<p>A series of novel methods for detection of known sequence variants in DNA, in particular single nucleotide polymorphism, using padlock probes and rolling circle replication are presented. DNA probes that can be circularized – padlock probes – are ideal for rolling circle replication. Circularized, but not unreacted probes, can generate powerful signal amplification by allowing the reacted probes to template a rolling circle replication (RCR) reaction. However, when hybridized and ligated to a target DNA molecule with no nearby ends, the probes are bound to the target sequence, inhibiting the RCR reaction is. This problem can be solved by generating a branched DNA probe with two 3’ arms such that the probes may be circularized while leaving the second 3’ arm as a primer for the RCR reaction. We describe how T4 DNA ligase can be used for efficient construction of DNA molecules having one 5’ end but two distinct 3’ ends that extend from the 2’ and 3’ carbons of an internal nucleotide. An even stronger approach to circumvent the topological problem that can inhibit RCR is to restriction digest the template downstream of the padlock recognition site. By using Phi 29 DNA polymerase with efficient 3’ exonuclease and strand displacement activity, the template strand can then be used to prime the RCR reaction. The amplified molecule is contiguous with the target DNA, generating an anchored localized signal. The kinetics of the reaction was investigated by following the reaction in real-time using molecular beacon probes. Localized RCR signal were obtained on DNA arrays, allowing detection of as little as 104-105 spotted molecules, of either single- or double-stranded M13 DNA, in a model experiment. We have also established a serial rolling circle amplification procedure. By converting rolling circle products to a second and even third generation of padlock probes the signal was amplified thousand-fold per generation. This procedure provides sufficient sensitivity for detection of single-copy gene sequences in 50 ng of human genomic DNA, and large numbers of probes were amplified in parallel with excellent quantitative resolution.</p>

A series of novel methods for detection of known sequence variants in DNA, in particular single nucleotide polymorphism, using padlock probes and rolling circle replication are presented. DNA probes that can be circularized – padlock probes – are ideal for rolling circle replication. Circularized, but not unreacted probes, can generate powerful signal amplification by allowing the reacted probes to template a rolling circle replication (RCR) reaction. However, when hybridized and ligated to a target DNA molecule with no nearby ends, the probes are bound to the target sequence, inhibiting the RCR reaction is. This problem can be solved by generating a branched DNA probe with two 3’ arms such that the probes may be circularized while leaving the second 3’ arm as a primer for the RCR reaction. We describe how T4 DNA ligase can be used for efficient construction of DNA molecules having one 5’ end but two distinct 3’ ends that extend from the 2’ and 3’ carbons of an internal nucleotide. An even stronger approach to circumvent the topological problem that can inhibit RCR is to restriction digest the template downstream of the padlock recognition site. By using Phi 29 DNA polymerase with efficient 3’ exonuclease and strand displacement activity, the template strand can then be used to prime the RCR reaction. The amplified molecule is contiguous with the target DNA, generating an anchored localized signal. The kinetics of the reaction was investigated by following the reaction in real-time using molecular beacon probes. Localized RCR signal were obtained on DNA arrays, allowing detection of as little as 104-105 spotted molecules, of either single- or double-stranded M13 DNA, in a model experiment. We have also established a serial rolling circle amplification procedure. By converting rolling circle products to a second and even third generation of padlock probes the signal was amplified thousand-fold per generation. This procedure provides sufficient sensitivity for detection of single-copy gene sequences in 50 ng of human genomic DNA, and large numbers of probes were amplified in parallel with excellent quantitative resolution.

Tumours are characterised by unorganised vasculature, which often results in hypoxic regions. Hypoxia is a common cause for photon radiotherapy (RT) treatment failure, as hypoxic cells require up to 2-3 times higher doses compared to well-oxygenated cells for the same effect in terms of cell kill. The increase in dose that would be required to treat the tumours of cancer patients is limited by the radiation sensitivity of surrounding normal tissues. Using carbon ions instead of photons, the radiation dose can be conformed to the tumour to a much higher degree, resulting in an improved sparing of normal tissues. In addition, carbon ions have a much higher radiobiological effectiveness near the end of their range, which is positioned in the tumour. Also, the radiation modes of action leading to cell death when carbon ions interact with living tissues, are less sensitive to the oxygen status compared with the action modes of photons. The focus of this thesis lies in the development of models for the computation of the cell surviving fraction and tumour control probability (TCP) in hypoxic tumours after photon and carbon ion RT. The impact of fractionation was evaluated with regard to possible spatial changes in oxygenation, both for stereotactic body RT and for carbon ion RT. The feasibility of a method to determine and deliver the optimal photon dose for achieving a high TCP according to spatial variations in radiation sensitivity was evaluated in a treatment planning study. The radiobiological models were finally used for the theoretical quantification of the gain in using carbon ions instead of photons. The results show that there are great possibilities to increase the number of positive outcomes of radiation treatment of tumours if the key influential factors are taken into account, such as level and distribution of hypoxia, radiation quality and choice of fractionation schedule. / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows; Paper 3: Manuscript; Paper 4: Epubl ahead of print; Paper 5: Manuscript</p>

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Abstract This research work focuses on how to improve the toughness of heat affected zones (HAZs) of low heat input welds in the case of high strength thermomechanically processed (TMCP) and recrystallization controlled rolled and accelerated cooled (RCR) plates with yield strengths of 355–500 MPa. Experimental work was aimed at the investigation of the intragranular nucleation of acicular ferrite or bainite in hot-rolled plates and the evaluation of the Charpy V and CTOD toughness of the most critical sub-zones of the weld HAZ using simulated specimens with a cooling time t8/5 = 5 s. The zones studied were the coarse grained HAZ (CGHAZ), the intercritically reheated coarse-grained HAZ (ICCGHAZ) and the intercritical HAZ (ICHAZ), the metallographical analyses consisted of microstructural investigations complemented with hardness measurements. Optical, scanning and transmission electron microscopy techniques were employed together with image and electron backscatter diffraction (EBSD) analysis. The test results showed that the toughness of the various sub-zones of the HAZ is improved by promoting intragranularly nucleated ferritic-bainitic (acicular) microstructure in both the CGHAZ and in the base plate. In this way, the sub-zones subjected to intercritical thermal cycles (the ICCGHAZ and the ICHAZ) develop evenly distributed M-A constituents between ferrite and bainite laths. These favourable microstructures can be achieved by using titanium killing or by avoiding niobium microalloying by using copper plus nickel alloying instead. In the laboratory experiments titanium killed steel, containing titanium-manganese oxide/manganese sulphide inclusions with a number density of 300–750 particles/mm2, develops a largely acicular ferritic microstructure in the base plate provided the austenite grain size is greater than about 120 μm and the cooling rate is in the range 6–11 °C/s down to 500 °C. Under plate mill conditions, no significant amount of acicular ferrite could be obtained, because it was not possible to achieve austenite grain sizes larger than about 70 μm after rolling. However, a significant fraction of acicular ferritic-bainitic microstructure was achieved in the CGHAZ, when the austenite grain size exceeded 90 μm. When achieved, a uniform distribution of M-A particles in an acicular ferritic-bainitic microstructure improves toughness. Cracks nucleate at numerous sites on M-A/ferrite boundaries or bainite packet interfaces, but they are initially arrested in the acicular matrix. Crack growth finally occurs by linking of the numerous arrested microcracks.

Magister Scientiae - MSc (Biotechnology) / The vaginal mucosa is dominated by Gram positive, rod shaped lactobacilli which serve as a natural barrier against infection. In both healthy and BV infected women Lactobacillus crispatus and Lactobacillus jensennii has been found to be the predominant Lactobacillus species. Many studies have been conducted to assess factors influencing lactobacilli dominance in the vaginal microbiome. However, no study has evaluated the impact of plasmids on the vaginal lactobacilli. In the present study two plasmids, pLc17 and pLc4, isolated from vaginal Lactobacillus species of both healthy and BV infected women were characterized. pLc4 was present in both Lactobacillus crispatus and Lactobacillus jensennii while pLc17 was only present in Lactobacillus crispatus. pLc17 (16663 bp in size) encoded a ribonucleotide diphosphate reductase (RNR), a filamentation induced by cAMP-like (FIC-like) protein and numerous mobile elements. The FIC-like protein may assist pLc17 to persist within the bacterial population, while RNR is commonly associated with phages and may indicate phage infection. pLc4 (4224 bp in size) encodes for a replication initiator protein and a plasmid partitioning protein. The replication protein on pLc4 shows 44% identity with the replication initiation protein of pSMQ173b_03. On further phylogenetic and sequence analysis with other Rolling Circle Replication (RCR) plasmids, pLc4 appears to be novel as the plasmid shows a low degree of similarity to these RCR plasmids. pLc17 appears to carry both a RCR replicon as well as a theta replicon, similar to pIP501, the broad-host-range plasmid from Bacillus subtilis. The relative Plasmid Copy Number (PCN) for pLc4 and pLc17 was analysed using quantitative polymerase chain reaction (qPCR) for the healthy state relative to the disease state from twentyeight vaginal swab samples obtained from the National Institute for Communicable Diseases (NICD). The relative PCN for pLc4 and pLc17 had a fold increase of ~2.803 and ~1.693, respectively in the healthy patient samples relative to BV infected patient samples. However, there were not found to be significant differences when taking the standard error into account Due to the novelty of these plasmids further analysis and characterisation is required for both plasmids, to establish what role they may play in the health of the vaginal milieu.