-54::CV (I)) control strains. Confocal images were taken to confirm the presence of FRET. FRET signals increase markedly during early adult life in unc-54::SC+SV (I) worms. RNA interference by feeding was performed in unc-54::SC+SV (I) worms to knock out the Hip-1 co-chaperone function, thereby increasing the FRET signal. unc-54::SC+SV (I) fusion worms were also exposed to pesticides such as chlorpyrifos and rotenone, and we observed an increase in the size and intensity of fluorescent aggregates thereby increasing the FRET signal. Finally we have quantified reactive oxygen species (ROS) for unc-54::SC+SV (I) fusion worms and NL5901 strains by using the H2DCF-DA assay, showing that ROS levels were increased by pesticide exposure.

Background: Osteoarthritis (OA) is a highly prevalent joint degenerative disorder among the older population. The main symptoms of OA are chronic pain, swelling and stiffness of joint. OA histopathology is characterized by cartilage damage, synovial inflammation and remodelling of subchondral bone. Resolvins are endogenous lipid mediators produced from Ω-3 poly-unsaturated fatty acids (PUFAs) during resolution of inflammation. The main biological functions of resolvins include anti-inflammation and resolution of inflammation. Currently, the emerging anti-nociceptive roles of some resolvins have been reported in various models of pain. However, roles of resolvins and the resolvin receptor system on osteoarthritic pain are unknown. Objectives: This thesis assesses the therapeutic potential of a resolvin precursor on OA pain and investigates the underlying mechanisms of action and resolvin receptor system in OA. Methods: Monosodium iodoacetate (MIA) and medial meniscus transection (MNX) -induced joint damage was used as models of OA pain. 17(R)-HDoHE (300ng/300μl) or vehicle (1% ethanol in saline, 300μl) was acutely or chronically administered at day 14 post model induction and pain behaviour was measured to determine the analgesic effects of the drug in these models. Haematoxylin and eosin (H&E) staining was used to assess joint histopathology. Gene expression of resolvin receptors, inflammatory cytokines and metabolic enzymes were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in associated tissues from the models and human OA samples. Results: Pain behaviour and joint histopathology were established in both the MIA and MNX models. Expression of chemokine-like receptor 1(ChemR23) was lower in the synovia and higher in the spinal cord in the MIA model. 15-lipoxygenase (15-LOX) was expressed at a lower level in both synovia and spinal cord in the MIA model. Negative correlations were revealed between synovial ChemR23 expression and pain behaviour at both day 14 and 35 in the MIA model. ChemR23 expression in the spinal cord was positively correlated with pain behaviour at day 35 in the MIA model. Expression of formyl peptide receptor 2 (ALX), some inflammatory cytokines and metabolic enzymes was lower in the synovia in the MNX model but expression of 5-lipoxygenase-activating protein (FLAP) was higher. Expression of ALX in the synovia was positively correlated with tumor necrosis factor alfa (TNFα), interleukin 1 beta (IL1β) and cyclooxygenase 2(COX2) but negatively correlated with 5-LOX expression in the MIA model. Expression of ALX in the spinal cord was positively correlated with pain behaviour at day 14 but then the converse was true at day 35. Expression of ALX in the spinal cord was negatively correlated with IL6 in the MIA model. 17(R)HDoHE attenuated pain behaviour in both the MIA and MNX models following acute, chronic and discontinuous administration. Effects of acute administration of 17(R)HDoHE on pain behaviour were associated with an up-regulation in the expression of IL6 and decreased 5-LOX expression in the synovia of MIA model. A trend towards down-regulation of pro-inflammatory cytokines and associated enzymes by 17(R)HDoHE was observed in the acute study in the MIA model. Repeated administration of 17(R)HDoHE produced robust and sustained inhibitory effects on pain behaviour, but no change in joint histopathology. Pain behaviour was attenuated when 17(R)HDoHE was administered but returned to levels seen in vehicle treated rats after 7 days after drug cessation. In human OA samples, expression of ChemR23 was significantly higher than expression of ALX in both synovia and medial tibial plateau. ChemR23 expression was positively correlated with expression of 5-LOX in both synovia and medial tibial plateau and negatively correlated with 15-LOX2 expression in the medial tibial plateau from OA patients. There was a significantly positive correlation between ChemR23 expression and IL6 and 15-LOX1 expression in the medial tibial plateau. In addition, there was a significantly positive correlation between ALX and IL6 and 15-LOX1 expression in both synovia and medial tibial plateau. Expression of ALX, TNFα, IL6, COX2 and 5-LOX in the medial tibial plateau from OA patients was lower, compared to expression in bone from femoral heads obtained from trauma patients. Conclusions: These findings support anti-nociceptive and anti-inflammatory roles of resolvins and provide evidence that resolvins may be potential novel drugs to treat OA pain.

Antisense oligonucleotide therapy is one of the most promising strategies for treatment of myotonic dystrophy type 1 (DM1), type 2 (DM2) and Duchene muscular dystrophy (DMD). These three diseases have nuclear retained mRNA, suitable for antisense therapy targeting. The delivery of oligonucleotides to their desired target has long been an obstacle in antisense therapy with a large number of delivery reagents or methods having adverse side effects. Promising work published detailing the successful delivery of various chemically modified oligonucleotides (CMOs) naked, via gymnosis, led to us investigating a number of these CMOs: deoxyribonucleic acids (DNA), Peptide nucleic acids (PNAs), 2’OMethyl (2’OMe), and Phosphorodiamidate morpholino (PMO) oligonucleotides. In DM1 expanded CUG repeat (CUGexp) mRNAs aggregate in the nucleus forming “foci”. Testing the CMOs effectiveness at disrupting nuclear foci in a cell based assay, using high content imaging to visualise the number, size and intensity of foci we initially discovered that PNA and 2’OMe, were seemingly taken up via gymnosis by DM1 cells, and removed nuclear foci at nanomolar concentrations. However further experimentation using live cell imaging indicated that although all CMOs could enter the cell, in all disease models tested, via gymnosis, the CMOs could not penetrate the nuclear membrane. In depth analysis led us to identify an artefact of the in-situ process used to identify these foci, explaining earlier positive results. As the target mRNA is trapped within the nuclear compartment we investigated several transfection reagents for their ability to deliver 2’OMe oligonucleotides to the nucleus using live cell fluorescent imaging and a modified northern blot based method. It was established that polyethylenimine could successfully deliver 2’OMe oligonucleotides to the cell, with a high abundance of the oligonucleotide residing within the nuclear compartment. It was observed that PEI degrades the expanded nuclear retained repeat in the DMPK transcript of a DM1 patient cell line alone, without the addition of an antisense agent, in a concentration dependent manner.

The diploma thesis deals with the problem of muscle imbalances of middle school aged children and its relationship with their free time activities. The selected exercises were tested for the muscles involved in the lower and upper cross syndrome. On the basis of the measured results, the frequency of shortened / weakened muscles and muscles in the physiological standard were compared. A questionnaire survey was used to identify preferences of children concerning leisure activities. Based on the data obtained from the comparison of the data found in the questionnaire and the results measured during the testing of the muscles, the following topics were addressed: Whether the muscular condition is influenced by the sex, the frequency of sport activities performed and the type of sport practiced. In addition, the influence of parents' and their lifestyle. KEYWORDS muscoskeletal system, muscle imbalance, upper crossed syndrome, lumboischiatic syndrome, gender, middle aged children

The diploma thesis deals with the problem of muscle imbalances in pre-pubertal children and its relationship with their free time activities. The selected exercises were tested for the muscles involved in the lower and upper cross syndrome. On the basis of the measured results, the frequency of shortened / weakened muscles and muscles in the physiological standard were compared. A questionnaire survey was used to identify preferences of children concerning leisure activities. Based on the data obtained from the comparison of the data found in the questionnaire and the results measured during the testing of the muscles, the following topics were addressed: Whether the muscular condition is influenced by the sex, the frequency of sport activities performed and the type of sport practiced. In addition, the influence of parents' and their lifestyle.