MINOS is a long baseline neutrino oscillation experiment. It measures the flux from the predominately muon neutrino NuMI beam first 1 km from beam start and then again 735 km later using a pair of steel scintillator tracking calorimeters. The comparison of measured neutrino energy spectra at our Far Detector with the prediction based on our Near Detector measurement allows for a measurement of the parameters which define neutrino oscillations. This thesis will describe the most recent measurement of muon neutrino disappearance in the NuMI muon neutrino beam using the MINOS experiment. The general method of a disappearance analysis at the MINOS experiment will be outlined, the selection of events, extrapolation between detectors, and fitting the data to the atmospheric mixing parameters. An analysis of the full MINOS Forward Horn Current charged current muon neutrino interactions sample is detailed, with a best fit to the atmospheric mixing parameters in a two flavour approximation of $\Delta |m^2_{atm}| = 2.42\times10^{-3}~\mathrm{eV}^2$ and $\sin^2(2\theta_{23}) = 0.936$. The change to a three flavor analysis with matter effects from a simple two flavour approximation is described, with a very slight preference of $-2\Delta \log(L)=0.01$ for the inverted hiearchy found. A study of the NuMI beam is also shown, with potential locations for new oscillation experiments in the NuMI beam discussed.

Hypoxic signaling is involved in homeostatic and developmental angiogenic and neurogenic proliferation and differentiation as well as modulating many disease processes that result in or from restricted blood flow. A better understanding of this pathway and how other pathways interact with it is important for progression towards targeted therapeutics. Von Hippel Lindau (vhl-/-) zebrafish embryos demonstrated hypoxic phenotypes such as hyperventilation, polycythemia and an altered vascular network (van Rooijen et al., 2009). The fih-/- embryos showed no morphological phenotype and the adults are fully viable and fertile. By combining the two lines it was possible to investigate the effect of a double knock out of these genes and the effect this had on downstream targets. The first hypothesis to test, the first observation to make, was to assess the effect of the loss of fih on both wild type embryos and on vhl null embryos. Given the roles of fih and vhl are independent of each other in regulating HIF function (FIH regulating HIF function, while VHL regulates HIF turnover), the hypothesis in this case would be that the loss of fih alone may not show dramatic phenotypes, however in the context of the loss of vhl it may alter or exacerbate phenotypes observed in the organisms where vhl was lost. This was proved to be correct when it came to assessment of global and morphological phenotypes. The loss fih resulted in no significant changes compared with wild type embryos in initial observations; however the loss of both fih and vhl altered the known vhl-/- phenotype, which indicated a role for fih, under conditions of de-regulated hypoxia inducible factor (HIF), in enhancing hypoxic signaling. Expression levels of Hif targets, phd3 and vegfa, were assessed and showed that these were elevated in the vhl-/- zebrafish line and further elevated in fih-/-;vhl-/- embryos, by in situ hybridisation. Further analyses of the downstream effects of the loss of fih were assessed using microarray analysis and further validated using qRT-PCR. Preliminary results suggested that on a transcriptional level, fih in isolation was not influencing expression and that fih functioned mainly through HIF. Vascular branching was altered in the vhl-/- zebrafish line compared to wild-type siblings, and this was exacerbated in the fih-/-;vhl-/- embryos. This correlated with increased vegf expression and a corresponding increase in endothelial cell numbers. The fih-/-;vhl-/- embryos exhibited decreased blood flow along the aorta, which ceased at 5dpf despite continuation of the heart beat. The fih-/- embryos exhibited an increase in blood velocity along the aorta with a corresponding increase in heart rate at the same stage. Our data suggested that Fih had an essential role in the regulation of heart rate in the zebrafish and, in combination with vhl; it influenced formation and functionality of blood vessels by affecting the hypoxic signaling pathway. Also investigated were correlations between the zebrafish model of fih loss and those published in the mouse, and several connections and corroborations were made. However there was also scope, in the zebrafish, for the proposal of a potential mechanism of action which was not proposed in the mouse model. Microarray analysis revealed that, specifically in the fih null embryos, two key genes in the renin angiotensin system were down regulated. Since this is a mechanism by which fluid dynamics are regulated, and since there have been observations of altered blood flow dynamics in double mutants, further investigation as to the potential role of this homeostatic mechanism and its potential regulation by fih are needed to further investigate these findings. A second hypothesis that was proposed was that lead by a study by Lee et al, which proposed a model whereby the three proteins, FIH, VHL and HIF are required to bind together with VHL acting as a functional bridge in order to result in downstream effects (Lee et al., 2003). In this case a loss of vhl would disrupt the hydroxylation of HIF-a by fih as well. The hypothesis in this case would therefore be that the loss of fih as well, in an individual that had lost vhl function, would result in no alteration in the phenotypes observed. This hypothesis was shown not to be supported by the data and further investigation into possible interactions between the proteins would need to be assessed in order to ascertain the discrepancy.

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Optical coherence tomography is a biomedical imaging technique employed to visualize tissue structure, ocular vasculature and blood flow. An OCT system is a non-invasive biomedical imaging system that provides bi-dimensional and three-dimensional images of biological tissue with micrometer scale resolution and millimeter scale depth range. In clinical application, OCT is employed in vivo imaging of the human eye. Swept source optical coherence tomography (SS-OCT) is the latest and fastest method of scanning. However, there are several disadvantages of the SS-OCT such as: the decrease of the roll-off sensitivity as the depth of scanning increases and the presence of mirror terms in the OCT images. The main objective of the work presented throughout this thesis was to evaluate the effects of dispersion on the performance of OCT. This study extends the research from the optical configurations at the core of OCT systems to the optical sources, where we show that dispersion can be usefully employed to obtain sweeping. We prove that an akinetic swept source (AKSS) can be devised for the important band of OCT, 800 nm, where there is still no MHz swept source available. In a Michelson interferometer a Fourier domain optical delay line was used for dispersion compensation that subsequently was employed to control the dispersion in an OCT system. A first goal of this thesis was to increase dispersion in order to evaluate the possibility of removing mirror terms from the OCT images. Therefore, three methods of dispersion measurement were evaluated. The first method measures the full width half maximum of the autocorrelation function. The second method uses a super continuous laser and an acoustic-optic tunable filter to measure the path dispersion in the position of the autocorrelation peak. A third method consists in a fitting method applied to channeled spectra collected from the interferometer when using a mirror. A second goal of this thesis was to prove the usefulness of employing dispersion in building a SS. In the process of akinetic swept source optimization, several types of dispersive fibres were tested and the most optimum conditions for driving a semiconductor optical amplifier were established. A dual mode locking scheme was used to tune the akinetic swept source at MHz rates. The axial range of the swept source was evaluated by scanning through the channeled spectrum of a Michelson interferometer.

Translational reprogramming and mRNA translation e ciency greatly in uence global protein synthesis, cell proliferation and growth; important parameters in de ning recombinant protein expression yields. Polysome pro ling is a widely-used technique to analyse mRNA transla- tion and its e ciency that provides a snapshot of ribosomes loaded on mRNA transcripts at any particular time. A higher number of polysomes present on a given mRNA suggests that the mRNA is being more heavily translated than those mRNAs with few ribosomes. Fur- ther, a large pool of sub-polysomes (40S, 60S and 80S) compared to polysomes in a sample suggests low translational activity. Here, polysome pro ling has been applied to investigate translational reprogramming in multiple recombinant monoclonal antibody (mAb)-producing Chinese hamster ovary (CHO) cell lines, and to determine how reprogramming re ects the ability of such cells to proliferate and make recombinant proteins in stable and transient mAb expression systems, in batch and fed-batch culture mode. The impact of culture temperature on the polysome pro le and hence on reprogramming was also investigated in transient studies. Polysome pro ling revealed reprogramming di ered between recombinant cell lines. Those with the highest global translational e ciency generally had the fastest cell speci c growth rates, although total ribosome capacity did not directly relate to those with the fastest growth rates or mAb productivities. This suggests it is the ability to utilise available machinery that determines protein synthetic capacity. Recombinant cell lines with higher cell speci c produc- tivities generally maintained a higher polysome to monosome (P:M) ratio during stationary phase and had elevated recombinant mRNA copy numbers localised to translationally active heavy polysomes. In transient systems, the P:M ratio was maintained longer at reduced tem- perature cultivation and related to higher mAb yields being obtained. A number of endogenous transcripts were found to be more or less abundant on polysomes at di erent times of culture, indicative of changes in the cellular requirements of the encoded proteins. Such transcripts could be potential cell engineering targets to help tune the needs of the cell to the demands of a culture process or recombinant protein, or alternatively their untranslated regions harnessed to help preferentially load target mRNAs onto ribosomes. When upstream open reading frames (uORFs) or alternative translation start sites were engineered into recombinant transcripts a range of mAb expressions were observed allowing the tuning of mAb expression, including improvement over a standard untranslated region used industrially as a control. The ndings described in this thesis therefore reveal insights into the mechanisms involved in translational regulation and reprogramming in CHO cells during bioprocessing. These can be utilised for further improvement via targeted cell engineering strategies, cell line screen- ing approaches or modi cation of recombinant transcripts for enhanced industrial host and recombinant cell lines.

The polymorphic nature of amyloid fibrils is important in the understanding of structural based relationships, such as a morphology influence on cytotoxicity and disease progression. The work reported here uses Atomic force microscopy (AFM) to enhance the understanding of fibril morphology in addition to the relationship between structure and stability towards breakage. A novel quantitative cluster analysis was developed here to identify the vast range of fibril morphologies present within a population. Using fibrils formed from three peptide sequences identified by the WALTZ algorithm, we have characterised the polymorphism displayed by each fibril population and provided structural models to predict the likely filament arrangements accessible to each. The range of fibril polymorphism also conveys mechanical differences, defined here by persistence length values for each respective population. These mechanical differences subsequently affect fibrils stability towards breakage, quantified here using AFM and subsequent image analysis. Additionally, using AFM, a structural comparison was performed between Sup35NM amyloid fibrils formed in vitro and those formed in situ using a synthetic biology approach with the Curli-dependent amyloid generator (C-DAG) in E. Coli. Structural similarities between fibrils formed using this system and those formed in vitro is of great value given the importance of a sequence-structure relationship. The work in this thesis expands on possible fibril morphologies and the related mechanical properties, which has implications in the understanding of disease enhancing structural motifs and the utilisation of amyloid fibrils in a biotechnology role.

MicroRNAs (miRs) are a class of non-coding RNA that function to regulate global mammalian gene expression by mediating the translational repression of mRNAs harbouring a complementary target region sequence within their 3'UTR. The RNA-RNA binding event between the miR seed region and mRNA target region disrupts mRNA translation and hence repression of protein synthesis from multiple mRNA targets encompassing a variety of cellular processes and pathways. In addition to this multiplicity, miRs also exhibit a high level of promiscuity as a single miR may silence many mRNA targets and a single mRNA transcript may be under the regulation of multiple miRs. miR activity may therefore be manipulated to favour/inhibit cellular processes/pathways of interest to yield desirable cell phenotypes such as enhanced secretion, metabolism or growth. The engineering of miR activity has been applied to enhancing the production of recombinant proteins in the industrially-relevant Chinese hamster ovary (CHO) cell line. In the industrial setting, the utilization of CHO cells has become the dominant mammalian system for manufacturing biotherapeutic recombinant proteins due to their aptitude for accurate protein folding, assembly and performing 'human like' post-translational modifications. A historical Lonza microarray conducted on a range of GS-CHOK1SV IgG-producing cell lines identified changes in miR abundance throughout culture that correlated with growth or recombinant IgG productivity. From this screen, three miRs in particular (miR-15b, -16-1 and -34c) were selected for further study with regard to the impact of targeted miR knockdown and engineered pri-miR over-expression in recombinant and host CHO cell lines and transfectants with respect to establishing whether changes in the amounts of these miRs impacted upon CHO cell growth and productivity. The studies undertaken here have shown that the engineered over-expression of pri-miRs can improve the longevity (e.g. when over-expressing pri-miR-16-1-34c and -15b-34c-16-1) and maximum viable cell concentration (e.g. when over-expressing pri-miR-15b, -16-1 and -16-1-34c) of a CHOK1SV-GSKO host cell line whilst the expression of miR-sponges for targeted miR knockdown can enhance the cell specific productivity (e.g. when expressing miR-sponge-S6) in a variety of GS-CHOK1SV IgG-producing cell lines. In particular, miR-sponges derived from the 3'UTR of the SRPRα mRNA, which should be targeted by miR-34c, reduced miR-mediated repression of SRPRα expression and hence an increase in SRPRα mRNA and protein expression was observed. Specifically, the application of a SRPRα-derived miR-sponge construct constituting 6 miR-binding site motifs was shown to be both functional in relieving endogenous SRPRα from miR-mediated translational repression as well as potentially enabling an increase in cell specific productivity in selected recombinant CHO cell lines, suggesting that secretory capacity was limited by the availability of SRPRα. In conclusion, the studies presented here have demonstrated that the exploitation of miR activity can be an effective tool for CHO cell engineering for the tuning of recombinant protein production without placing any additional translational burdens on the cell.

In vitro production (IVP) in agricultural species such as pigs and cattle is performed in a similar manner to human IVF, and involves ovum pick up or oocyte maturation, fertilization outside the body and subsequent transfer of viable embryos. Its application in agricultural breeding can significantly speed up genetic gain rates by reducing generational intervals, increasing selection intensity, and facilitating the dissemination of valuable genetics. IVP is an increasingly important tool for breeding companies given the ongoing emphasis on feeding a growing population with fewer resources. With the above in mind, the aim of this work was to improve the yield of competent, euploid, and high genetic merit IVP embryos available for transfer. Oocytes destined for IVP are commonly assessed for developmental competence by morphological screening. To determine whether it would be possible to improve the utilisation of oocytes per donor, the developmental competence of porcine oocytes with decreasing cumulus oocyte complex (COC) investment was examined. It was found that current IVP practice is wasteful, through the elimination of oocytes with slightly impaired morphology, which still have remarkable developmental potentials. Moreover, whilst it is accepted that embryo splitting (generating more than one embryo from a single fertilized zygote) could benefit the breeding industry by increasing the offspring of the most desirable parents, a comparative analysis of the different splitting methodologies available, including stage of the split and single versus serial splitting strategies, is currently missing in the literature. Here, the splitting of an 8-cell stage embryo into four identical twins was identified as the strategy producing the greatest output of good quality embryos. Additionally, time-lapse investigation of the embryo splits found evidence of the existence of a developmental clock that tightly regulates early cleavage events. Normally, only embryos that display satisfactory morphology are selected for transfer. However, in cattle and pigs, this assessment is complicated by the accumulation of lipid droplets within the embryo, which renders it opaque. Consequently, there is scope for the application of new imaging modalities, such as optical coherence tomography (OCT), which are able to image an embryo in full depth and non-invasively. In this work, swept source OCT was successfully tested for use in early stage bovine embryos to obtain both structural and functional imaging. Moreover, micron-scale movements were measured within blastocysts by OCT as a way to rapidly discriminate between living and deceased embryos, representing a novel application of this methodology. Embryo biopsies can be used to establish the genetic merit of an embryo through single nucleotide polymorphism (SNP) analysis, allowing the application of genomic selection soon after fertilisation rather than at birth. Moreover, SNP information can be analysed by karyomapping to select the most chromosomally normal embryos for transfer. Here, the birth of the first five karyomapped calves in the world is reported. Additionally, karyomapping was used to measure the incidence of aneuploidy in bovine blastocysts by parent of origin and to determine the recombination frequency for each chromosome, demonstrating the applicability of this methodology to basic research.

Enhancing the catalytic efficiency of enzymes has been an aspiration for many years and protein engineering has been quite successful in this area. However, researchers are now turning their attention to the organisation of enzymes within cells to enhance whole pathway productivity. As such, bacterial microcompartments (BMCs) have attracted significant interest due to their ability to not only concentrate enzymes and metabolites but also prevent the release of toxic intermediates into the cell owing to their semi-permeable protein shell. The aim of the research described in this thesis was to develop a number of mechanisms to enhance the spatial organisation of proteins within E. coli and thereby increase metabolic productivity. The ability of BMC encapsulation peptides to target foreign proteins to a BMC was initially investigated. However, we found that the fusion of BMC encapsulation peptides to the enzymes of a 1,2-propanediol (1,2-PD) synthesis pathway resulted in the aggregation of the tagged enzymes rather than their localisation to a BMC. Nonetheless, the tagged enzymes were found to produce significantly more 1,2-PD than strains producing untagged enzymes, irrespective of the presence of BMCs, suggesting that it is the enzyme aggregate that results in enhanced substrate channelling. Furthermore, we showed that a highly conserved hydrophobic motif is conserved across a wide range of BMC targeting peptides and suggest that it is the amphipathic nature of these peptides that results in aggregation of tagged components. The ability of PduA, a component of the BMC shell, to form filamentous structures was investigated for its potential as a cytoplasmic scaffold. Targeting proteins to the cytoscaffold was explored through the use of de novo designed coiled-coil peptides. Localising the enzymes pyruvate decarboxylase and alcohol dehydrogenase on this scaffold by tagging them with the cognate coiled-coil peptide resulted in a significant enhancement in ethanol production in comparison to a strain lacking the scaffold. Additionally, we showed that it was possible to locate this intracellular scaffold to the inner membrane of the cell, further demonstrating the flexibility of this system. Finally, we utilised the same coiled-coil peptide technology to target fluorescent proteins to BMCs, overcoming the aggregation behaviour of the native targeting peptides. Through the re-design of a BMC shell protein we showed targeting occurs to both the external and luminal faces of these BMCs. Taken together, the evidence presented in this thesis provides a number of alternative strategies to not only enhance spatial organisation within a cell but also to increase productivity of engineered metabolic pathways.