This dissertation describes the benzylation of adenine under basic conditions, the unequivocal determination of the identity of the products of this reaction, an exploration of the effect of solvent on the reaction, a thorough computational study of the reaction mechanism and an investigation into the hydrogen-deuterium exchange reaction of the N-benzyladenine products and related compounds. The preferential sites of alkylation of adenine under basic conditions in DMSO were proven to be the N9 and N3 positions. X-ray crystal structures were obtained for both compounds. Formation of the N9-benzyladenine product is the most favoured in polar aprotic solvents, such as DMSO, and as the proportion of polar protic solvents, such as water, increases, so does the formation of the N3-benzyladenine product. Characteristic 1H NMR  chemical shifts of the purine ring protons and HMBC 1H-13C correlation NMR spectroscopy were useful tools to assign the 1H and 13C NMR spectra chemical shifts and confirm that the solution structures were the same as the isolated crystals. Simulating the SN2 mechanism for the N1-, N3-, N7- and N9-pathways computationally, employing DMSO as the simulated solvent, resulted in ambiguous results when considering the electronic energies of initial, TS and final products alone. However, a novel approach was developed (employing IQA-defined energy terms) to study fragment interactions along the reaction paths. It provided a full explanation of the reaction mechanism and yielded results which supported the N3/N9 positions of alkylation over the N1/N7 sites. The preference for the sites of alkylation occurs after the transition state, in which the N1/N7 reaction paths fail to proceed favourably to the end product, N1- and N7-benzyladenine, respectively. The N9-pathway dominates the N3-pathway at the product formation step, which corresponds to the N9- benzyladenine being the major product, as shown in Figure 1, and the N3-benzyladenine being the minor product from the benzylation of adenine. The faster rate of deuteration at the C8 position of N9-benzyladenine as compared to the deuteration rates at the C2 and the C8 of N3-benzyladenine, have shown support for a sp3 mediated mechanism and a carbene mediated mechanism of deuteration based on the “push” and “pull” mechanisms proposed for the C8 proton transfer of ATP in kinase enzymes. The deuteration of the C8 proton of 2,6-dichloropurine derivatives supports the existence of the carbene mediated mechanism since these compounds lack the amine moiety necessary for the sp3 mediated mechanism. These results demonstrate how experimentation and computation have led to greater insights into the reactivity of adenine and its derivatives. This strategy provides a useful platform for future research into adenine reaction mechanisms and the role adenine plays in kinase catalysis. / Dissertation (MSc)--University of Pretoria, 2015. / National Research Foundation (NRF) / Chemistry / MSc / Unrestricted

Morphological plasticity of brown knapweed (Centaurea jacea L.) was examined. South bohemian populations of subsp. jacea were compared to plasticity in larger area. The plasticity of local population overlaps with both subspecies. The correlation between abiotical factors and determination characteristics were found. Molecular survey using ISSR method shows no difference between subspecies. The final resolution of subspecies existence will be questioned in following study.

<p>Gilbert’s syndrome (GS) occurs in approximately 10% of the European population. The most common cause is homozygosity for UGT1A1*28, which is a TA repeat expansion in the promoter of UGT1A1. It is characterised by intermittent hyperbilirubinemia due to reduced hepatic activity of the  enzyme UDP-glucuronosyl-transferase 1A1(UGT1A1). GS also  alteres the pharmacokinetics of some drugs and increases the risk of drug toxicity. Irinotecan (Camptosar®, Campto®) is used in metastatic colorectal cancer and the active metabolite is inactivated by UGT1A1. Studies have shown that GS can be a risk factor for toxicity during irinotecan therapy.</p><p>Three different methods for genotyping of UGT1A1*28 have been tested.</p><p>PCR with electrophoresis used for size separation, melting temperature analysis and fluorescent PCR followed by fragment analysis on a capillary sequencer.</p><p>The last method was found to be superior. This method was used for genotyping of patients with colorectal cancer treated with irinotecan and 5-fluorouracil in the Nordic VI study. A significant association between UGT1A1 genotype and plasma bilirubin level before the start of irinotecan treatment was seen (ANOVA p<0.0001). Patients with GS had an overall increased risk of adverse drug reactions (Fishers Exact test p=0.02).</p><p>Gilbert’s syndrome can be diagnosed by genotyping UGT1A1*28 with a fragment analysis method. Genotyping of UGT1A1*28 can be used to identify patients with an increased risk of adverse reactions to irinotecan.<strong> </strong></p><p><strong> </strong></p> / <p>Gilberts syndrom (GS) drabbar upp till 10% av befolkningen i Västeuropa. GS beror på nedsatt aktivitet av enzymet UDP-glukuronosyltransferas 1A1 (UGT1A1) i levern. Den vanligaste orsaken är att individen är homozygot för en insertion av två baser i promotorn för genen UGT1A1. Denna genvariant kallas (TA)7TAA  eller UGT1A1*28. GS leder till intermittent stegring av bilirubin vid infektioner, men bilirubinstegring kan förekomma även utan utlösande agens. GS kan också leda till bilirubinstegring vid viss läkemedelsbehandling. Irinotekan (Campto®) används vid metastaserande colorektal cancer och dess aktiva metabolit inaktiveras av UGT1A1. Det finns rapporter om att GS ger ökad risk för toxiska biverkningar av irinotekan.</p><p>Tre metoder för att bestämma UGT1A1 har jämförts: PCR med elfores, PCR med smältpunktsanalys och PCR med fragmentanalys på sekvensator. Den sista metoden var bäst och användes för att genotypa UGT1A1 hos patienter med colorektal cancer från Nordic VI-studien. De behandlades med irinotekan i kombination med bolusinjektion eller infusion av 5-fluorouracil. Vi fann att  patienter med GS hade signifikant högre S-bilirubin före behandling jämfört med övriga patienter. De hade även ökad frekvens biverkningar av irinotekan (Fishers exakta test p=0,02).</p><p>Genotypning av UGT1A1 kan således användas för att diagnostisera Gilberts syndrom hos patienter med oförklarad bilirubinstegring. Det kan även användas för att identifiera patienter med ökad risk för biverkningar av irinotekan.</p>

Detecting mutations and genomic variations is fundamental indiagnosis, isolating disease genes, association studies,functional genomics and pharmacogenomics. The objective hasbeen to use and further develop a variety of tools andtechnologies to analyze these genetic alterations andvariations. The p53 tumor suppressor gene and short arm of chromosome 9have been used as genetic markers to investigate fundamentalquestions concerning early events preceding non-melanoma skincancers, clonal progression and timing of different mutationsand deletions. Conventional gel based DNA sequencing andfragment analysis of microsatellite markers were utilized forthis purpose. In addition, a sequence-specific PCR-mediatedartifact is discussed. Pyrosequencing, a bioluminometric technique based onsequencing-by-synthesis, has been utilized to determinemutation ratios in the p53 gene. In addition, in the case ofmultiple mutations, pyrosequencing was adopted to determineallelic distribution of mutations without the use of cloningprocedures. Exons 5 to 8 of the p53 gene were also sequenced bythis method. The possibility of typing single base variations bypyrosequencing has been evaluated. Two different nucleotidedispensation orders were investigated and data were comparedwith the predicted pattern for each alternative of the variableposition. Analysis of loss of heterozygosity was possible byutilizing single nucleotide polymorphisms. A modified allele-specific extension strategy for genotypingof single nucleotide polymorphisms has been developed. Throughthe use of a real-time bioluminometric assay, it has beendemonstrated that reaction kinetics for a mismatchedprimer-template is slower than the matched configuration,butthe end-point signals are comparable. By introduction ofapyrase, the problems associated with mismatch extensions havebeen circumvented and accurate data has been obtained. Keywords:fragment analysis, microsatellite, loss ofheterozygosity, DNA sequencing, pyrosequencing, cancer,mutation, variation, single nucleotide polymorphism,allele-specific extension, bioluminescence, apyrase. / QC 20100415

Gilbert’s syndrome (GS) occurs in approximately 10% of the European population. The most common cause is homozygosity for UGT1A1*28, which is a TA repeat expansion in the promoter of UGT1A1. It is characterised by intermittent hyperbilirubinemia due to reduced hepatic activity of the  enzyme UDP-glucuronosyl-transferase 1A1(UGT1A1). GS also  alteres the pharmacokinetics of some drugs and increases the risk of drug toxicity. Irinotecan (Camptosar®, Campto®) is used in metastatic colorectal cancer and the active metabolite is inactivated by UGT1A1. Studies have shown that GS can be a risk factor for toxicity during irinotecan therapy. Three different methods for genotyping of UGT1A1*28 have been tested. PCR with electrophoresis used for size separation, melting temperature analysis and fluorescent PCR followed by fragment analysis on a capillary sequencer. The last method was found to be superior. This method was used for genotyping of patients with colorectal cancer treated with irinotecan and 5-fluorouracil in the Nordic VI study. A significant association between UGT1A1 genotype and plasma bilirubin level before the start of irinotecan treatment was seen (ANOVA p&lt;0.0001). Patients with GS had an overall increased risk of adverse drug reactions (Fishers Exact test p=0.02). Gilbert’s syndrome can be diagnosed by genotyping UGT1A1*28 with a fragment analysis method. Genotyping of UGT1A1*28 can be used to identify patients with an increased risk of adverse reactions to irinotecan. / Gilberts syndrom (GS) drabbar upp till 10% av befolkningen i Västeuropa. GS beror på nedsatt aktivitet av enzymet UDP-glukuronosyltransferas 1A1 (UGT1A1) i levern. Den vanligaste orsaken är att individen är homozygot för en insertion av två baser i promotorn för genen UGT1A1. Denna genvariant kallas (TA)7TAA  eller UGT1A1*28. GS leder till intermittent stegring av bilirubin vid infektioner, men bilirubinstegring kan förekomma även utan utlösande agens. GS kan också leda till bilirubinstegring vid viss läkemedelsbehandling. Irinotekan (Campto®) används vid metastaserande colorektal cancer och dess aktiva metabolit inaktiveras av UGT1A1. Det finns rapporter om att GS ger ökad risk för toxiska biverkningar av irinotekan. Tre metoder för att bestämma UGT1A1 har jämförts: PCR med elfores, PCR med smältpunktsanalys och PCR med fragmentanalys på sekvensator. Den sista metoden var bäst och användes för att genotypa UGT1A1 hos patienter med colorektal cancer från Nordic VI-studien. De behandlades med irinotekan i kombination med bolusinjektion eller infusion av 5-fluorouracil. Vi fann att  patienter med GS hade signifikant högre S-bilirubin före behandling jämfört med övriga patienter. De hade även ökad frekvens biverkningar av irinotekan (Fishers exakta test p=0,02). Genotypning av UGT1A1 kan således användas för att diagnostisera Gilberts syndrom hos patienter med oförklarad bilirubinstegring. Det kan även användas för att identifiera patienter med ökad risk för biverkningar av irinotekan.

Hematopoietic stem cell transplantation (HSCT) is used to treat patient with hematological diseases such as leukemia and genetic conditions such as sickle cell anemia. After HSCT the patients are supervised for signs of relapse of disease or rejection of transplanted cells. This is done by using chimerism analysis. At the department of clinical genetics at Akademiska sjukhuset fragment analysis of short tandem repeats is used for chimerism analysis, which is to be replaced by a Next generation sequencing (NGS) based method called Devyser chimerism, which includes an IVDR labelled kit. The aim of this project was to validate the new method for chimerism analysis. DNA samples from twelve HSCT patients and their donors were analyzed with Devyser chimerism and the results were compared to the results from the current method. The sensitivity of the new method was tested by analysis of artificial chimerism samples from blood donors. The results from the comparison showed a good correlation between methods (R2 = 0,9864) and the sensitivity of the method was confirmed to be 0,1% mixed chimerism. There was some difficulty in identifying enough informative markers for re-transplanted patients two had separate donors. This is a known problem for chimerism analysis in general and not a specific problem to the new method and will not be a hindrance for the implementation of Devyser chimerism at the clinical laboratory.

Squamous cell carcinoma is the most frequent malignant tumour of head and neck. Prognostic and predictive information as an individual imprint of molecular-genetic analysis of HNSCC will help to determine the best indivicual treatment. And in case of surgical appraoch the optimal resection with adequate quality of life and long-term survival. Study of early cancerogenesis in our project is based on knowledge, that histological normal mucosa next to tumor shows preneoplastic molecular alterations. Molecular genetic changes in a histological normal mucosa harbouring a tumor may play a principal role in revealing of early cancerogenesis process. Molecular-genetic analysis of cancerogenesis in HNSCC reveals prognostic and predictive factors, which are necessary for evaluation and decission for the best individual treatment. This is the concept of tailored medicine The text summarizes current knowledge of early cancerogenesis in HNSCC and presents molecular-biological trends, which are necessary to discover details of early cancerogenesis and thus to get a tool for better detection as well as treatment of malignant disease. The study is based on fragment analysis of microsatelites lesions in tumor tissue in comparison to adjacent mucosa and the healthy mucosa. Results show significant molecular-biological...

Microsatellites are repetitive DNA characterized by tandem repeats of short motifs (2 – 5 bp). High mutation rates make them ideal for population level studies. Microsatellite allele genesis is generally attributed to strand slippage, and it is assumed that alleles are caused only by changes in repeat number. Most analyses are limited to alleles (electromorphs) scored by mobility only, and models of evolution rarely account for homoplasy in allele length. Additionally, insertion/deletion events (indels) in the flanking region or interruptions in the repeat can obfuscate the accuracy of genotyping. Many investigators use microsatellites, designed for a focal species, to screen for genetic variation in non-focal species. Comparative studies have shown different mutation rates of microsatellites in different species, and even individuals. Recent studies have used reciprocal comparisons to assess the level of polymorphism of microsatellites between pairs of taxa. In this study, I investigated the evolution of microsatellites within a phylogenetic context, using comparisons within the rodent family Bathyergidae. Bathyergidae represents a monophyletic group endemic to sub-Saharan Africa and relationships are well supported by morphological and molecular data. Using mitochondrial and nuclear DNA, a robust phylogeny was generated for the Bathyergidae. From my results, I proposed the new genus, Coetomys. I designed species-specific genotyping and microsatellite flanking sequence (MFS) primers for each genus. Sequencing of the MFS provided direct evidence of the evolutionary dynamics of the repeat motifs and their flanking sequence, including rampant electromorphic homoplasy, null alleles, and indels. This adds to the growing body of evidence regarding problems with genotype scores from fragment analysis. A number of the loci isolated were linked with repetitive elements (LTRs and SINEs), characterized as robust phylogenetic characters. Results suggest that cryptic variation in microsatellite loci are not trivial and should be assessed in all studies. The phylogenetic utility of the nucleotide variation of the MFS was compared to the well-resolved relationships of this family based on the 12S/TTR phylogeny. Variation observed in MFS generated robust phylogenies, congruent with results from 12S/TTR. Finally, a number of the indels within the MFS provided a suite of suitable phylogenetic characters.

No description available.