Ovarian stimulation plays a pivotal role in assisted reproductive therapies, to increase the number of embryos available for treatment; however, there is no clear consensus from meta-analyses in the literature which, if any, of the preparations in use are superior in terms of clinical outcomes. The aim of this thesis was to examine the effect of common human gonadotrophin preparations with different half lives and LH activity (hMG, rFSH and Pergoveris) on embryo quality and resulting offspring, compared to non- stimulated negative controls and positive PMSG treated controls, using the mouse model. The studies in this thesis indicated that an LH ceiling threshold is evident during folliculogenesis, where the use of long acting LH preparations resulted in higher numbers of fragmented oocytes, absent of cumulus cells (P<0.001), reduced expression of the pro and anti-angiogenic factors, MYHII and PEDF in cumulus cells (P<0.05), increased embryonic developmental arrest (P<0.001) and perturbed IGF2 (P<0.05) and VEGFA gene expression in resulting blastocysts (P<0.01), compared to negative controls. Use of preparations containing LH bioactivity resulted in offspring with altered total body weight trajectories and internal organ weight abnormalities (P<0.05), which were, in some instances, compounded by in vitro culture. In addition, we elucidated a relationship between FSH half life differences between urinary and recombinant preparations and embryo quality. The urinary human gonadotrophin preparation, hMG, could yield developmentally competent embryos at lower concentrations, than the recombinant Pergoveris treatment. In addition to FSH, these preparations contain LH and both low doses of preparations composed of short half life rFSH and rLH and high doses of preparations containing long acting LH bioactivity, resulted in the highest rates of developmental arrest. These groups were observed to have complete absence of H19 expression. The results of this thesis provide clear evidence that ovarian stimulation does negatively impact the embryo and subsequent offspring and provides support for an LH ceiling threshold, above which detrimental effects occur, both on in vitro embryo development and in vivo foetal development, which later effects postnatal growth.

Understanding how the germ layers are formed is one of the key questions of developmental biology. Abundant studies in the anuran amphibian Xenopus laevis have described that maternal and vegetally localised mRNAs for VegT and Vg1 contribute greatly to the formation of mesoderm and endoderm in the developing embryo. Within Xenopus mesendoderm gene-regulatory network (GRN), Wnt/β-catenin as well as Nodal and Mix family members have been shown to play important roles. The involvement of several members of the Nodal and Mix gene families with redundant functions makes the mesendoderm GRN surprisingly complex and difficult to study in Xenopus laevis. By contrast, mouse and humans have only single copies of Nodal and Mix. Since urodeles have an embryology that is basal to amphibians and that has most likely also been conserved during the evolution of amniotes, including mammals, we have investigated the Mix and Nodal genes in the urodele Axolotl in the hope that their gene families contained fewer members. We cloned one Mix and two Nodal orthologs from the axolotl and showed by Southern blot analysis that there are likely no further copies in the axolotl genome. Morpholino and rescue experiments furthermore showed that AxNodal-1, Mix and Brachyury play essential roles in mesoderm specification in axolotl embryos, suggesting that the urodele Axolotl has a more simplified mesendoderm GRN. In this context, we demonstrate that Mix acts to induce Brachyury expression during mesoderm induction. Mixl1 shRNA knowdown in mouse embryonic stem cells (mESCs) shows that Mixl1 is involved in the production of mesoderm in mESCs too. Analysis of the localisation of the VegT and Vg1 mRNAs in oocytes revealed that they are neither vegetally localised in the Axolotl, nor in the basal fish species lungfish and sturgeon. Furthermore, gain and loss of function assays examining the roles of maternal VegT and β-catenin demonstrated that VegT is not required for mesoderm induction, whereas β-catenin is necessary and sufficient for mesoderm induction by activating AxNodal-1 expression in the axolotl. As these results reveal additional similarities to the GRN in mammals they further support our hypothesis that the regulatory network in the axolotl is more closely related to that in amniotes rather than anuran amphibians.

A good QSAR model comprises several components. Predictive accuracy is paramount, but it is not the only important aspect. In addition, one should apply robust and appropriate statistical tests to the models to assess their significance or the significance of any apparent improvements. The real impact of a QSAR, however, perhaps lies in its chemical insight and interpretation, an aspect which is often overlooked. This thesis covers three main topics: a comparison of contemporary classifiers, interpretability of random forests and usage of interpretable descriptors. The selection of data mining technique and descriptors entirely determine the available interpretation. Using interpretable approaches we have demonstrated their success on a variety of data sets. By using robust multiple comparison statistics with eight data sets we demonstrate that a random forest has comparable predictive accuracies to the de facto standard, support vector machine. A random forest is inherently more interpretable than support vector machine, due to the underlying tree construction. We can extract some chemical insight from the random forest. However, with additional tools further insight would be available. A decision tree is easier to interpret than a random forest. Therefore, to obtain useful interpretation from a random forest we have employed a selection of tools. This includes alternative representations of the trees using SMILES and SMARTS. Using existing methods we can compare and cluster the trees in this representation. Descriptor analysis and importance can be measured at the tree and forest level. Pathways in the trees can be compared and frequently occurring subgraphs identified. These tools have been built around the Weka machine learning workbench and are designed to allow further additions of new functionality. The interpretability of a model is dependent on the model and the descriptors. They must describe something meaningful. To this end we have used the TMACC descriptors in the Solubility Challenge and literature data sets. We report how our retrospective analysis confirms existing knowledge and how we identify novel C-domain inhibition of ACE. In order to test our hypotheses we extended and developed existing software forming two applications. The Nottingham Cheminformatics Workbench (NCW) will generate TMACC descriptors and allows the user to build and analyse models, including visualising the chemical interpretation. Forest Based Interpretation (FBI) provides various tools for interpretating a random forest model. Both applications are written in Java with full documentation and simple installations wizards are available for Windows, Linux and Mac.

One hallmark of embryonic stem cells (ES) is their ability to renew themselves indefinitely and still retain the potential to develop into any specialized cell type once triggered by specific exogenous signals. This very versatile nature has made them an attractive model to study developmental events occurring during embryogenesis and also to employ them for regenerative medicine. The idea to exploit their developmental potential for intended therapeutic applications requires a detailed knowledge of the molecular regulation of differentiation. Thus pluripotent embryonic stem cells can be employed to investigate the molecular framework regulating pluripotency. The major aim of this research endeavour is to explore the role of DNA methylation in regulation of endogenous Sox2 transcription factor in context of mouse ES cells following their transition from the pluripotent to a differentiated state. An insight into molecular regulatory mechanisms will shed light on developmental programming and also aid in refining of methodologies for differentiation and nuclear reprogramming increasing their chances of success and efficiency. Mouse embryonic stem cells were differentiated towards osteogenic and neural cell types through the formation of embryoid bodies (EBs) – cellular aggregates partially recapitulating the early embryonic development. These EBs were then disaggregated and single cells plated in medium containing supplements to promote osteogenic or neural differentiation while control cells were grown in medium lacking those factors. Cells were harvested undifferentiated and at different time points during differentiation. Molecular characterization was carried out by expression profiling of lineage specific genes and proteins using RT-PCR and immunofluorescence respectively. DNA methylation analysis of two regulatory regions of Sox2 i.e. SRR1 and SRR2 was carried out by MS-PCR and bisulphite sequencing. Embryonic stem cells were observed to be differentiating as evidenced by changes in cellular morphologies and lineage-specific markers expression. Two regulatory regions of Sox2, namely SRR1 and SRR2, were found to be methylated by methylation sensitive PCR at all time-points chosen for analysis in differentiating cells. Three individual CpGs in SRR2 region were then analysed further by bisulphite sequencing which appeared unmethylated in both undifferentiated and differentiated embryonic stem cells. This hints towards the possible role of DNA methylation in regulating the expression of Sox2 in differentiating embryonic stem cells and need further investigation.

This thesis deals with a two component reaction-diffusion system (RDS) for competing and cooperating species. We have analyse in detail the stability and bifurcation structure of equilibrium solutions of this system, a natural extension of the Lotka-Volterra system. We find seven topologically different regions separated by bifurcation boundaries depending on the number and stability of equilibrium solutions, with four regions in which the solutions are similar to those in the Lotka-Volterra system. We study RDS in the small parameter of the range $0< \lambda \ll 1 $ (fast diffusion and slow reaction), and in a few cases we assume $\lambda=O(1)$. We consider three types of initial conditions, and we find three types of travelling wave solutions using numerical and asymptotic methods. However, neither numerical nor asymptotic methods were able to find a particular travelling wave solution which connects a coexistence state say, $(u_0,w_0)$ to an extinction state $(0,0)$ when $0< \lambda \ll 1 $. This type can be found when the reaction-diffusion system satisfy the symmetry property and $\lambda=1$.

In vertebrates, haematopoiesis is maintained by self-renewing multipotent haematopoietic stem cells (HSCs) in the adult bone marrow. HSCs are specified and generated during early embryonic development from arterial endothelial cells in the ventral wall of the dorsal aorta (vDA) that become haemogenic. Examination of mib mutants and rbpja/b morphants zebrafish embryos, that have defects in Notch signalling show that both arterial specification and HSCs development require an intact Notch signalling pathway. Utilizing in-house generated zebrafish Notch reporter lines, we show that Notch signalling initiates in the migrating hEC progenitors before their arrival to the midline to form the DA. Following arterial specification, Notch activity rapidly increases in the DA cells and persists at high levels until the time point of HSC emergence from the vDA, then sharply falls to hardly detectable levels. Embryos treated with the γ-secretase inhibitor DAPT or DAPM lose HSC development but retain arterial specification. Quantification of the residual Notch activity in our reporter lines by RT-PCR, revealed that low levels of Notch signalling retained in treated embryos are sufficient for arterial gene expression, while high levels of Notch signalling are required for hECs induction. Additional to these loss of function experiments, our endothelial specific gain of function studies suggested that continuous expression of nicd in the ECs in stable transgenic lines prevent cells from differentiation into mature HSCs that can leave the vDA to seed the CHT. By contrast, transient expression of nicd in ECs appeared to expand HSC fate and allow cells to seed the CHT, suggesting that down-regulation of Notch signalling is essential to enable cells committed to the blood lineage to leave the vDA and seed the CHT. These data represent the first demonstration, to our knowledge, that cell autonomous Notch induction in ECs in vivo is sufficient to expand HSC formation in vertebrates. Ten-Eleven-Translocation (Tet) proteins are a family of enzymes known to be actively involved in DNA de-methylation by 5-mC oxidation. This process is essential for proper development and cell lineage specification. Here, we used zebrafish tet1/2/3 morpholinos to study the role of Tet enzymes in zebrafish development and organogenesis. Our results showed that individual depletion of Tet1 or Tet3 enzymes is sufficient to arrest zebrafish development at the onset of somitogenesis, whereas tet2 morphants appear normal. Moreover, analysis of mildly affected morphants shows that Tet1 knockdown leads to atypical neurogenesis in zebrafish embryos. Global 5hmC levels are dramatically reduced in both tet1 and tet3 morphants, indicating the importance of Tet proteins in vertebrate organogenesis.