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Meiotic gene conversion has a major impact on genome diversity. Both crossovers and non-exchange conversions cluster into distinct recombination-active regions that we call hotspots. Hotspot analysis in humans has focused on the description of crossover profiles and only few hotspots had been tested for crossover and non-exchange gene conversion. Therefore, very little was known about the frequency and distribution of non-exchange conversions and how well they correlate with crossing over. Six extremely active recombination hotspots were analysed by using small pool PCR approaches on sperm DNA to detect both types of recombinant molecules. Non-exchange conversions were detectable and arose at high frequencies (0.01-0.47%) per sperm, in addition to crossovers (0.2-0.70%). Conversion tracts were short and their distribution defined a steep conversion gradient, centred at the peak of crossover activity and probably marking the zone of recombination initiation. It was also observed that non-exchange gene conversion and crossover frequencies were positively correlated, not just between men at the same hotspot but equally when compared across several hotspots. On average, non-exchange gene conversions spanning a marker close to the centre of the hotspot occurred at 50% of the crossover frequency. Hotspot regulation in cis had been described previously to results in different initiation efficiencies between interacting haplotypes. Preferential initiation on a more active haplotype, in turn leads to the overtransmission of alleles from the less active haplotype. Additional hotspots that showed a phenomenon of biased gene conversion were described in this study, with crossovers and non-exchange gene conversions influenced to the same degree. More unusually, biased gene conversion specifically affecting non-exchange events was also observed at two hotspots. Here single SNP heterozygosities appear responsible for triggering the bias in cis. Crossovers were not affected, which may provide evidence for distinct crossover and non-crossover pathways operating at human hotspots. Inter-individual differences in recombination frequencies between men at a given hotspot established PRDM9 as major trans-regulator of hotspot activity. PRDM9 regulation was characterised at two hotspots activated by specific sets of PRDM9 variants. At both hotspots a sequence motif, proposed to be the PRDM9 binding site in vitro, was not found to be responsible for hotspot activation as had been predicted previously. Curiously, better motif matches were not correlated with higher crossover frequencies, and PRDM9 can in fact activate hotspot without the proposed binding motif.

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In this project I have used Xenopus laevis to address the spatial and temporal requirements for various signaling pathways in organogenesis, specifically craniofacial development. Craniofacial structures of Xenopus, such as the cartilages, are analogous to those in other vertebrates. A significant portion of the vertebrate head is derived from the neural crest. Thus, this is also a study of neural crest development. The vertebrate head can be defined by the presence of a number of specialised features including a segmented brain, complex sensory organs and others such as skull bones, teeth and facial muscles. This developmental biology project strives to understand how various signaling pathways regulate the development of this complex, multicellular, three-dimensional region. Moreover, I am interested in how the neural crest can contribute to these wide-ranging structures. The unique features of the head help set vertebrates apart from cephalochordates, their closest non-vertebrate relatives (Stone and Hall, 2004) (Wada, 2001). One such close relative is Amphioxus, which has a notochord and a neural tube, but does not possess a complex brain or any complex facial structures. Crucially, it lacks the neural crest cells that comprise the head structures of vertebrates. In cephalochordate and urochordate embryos the cells in the border between the neural plate and the epidermis express several key genes required for vertebrate crest development (Snail, Msx, Bmp2, Pax3) (Meulemans and Bronner-Fraser, 2007; Yu et al., 2008). Unlike vertebrate crest, these cells are not migratory and do not have the same pluripotency of vertebrate neural crest cells (Holland and Holland, 2001). As such, the innovation of migratory neural crest cells is integral to the development of the head.

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Impaction allografting is used to fill osteolytic defects during revision total hip replacements (rTHR). However clinical results are inconsistent as bone fracture and excessive implant migration are a major complication. Most importantly allograft does not adequately enhance new bone formation. As a consequence the results of rTHR are often inferior to primary THR due to lack of bone stock. Other studies indicate that tissue engineering (TE) using Mesenchymal Stromal Cells (MSCs) seeded onto a resorbable scaffold can regenerate new bone. The overall aim of my thesis was to test the hypothesis that TE of autologous MSCs and MSC derived osteoblast cells incorporated with impaction allografting will significantly enhance new bone formation in revision THRs. The study is divided into in vitro and in vivo phases. In the in vitro phase the technique of osteogenic differentiation and seeding of the autologous ovine MSCs onto allograft was optimised. Previously determined normal impaction forces of 3, 6 and 9kN were used to study the viability of these impacted MSCs and osteogenically seeded cells on the allograft. The results showed that both MSCs and osteoblast cells are affected by the impaction forces. However the MSCs can survive normal impaction forces of 3-6kN while osteoblast cells can only survive impaction forces under 3kN. The in vivo phase was an ovine model used to compare new bone formation between MSCs, osteoblasts and a control group in an orthotopic, ectopic and revision hip. The results show that overall MSCs enhance new bone formation in all the scaffolds compared with the osteoblast and control group. However osteoblast cells do not contribute to new bone formation. The conclusion from the study was that MSCs in conjunction with the impaction allografting technique enhance new bone formation. The tissue engineering technique can be developed into a clinical application in revision THRs where large bone defects have become problematic.