Search

The tribological performance of internal joint prostheses is a fundamental influence on their longevity. The aim of this study is to characterise the tribological performance of the Porous Coated Anatomic total hip replacement by the analysis of 119 explanted prostheses. Investigations of the friction, wear, surface topography and wear debris were made and related to the joint's clinical performance. The friction of the joints at explant was similar to that of new prostheses. The median total wear volume (419mm(^3)) was found to agree with previous wear studies suggesting the existence of a threshold wear volume which promotes osteolysis. Clinical wear factor for the whole cohort matched that of alternative joint designs. The femoral head finish was shown to degrade but not in proportion to implant duration. The roughness of the UHMWPE liner was shown to fall but no relationship with any head roughness, or temporal, parameter could be distinguished. Simulator studies confirmed that the wear factor of a joint is likely to change over its lifespan. Wear models published previously describing the influence of femoral head roughness on wear could not predict the performance of explanted prostheses. An alternative relationship was observed indicating that head roughness is not as powerful a predictor of wear as previously held. A novel technique for the characterisation of the size distribution of ex vivo and in vitro wear debris was developed. A Low-Angle Laser Light Scattering Particle Analyser was used to size particles continuously over a range from 0.5 to 1000μm. This technique offers considerable unprovement over existing microscope-based methods in terms of the detail of the information and does so with less experimental effort. It was shown to be highly accurate and repeatable in preliminary investigations. Case studies of five tissue samples revealed the potential of this method.

Autophagy is a highly conserved biological process in eukaryotic cells with an essential role in cellular homeostasis, where dysfunction in this process is a key factor in a multitude of diseases. To this end, accuracy and reliability in the detection and monitoring of autophagy is critical for medicine and research. Current methods for autophagy detection can be costly, time consuming and labour intensive, which hampers their use outside of research laboratories. Raman spectroscopy is a spectrographic technique which allows visualisation of the chemical environment within a sample in a non-destructive and non-invasive manner. The use of Raman spectroscopy has never been fully realised for the detection of autophagy and can offer key benefits to the process. By using Raman spectroscopy to assess the chemical changes in cells undergoing autophagy key changes can be mapped out and used to create a predictive model via the use of machine learning. Raman collection was optimised via the use of drying cells onto a gold coated mirror, to facilitate high Raman scattering intensities with relatively short collection times. The processing of the spectra was optimised with a focus on creating a more generalisable process. Testing different baseline correction algorithms it was found that the asymmetric least squares algorithm performed the best across multiple distinct Raman spectra from different cell lines and different experiments. Raman spectra of cells after 4 hours complete amino acid deprivation show little difference in their Raman spectra, whereas the spectra of cells after 1, 2 and 5 days without l-glutamine show significant changes. These changes are often cell line specific but a commonly seen change is a drop in the 1675 cm-1 shoulder region associated with the protein secondary structure β-pleated sheets. Many of these changes however are also seen in ATG5(-/-) cells suggesting the changes are not autophagy specific. Using an artificial neural network, trained on the first 20 principal components of the Raman spectrum and the cell type, could distinguish between control cells, autophagy induced ATG5(+/+) cells and autophagy induced ATG5(-/-) cells with a 98.2% accuracy. This research lays the groundwork for the use of Raman spectroscopy as a method of detection for autophagy. Larger and more comprehensive data sets would need to be collected to further the applicability of this method.

Mitigating climate change calls for a reduction in emissions of greenhouse gases, mainly CO2. Twenty-seven per cent of all CO2 emissions come from sources hard to eliminate, including aviation, shipping, and long-distance land transportation. To scale-up production, a transition from first-generation biofuels (edible plant biomass feedstock) to second-generation biofuels (non-edible lignocellulosic plant biomass) is required. However, lignocellulosic bioprocesses struggle to reach economic viability due to the high cost of added enzymes required to digest cellulose. A microbe that was able to generate the cellulase to digest cellulose and able to generate desired products (in what is called a consolidated bioprocess) is not known to exist in nature. Decades of research in synthetic biology have not been successful in creating it due to multiple challenges, such as generating candidate microbial strains and testing them. In this work, novel methodological approaches were developed to overcome these challenges, thus increasing the likelihood of future research finding the ideal microbe. Firstly, the generation of candidate strains was improved by a re-design of the paradigm of modular cloning, based on a vector design called JUMP (Joint Universal Modular Plasmids). Complex multi-gene plasmids can be built from standard DNA parts in a reliable and automation-friendly way using modular cloning systems, based on Golden Gate cloning (which uses type IIS restriction enzymes). However, current standards lack the flexibility to change the microbial host and to perform assemblies to optimise genes of a group. JUMP vector backbones are based on the Standard European Vector Architecture (SEVA), and also have additional modular cloning sites to modify vectors for specific purposes. The experimental results presented here showed that these features allowed use of the vector system in different organisms and reduced the number of assemblies required to optimise and test multi-gene constructs. Secondly, among different explored approaches to test strains, using fluorescent growth reporters was found to have the properties required to screen strains in a faster and more relevant way. Cellulose is insoluble in water, and consequently, previous analytical methodologies to assess the cellulolytic capacity of microorganisms used soluble analogues of cellulase substrates or depended on separation steps which are difficult to do in a fast and high-throughput way. The data presented here showed that expression of fluorescence genes, providing a direct measure of growth, could be measured without separation of cellulose. An Escherichia coli strain expressing cellulases CenA and Chu2268, previously shown to bestow cellulolytic ability on E. coli, was confirmed to be able to grow using cellulose as sole carbon source, which demonstrated the use of fluorescent growth reporters to detect cellulolytic activity. Finally, a strain benchmark was built and characterised to allow screening of expression of cellulolytic genes. A collection of modular parts encoding cellulolytic proteins and secretion signal peptides was built to allow future screening, and the effect of genetic and environmental factors in the measurement of fluorescence as a reporter of growth was investigated. It was found that volumetric conditions and medium additives critically affected the capacity of E. coli to grow at the expense of cellulose. A methodology was developed to allow growth measurement in 96-well plates. In conclusion, this study lays the foundations to establish a faster research cycle to generate and screen microbes that can utilise cellulosic biomass to produce valuable bioproducts and biofuels.

In any biotechnological application that requires a transgene to be expressed long-term in mammalian cells, preventing its silencing by epigenetic processes can be a challenge. This includes the biopharmaceutical production of therapeutic proteins in Chinese Hamster Ovary cells and gene therapies currently being trialled for diseases such as Severe Combined Immunodeficiency. One of the strategies available for ensuring the stable expression of a trans-gene from the mammalian genome is to include chromatin control elements, such as barrier insulators, in the vector it integrates with. These sequences act in cis to maintain nearby genes within an epigenetic environment permissive to transcription. Barrier insulators are not a particularly well-catalogued variety of genetic part, so new examples could help improve our understanding of how they function and are desired for building genetic circuits. This thesis discusses an effort to develop a high-throughput barrier insulator testing platform, based on a Bxb1 integrase landing pad. Landing pads are loci in a genome that allow highly efficient targeted integration of DNA cargoes by site-specific recombination. In this case, the landing pad would be used to introduce many prospective barrier insulators into the genome of a human cell line, where they would then be evaluated. Barrier activity would be assessed at the landing pad using an epigenetic silencing assay that would follow the expression of a fluorescent reporter gene. The reporter would be silenced experimentally by tethering repressor domains, involved in heterochromatin formation, to a site downstream in the genome. This should seed the formation of heterochromatin that should spread into, and repress the transcription of a reporter. Placing a functional barrier insulator in the path of this spreading has previously been shown to mitigate silencing. Chapter 3 describes the development of an experimental setup for inducing and monitoring gene silencing in the genome of HEK293-FT cells. In this work, a cell line containing a CAG driven EGFP reporter and downstream TetO7 recruitment site at the AAVS1 locus was generated. It was found that targeting several Streptococcus pyogenes (Sp) dCas9 repressors to the TetO7 site could effectively silence EGFP expression within six days. This chapter also describes the development of Ouabain co-selection and Double Cutting (ODC), a CRISPR-Cas9 method for targeted integration, which proved to be an effective means for generating edited HEK293-FT cell lines. In chapter 4, the silencing assay was utilised to screen for new CRISPR repressor tools based on the CRISPR systems from Staphylococcus aureus (Sa dCas9), the commercially relevant Eubacterium rectales (Er dCas12a), Acidaminococcus sp. BV3L6 (As dCas12a) and Lachnospiraceae bacterium ND2006 (Lb dCas12a). It was anticipated that these might be applied as complementary, orthogonal repressors to Sp dCas9 repressors. Three novel repressors were identified that are able to silence EGFP expression: As dCas12a-csHP1a, Sa dCas9-KRAB-MeCP2 and Sa dCas9-REPR. Lb dCas12a and Er dCas12a repressors appeared to be too poorly expressed to detect any silencing effects. Er dCas12a, despite not being suitable for repression, was demonstrated to be suitable as a scaffold for activating gene expression for the first time. In chapter 5, the experimental set up from chapter 3 was used to inform the design of a Bxb1 landing pad platform for testing insulators. This design utilised a promoter trap for selecting cells which had undergone Recombinase Mediated Precision Integration (RMPI). HEK293-FT cell lines containing a single copy of the landing pad were isolated, and a set of cargo vectors that could recombine into the landing pad were generated. These vectors each contained a promoter-less mNeonGreen reporter cassette, a different insulator sequence and the TetO7 array. Unexpectedly, when the cargo vectors were recombined into landing pad cell lines, the recombinant cells were found to express different amounts mNeonGreen depending on the insulator sequence they now harboured. This hampered the planned epigenetic silencing assay, and the cause of this phenomenon could not be conclusively determined. Despite this complication, two of the novel candidate insulator sequences introduced into the landing pad exhibited characteristics that could indicate barrier activity. Both ensured high levels of mNeonGreen expression from the landing pad up to 42 days post-transfection. This was comparable to the level observed from a cargo plasmid containing two copies of the well-characterised cHS4 barrier insulator sequence. In summary, this work made progress towards developing a high-throughput insulator testing platform. Two sequences linked to long-term enhanced trans-gene expression were identified, which will require further exploration and validation. Along the way, new CRISPR tools for controlling transcription based on the As dCas12a, Er dCas12a and Sa dCas9 scaffolds were also demonstrated. Further pursuing this barrier assay may yield additional sequences with insulator activity. This may eventually help to determine the design rules necessary for building improved, synthetic barrier insulator sequences de novo.

There is growing interest in copper nanoparticles for their potential role in industrial catalysis and as antimicrobial agents. However, their chemical synthesis requires strong reducing agents and high temperatures. Using living cells to synthesise copper nanoparticles can overcome these requirements, but the mechanism of this biological synthesis is not well understood. Better understanding and control of the synthesis mechanism will allow the scale up of biological production methods, and presents opportunities in the remediation of copper ions from industrial waste streams, such as those produced by the distillery industry. Morganella psychrotolerans has previously been shown to convert copper (II) ions to stable copper (0) nanoparticles at room temperature. It can tolerate high concentrations of copper and grow in whisky co-products. The aim of this study was to better understand the mechanism of copper nanoparticle formation in Morganella psychrotolerans through the investigation of the proteins involved. For the first time, proteomic analysis of the cell’s response to copper exposure was carried out using mass spectrometry. The cellular proteins stabilising the copper nanoparticles were also identified and described. Selected proteins from these analyses were constitutively overexpressed to quantify their effect on nanoparticle production and their effect on the reductive capacity of the cell milieu. In order to develop genetic tools to modify these proteins, both homologous recombination and CRISPR-Cas9 were trialled in Morganella psychrotolerans.

There is a pressing need for further advancement in tissue engineering of functional organs with a view to providing a more clinically relevant model for drug development and reduce the dependence on organ donation. Polymer based scaffolds, such as polycaprolactone (PCL), have been highlighted as a potential avenue for tissue engineered kidneys, but there is little investigation down this stream. The focus within kidney tissue engineering has been on two-dimensional cell culture and decellularised tissue. The aim of this project is to utilise electrospun scaffolds within a three-dimensional printed bioreactor system to create an ex vivo environment, to be used as either a conditioning tool for kidney tissue engineering scaffolds or as a model for disease. Electrospun polymer scaffolds can be created with a variety of fibre diameters and the variation in morphology of tissue engineered scaffolds has been shown to affect the way cells behave and integrate. The first study of this thesis examined the cellular response of a kidney cell-line to scaffold architecture using novel electrospun scaffolds. Two fibre diameters were used and three distinct scaffold architectures: random, aligned and cryogenic. The results showed that architecture of the scaffold has a profound effect on kidney cells; whether that is effects of fibre diameter on the cell attachment and viability or the effect of fibre arrangement on the distribution of cells and their alignment with fibres, overall there was a preference for a larger fibre diameter of around 4 μm. Following this, electrospun scaffolds were investigated for their potential to host a multicell population. Rat primary kidney cells were used, and results showed that the scaffolds were capable of sustaining a multi-population of kidney cells, determined by the presence of: aquaporin-1 (proximal tubules), aquaporin-2 (collecting ducts), synaptopodin (glomerular epithelia) and von Willebrand factor (glomerular endothelia cells). Viability of cells appeared to be unaffected by fibre diameter. Overall, the ability of electrospun polymer scaffolds to act as conveyors for kidney cells is a promising strategy for kidney tissue engineering and one that should be explored further; the 'non-woven path' provides benefits over decellularised tissue by offering a high degree of morphological control with a scalable fabrication process and a tuneable rate of degradation. Investigation continued with the development of a 3D printed bioreactor. This was a proof of concept device aiming to represent the in vivo environment. The device was designed to be simple to use, delivering a finely controlled shear stress to cells adhered to the scaffold in a dual chamber system, using a modified cell culture media. Computational fluid dynamics was used to gain a better insight into the forces experienced by cells and a modified cell culture media was used to give a better shear distribution across the scaffold, whilst keeping the flow chamber height large. Our investigation demonstrated the ability of the lab scale system to sustain cell life whilst upregulating key transmembrane (AQP-2), cytoskeletal (KRT-8, KRT-18) and tight junction (E-CAD) proteins. Development continued, consolidating the positive characteristics of the bioreactor whilst providing a simplified redesign. In this second-generation bioreactor, the shear profile delivered to each side of the scaffold was mirrored with a flow regime intended to have a low Reynolds number, allowing for the use of two different media without mixing by convection. The device produced a shear stress of 18 to 21 mPa over 80% of the scaffold surface on both superior and inferior sides. The bioreactor maintained a co-culture of endothelial (HUVEC) and epithelial (RC-124) cells, showing the distinct cell types localised on opposite sides of the bioreactor, identified by aquaporin-2 and von Willebrand factor. This bioreactor is a useful tool for modelling of kidney tubules, but has applications in any area where a dual environment with a controlled shear stress is needed. Overall, this work has expanded the breadth of kidney tissue engineering potentially guiding new potential areas of research.

Fluorinated products are widely used in pharmaceutical and agrochemical applications where a large portion of the commercial compounds being developed contain at least one atom of fluorine. Most of the conventional fluorination reactions require volatile chemicals and are highly exothermic requiring controlled procedures which are rather costly, produce low yields and exhibit low selectivity of the moiety accepting fluorine. As a result, much attention has been drawn on enzymatic fluorination in a quest to ameliorate these issues. The only characterized enzyme that mediates direct fluorination is the 5'-fluoro- 5'-deoxyadenosine (5'-FDA) synthase (a.k.a. fluorinase), which uptakes an S- adenosyl-L-methionine (SAM) molecule and a fluorine atom as substrates and yields 5'-FDA and methionine. Variants of this enzyme have been extensively studied structurally and functionally with in vitro assays and they exhibit very high homology, and a similarly slow activity. In this work, a bacterial in vivo bioreactor is presented, based on a variant of the enzyme, which is found in Streptomyces sp. MA37 (FlA1). The base host that has been chosen for the implementation of modiflcations is Escherichia coli BL21(DE3), which is a host of choice for the over-expression of recombinant proteins and in some occasions utilized for the construction of novelty bioreactors. In the case of enzymatic fluorination, a few challenges had to be addressed. First of all, E. coli contains an ion channel protein, termed CrcB, which expels fluoride anions out of the cell. Secondly, SAM cannot passively enter E. coli cells rendering the intracellular molarity of SAM a bottleneck. Last but not least, the E. coli purine nucleoside phosphorylase, DeoD, has been suggested to degrade the fluorination product, 5'-FDA. Therefore, the modified E. coli strain, consists of 2 deleted genes (∆crcB∆deo∆), the expression of a SAM transporter from Rickettsia prowazekii and the over-expression of flA1. Using this strain, in vivo production of 5'-FDA to near millimolar concentrations has been confirmed using an array of chemical analysis methods including High Performance Liquid Chromatography (HPLC), Fluorine Nuclear Magnetic Resonance (F-NMR) and High Resolution Mass Spectrometry (HRMS).

This project set out to examine the possibility that by acquiring Raman spectra and performing multi-photon imaging we can get better diagnosis and understanding of the biochemistry of an individual cancerous tumour and distinguish it from the healthy tissue. Within the frame of this study, colorectal primary and secondary cancer cells are examined with Raman spectroscopy in order to (i) study and distinguish them according to their chemical composition by applying multivariate methods and (ii) determine whether Raman spectroscopy can identify the cells which are the link between primary and secondary colorectal cancer cells, the so-called Cancer Stem Cells. The second part of this thesis is based on tissue studies. Human colorectal tissue sections are examined in a label-free manner with the use of multi-photon imaging modes (i) Two photon excitation fluorescence, (ii) stimulated Raman scattering and (iii) second harmonic generation, in order to determine whether these can provide fast and accurate diagnosis of colorectal cancer. These techniques were able to distinguish between healthy and cancerous tissue regions, based on the chemically-specific images of the tissue microenvironment and architecture. The hypothesis of Cancer stem cell is examined with the use of Raman spectroscopy shown that the CSCs have some small differences according to their tissue origin.

Magnetic hyperthermia (MH) is an anti-cancer treatment which exploits the heat produced by tumour-targeted magnetic nanoparticles (MNPs) subjected to an alternating magnetic field (AMF). A problem limiting the clinical use of MH, however, is the inability to adequately localise the MNPs at the tumour site. A cellular approach using mesenchymal stem cells (MSCs) as carriers has been proposed as these cells are believed to home to sites of tissue injury and tumour growth, however problems with MNPs uptake and toxicity retard progress and need to be overcome. The aim of this project was to find an alternative approach in MH treatment, creating engineered human MSCs able to biosynthesise MNPs. To achieve this goal, MSCs were transfected with either, or both, M. magneticum AMB-1 mms6 and mmsF genes. M. magneticum AMB-1 is a genus of magnetotactic bacteria, containing magnetosomes, which are lipidic organelles containing single crystals of magnetite. M. magneticum-AMB1 mms6 and mmsF genes are important for final crystal morphology and are known to play a role in crystal synthesis and growth respectively. The originality of this study was in using mms6 and mmsF genes, which were codon-optimized for mammalian expression, alone or in combination, for transfection of human MSCs, which have known tumour homing capacity. The transfected MNPs-bearing MSCs, able to migrate into the tumour tissue, were subjected to AMF in MH experiments in an attempt to induce cancer cell death. mms6 and mmsF gene expression, following transfection, was investigated in the human osteosarcoma cell line MG63 by reverse transcription polymerase chain reaction (RT-PCR). The cellular ultrastructure of transfected MG63 cells was investigated by transmission electron microscopy (TEM), revealing the presence of nanoparticles. The magnetism of transfected MG63 cells was proved by superconducting quantum interference device (SQUID) and supported by in vitro MH experiments. Then, human MSCs were transfected with mms6 and mmsF genes, alone or in combination. The effect of transfection experiments and MNPs synthesis on MSCs markers of stemness, cell proliferation and differentiation ability were investigated. The MTB genes expression in human MSCs was assessed by RT-PCR and cell magnetism was confirmed by SQUID, in vitro MH experiments and by magnetic force microscopy (MFM). Then, in vitro studies of MH were undertaken to establish whether mms6 transfected MSCs expressing MNPs supported a MH effect when exposed to an AMF. Cells were initially exposed to an AMF of 565.3 kHz frequency in monolayers and in 3D arrangements and cell death/viability was assessed. Subsequently, the effect of the same AMF on 3D models of mixed populations of mms6-expressing MSCs and cancer cells was assessed. The results indicate that viability of MNPs-expressing MSCs and adjacent cancer cells is reduced following AMF exposure. In vivo studies of MH were undertaken following intracardiac injection of mms6-expressing MSCs in tumour-bearing mice (epidermoid carcinoma). The expression of mms6-expressing MSCs inside mice organs was confirmed by RT-PCR, fluorescence microscopy and immunohistochemistry. The effect of the application of an AMF of 565.3 kHz on mice tumours was studied with different techniques (tumour size and volume measurement, multiphoton microscopy, haematoxylin and eosin staining, and activated Caspase 3 expression), to understand if MNPs created inside mms6- expressing MSCs, following AMF exposure, could lead to cancer cell death. Results indicate that mice tolerate the treatment well, however no appreciable tumour reduction or necrosis was evident. Overall the results suggest that mms6 transfection alone confers the highest magnetisation to MSCs compared to mmsF alone or mms6+mmsF co-transfected, and that mms6 expression in human MSCs does not have an adverse effect on important cell functions. mms6-expressing MSCs, when exposed to an AMF, show reduced viability and enhanced cell cytotoxicity in vitro. When co-cultured with cancer cells in 3D models in vitro, mms6-expressing MSCs are able to reduce viability of adjacent cancer cells confirming the potential applicability of mms6- expressing MSCs for MH treatment. In vivo proof of concept experiments show that mms6-expressing MSCs can locate to the tumour tissue, and mms6-expressing intracardiac injected MSCs mice exposed to AMF tolerate the treatment well. However, the number of mms6-expressing MSCs able to localize to the tumour tissue in this experiment was too low to give an appreciable tumour reduction, so more experiments are needed to enhance the experimental protocol. A number of improvements are required to progress this novel technique towards clinical application. Gene transfection and MNPs production need to be optimised, the best frequency for MH needs to be established and MSCs delivery to the tumour has to be significantly increased to allow concentration of MNPs. The study has helped to increase our knowledge on the creation of magnetic human MSCs to potentially use these cells in MH cancer treatment.

The mammalian testis is divided into two distinct compartments which carry out its principal functions. Spermatogenesis occurs within the seminiferous tubules and androgen biosynthesis primarily occurs in the interstitial space. Both these processes are entirely dependent upon the two major testicular somatic cell populations - the Sertoli and Leydig cells respectively. In human males, testicular spermatogenic and endocrine function declines during the ageing process. Of particular significance is the reported age-related decrease in Leydig cell androgen production as androgens have been suggested to play a crucial role in supporting lifelong general health in men, with low circulating testosterone linked to an increased risk of developing chronic age-related cardiometabolic diseases. However, the relationship between ageing, testicular function and disease is not fully understood, impeding the development of novel therapeutic strategies to treat age-related testicular dysfunction. In one set of studies undertaken herein, a series of novel mouse models of premature ageing were utilised to begin to dissect the process of age-related testicular degeneration. Firstly, a novel knockout-first conditional allele of a previously reported premature-ageing model driven by Cisd2 (CDGSH Iron Sulphur Domain 2) deficiency was validated and the testicular phenotype characterised and compared to that of naturally aged mice at 18-months of age. Histological analyses revealed premature testicular atrophy at 6-months of age in CISD2 deficient mice, consistent with observations of the naturally aged testis. Circulating testosterone was significantly lower in CISD2-deficient mice compared to wild-type controls at 6-months of age and the luteinising hormone/testosterone ratio was significantly elevated, indicative of compensated Leydig cell failure. mRNA expression of key genes involved in androgen production were also significantly reduced in the CISD2-deficient testis, pointing to Leydig cell dysfunction in this model of premature aging. Next, Cre/LoxP technology was used to delete Cisd2 from specific testicular cell populations to determine which cell types control/support Leydig cell function during the ageing process. Testosterone production was unaffected when Cisd2 was disrupted in either the Leydig cell population or Sertoli cell population. These observations suggest that disruption to the testicular microenvironment in which Leydig cells reside, rather than intrinsic Leydig cell ageing, may play a significant role in age-associated Leydig cell dysfunction. A second set of studies were carried out to investigate the role of leukemia inhibitory factor (LIF) signalling in the maintenance of testicular function. LIF is a pleiotropic cytokine belonging to the interleukin-6 family. In the rodent testis, LIF is expressed in fetal life and adulthood; the peritubular myoid cells thought to be the main site of production. Given their anatomical location within the testis, LIF produced by peritubular myoid cells may act on both intratubular and interstitial cells to influence spermatogenesis and steroidogenesis respectively. Indeed, LIFR is expressed in germ cells, Sertoli cells, Leydig cells as well as testicular macrophages suggesting that LIF may be a key paracrine regulator of testicular function. However, the precise role of LIF/LIFR signalling in the testis is largely unknown. As such, models of testicular cell-specific Lifr deletion were generated using Cre/LoxP technology. Analysis of these novel models of conditional LIFR ablation revealed that LIFR is dispensable in germ cells for normal spermatogenesis. However, LIFR ablation from Sertoli cells resulted in a progressive degenerative phenotype, characterised by abnormal germ cell loss, sperm stasis, seminiferous tubule distention and subsequent atrophy of the seminiferous tubules. In a final set of studies, a rat model of Leydig cell ablation-regeneration was used to determine the regenerative capacity of human adipose-derived perivascular stem cells (hAd-PSC) as a potential therapy for testicular dysfunction. Following ethane dimethanesulphonate (EDS) mediated Leydig cell ablation, primary hAd-PSCs, cultured with or without LH, IGF-1, PDGFBB, T3 and ITS supplement, were transplanted into the rat testis and Leydig cell regeneration was monitored via serial measurements of circulating luteinising hormone (LH) and testosterone. Overall, hAd- PSCs had no impact on the recovery of circulating testosterone levels. However, when pre-cultured with the cocktail of hormone/growth factor supplements, the LH spike induced by the removal of testosterone negative feedback was dampened, suggesting the transplanted cells may promote Leydig cell regeneration. Whether these cells differentiate into Leydig cells, or simply provide paracrine support to the regenerating Leydig cells remains to be determined. Although Ad-PSCs may enhance regeneration kinetics, the transplanted cells were undetectable in the testis 5 weeks post transplantation suggesting they may not survive in the context of long term xenogeneic transplantation.

Advances in DNA synthesis technology have led to rapid growth in the field of synthetic biology, heralding a nascent era of synthetic genomics. Sc2.0 (Saccharomyces cerevisiae version 2.0) is an international consortium with the aim of designing and constructing a fully‐synthetic eukaryotic genome. Fundamental design changes to the synthetic genome include the removal of unstable tRNA genes and their intended collation onto a “tRNA neochromosome”, with the aim of producing a more robust and stable synthetic genome structure. To maintain viability of a synthetic yeast, the tRNA neochromosome is therefore considered an important if not essential aspect of this project. The application of engineering principles is synonymous with synthetic biology, regularly employing the recursive Design‐Build‐Test cycle to improve experimental approach. This doctoral study explores the design, construction and characterisation of a tRNA neochromosome in Saccharomyces cerevisiae. A series of design principles influenced by engineering concepts were used to rationalise the complexities of de novo chromosome engineering, maximise its stability and ensure function in vivo. A methodology based on in vivo homologous recombination was then developed and shown to reliably construct the neochromosome from its constituent parts. Experimental characterisation revealed that genetic elements function as expected, and that the parental strain can tolerate the sole presence of one each of three single‐copy, essential tRNA genes (SUP61, TRT2 and TRR4), although Northern blot revealed potential precursor accumulation of the SUP61 tRNA caused by the presence of a synthetic 5’ flanking sequence. Following the addition of synthetic telomere seed sequences, pulsed‐field gel electrophoresis (PFGE) and deep sequencing revealed complex structure variations in two independent strain backgrounds. Except for these structural variations, successful neochromosome construction demonstrated the applicability of the approaches used and the remarkable ability of the yeast model to support the presence of a 17th chromosome housing an additional 275 tRNA genes. The research in this thesis has for the first time described the design, construction and characterisation of a eukaryotic neochromosome de novo. It is hoped that the findings presented will further our understanding of tRNA biology and enhance the aims of the Sc2.0 project.

Heavy metals from natural and man-made sources can be a great threat to human and animal life. As small inorganic ions they are challenging to detect, usually requiring expensive and complicated machinery. Several heavy metals can accumulate in the human body, leading to long term toxic effects on the nervous system. Many bacteria have developed strategies to survive in heavy metal rich environments. One of these strategies is a bacterial operon containing genes for detoxification mechanisms controlled by a promoter and a regulatory protein. In this work some of these promoter-protein pairs, Pars-ArsR, PcopA-CueR, PmerTPAD-MerR and PzntA-ZntR from Escherichia coli have been employed in the design and construction of a set of biosensors aimed at the detection of heavy metals in drinking water. Biosensors usually employ biological recognition elements, transducing the signal from these to produce an output that can be integrated into electronic circuitry. The sensors presented in this work focus on reducing complexity and on providing a controlled sensor reaction. The arsenic biosensor ‘AsGard’ is based on the Pars-ArsR pair and functions by making the dissociation of an ArsR-mCherry fusion protein from its binding site in the Pars promoter visible. In the cell, ArsR dissociates from Pars upon binding of trivalent arsenic ions. Immobilising the relevant part of the Pars sequence on a solid plastic support allows for the mobilisation of previously bound ArsR-mCherry proteins in the presence of arsenic to become the sensor output. The AsGard sensor detects arsenic within minutes in a concentration range overlapping with the arsenic thresholds for drinking water as set by the World Health Organisation. Additional prototype sensors are presented bringing a reporter gene under the control of the aforementioned promoters. These sensors have been tested in vivo and in vitro in a cell free transcription translation system and partially detect metal concentrations close to relevant ranges. The Pars based sensor is tuneable in vitro by modifying the ratio of the supplied regulatory protein ArsR and is able to detect arsenic well within the relevant range. Spinach2, a fluorescent RNA aptamer, may make future designs independent from translation, drastically reducing complexity of cell free biosensors based on cis-trans transcriptional regulation.

The creation of molecular machines has been one of the goals of modern nanotechnology for a few decades. Such machines can be assembled from small molecules, as well as DNA. Of particular interest are mechanically interlocked nanoconstructs – catenanes and rotaxanes. These structures offer developments such as nanoswitches and rotational motors. DNA nanotechnology has produced numerous systems that consist of catenanes that could perform programmable switching and stimuli-responsive behaviour, as well as switching between stations in a semi-autonomous, rotary, motor-like behaviour. Energy transduction and the speed of such Brownian ratchet motors are negligible when compared to natural enzymatic activity. Bridging the gap between enzymology and structural DNA nanotechnology, we propose a method to assemble and operate a prototype system of fully complementary interlocked ssDNA rings that can roll against each other as a pair of gears with a ratio 1:2. The directionality and force is proposed to be generated by a strand-displacing polymerase enzyme performing a rolling circle amplification reaction on one of the members of this catenane, generating torque. Computer modelling of this system using oxDNA script package has been carried out, enabling both the topological visualisation and structural inquiry into the system prior to experimental development. Variations to the system such as changing the overall size, gearing ratio and developments towards integration into larger assemblies have also been described and are discussed in detail. Several experimental assembly strategies are described, together with experimental evidence of their outcomes. A method for operation of the single-stranded DNA catenane as a pair of continuously rolling gears has been investigated using strand displacing polymerases. Applications and suggestions for future developments is provided, addressing integration into complex systems. Additional methods of assembly and operation are discussed and compared.

p53 is a key component of the protein network that regulates cell cycle progression and prevents cancer. Under non-stressed conditions, its activity is controlled by an autoregulatory feedback loop with MDM2 that maintains low levels of the p53 protein. However, in response to stress signals, p53 is triggered to become active. MDM2 has been reported to regulate p53 by a combination of mechanisms: ubiquitination using its E3-ligase capability, chaperone activity in an ATP-dependent manner and directly transrepressing p53. Because of MDM2's central role in the control of p53, it has been the target of intense drug development efforts. A family of small molecules, the Nutlins, can bind to an MDM2 pocket modulating the p53: MDM2 complex. This leads to p53 activation and growth inhibitory effects. The aim of our study was to analyse the interactome of endogenous MDM2 and to determine whether anti-cancer drugs, such as Nutlin-3, could stabilise or disrupt sets of MDM2 interactions in order to better understand the p53- dependent and independent functions of MDM2 as a signalling hub, as well as the p53-independent activity of Nutlin-3. Results show a remarkable difference in the sets of proteins found in MDM2 complexes in control and Nutlin-3 treated cells. Two proteins, TRIM25 and OTUB2, were selected from the output list for validation based on their known functions in the ubiquitin signalling network. Binding has been studied in detail and confirmed using both in cell and in vitro techniques. The data highlight potentially novel functions for MDM2 and provides insight into the on-target p53-independent activities of Nutlin-3. Additionally, and with the aim of blocking p53 ubiquitination by MDM2, I have developed probes that are able to inhibit the ubiquitylation of p53 in vitro.

Enhanced Biological Phosphorus Removal (EBPR) is an established process in wastewater treatment that uses bacteria to reduce phosphorus levels below regulatory discharge limits. Recently, in light of growing political concern over phosphorus sustainability, EBPR has also been recognised as a platform from which phosphorus may be recovered and recycled onto land as fertiliser. Operating EBPR to optimise performance and efficiency is therefore extremely important, but remains a challenge due to poor understanding of these bacterial ecosystems. Raman spectroscopy is a non-invasive, label-free, culture-independent technique capable of analysing live, single cells. Despite its advantages, Raman spectroscopy has been applied to study EBPR bacteria in just a handful of studies and thus has a low profile in this field of research. More work is required to investigate potential areas of application for Raman spectroscopy in EBPR research. The principal thesis presented here is that Raman spectroscopy could be used as a tool to improve EBPR. The Raman spectra used for this investigation were acquired from individual EBPR bacteria dried onto a calcium fluoride substrate. The bacterial samples were collected from three different sources, namely lab-scale sequencing batch reactors located in Edinburgh (University of Edinburgh, UK) and Boston (Northeastern University, USA), and a full-scale EBPR plant in Slough (Thames Water, UK). Using these spectra, some potential applications and limitations of Raman spectroscopy for improving EBPR were explored. In this foundation work, a particular emphasis on spectral analysis methods was kept in light of the benefits of automating analysis as well as the need for standardisation to be able to compare results between different studies and groups. Nine methods were compared for baselining Raman spectra of individual EBPR bacteria. From these, the “small-window moving average” (SWiMA) method was determined to be the best baselining technique for our purposes at the current time. In agreement with earlier studies, the Raman spectroscopic signatures of three key EBPR metabolites – polyphosphate, polyhydroxyalkanoate (PHA) and glycogen – were shown to be clearly identifiable in individual EBPR bacteria when present. The Raman shifts of characteristic spectral bands arising from polyphosphate were shown to vary significantly between samples and the implications of this were discussed. Examples of how the Raman spectra of individual bacteria can be modelled with multivariate tools to open up new areas for research were given. MCR modelling was demonstrated to offer a novel way to normalise the Raman spectra of individual EBPR bacteria prior to quantitative analysis. With the instrumental set-up in this work, the limit of detection (LOD) of aqueous polyphosphate samples was estimated to be approximately 0.08 M and 0.02 M for 10 second and 200 second acquisitions respectively. Future work is required to research ways in which a more comparable form of polyphosphate ‘standard’ might be prepared so that direct correlation can be drawn between measurements made on such a standard and measurements made in bacterial cells. Overall, several applications and challenges of Raman spectroscopy for the investigation of EBPR bacteria are presented in this work together with recommendation for how to process the spectral data. The conclusions drawn from this work indicate that Raman spectroscopy could be used as a tool to improve EBPR but further work is required to refine and apply these methods.

The cell membrane is a highly complex structure consisting of a large diversity of phospholipids and macromolecules. There exist a variety of diseases that compromise the integrity of this key component of the cell. This thesis considers the investigation of interactions between β-amyloid peptide (Aβ₄₂) and lipid bilayers. To facilitate understanding of this complex system, it is advantageous to employ a model sample; supported lipid bilayers (SLB) and giant multilamellar vesicles (MLVs) are used as proxy cell membranes. These nanostructures are widely used as models of cellular membranes in many areas of scientific research. Phospholipid molecules self-organise into bilayer structures containing phase-separated microdomains, which are believed to be important in many biological processes. This study aims to develop model systems and experimental tools to explore hypothetical mechanisms through which the β-amyloid interacts with the lipid membranes. A lack of mechanistic understanding is the major challenge to our efforts to elucidate not only the interactions of the Aβ42 with the lipid membranes, but also the behaviour of these systems towards the changes of the environmental conditions (pH, concentration, temperature). Our results suggest that there are various different methods, such as AFM, CARS microscopy and Raman spectroscopy as well as neutron scattering that are capable of fast imaging. Overall, all these techniques contributed in a complementary study of Aβ₄₂ aggregation states under extreme and physiological conditions as well as to image Aβ₄₂ interactions with lipid bilayers consisted of specific lipids.

Purpose: Sperm nuclear proteins and DNA integrity have been implicated in infertility and treatment failures. High stallion to stallion variability is observed in sperm cryopreservation protocols. The cells are destroyed with harsh chemicals prior to using biochemical assays to test sperm DNA quality. The feasibility of using Raman spectrometry in combination with a laser trap for non-destructive micromanipulation and characterization of DNA damage in motile stallion and human sperm is experimentally investigated in this thesis. Methods: Live stallion sperms were subjected to controlled cellular damage: (a) four grades of chemically induced oxidative stress using Xanthine – Xanthine Oxidase (b) three grades of osmotic stress using PBS and (c) membrane damage using thermal shock. Live human sperm DNA disintegration with time and oxidative stress were explored on fresh, cryopreserved and swim-up categories. The specimens ranged from sub-fertile patients to fertile donors in a limited study. Post-treatment sperms resuspended in sperm media, placed on a quartz coverslip were trapped with a 785 nm, 25 mW laser, using a 1.4 NA, 60X, water immersion microscope objective. A Raman spectrum of a trapped cell was acquired for 20 – 30 seconds. The spectra from 20 – 40 cells from each specimen were analysed in the 630 cm-1 – 1630 cm-1 region using statistical variance and PCA. Results: The Raman spectra from trapped motile sperm head contain intense peaks that did not require smoothing prior to analysis. PCA of the Raman spectra could not resolve the different grades of applied osmotic and oxidative stress in stallion cells. PCA showed high variability between specimens from the same stallion and between stallions, with distinct clustering by ejaculate. Membrane damage study and spectra from extended trapping also showed distinct specimen to specimen difference within and between stallions. Specimen to specimen variability is observed in motility and viability tests on 1000s of stallion cells using CASA and flow cytometry. Human sperms showed some clustering by category, time, stress and motility and appeared more sensitive to the tests than stallion sperms. Conclusions: Raman spectra originate from the dense region of the trapped sperm head and resemble the fingerprint of dense calf thymus DNA. The cells show species specific response to the applied stress/damage. Stallion sperms show high variability between ejaculates that could not be differentiated by stallions. Human cells appear more sensitive to the applied processes. LTRS of live sperms needs further detailed research, cross correlated with other established complementary techniques, to identify spectral bands that are most sensitive to the various grades of induced DNA and membrane damage.

Aseptic loosening of artificial joints is caused by an osteolytic reaction to wear debris mediated by macrophages and other cells. Imaging these wear particles within cells can be a key process in understanding particle-cell interactions. However, the compounds used in surgical implants are not easily visualised as no tagging molecule can be added without altering the properties of the material. We were interested in using a label free optical technique known as coherent anti-Stokes Raman scattering spectroscopy (CARS) to image these particles in cells. In this thesis we studied how to use CARS to image physiologically relevant wear particles within cells. We characterised the responses from our CARS system and found them to be in good agreement to the Raman spectra we obtained for the same materials. We showed that the forward scattered CARS signal was consistently larger than the backwards scattered signal for the same size particles, and also generated a larger contrast, especially between sub-micron sized particles and the non-resonant background. Wear particles of polyethylene isolated from a pin-on-plate wear simulator were shown to be in a similar size range to those retrieved from revision tissue. When incubated in our model macrophage cells we were able to image areas of CARS signal that indicated the location of these particles in the cell. Furthermore, using multiple CARS images taken at different Raman resonances we were able to distinguish between three different polymeric compounds added to cells, showing the specificity of the technique. The inherent 3D sectioning capabilities of multiphoton microscopy were used to generate projected images of the cells and contents, as well as estimating the particle loads within cells. These results show that CARS could be an important tool in imaging intra-cellular polyethylene and characterising the interactions of wear particles with cells and the surrounding tissue.

The enzymatic deconstruction of lignocellulosic plant biomass is performed by specialist microbial species. It is a ubiquitous process within nature and central to the global recycling of carbon and energy. Lignocellulose is a complex heteropolymer, highly recalcitrant and resistant to hydrolysis due to the major polysaccharide cellulose existing as a crystalline lattice, intimately associated with a disordered sheath of hemicellulosic polysaccharides and lignin. In this thesis I aim to transfer the highly efficient cellulolytic mechanism of the bacterium Cellulomonas fimi, to that of a suitably amenable and genetically tractable expression host, in the hopes of better understanding the enzymatic hydrolysis of lignocellulose. Using tools and concepts from molecular biology and synthetic biology, I constructed a library of standardised genetic parts derived from C. fimi, each encoding a known enzymatic activity involved in the hydrolysis of cellulose, mannan or xylan; three of the major polysaccharides present in lignocellulose. Characterization assays were performed on individual parts to confirm enzymatic activity and compare efficiencies against a range of substrates. Results then informed the rational design and construction of parts into modular devices. The resultant genetic devices were introduced into the expression hosts Escherichia coli and Citrobacter freundii, and transformed strains were assayed for the ability to utilize various forms of xylan, mannan and cellulose as a sole carbon source. Results identified devices which when expressed by either host showed growth on the respective carbon sources. Notably, devices with improved activity against amorphous cellulose, crystalline cellulose, mannan and xylan were determined. Recombinant cellulase expressing strains of E. coli and C. freundii were shown capable of both deconstruction and utilization of pure cellulose paper as a sole carbon source. Moreover, this capacity was shown to be entirely unhindered when C. freundii strains were cultured in saline media. These findings show promise in developing C. freundii for bioprocessing of biomass in sea water, so as to reduce the use of fresh water resources and improve sustainability as well as process economics. Work presented in this thesis contributes towards understanding the complementarities and synergies of the enzymes responsible for lignocellulose hydrolysis. Moreover, the research emphasizes the merits of standardizing genetic parts used within metabolic engineering projects and how adopting such design principles can expedite the research process.

The plant hormone salicylic acid (SA) is an essential activator of plant immune responses directed against biotrophic pathogens. The transcription cofactor NPR1 (Nonexpressor of pathogenesis- related (PR) genes 1) functions to transduce the SA signal into an operational response directed to limited pathogen damage. In the absence of pathogen, NPR1 protein resides in the cytoplasm as a large molecular weight oligomer held together by disulphide bonding. Initiation of defence signalling leads to changes in intracellular redox conditions that promote NPR1 momomer release. Translocation of monomeric NPR1 to the nucleus results in the activation of over 2200 immune-related genes in Arabidopsis. NPR1 lacks a canonical DNA-binding domain but is known to perform part of its regulatory function through engagement of TGA factors (bZIP transcription factor). Induction of SA-dependent signalling is invariably associated with PR-1 gene expression and accumulation of mRNA for this gene serves as a useful marker of defence activation. However, both functional redundancy and stochastic factors limit the effectiveness of standard genetic approaches used in plant research, and thus much of the hierarchal processes surrounding NPR1-dependent gene activation are not fully understood. Using a synthetic biology approach we aim to complete exploratory work and set the foundations for the development of a yeast tool that can be used to manipulate and subsequently understand NPR1 function in relation to interacting partners and gene activation. Accordingly, using this tool we sought to create a conceptual protein circuit based on theoretical plant immunity. In completing this work we have developed a Saccharomyces cerevisiae strain that exhibits a highly oxidising intracellular redox environment. This was achieved by knocking out genes encoding S-nitrosoglutathione reductase (SFA1), flavohemoglobin (YHB1) and YAP1 (bZIP transcription factor), all important components in regulating cellular redox homeostasis and protein S-nitrosylation state in S. cerevisiae. Characterisation of this cell (designated Δsfa1yap1yhb1) reveals a high tolerance to such redox perturbations. Importantly, NPR1 is by default, assembled predominantly in the oligomeric form in this biological chassis. By activating two inducible inputs in the form of Arabidopsis S-nitrosoglutathione reductase (AtGSNOR) and Thioredoxin (AtTRXh5) which both function to promote NPR1 monomerisation, we have created a switch to selectively control NPR1 oligomer-monomer equilibrium. To complete the synthetic circuit, TGA3 was included, along with a modified yeast MEL1 promoter that has been customised to contain the TGA-responsive upstream activation sequence (termed the as-1 element) present in the promoter region of the PR-1 gene. Using FRET tools we were able to confirm nuclear interaction between monomeric NPR1 and TGA3, with this association appearing to induce as-1 element binding. However this process is not sufficient to activate a Luciferase (LUC) reporter gene, even when the GAL4 activation domain (GAL4 AD) is fused to NPR1. Ordinarily, a CUL3-dependent proteolysis-coupled transcription cycle is necessary to maintain efficient NPR1-dependent gene transcription in Arabidopsis. Although S. cerevisiae encodes an evolutionarily related CUL3 ortholog, examination by western blot demonstrates that NPR1 protein is stable in this cell, indicating an endogenous mechanism to degrade NPR1 is either not present or not functional in yeast. As such, this synthetic yeast tool represents a completely novel approach to identify missing components functioning in NPR1-mediated transcriptional regulation. Furthermore, in collaboration with a skilled bioinformatician, and using a rule-based stochastic modeling tool known as Kappa, we have been able to develop, for the first time, a preliminary mathematical simulation representative of NPR1-dependent gene activation that can be used as a foundation for future works.

The confluence of next generation DNA sequencing and synthesis when combined with the application of concepts such as standardization and modular design has led to the genesis of a new discipline. The nascent field of Synthetic Biology concerns the rational design and construction of genetic circuits, pathways, machines and eventually whole organisms. The immaturity of this field dictates that early research efforts, including this Thesis, describe foundational work towards the creation of tools which make biology more amenable to being engineered. The first part of this Thesis describes an attempt to standardize the measurement of transcriptional promoter activity in E. coli. A method to measure in vivo promoter activity was developed for E. coli and tested in a multi-institution trial. Comparable results were achieved with less than a two-fold range for the measured promoters across eight laboratories. A standardized measurement kit was created and distributed for use by the teams participating in the 2008 international Genetically Engineered Machines competition. Techniques learned measuring the activity of E. coli promoters were applied to a collection of S. cerevisiae strains. Several promoters were measured in synthetic dextrose media and ADH1 was measured in multiple media conditions. The outcome of these experiments is to consider proposing ADH1as the reference promoter in S. cerevisiae. The second aspect of this Thesis describes the construction of artificial organelles in S. cerevisiae. Artificial organelles hold the prospect of being able to insulate synthetic genetic pathways from the cell. Two proteins are essential for the biogenesis of the peroxisome organelle in humans and yeast, Pex3p and Pex19p. Pex3p functions as a peroxisomal membrane receptor for Pex19p, while Pex19p shuttles other peroxisomal proteins to the membrane, including Pex3p, creating a feedback loop. Human Pex19p has previously been shown to dock to yeast Pex3p and a version of yeast Pex19p has been shown to work with human Pex3p as a high degree of evolutionary conservation exists between these proteins. Because of these inter-species protein docking characteristics, there exists the possibility of creating bimodality: the ambition of the work was therefore to create a cell strain which possessed both synthetic “humanized” and natural yeast peroxisomes. An S. cerevisiae BY4741a derivative strain was engineered with fluorophore tagged versions of human (CFP) and yeast (YFP) Pex3p and untagged yeast and human Pex19p proteins. The results indicated the creation of a single population of peroxisomes when a measure of fluorescently imaged CFP and YFP peroxisomes were plotted on a scatter plot. A log of the ratio of CFP to YFP peroxisomes was plotted on a histogram and a normal distribution was found to best fit the curve, indicating a lack of bimodality. Finally, microscopy images of this strain were reviewed and by visual inspection, showed no evidence of distinct human or yeast peroxisomes. This experiment therefore produced no evidence of bimodality when examining the interactions of human and yeast Pex3p and Pex19p proteins. However, the four proteins were shown to interact closely to produce a single population of chimeric human-yeast peroxisomes. The peroxisome-deficient mutant phonotype strain was rescued using human Pex3p and Pex19p.

Over recent years, the application of assisted reproductive techniques in the treatment of infertility has increased exponentially, yet these methodologies still remain inherently inefficient. It has long been established that the single greatest obstacle to improving the success of these treatments is determining the quality of the oocytes used. However, currently the methods available for oocyte assessment are mainly qualitative, and suffer due to a lack of standardisation. As such, the efficiency of fertility treatments could benefit from the introduction of a rigorous quantitative measure of oocyte quality and maturation. The principal aim of this thesis was to determine the potential of Raman spectroscopy when applied to the field of oocyte biology. Consequently, this thesis addressed three main areas of investigation: I. the intra-oocyte biochemical variation; II. the biochemistry of oocyte maturation; and finally, III. the effect of environment on the mature oocyte in vivo and in vitro. I. To investigate the presence of intra-oocyte biochemical variation, oocytes from various stages of development were analysed using high resolution Raman mapping, in combination with univariate and multivariate analysis. Images revealed variation between the germinal vesicle and ooplasm in immature oocytes, as well as intra-ooplasmic variation in all oocytes. II. The spectral analysis of oocytes derived from pre-antral and in vitro cultured follicles revealed significant variation: It was found that Raman spectroscopy could successfully discriminate between immature and mature oocytes. III. Finally, the spectral analysis of oocytes derived from unstimulated and stimulated ovulation cycles, as well as those derived from in vitro follicle cultures, revealed that although biochemically similar, in vitro matured oocytes demonstrated reduced protein content. Furthermore, greater spectral variation was observed in superovulated oocytes, which was found to describe the corresponding morphological quality. In conclusion, this thesis has demonstrated the effective application of Raman spectroscopy to the study of fixed murine oocytes. Raman mapping experiments have demonstrated this technique for the visualisation of biochemical variation which exists within the oocyte. Furthermore, using Raman spectroscopy, the identification of the biochemical variation resulting from different maturation mechanisms has been achieved, as has the discrimination of immature and mature oocytes. These results indicate that Raman spectroscopy holds promise as a quantitative analysis method in the field of fertility treatment.

Total hip arthroplasty commonly involves a hard metallic/ceramic femoral ball component articulating against an acetabular ultra-high molecular weight polyethylene (UHMWPE) counter-bearing. A novel six-station, wear tribometer, featuring programmable load and motion, was designed to further the investigation into wear, sub-surface plasticity and debris generation of UHMWPE. This thesis describes the pin-on-plate device, its validation and subsequent use to assess unirradiated (-PE) and gamma-irradiated highly crosslinked (+PE) UHMWPE wear behaviour. With the emphasis on dynamic loading and a closer gait matched open wear path, the tribometer improves on the clinical relevance of pin-on-plate testing. There is a requirement for this type of machine in order to investigate the directional dependence of wear and debris generation of UHMWPE more accurately, where “simplified” tribometers (adequate for constant load/constant velocity and constant load/sinusoidal velocity work) are not capable or suitable. For the first time in orthopaedic pin-on-plate studies, tests were conducted using an advanced dynamic load synchronised to a more physiologically accurate elliptical motion path. To validate the machine, three orthopaedic polymers of clinical relevance; Polytetrafluroethylene, Polyacetal, and UHMWPE were subjected to linear-reciprocating (LR) and novel elliptical motion paths under a Paul-type load profile. All three polymers showed higher wear factors under elliptical motion, by up to 2 orders of magnitude, agreeing well with explanted values. The UHMWPE elliptical wear factor was comparable to that reported for clinical, where kelliptical = 1.56 x 10-6 mm3/Nm. In the crosslinked study, the mean steady state wear of -PE and +PE groups under linear reciprocating motion was not significantly different. However, under elliptical motion, crosslinking reduced UHMWPE wear by up to 92% when compared to the unirradiated group. In –PE pins worn under LR motion and in +PE pins subjected to both motion paths a sub-surface damage zone with reduced crystallinity and increased strain was measured using Raman spectroscopy. This was attributed to large strain accumulation in the slower wearing surfaces providing a mechanism for de-crystallisation. The discovery of such a near-surface layer is in good agreement with critical strain wear models. In disagreement, however, we found the sliding induced layer to extend to greater depths than previous reported.