The World Health Organization estimates that vector-borne diseases account for 17% of the global burden of all infectious diseases and has identified the mosquito as the most dangerous of all disease-transmitting insects, being responsible for several million deaths and hundreds of millions of cases each year. The study of mosquito genomics provides a deeper understanding of the molecular mechanisms involved in every aspect of vector biology, such as sex determination, host-parasite interaction, ecology, feeding behavior, immunity and evolutionary trends and can be used for the development of new strategies for vector control. We developed the first map of the mitotic chromosomes of the major vector for West Nile fever and lymphatic filariasis, Culex quinquefasciatus. The map was then successfully utilized for mapping of approximately 90% of available genetic markers to their precise positions on the chromosomes. Idiograms were integrated with 140 genetic supercontigs representing 26.5% of the genome. A linear regression analysis demonstrated good overall correlation between the positioning of markers on physical and genetic linkage maps. This will improve gene annotation and help in distinguishing potential haplotype scaffolds and regions of segmental duplications. It will also facilitate identification of epidemiologically important genes that can be used as targets for the vector control and provide a better framework for comparative genomics that will help understanding of the evolution of epidemiologically important traits. In another study, we confirmed the presence of the newly described species, Anopheles daciae, in regions of Russia using molecular data. Although sympatric with its sibling species, Anopheles messeae, five nucleotide substitutions in the internal transcribed spacer 2 of ribosomal DNA can be used to distinguish the morphologically similar species. Chromosome rearrangements have a significant impact on mosquito adaptation and speciation. Using sequencing data in combination with karyotyping, we demonstrated that significant differences in inversion frequencies distinguish An. messeae from An. daciae, suggesting that these inversions are actively involved in adaptation and speciation. It is essential to have reliable toolbox for correct identification of these species and to know their range for future possible malaria outbreaks prevention. / Ph. D.

Foodborne diseases are a growing health problem today and can be caused by eating food contaminated with bacteria. To monitor known foodborne diseases, institutions keep track of bacteria in surveillance projects. Whole genome sequencing is becoming the new standard method for comparing isolates, which generates large amounts of data. Today, the standard analyses are focused on conserved regions in genomes. The dynamics in less conserved regions can be studied by creating pan-genomes. A pan-genome consists of conserved genes, called core genes, and genes of varied conservation grade, called accessory genes. This thesis aimed to analyse pan-genomes of large datasets from six bacterial species coming from surveillance projects: Campylobacter coli, Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Salmonella enterica, and Streptococcus pneumoniae. The purpose was to investigate the species dynamics in the genomes and to look at properties of the genomes not included in the standard analyses that are used in surveillance projects today. Bacterial Pan Genome Analysis tool was used for the pan-genome analysis of the six species and datasets of 1,000-2,000 genomes per species were analysed. All species were estimated to have open pan-genomes, meaning the pan-genomes are increasing in size as more genomes are added. Escherichia coli and Salmonella enterica had more dynamic and open genomes compared to the other species. They had the highest number of accessory genes relative to their genome sizes and had the largest accessory segments between core genes. The synteny of the core genes showed high conservation for a part of the core genes in all species. Some core genes always sat directly after each other in the analysed genomes, never having accessory genes between them. Other core genes always had accessory genes between them, indicating very open regions in the genomes. The core genes were evenly distributed through the reference genomes with some regions showing increased gene density for all species. Some regions had a higher gene density for core genes often followed by core genes, and others for core genes often followed by accessory genes. However, the placement of genes needs to be investigated further with more reference genomes to be able to draw confident conclusions.

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Primary rhegmatogenous retinal detachment (RRD) is one of the most common ophthalmic emergencies. RRD is caused by a full thickness break in the retina which initiates separation of the neurosensory retina from the underlying retinal pigment epithelium. The subsequent accumulation of fluid within this potential space extends the area of detachment and causes visual loss. Previous assessments of RRD incidence have demonstrated large differences in case definition and methodology, with incidence estimates varying 3-fold geographically and in different time periods. To date there have been no systematic or prospective incidence estimates of primary RRD in the U.K. In this thesis I present the findings of a 2-year epidemiology study that prospectively aimed to recruit all incident cases of primary RRD diagnosed in Scotland. Case recruitment from consenting participants comprised a detailed questionnaire and a blood sample. In this thesis, I present the findings of the Scottish retinal detachment study that examined the incidence, demographic features, temporal incidence trends, as well as clinical and socio-economic associations of primary RRD in Scotland. From the clinical and genetic resource I assembled, I calculated the first population based estimate of the sibling recurrence risk ratio for RRD and designed and assisted in the analysis of the first case-control genome wide association study of this condition. Results from this study have estimated the annual incidence of primary RRD in Scotland to be 12.05 per 100,000 population. Based on this estimate, there are approximately 7,300 new cases annually in the United Kingdom. RRD incidence increases with age, is more common in men and right eyes, and is strongly associated with socio-economic affluence. In addition, using hospital episode data, the overall age-standardised incidence of RRD in Scotland was shown to be steadily increasing since 1987 with an average annual increase of 1.9%. Analysis of the clinical findings highlighted that the majority of RRD cases are caused by more than one retinal break; an important consideration for appropriate surgical management. Ocular trauma, previous cataract surgery, family history, and retinal degeneration are important predisposing features. In addition, over a 2 year period approximately 7% of individuals will suffer a RRD in the fellow eye representing an important risk of bilateral visual loss. Furthermore, I demonstrate that the risk of having an affected sibling with RRD is increased 2-fold given that one sibling has had the condition, substantiating a genetic component to the pathogenesis of this condition. In the final aspect of this thesis I will present the design and analysis of a two stage case-control genome-wide association study examining the role of common genetic variants and selected candidate genes in predisposing to RRD development.

Comparisons of flowering plant genomes reveal multiple rounds of ancient polyploidy characterized by large intragenomic syntenic blocks. Three such whole-genome duplication (WGD) events, designated as rho (rho), sigma (sigma), and tau (tau), have been identified in the genomes of cereal grasses. Precise dating of these WGD events is necessary to investigate how they have influenced diversification rates, evolutionary innovations, and genomic characteristics such as the GC profile of protein-coding sequences. The timing of these events has remained uncertain due to the paucity of monocot genome sequence data outside the grass family (Poaceae). Phylogenomic analysis of protein-coding genes from sequenced genomes and transcriptome assemblies from 35 species, including representatives of all families within the Poales, has resolved the timing of rho and sigma relative to speciation events and placed tau prior to divergence of Asparagales and the commelinids but after divergence with eudicots. Examination of gene family phylogenies indicates that rho occurred just prior to the diversification of Poaceae and sigma occurred before early diversification of Poales lineages but after the Poales-commelinid split. Additional lineage-specific WGD events were identified on the basis of the transcriptome data. Gene families exhibiting high GC content are underrepresented among those with duplicate genes that persisted following these genome duplications. However, genome duplications had little overall influence on lineage-specific changes in the GC content of coding genes. Improved resolution of the timing of WGD events in monocot history provides evidence for the influence of polyploidization on functional evolution and species diversification.

Background: Gene expression is regulated via highly coordinated epigenetic changes, the most studied of which is DNA methylation (DNAm). Many studies have shown that DNAm is linearly associated with age, and some have even used DNAm data to build predictive models of human age, which are immensely important considering that DNAm can predict health outcomes, such as all-cause mortality, better than chronological age. Nevertheless, few studies have investigated non-linear relationships between DNAm and age, which could potentially improve these predictive models. While such investigations are relevant to predicting health outcomes, non-linear relationships between DNAm and age can also add to our understanding of biological responses to late-life events, such as diseases that afflict the elderly. Objectives: We aim to (1) examine non-linear relationships between DNAm and age at specific loci on the genome and (2) build upon regularization methods by comparing prediction errors between models with both non-transformed and square-root transformed predictors to models that include only non-transformed predictors. We used both the sparse partial least squares (SPLS) regression model and the lasso regression model to make our comparisons. Results: We found two age-differentially methylated sites implicated in the regulation of a gene known as KLF14, which could be involved in an immunosenescent phenotype. Inclusion of the square-root transformed variables had little effect on the prediction error of the SPLS model. On the other hand, the prediction error increased substantially in the lasso regression model, particularly when few predictors (70) were included. Conclusion: The growing amount and complexity of biological data coupled with advances in computational technology are indispensable to our understanding of biological pathways and perplexing biological phenomena. Moreover, high-dimensional biological data have enormous implications for clinical practice. Our findings implicate a possible biological pathway involved in immunosenescence. While we were unable to improve the predictive models of human age, future research should investigate other possible non-linear relationships between DNAm and human age, considering that such statistical methods can improve predictions of health outcomes.

Given the explosion in human genetic data, new high-throughput genetic methods are necessary for studying variants and elucidating their role in human disease. In Chapter I, I will expand on this concept and describe current methods for genetically modifying human cells. In E. coli, Multiplex Automatable Genome Engineering (MAGE) is a powerful tool that enables the targeting of multiple genomic loci simultaneously with synthetic oligos that are recombined at high frequencies in an optimized strain. MAGE as a method has two components: organism-specific optimization of oligo recombination parameters and a protein capable of increasing recombination frequencies.

Tiger salamanders, and especially the Mexican axolotl (Ambystoma mexicanum), are important model organisms in biological research. This dissertation describes new genomic resources and scientific results that greatly extend the utility of tiger salamanders. With respect to new resources, this dissertation describes the development of expressed sequence tags and assembled contigs, a comparative genome map, a web-portal that makes genomic information freely available to the scientific community, and a computer program that compares structure features of organism genomes. With respect to new scientific results, this dissertation describes a quantitative trait locus that is associated with ecologically and evolutionarily relevant variation in developmental timing, the evolutionary history of the tiger salamander genome in relation to other vertebrate genomes, the likely origin of amniote sex chromosomes, and the identification of the Mexican axolotl sex-determining locus. This dissertation is concluded with a brief outline of future research directions that can extend from the works that are presented here.

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No description available.