Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.

We describe the use of a tensor mode-1 higher-order singular value decomposition (HOSVD) in the analyses of alignments of 16S and 23S ribosomal RNA (rRNA) sequences, each encoded in a cuboid of frequencies of nucleotides across positions and organisms. This mode-1 HOSVD separates the data cuboids into combinations of patterns of nucleotide frequency variation across the positions and organisms, i.e., "eigenorganisms"' and corresponding nucleotide-specific segments of "eigenpositions," respectively, independent of a-priori knowledge of the taxonomic groups and their relationships, or the rRNA structures. We show that this mode-1 HOSVD provides a mathematical framework for modeling the sequence alignments where the mathematical variables, i.e., the significant eigenpositions and eigenorganisms, are consistent with current biological understanding of the 16S and 23S rRNAs. First, the significant eigenpositions identify multiple relations of similarity and dissimilarity among the taxonomic groups, some known and some previously unknown. Second, the corresponding eigenorganisms identify positions of nucleotides exclusively conserved within the corresponding taxonomic groups, but not among them, that map out entire substructures inserted or deleted within one taxonomic group relative to another. These positions are also enriched in adenosines that are unpaired in the rRNA secondary structure, the majority of which participate in tertiary structure interactions, and some also map to the same substructures. This demonstrates that an organism's evolutionary pathway is correlated and possibly also causally coordinated with insertions or deletions of entire rRNA substructures and unpaired adenosines, i.e., structural motifs which are involved in rRNA folding and function. Third, this mode-1 HOSVD reveals two previously unknown subgenic relationships of convergence and divergence between the Archaea and Microsporidia, that might correspond to two evolutionary pathways, in both the 16S and 23S rRNA alignments. This demonstrates that even on the level of a single rRNA molecule, an organism's evolutionary pathway is composed of different types of changes in structure in reaction to multiple concurrent evolutionary forces. / text

An essential tool in biology is the alignment of multiple sequences. Biologists use multiple sequence alignments for tasks such as predicting protein structure and function, reconstructing phylogenetic trees, and finding motifs. Constructing high-quality multiple alignments is computationally hard, both in theory and in practice, and is typically done using heuristic methods. The majority of state-of-the-art multiple alignment programs employ a form and polish strategy, where in the construction phase, an initial multiple alignment is formed by progressively merging smaller alignments, starting with single sequences. Then in a local-search phase, the resulting alignment is polished by repeatedly splitting it into smaller alignments and re-merging. This merging of alignments, the basic computational problem in the construction and local-search phases of the best multiple alignment heuristics, is called the Aligning Alignments Problem. Under the sum-of-pairs objective for scoring multiple alignments, this problem may seem to be a simple extension of two-sequence alignment. It is proven here, however, that with affine gap costs (which are recognized as necessary to get biologically-informative alignments) the problem is NP-complete when gaps are counted exactly. Interestingly, this form of multiple alignment is polynomial-time solvable when we relax the exact count, showing that exact gap counts themselves are inherently hard in multiple sequence alignment. Unlike general multiple alignment however, we show that Aligning Alignments with affine gap costs and exact counts is tractable in practice, by demonstrating an effective algorithm and a fast implementation. Our software AlignAlign is both time- and space-efficient on biological data. Computational experiments on biological data show instances derived from standard benchmark suites can be optimally aligned with surprising efficiency, and experiments on simulated data show the time and space both scale well.

Introduction: As a part of the Gene Regulators in Disease project (GRID), this study aims to create a novel high throughput method to discover the genetic effect on gene translation, taking advantage of the rationale that efficiently translated mRNAs associate with multiple ribosomes, while less active ones with fewer or none. / Methods: Lymphoblastoid cell lines (LCLs) from 44 HapMap European individuals were used for polyribosomal fractionation and establishing the sample bank for the future study. The fractionated mRNA samples of 10 out of the 44 individuals were run on an Illumina GoldenGate Beadarray to detect allelic imbalance (developed by the group of T.J. Hudson and T.M. Pastinen). / Results: This study established a high-quality RNA bank, including 1,100 RNA fraction samples. By the Illumina chip, translational imbalance was detected in 75 out of 1483 (5.06%) assays, and 63 out of269 (23.4%) genes. The translational effect was well replicable by the resequencing method. / Conclusion: This study found that genetic effect on gene translation is a common mechanism of expression regulation. Our best hit found in the integrin beta 1 binding protein 1 gene (ITGB1BP1 ) highlights the role of mRNA 3'UTR secondary structure in gene translation. / Keywords: Gene translation, High throughput genotyping, Human genetics, Polyribosome, RNA, Single nucleotide polymorphism

Uveal melanoma is the most common primary intraocular malignant tumour in adults. Despite improvements in the diagnosis and treatment of the primary tumour, patients continue to have the same mortality rate as several decades ago, reflecting our poor understanding of the mechanisms behind the formation of metastases in this disease. The purpose of this study was therefore to characterize an animal model of uveal melanoma and use this model to study the transcriptional changes that cells undergo from culture to intraocular tumour, to circulation and finally to the formation of a metastatic nodule. / Using microarrays we identified 314 changes in transcript abundance between the intraocular tumour and metastatic lesions. Principal Components Analysis was used to cluster these transcripts into four distinct groups. A further 61 gene transcripts showed statistically significant changes between re-cultured cells isolated from the model, with the circulating malignant cells representing an intermediate step between cells isolated from intraocular tumours and metastatic lesions. We have produced a detailed analysis of the molecular changes that take place as human uveal melanoma cells evolve from a primary tumour to metastasis in an animal model, including the decrease in expression of specific melanoma markers. These changes were verified using quantitative real time polymerase chain reaction and three different functional assays. / In addition we sought to describe the genetic changes that are present in these cells. Using comparative genomic hybridization arrays we were able to successfully describe the deletions and amplifications that are present in genomic DNA extracted from paraffin embedded sections of the primary tumour. This represents the first time that archival tissue has successfully been used for this sort of analysis in uveal melanoma. We identified several genomic amplifications and deletions including an area of amplification of Wnt2, which is involved in beta-catenin regulation and C-Met, which plays a role in tumour cell homing to the liver in patients. / To the best of our knowledge, this is the first time that a detailed genetic analysis has been carried out on the progression of uveal melanoma from intraocular tumour, to circulation, to the formation of metastases.

The sequencing of the human genome revealed that the number of genes does not explain why humans are different from other organisms like mice and dogs. Instead, it is how genes interact with each other and the environment that separates us from other organisms. This motivates the study of genetic networks and, consequently, my research. My work delves into the roles that simple genetic networks play in a cell and explores the biotechnological aspects of how to uncover such genes and their interactions in experimental models. / Cells must respond to the extracellular environment to contract, migrate, and live. Cells, however, are subject to stochastic fluctuations in protein concentrations. I investigate how cells make important decisions such as gene transcription based on noisy measurements of the extracellular environment. I propose that genetic networks perform Bayesian inference as a way to consider the probabilistic nature of these measurements and make the best decision. With mathematical models, I show that allosteric repressors and activators can correctly infer the state of the environment despite fluctuating concentrations of molecules. Viewing transcriptional networks as inference modules explains previous experimental data. I also discover that the particular inference problem determines whether repressors or activators are better. / Next, I explore the genetic underpinnings of two canine models of atrial fibrillation: atrial tachypacing and ventricular tachypacing. Using Affymetrix microarrays, I find that the genetic signatures of these two models are significantly different both in magnitude and in class of genes expressed. The ventricular tachypacing model has thousands of transcripts differentially expressed with little overlap between 24 hours and 2 weeks, suggesting independent mechanisms. The atrial tachypacing model demonstrates an adaptation as the number of genes found changed decreases with increasing time to the point that no genes are changed at 6 weeks. I use higher level analysis to find that extracellular matrix components are among the most changed in ventricular tachypacing and that genes like connective tissue growth factor may be responsible. / Finally, I generalize the main problem of microarray analysis into an evaluation problem of choosing between two competing options based on the scores of many independent judges. In this context, I rediscover the voting paradox and compare two different solutions to this problem: the sum rule and the majority rule. I find that the accuracy of a decision depends on the distribution of the judges' scores. Narrow distributions are better solved with a sum rule, while broad distributions prefer a majority rule. This finding motivates a new algorithm for microarray analysis which outperforms popular existing algorithms on a sample data set and the canine data set examined earlier. A cost analysis reveals that the optimal number of judges depends on the ratio of the cost of a wrong decision to the cost of a judge.

The larger grain borer (LGB) Prostephanus truncatus (Horn) is the most important pest of farm stored maize and cassava in Africa. This alien invasive species was introduced into the continent from Mesoamerica in the late 1970s and by 2008 had spread to at least 18 countries. In contrast to indigenous primary storage pests, LGB exists as on-farm and as wild populations, hence, sustainable control must target both environments. Biological control is especially attractive for wild populations to reduce early season grain store infestation, while cultural and chemical methods are useful to protect stored produce directly. Two populations of the predator Teretrius nigrescens Lewis were introduced into several African countries' as a biocontrol agent. It has shown long-term success and cost effective control in warm-humid areas. Control has however not been successful in cool and hot-dry zones. The aim of this study was to investigate the possible underlying genetic and ecological explanations for these observations and the possibility of joint use of molecular markers and ecological parameters in the development of sustainable control strategies. A 28-month baseline monitoring and recovery activity was done in from 2004 in five regions in Kenya along an east-westerly transect. Monitoring and live sample collection was also done in the original outbreak area in eastern Kenya. There was greater LGB flight activity in western Kenya (high potential maize production area) than the low potential areas. Very few T. nigrescens were recovered, solely in the eastern regions. LGB flight activity followed a seasonal pattern mostly related to changes in the relative humidity at 12:00, rainfall and dew point temperature but with a 3 - 4 week lag. A linear predictive model based on these factors predicted 27 % of the observed flight activity. The survival and predation of five strains of T. nigrescens were compared at eight temperature levels between 15 °C and 36 °C at low and high humidity. All the strains of T. nigrescens exerted a significant reduction of LGB population build-up between 21 °C and 33 °C with generally better performance under humid conditions. There was no evidence of T. nigrescens development at 15 °C. At 18 °C, T. nigrescens oviposition and development was observed but the effect on LGB did not differ significantly from the control. The KARI population was the least effective in preventing grain damage at lower temperatures, but performed better than other strains above 30 °C at low humidity conditions. There was no control at 18 °C and 36 °C under both high and low humidity conditions. Since the extent of genetic differentiation in T. nigrescens was unclear from prior studies, several molecular marker techniques were progressively used. The RAPD-PCR did not reveal any genetic diversity between geographical populations. A 1000bp region of the mitochondrial mtCOI gene revealed two distinct clades differing consistently at 26 segregating sites. The two clades can be identified by simple PCR-RFLP procedure using single or double sequential restriction with EcoR1, HincII, RsaI and DdeI digestion. However, the two lineages co-exist among the mid-altitude Central American populations. The internal transcribed spacer regions ITS1 and ITS2 with some neighbouring coding sequences of the ribosomal DNA were cloned and sequenced. The spacer regions were so variable in length and sequence between T. nigrescens and related Histeridae species that direct sequence alignment was not meaningful. Within T. nigrescens, there was intragenomic variability of the spacer regions mostly involving insertions and deletions of variable tandem repeat units predominantly within the ITS regions. The short flanking coding (18S, 5.8S and 21S) regions were conserved across populations and six other Histeridae species. There was no significant secondary structure variation of the ITS regions among populations of T. nigrescens. Twenty-four novel variable microsatellite markers were developed and tested on the Honduras populations. Alleles per locus ranged between two and twelve with observed heterozygosity between 0.048 and 0.646. Six loci deviated significantly from Hardy Weinberg Equilibrium and possibly had null alleles. The success of microsatellite amplification in outgroup species and variability of markers declined with an increase in the phylogenetic distance between the test species and T. nigrescens. Genotyping 432 individuals from 13 geographic populations revealed a comparatively higher genetic diversity in field populations. Partial isolation by distance and time was observed. Population bottlenecks were not detected, but recent expansion was evident in laboratory populations. Although five dominant genetic clusters were identified by Bayesian methods, meaningful hierarchical population structure was observed at between two and nine population groups (p < 0.01; 10,000 iterations). Biological control of the larger grain borer using T. nigrescens seems an important aspect of the sustainable integrated control approach of the pest. Ecological adaptations, appropriate release strategies and genetic diversity are all essential considerations in these efforts and could be responsible for the variable success already observed. There is some genetic differentiation between populations of T. nigrescens but, further studies would be necessary to ascertain the contribution of such diversity to its predatory performance. The effect of laboratory culturing in aggravating genetic drift should be accommodated to avoid loss of diversity during sampling, quarantine, rearing and release of the predator. / Thesis (Ph.D. (Environmental Science))--North-West University, Potchefstroom Campus, 2009.

The larger grain borer (LGB) Prostephanus truncatus (Horn) is the most important pest of farm stored maize and cassava in Africa. This alien invasive species was introduced into the continent from Mesoamerica in the late 1970s and by 2008 had spread to at least 18 countries. In contrast to indigenous primary storage pests, LGB exists as on-farm and as wild populations, hence, sustainable control must target both environments. Biological control is especially attractive for wild populations to reduce early season grain store infestation, while cultural and chemical methods are useful to protect stored produce directly. Two populations of the predator Teretrius nigrescens Lewis were introduced into several African countries' as a biocontrol agent. It has shown long-term success and cost effective control in warm-humid areas. Control has however not been successful in cool and hot-dry zones. The aim of this study was to investigate the possible underlying genetic and ecological explanations for these observations and the possibility of joint use of molecular markers and ecological parameters in the development of sustainable control strategies. A 28-month baseline monitoring and recovery activity was done in from 2004 in five regions in Kenya along an east-westerly transect. Monitoring and live sample collection was also done in the original outbreak area in eastern Kenya. There was greater LGB flight activity in western Kenya (high potential maize production area) than the low potential areas. Very few T. nigrescens were recovered, solely in the eastern regions. LGB flight activity followed a seasonal pattern mostly related to changes in the relative humidity at 12:00, rainfall and dew point temperature but with a 3 - 4 week lag. A linear predictive model based on these factors predicted 27 % of the observed flight activity. The survival and predation of five strains of T. nigrescens were compared at eight temperature levels between 15 °C and 36 °C at low and high humidity. All the strains of T. nigrescens exerted a significant reduction of LGB population build-up between 21 °C and 33 °C with generally better performance under humid conditions. There was no evidence of T. nigrescens development at 15 °C. At 18 °C, T. nigrescens oviposition and development was observed but the effect on LGB did not differ significantly from the control. The KARI population was the least effective in preventing grain damage at lower temperatures, but performed better than other strains above 30 °C at low humidity conditions. There was no control at 18 °C and 36 °C under both high and low humidity conditions. Since the extent of genetic differentiation in T. nigrescens was unclear from prior studies, several molecular marker techniques were progressively used. The RAPD-PCR did not reveal any genetic diversity between geographical populations. A 1000bp region of the mitochondrial mtCOI gene revealed two distinct clades differing consistently at 26 segregating sites. The two clades can be identified by simple PCR-RFLP procedure using single or double sequential restriction with EcoR1, HincII, RsaI and DdeI digestion. However, the two lineages co-exist among the mid-altitude Central American populations. The internal transcribed spacer regions ITS1 and ITS2 with some neighbouring coding sequences of the ribosomal DNA were cloned and sequenced. The spacer regions were so variable in length and sequence between T. nigrescens and related Histeridae species that direct sequence alignment was not meaningful. Within T. nigrescens, there was intragenomic variability of the spacer regions mostly involving insertions and deletions of variable tandem repeat units predominantly within the ITS regions. The short flanking coding (18S, 5.8S and 21S) regions were conserved across populations and six other Histeridae species. There was no significant secondary structure variation of the ITS regions among populations of T. nigrescens. Twenty-four novel variable microsatellite markers were developed and tested on the Honduras populations. Alleles per locus ranged between two and twelve with observed heterozygosity between 0.048 and 0.646. Six loci deviated significantly from Hardy Weinberg Equilibrium and possibly had null alleles. The success of microsatellite amplification in outgroup species and variability of markers declined with an increase in the phylogenetic distance between the test species and T. nigrescens. Genotyping 432 individuals from 13 geographic populations revealed a comparatively higher genetic diversity in field populations. Partial isolation by distance and time was observed. Population bottlenecks were not detected, but recent expansion was evident in laboratory populations. Although five dominant genetic clusters were identified by Bayesian methods, meaningful hierarchical population structure was observed at between two and nine population groups (p < 0.01; 10,000 iterations). Biological control of the larger grain borer using T. nigrescens seems an important aspect of the sustainable integrated control approach of the pest. Ecological adaptations, appropriate release strategies and genetic diversity are all essential considerations in these efforts and could be responsible for the variable success already observed. There is some genetic differentiation between populations of T. nigrescens but, further studies would be necessary to ascertain the contribution of such diversity to its predatory performance. The effect of laboratory culturing in aggravating genetic drift should be accommodated to avoid loss of diversity during sampling, quarantine, rearing and release of the predator. / Thesis (Ph.D. (Environmental Science))--North-West University, Potchefstroom Campus, 2009.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Stockholms universitet, 2007. / Härtill 4 uppsatser.