Ulm, Univ. Diss., 2006.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 88-93). Also available via the Internet.

Whole-genome sequencing has shown that the large (8-12 Mbp) genomes of Streptomyces and allied genera of Gram-positive filamentous bacteria house a rich and previously underestimated repertoire of gene clusters for biosynthesis of specialised metabolites, including antibiotics, immunosuppressants and anticancer compounds. Many of these clusters remain uncharacterised because they are not expressed under the culture conditions used. Even for strains from which a specific compound has been identified, the challenge remains to link the compound to its gene cluster, and to develop procedures for analysing and manipulating the biosynthetic pathway. In this work, three strains have been studied that address different aspects of this challenge. Streptomyces sp. DSM4137 is a genetically amenable strain and a notably prolific producer of diverse natural products, but also has multiple biosynthetic gene clusters that remain uncharacterised. In an attempt to differentiate those clusters where the genes are expressed from those that are essentially silent, the transcriptome of DSM4137 was analysed using total RNASeq and the results were used to inform analysis of HPLC/MS data of extracts under the same conditions. There was shown to be good correlation between the RNASeq results and the pattern of metabolites produced, suggesting that RNASeq may be a useful complement in the search for novel gene clusters. In contrast, Saccharopolyspora spinosa, producer of the valuable insecticidal spinosyns, is not genetically amenable. A new technique has been developed for the mobilisation of an entire biosynthetic gene cluster and refactoring attempted to increase the production of spinosyns in a heterologous strain. Total transcriptome was analysed by RNAseq to give an insight into the regulation of the WT strain, helping identify future methods for strain manipulation for increase yields.

Microorganisms are a goidmine for evolutionary genetics as their genomes can evolve at an extraordinary rate which results in some of the most extravagant adaptations in terms of genome structure and function as well as survival in the most unusual environments. One trend observed in several evolutionary scenarios is genome degeneration. It is most prominent in endosymbionts and obligate intracellular parasites and is a consequence of many constraints encountered in the intracellular environment. The process involves loss of many protein-coding genes, resulting in greater dependence on the host, and loss of non-coding DNA such as intergenic regions, which has a direct impact on regulation of genome function. I have chosen two evolutionarily distinct systems to analyze the stages and functional consequences of genome degeneration, namely the impact of genome compression on transcription in an obligate parasite Antonospora locustae (genus Microsporidia), and gene content in the mitochondrion of a diatom endosymbiont found in the dinoflagellate Durinskia baltica. I have successfully mapped transcriptional start and termination sites from 14 loci in Antonospora locustae, and cloned fragments of two genes that are part of the electron transport chain from the mitochondrion of the diatom endosymbiont in Durinskia baltica. My analysis reveals that transcription in A. locustae is always initiated immediately upstream of the open reading frame at a single point for every locus, whereas transcriptional termination can occur at several points for a single gene and, in some instances overlaps with a downstream reading frame. The identification ofNADH5 and ATPase9 from the mitochondrion of the endosymbiont in D. baltica is further evidence for the preservation of function in this enigmatic organelle. / Science, Faculty of / Botany, Department of / Graduate

With an increasing amount of whole genome sequence data becoming available on a daily basis we have an opportunity to study the interactions and dynamics of different organisms on a whole genome level. In the past, reports of horizontal gene transfer have focused mainly on the identification of single genes that show distorted phylogenetic profiles to that of the organism it was isolated from. This study firstly did whole genome comparisons between the rice nuclear and plastid genomes to determine the level and dynamics gene transfer and insertion of the chloroplast ad mitochondrial genomes into that of the nuclear genome of rice. Secondly, it looked to identify sequence similarities between the rice genome and microbial genomes by performing whole genome comparisons between the rice genome and that of several microbial genomes. These sequences were analyzed further to identify possible instances of horizontal transfer of DNA from microbes to the rice genome. Using this approach, this study reports several fragments in the rice genome with significant sequence similarity to that of microbial DNA fragments. This study also provides evidence supporting horizontal transfer of several of these fragments. This study provides valuable information regarding intra- as well as inter-genome DNA transfer dynamics. / Thesis (PhD)--University of Pretoria, 2009. / Plant Science / unrestricted

Introduction The present study evaluated reported methodological characteristics of GWAS, investigating relationships between reported methodological characteristics and outcomes observed. Methods GWAS were identified from NHGRI’s catalogue of GWAS (2005 to 2009). Multivariate meta-regression models (random effects) were produced to identify the impact of reported study characteristics and the strength of relationships between the variables and outcomes. Results The summary odds ratios for replication components of GWAS in cancer was 1.34 (95% CI 1.25, 1.43) and neuropsychiatric disorders was 1.43 (95% CI 1.30, 1.57). Heterogeneity was accounted for by nature of the control group, relationship between case/control groups, whether cases/controls were drawn from the same population, if data was a primary collection or a build on pre-existing data, if quality assurance was reported, and if the study reported power/sample size. Conclusion Evidence supports the existence of variability in reporting, with index components demonstrating less variability than replication components in the GWAS.

Peru has the highest burden of multidrug-resistant tuberculosis in the Americas region. Since 1999, the annual number of extensively drug-resistant tuberculosis (XDR-TB) Peruvian cases has been increasing, becoming a public health challenge. The objective of this study was to perform genomic characterization of Mycobacterium tuberculosis strains obtained from Peruvian patients with XDR-TB diagnosed from 2011 to 2015 in Peru. Whole genome sequencing (WGS) was performed on 68 XDR-TB strains from different regions of Peru. 58 (85.3%) strains came from the most populated districts of Lima and Callao. Concerning the lineages, 62 (91.2%) strains belonged to the Euro-American Lineage, while the remaining 6 (8.8%) strains belonged to the East-Asian Lineage. Most strains (90%) had high-confidence resistance mutations according to pre-established WHO-confident grading system. Discordant results between microbiological and molecular methodologies were caused by mutations outside the hotspot regions analysed by commercial molecular assays (rpoB I491F and inhA S94A). Cluster analysis using a cut-off ≤ 10 SNPs revealed that only 23 (34%) strains evidenced recent transmission links. This study highlights the relevance and utility of WGS as a high-resolution approach to predict drug resistance, analyse transmission of strains between groups, and determine evolutionary patterns of circulating XDR-TB strains in the country. / Revisión por pares

Technological advancements have lead to the generation of enormous amounts ofdata. In order to capitalize on this trend, however, both computational and sta-tistical challenges must be tackled. While computational efficiency is important,interpretability of models and algorithms are essential to ensuring the validity of anyconclusions drawn. Nowhere is this more clear than in the case of biomedical data,where inferences drawn from large datasets are used to inform future directions ofresearch, diagnose diseases, and generate leads for the development of new pharma-ceuticals. This work examines the interplay between statistical concepts and compu-tational models in three applications. Specifically, quantifying protein expression offluorescent images, classifying somatic mutations in cancer, and combining p-valuescomputed from genomic summary statistics. Across these applications, there are threerecurring themes: accounting for technical and biological variation in data process-ing, evaluating the performance of a model in its end use case, and integrating resultswith outside data. Within these applications and themes, many statistical conceptsare employed including Bayes theorem, and type I error rate control alongside com-putational models such a convolutional neural networks and Monte Carlo samplingalgorithms. The results of these investigations inform much broader application ar-eas such as biomedical imaging, modeling genomic sequences, and hypothesis testingin high-dimensions. Specific contributions in the application of Convolutional NeuralNetworks include demonstrating their ability to replicate the quantification of proteinexpression images from various manually-generated or deterministic label sets as wellas the creation of a modeling framework for sequencing-based cancer diagnostics and the prioritization of unvalidated somatic mutations. In the area of hypothesis test-ing, novel algorithms are proposed that enable the use of a powerful and interpretabletechnique of combining p-values in the large-scale setting of genome-wide association studies.

A major disease damaging seedlings of Pinus palustris is caused by the fungus Mycosphaerella dearnessii. Population structure of this pathogen was studied in a population in Mississippi. High genetic diversity (0.65) was measured using microsatellite markers and coincides with the high number of vegetative compatibly groups observed. A 30 Mb genome sequence for a single isolate of M. dearnessii was assembled, representing 65% of the estimated genome size. Nearly all (93%) of the core set of genes present in eukaryotes were detected from a total of 10,996 predicted genes. A total of 853 enzymatic associations were identified along with several genes homologous to pathogenic genes in other fungal pathogens. These results provide insights into the infection process and host-pathogen interactions. Further investigating this pathosystem will lead to effective disease management strategies.

This thesis considers the moral permissibility of heritable non-therapeutic human genome editing. That is, genetic changes that seek to alter the genes of future generations for enhancement and aesthetic reasons. Some examples include genetic changes to muscle mass, cognitive abilities, eye colour, hair texture, skin colour, and so on. Given relevant moral considerations, I argue that the case against heritable non-therapeutic human genome editing is stronger than the case in favour. / Thesis / Master of Philosophy (MA)