DNA triple helices are an important tool in a variety of medicinal and biotechnological applications, such as gene therapy and chemotherapeutics. DNA triple helices are formed by binding of a triplex-forming oligonucleotide (TFO) to a DNA duplex, via specific recognition of the individual base pairs in the target sequence. Mixed-sequence recognition of duplex DNA by TFOs is therefore an essential requirement for successful targeting. However, achieving strong, yet specific binding to the pyrimidine.purine (Py.Pu) base pairs CG and TA, by TFOs is a greater challenge than to the purine.pyrimidine (Pu.Py) base pairs (GC, AT), as fewer hydrogen bonds are presented for binding in the major groove of the double helix. Selective recognition of CG, could be achieved by utilising additional interactions across the CG base pair, via amino-modified nucleosides, to form more stable, selective triplets than those which can be formed by the natural base T. Four modified phosphoramidite monomers, meta-aminophenyl-modified analogues of the bicyclic nucleosides, (2,3H)-furano[2,3-d]pyrimidin-2(7H)-one and N-methyl-(2,3H)-pyrrolo- [2,3-d]pyrimidin-2(7H)-one, were synthesised to address this potential hydrogenbonding motif. Biophysical studies demonstrate selective recognition of the CG base pair. Results indicate selectivity for CG and binding affinity are much improved on previous modifications. Their fluorescence properties and general oligonucleotide deprotection conditions were also studied. In addition, the synthesis of a bis-amine modified 6-oxocytidine phosphoramidite monomer for GC recognition was re-investigated. This research shows significant advances in the field of triplexes for therapeutic use.

With an ageing population putting ever increasing demands on the musculoskeletal system there is a growing need for the development of regenerative medical strategies to provide for the healthcare needs of the future. With increasing numbers of joint arthroplasty occurring in younger and younger patients there is likely to be a growing need for therapeutic strategies to replace lost bone stock in the coming decades This thesis aims to explore strategies to enhance the biological and mechanical properties of impaction bone grafting and the effects of skeletal stem cell (SSC) concentration. In order for SSC to be effective in replacing new bone stock new strategies looking to enhance osteogenic differentiation have been examined. Section I: An in vitro Impaction Bone Graft (lBG) model of SSC seeded onto human allograft was used to study the biomechanical effects of altering SSC concentration. The use of concentrated SSC was then used in the treatment of patients with avascular necrosis (AVN) of the femoral head and fracture non union with parallel in vitro analysis of the samples. Section 11: In vitro and murine in vivo analysis of the biomechanical effects of type 1 Collagen and Hydroxyapatite nanoparticles primarily looking at differences in shear strength and osteogenic differentiation compared to plain allograft and basal cultured SSC. Section Ill: An in vitro acetabular model was used to study the effect of vibration IBG compared to standard techniques in revision hip surgery. The graft compaction, force of impaction, fracture risk and rate of subsidence post cyclical loading was assessed. This thesis has demonstrated in vitro and in vivo strategies that are clinically translatable and have demonstrated that: • Skeletal Stem Cell concentration plays a pivotal role in the biomechanical enhancement of Impacted bone graft (IBG) • Translation of these strategies into the successful treatment of fracture non union and Avascular necrosis of the hip. • Type 1 Collagen and Hydroxyapatite nanoparticles both enhance the osteogenic differentiation and shear strength of the IBG / SSC construct • Vibration impaction bone grafting is a novel technique that significantly reduces the intraoperative risk of acetabular fracture or catastrophic subsidence This thesis has demonstrated novel techniques for the biomechanical enhancement of I BG with most techniques being readily transferable to clinical practice with the potential to form part of a surgeon's armament for regenerative medical techniques of the future.

Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Much of the work in this area has focused on the effects of mechanical stimulation upon bone cells. Very little has been done to investigate the effects of mechanical stimulation on other cell types. Therefore, could mechanical stimulation be used to accelerate the wound healing process? Could mechanical stimulation be used to encourage muscle cell growth? The data generated in this thesis will be completely new knowledge which will help us develop our understanding of mechanotransduction and help us develop new approaches and devices to address medical challenges such as accelerating wound healing process, increasing muscle mass in diseases associated with muscle degeneration and aging. The aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of three different fibroblast cells and muscle cells namely; human lung fibroblast cells (LL24), subcutaneous areolar/adipose mouse fibroblast cells (L929), Human dermal fibroblast cells (HDF) and C2C12P12 muscle cells. Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5 and 20 minutes) on wound/scratch assays, the mean cell migration distance (µm), actin and vinculin organisation, cell viability, muscle fibre diameters and the effects of temperature were investigated. The results show that 100Hz acoustic vibration can enhance cell migration, wound healing in LL24 and L929 cells and possible increase muscle fibre diameters although there was no significance, the trends were repeatable. Mechanical stimulation was also found to promote changes to the morphology of LL24 and L929 cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Vinculin appeared to be more punctate in L929 cells compared to LL24 cells. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. However, as the frequency increased, the temperature also increased. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechano-sensitivity of cells to control cell migration and to enhance the diameters of muscle fibres.

Amorphous solid dispersions over the last decade or so have been widely investigated by the pharmaceutical industry as a formulation method to increase the effective solubility of poorly water soluble drugs and subsequently their bioavailability. Cryomilling is attractive technique to render crystalline materials amorphous without using heat or solvent as are typically used in current processes. The possibility of amorphous formation via cryomilling was studied for three different types of drugs with different glass forming ability (GFA); Felodipine (class III) an easy glass former, paracetamol (class II) a moderate glass former and aspirin (class I) a poor glass former. These drugs were cryomilled alone, cryomilled then mixed with cryomilled hydroxypropyl methylcellulose (HPMC) physically (i.e. cryomilled separately) or co-cryomilled with HPMC together. The subsequent formulations were characterised by DSC, XRPD and FTIR. It was found that when felodipine is cryomilled alone, it can be transformed into the amorphous form, however no amorphous formation was achieved when cryomilling paracetamol or aspirin alone. It is thought that the relatively higher Tg of felodipine compared to paracetamol,and aspirin enables this transformation, however, this transformation was difficult to achieve for paracetamol or aspirin due to their rapid recrystallisation directly after cryomilling due to their low Tg and their resistance to mechanical disorder. Although felodipine was rendered amorphous when milled alone it then recrystallised within a day. It was thought that the conversion of these three drugs into amorphous form or not depends on the glass forming ability of each drug. The amount of polymer required to stabilise the amorphous form of each drug varied according to their glass forming ability with more polymer required for poor glass formers. Felodipine was selected as a model drug for further study with different polymers. This is because felodipine is widely used for the production of amorphous solid dispersion by hot melt extrusion and spray drying. Secondly this drug is practically insoluble but it is an important drug in the emergency treatment of hypertension due its high selectivity and its lack of a negative inotropic effect. Felodipine was co-cryomilled with different polymers and polymer blends that varied in water solubility namely HPMC, HPMCAS, Soluplus R, PMMA, HPMC-HPMCAS, HPMC:PMMA and Soluplus R:HPMCAS at 5, 25, 50 and 75% (w/w) drug loadings. For each mixture the miscibility was predicted using the Gordon-Taylor equation and solubility parameter value. All these mixtures at 50% drug loading were further investigated in dissolution studies for 6 hours under sink condition. All co-cryomilled samples except PMMA showed a high level of drug release (> 90%) after 6 hours dissolution. Only PMMA, which is water insoluble, retarded the release of felodipine from the co-cryomilled mixtures but in ternary mixture felodipine-HPMC/PMMA it showed more than 95% drug release after 6 hours dissolution. As felodipine with Soluplus R showed a good miscibility and stability at 0% humidity and high drug release this co-cryomilled mixture with 50% drug loading was used in the formulation of an orodispersible tablets (ODTs). Six different formulations were manufactured using different superdisintegrants such as F-melt and Glycolate. All the co-cryomilled formulas showed a higher release of felodipine compared to the physical mixtures. Obtaining miscible and stable amorphous solid dispersions without heat or solvent through co-cryomilling is promising as a manufacturing method. Use of heat or solvents can lead to instability in the dispersions and degrade certain drugs. Following this work, amorphous solid dispersions formed via cocryomilling with a high drug loading can be considered for future development for the formulation of fast release dosage form and for drugs liable to thermal or solvent mediated degradation.

P.aeruginosa is one of the most important pathogens in nosocomial infections and fails to respond to standard treatment, particularly in the case of patients subjected to prolong antibiotic treatment. To generate a more comprehensive understanding of the failure of antimicrobial treatment, focusing especially on the adaptive resistance could be the key area that the bacterium develops in this phenomenon. Most studies on antibiotic resistance in P.aeruginosa have focused upon genotypic studies. This study set out to develop an in vitro model to examine the effect of continual exposure of P.aeruginosa PA01 to the antibiotics studied. Experiments were initially conducted to consider the factors that having a significant influence on antibiotic susceptibility using a novel fluorescence based assay (OxoPlate® system). P.aeruginosa was subjected to the action of tobramycin, amikacin and colistin under various environmental factors. The results of the in vitro analysis showed that, from among the three antibiotics used, amikacin was the antibiotic where resistance was most readily developed. From these results, chemostat studies were designed to examine prolonged exposure of the antibiotic to planktonic cells. Chemostat cultures were exposed to amikacin at sub-inhibitory concentrations using Evans defined synthetic medium at different dilution rates (D) under glucose limitation. Both cultures grown at 0.025h-1 and 0.06h-1 developed the following characteristics i. low-level amikacin resistance, which exhibited an increase in the MIC 4-fold. ii. a clear development of phenotypic resistance and this resistance was not acquired as evidenced by the loss of resistance on culture into fresh medium lacking antibiotic. iii. adaptive resistance to amikacin conferred low-level resistance to other aminoglycosides such as tobramycin and antibiotics with different modes of action such as colistin. Low oxygen availability was seen in the cultures grown at 0.099 h-1 and 0.125 h-1, which lead to i. the appearance of the so called “persister” phenotype. These persisters are sub populations of cells that showed a reduction in bacterial cell size as evidenced from the flow cytometry output as well as being slow growing and resistant ii. extracellular polymeric fibrils were produced in the cells derived after 72h incubation time. In all cases, continual exposure resulted in phenotypically distinct mucoid and non- mucoid colony morphotypes, which were clearly observed on amikacin-free nutrient agar. Some of these selected morphotypes showed from the MIC and MBC data a high-level resistance to the antibiotic when left without antibiotics. The biological responses resulting from these studies offer valuable clues underlying unsuccessful treatment. Conducting experiments using robust systems renders this project extremely novel in the field of microbiology and this will contribute to the development of viable treatment options and ultimately the reduction of the emergence of antibiotic resistance.

The most successful antimicrobial agents in clinical use are of microbial origin and of these the greatest variety has been found in the genus Streptomyces. However, the resistance of the pathogenic microbes to the commonly used antibiotics is increasing as a result of the wide-spread and long-term use of these antibiotics. Therefore, understanding the strategies that bacteria use to become resistant is of crucial need. Streptomycetes are Gram positive bacteria, commonly found in soil and are known antibiotic-producers. The focus of this thesis was to underpin the mechanism of resistance to penicillin G in isolated strains of streptomycetes that exhibit elevated resistance to penicillin G and to characterise these organisms. Moreover, to investigate the interaction between penicillin G and PBPs in Streptomyces strains and investigate the relationship between growth rate and penicillin G resistance in Streptomyces in vitro. Ninety six Streptomycetes were isolated and characterized. Morphological examination and the16s rRNA sequences of these strains indicated that strains belong to the species Streptomyces. The MICs and MBCs for penicillin G for the isolated Streptomyces strains were measured by plate culture. Some strains showed growth up to 400 μg/ml with penicillin G, which indicate that the strains were highly resistant against penicillin G. Some strains were unable to grow at penicillin concentrations above 200μg/ml. Also, The MICs of penicillin G for isolated Streptomyces strains were measured using a novel OxoPlates® system in 96-well culture format employing Mueller-Hinton broth culture. The MICs of all strains ranged from 1-100 μg /ml. Results indicate that the sensitivity of Streptomyces strains of penicillin G is not directly related to β-lactamase production in the panel of isolates examined. There was no correlation between the MICs of penicillin G and the growth rate in these isolates. Likewise, there was no association between the position of beta-lactamase producing and non-beta-lactamase producing strains on the phylogenetic tree and their beta-lactamase xii activity. Beta-lactamase producing and non-producing strains refers to the same ancestral origin clade. Additionally, the comparative analysis of 16S rRNA gene sequence and phylogenetic relationship of strain (W43) revealed that the isolate clustered with (W76) Streptomyces lividans strain YLA0. Bocillin (a penicillin binding protein stain) staining in β-lactamase producing strains showed staining throughout the mycelia whereas in non β-lactamase producing strains staining only occurred in certain parts of the mycelia. Bocillin also revealed that in spores PBPs were located on both poles of the spores. Streptomyces coelicolor has the ability to grow at high concentrations of penicillin G up to 640 μg/ml in continuous culture. It also has the capacity to grow at very low amounts of dissolved oxygen in continuous culture. Significantly, there was a correlation between the growth rate of S. coelicolor and the resistant to penicillin G. S. Coelicolor was more sensitive to penicillin G at a high dilution rate. Furthermore, our strategy of using the Bug-Lab for monitoring the progress of S. Coelicolor 1147 in continuous culture, even at low concentrations of cells in real time was successful.

Molecular recognition refers to the interaction between two or more molecules through complementary noncovalent bonding, for example, via hydrogen bonding, electrostatic interactions, van der Waals forces or hydrophobic forces. Molecular recognition plays an important role in biology and mediates interactions between receptors and ligands, antigens and antibodies, nucleic acids and proteins, proteins and proteins, enzymes and substrates, and nucleic acids with each other. Many cellular processes are governed by a group of proteins acting in a coordinated manner; such complicated mechanisms are closely regulated: changes in the populations of particular complexes or changes in concentrations of the products of protein mediated reactions can switch cells from one state to another (from replication to apoptosis, for example). These small variations in molecular populations are caused by very delicate differences in the thermodynamics or kinetics of reactions. This implies that in order to understand not only biological systems in terms of their molecular components, but also to be able to predict and model system response to stimuli (whether it is a natural substrate or a drug), characterisation of the thermodynamic and kinetic components of the binding process is of paramount importance. This combined computational-experimental project was focused on the development of new computational approaches able to predict the enthalpic component of ligand binding, using quantum mechanics. A concept of ‘theoceptors’ was developed, which are theoretical receptors constructed by computing the optimal geometry of ligands binding in the receptor. This project was supported by AstraZeneca, and it included an industrial placement in the Structural and Biophysical Sciences area, where the experimental data was generated to characterise the thermodynamics and kinetics of binding of a range of ligands to two biological targets, using two experimental techniques, isothermal titration calorimetry and surface plasmon resonance. The findings contribute greatly to the process currently underway of expanding our understanding of the relevance of both of these aspects of biochemistry to drug discovery.

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Since their inception the B-Iactam antibiotics have become one of the most important classes of phannaceutical agents, both therapeutically and economically, in modern day usage for the treatment of a wide spectrum of bacterial infections. However, due to the versatility of bacteria many previously treatable species are developing resistance to the antibiotics currently available and so there is ever a need to develop more ~-lactam antibiotics, which are effective and yet safe. A major drawback to the ~-lactams is the degree of immunologically adverse reactions they induce. It was the aim of this study to develop both mechanistic and immunological methods to enable the prediction of a B-lactam's potential to induce an allergic response and to determine if a relationship between these responses and the molecular properties of the ~-lactams was present. In this study a database pertaining to frequency by which 70 p-lactams induce adverse reactions has been compiled and used to produce 27 QSAR models. A highly sensitive assay for the quantitation of cross-reactivity between B-lactams and serum anti-benzylpenicillin antibodies has been developed and used to determine the cross-reactivity potential of 31 ~-lactams and to develop 18 QSAR models. All of the QSARs developed suggest that the shape and electron separation of the ~-lactams are crucial to the development and extent of adverse response or crossreactivity induced by a specific p-lactam antibiotic, new or old. The QSARs developed will enable the design and development of new ~-lactam antibiotics which present a significantly lower risk of inducing immunologically mediated adverse responses when used therapeutically. Two sensitive assays for the quantitative detennination of the cytokines IL2 and IL4 in lymphocyte culture supernatants have been developed, and have been shown to have a potential use in the prediction of the type of immunological response initiated following p-Iactam stimulation of a sensitised individual.