Bibliography: leaves 104-130. The aim of this thesis is to characterise subgenomic RNAs of cucumoviruses and the functions of their encoding genes. Strains of cucumber mosaic virus (CMV) are classified into two major subgroups (I and II) on the basis of nucleotide sequence homology. The V strain of tomato aspermy virus (V-TAV) and a subgroup I CMV strain (WAII) are chosen to determine whether the 2b genes encoded by these viruses are expressed 'in vivo'. For further investigation of the 2b gene function, cDNA clones of three genomic RNAs of V-TAV are constructed. Using the infectious cDNA clones of V-TAV, a mutant virus containing only one of the two repeats is constructed.

Background: Phylogenomic analyses recently became popular to address questions about deep metazoan phylogeny. Ribosomal proteins (RP) dominate many of these analyses or are, in some cases, the only genes included. Despite initial hopes, hylogenomic analyses including tens to hundreds of genes still fail to robustly place many bilaterian taxa. Results: Using the phylogenetic position of myzostomids as an example, we show that phylogenies derived from RP genes and mitochondrial genes produce incongruent results. Whereas the former support a position within a clade of platyzoan taxa, mitochondrial data recovers an annelid affinity, which is strongly supported by the gene order data and is congruent with morphology. Using hypothesis testing, our RP data significantly rejects the annelids affinity, whereas a platyzoan relationship is significantly rejected by the mitochondrial data. Conclusion: We conclude (i) that reliance of a set of markers belonging to a single class of macromolecular complexes might bias the analysis, and (ii) that concatenation of all available data might introduce conflicting signal into phylogenetic analyses. We therefore strongly recommend testing for data incongruence in phylogenomic analyses. Furthermore, judging all available data, we consider the annelid affinity hypothesis more plausible than a possible platyzoan affinity for myzostomids, and suspect long branch attraction is influencing the RP data. However, this hypothesis needs further confirmation by future analyses.

This thesis presents the mathematical modeling of two new Escherichia coli platforms with economical potential for the production of biofuels and chemicals, namely glycerol fermentation and the reversal of the β-oxidation cycle. With the increase in traditional fuel prices, alternative renewable energy sources are needed, and the efficient production of biofuels becomes imperative. So far studies have focused on using glucose as feedstock for the production of ethanol and other fuels, but a recent increase in glycerol availability and its consequent decrease in price make it an attractive feedstock. Furthermore, the reversed β-oxidation cycle is a highly efficient mechanism for the synthesis of long-chain products. These two platforms have been reported experimentally in E. coli but their mathematical modeling is presented for the first time here. Because mathematical models have proved to be useful in the optimization of microbial metabolism, two complementary models were used in this study: kinetic and stoichiometric. Kinetic models can identify the control structure within a specific pathway, but they require highly detailed information, making them applicable to small sets of reactions. In contrast, stoichiometric models require only mass balance information, making them suitable for genome-scale modeling to study the effect of adding or removing reactions for the optimization of the synthesis of desired products. To study glycerol fermentation, a kinetic model was implemented, allowing prediction of the limiting enzymes of this process: glycerol dehydrogenase and di-hydroxyacetone kinase. This prediction was experimentally validated by increasing their enzymatic activities, resulting in a two-fold increase in the rate of ethanol production. Additionally, a stoichiometric genome-scale model (GEM) was modified to represent the fermentative metabolism of glycerol, identifying key metabolic pathways for glycerol fermentation (including a new glycerol dissimilation pathway). The GEM was used to identify genetic modifications that would increase the synthesis of desired products, such as succinate and butanol. Finally, glucose metabolism using the reversal β-oxidation cycle was modeled using a GEM to simulate the synthesis of a variety of medium and long chain products (including advanced biofuels). The model was used to design strategies that can lead to increase the productivity of target products.

Hfq is a bacterial RNA-binding protein recently shown to contain the Sm motif, a characteristic of Sm proteins that function in RNA processing in archaea and eukaryotes. Hfq plays a major role in RNA-RNA interactions regulating translation. Comparative structural modeling and amino acid sequence alignment were used to predict the 3-D structure of Hfq and the model was in excellent agreement with the crystal structure which determined for S. aureus Hfq. The evolution of Hfq was explored by a BLAST search of microbial genomes followed by phyletic analysis. About half of the genomes examined contain at least one gene coding for Hfq. The presence and absence of Hfq closely followed major bacterial clades. The potential RNA binding residues on the two surfaces of the Hfq hexamer were proposed based on the bioinformatics studies and the mutant Hfq proteins with either single or double mutations on the two surfaces of the Hfq hexamer were generated. Their RNA binding properties was biophysically studied by gel-shift assay, fluorescence anisotropy and fluorescence quenching techniques. Results indicated that 1) point mutations on the distal surface of the Hfq hexamer, Y25A and K31A, have a major effect on A18 binding. Both reduce binding by about 1000 fold. Mutations on the proximal surface have a small or no influence on A18 binding. 2) Two mutations, F39A and R16A, on the proximal surface of the Hfq structure reduce binding to the DsrA domain II by 10 fold. Other mutations reduce binding by less than 2 fold. 3) An amino acid covariance was observed in L12 and F39. Mutation L12F can partially restore F39A in DsrA RNA binding. 4) It appears that two Hfq hexamers cooperatively bind one RNA for both DsrADII and A18.

Nearly one half of the human genome is composed of transposable elements (TEs). Once dismissed as 'selfish' or 'junk' DNA, TEs have also been implicated in a numerous functions that serve the needs of their host genome. I have evaluated the role of TEs in mediating the epigenetic mechanisms that serve to regulate human gene expression. These findings can be broadly divided into two major mechanisms by which TEs affect human gene expression; by modulating nucleosome binding in the promoter regions and by recruiting epigenetic histone modifications that enable them to serve as promoters and enhancers. Thus. the studies encompassed in this thesis elucidate the contributions of TEs in epigenetically regulating human gene expression on a global as well as local scale.

published_or_final_version / Computer Science / Doctoral / Doctor of Philosophy

The genome of living organisms is constantly subjected to the environmental influences that result in different negative, negligible or positive impacts. The ability to maintain the genome integrity and simultaneously provide its flexibility is the main determinant for the evolutionary success of any species. One of the important aspects of genome maintenance is the precise regulation of the DNA repair machinery. Results reported here indicate the existence of a tight, age-dependent regulation of homologous recombination, one of the two main DNA double-strand break repair pathways. We show that recombination is influenced by conditions such as the change of temperature (cold or warm), day length, water availability (drought or overwatering stress) and salinity. These stresses not only influence the genome stability of stress-subjected generations but also change the recombination in subsequent generations. This indicates the possible involvement of homologous recombination in plant evolution and development of plant stress tolerance. / xiv, 121 leaves ; 29 cm.

The prevailing metaphysics of modern medicine and bioethics have been largely dualistic and materialistic in nature. The person is conceived of as a duality of mind and body, where the body is treated simply as a material object. In taking such a perspective, the background is set from which people can come to feel distanced from their bodies and believe it acceptable to alienate and sell their body parts. This thesis argues that the dualistic (and to a lesser extent the materialistic) conceptions of the person have contributed significantly to the objectification and commodification of the body. A most disturbing example of this is the patenting of human genetic material. / In place of the dualistic metaphysic, an embodiment perspective is proposed that treats the person as a unique individual who is inseparably unified in mind, body, and soul. This view can help address the problem of patenting and commercialisation as it avoids the difficulties raised by the application of property language to the body. The body is not simply an object that can be bought or sold, but is an integral part of a person's identity. This does not mean that medical research must be prohibited, but simply that an individual's cells and any derivative cell lines should not be subject to patents. Above all, an embodiment perspective forces the medical and technological establishment, and society in general, to accept that people are unique unified individuals who cannot be objectified, commodified, or alienated from their bodies and selves.

A novel approach for screening metagenomic libraries by merging both metagenomic and microarray platforms was developed and optimized. This high-throughput screening strategy termed "metagenomic microarrays" involved the construction of two Arctic soil large-insert libraries and the high density arraying of the clone plasmid DNA (~50 kb) onto glass slides. A standard alkaline lysis technique used for the purification of plasmid DNA was adapted and optimized to function efficiently in a 96-well format, providing an economically viable means of producing sufficient high-quality plasmid DNA for direct printing onto microarrays. The amounts of printed material and probe, required for maximal clone detection, were optimized. To examine catabolic clone detection libraries were first screened by PCR for catabolic genes of interest. Two PCR-positive clones were printed onto microarrays, and detection of these specific clones in the printed libraries was achieved using labeled probes produced from PCR fragments of known sequence. Also, hybridizations were performed using labeled PCR fragments derived from the amplification of a catabolic gene from the total community DNA. The ability of selected probes to specifically target clones of interest was demonstrated. This merger of metagenomics and microarray technologies has shown great promise as a tool for screening the natural microbial community for catabolic potential and could also be used to profile microbial diversity in different environments.

Transposable elements (TEs) are mobile DNA sequences that can move from one location to another in the genome. These elements encode regulatory features including transcriptional promotion and termination signals facilitating the production of new transcripts (or elements). The elements thus produced are inserted back into the genome. Due to their insertional capacity and encoded regulatory features, TEs have, in recent years, been recognized as significant contributors to regulatory variation both within and between species. In comparing the human and chimpanzee genomes it has been hypothesized that the genetic basis of the phenotypic differences that distinguish them may be the result of regulatory differences existing between the two species. Since TEs inserted in proximity to genes can significantly alter gene expression patterns, this research aims at exploring the influence of TE sequences and retrotransposons in particular in the evolution of gene regulation between humans and chimpanzees. A first systematic search of one particular class of retrotransposons - endogenous retroviruses (ERVs) was carried out in the chimpanzee genome. Forty two families of ERVs were identified in the chimpanzee genome including the discovery of 9 previously unknown families in humans. The vast majority of these families were found to have orthologs in the human genome except for two (CERV 1/PTERV1 and CERV 2) families. The two CERV families without orthologs in the human genome display a patchy distribution among primates. Nine families of chimpanzee ERVs have been transpositionally active since the human-chimpanzee divergence, while only two families have been active in the human lineage. The genomic differences [INDEL variation (80-12,000 bp in length)] between humans and chimpanzees are laid out. The INDEL variation located in or near genes is categorized in detail and is correlated with differences in gene expression patterns in a variety of organs and tissues. Results indicate that the majority of the INDEL variation between the two species is associated with retrotransposon sequences and that this variation is significantly correlated with differences in gene expression most notably in brain and testes. These findings are consistent with the hypothesis that retrotransposon mediated regulatory variation may have been a significant factor in human/chimpanzee evolution.