Endosymbiosis in arthropods involves intracellular bacteria that supply an array of benefits to the host. Endosymbionts likely enhance the health of ticks by provisioning amino acids such as cysteine and tyrosine, and cofactors such as biotin and folic acid that are not available in blood--the sole nutrient source of ticks. Endosymbionts of ticks are of special interest due to their close evolutionary relationship with tick-vectored pathogens that impact livestock and human health. For example, ticks typically contain Coxiella-like endosymbionts (CLEs) that are the closest relatives of the human pathogen Coxiella burnetii. In order to understand the evolutionary relationship between the mammalian pathogen Francisella tularensis, which is vectored by ticks, and the Francisella-like endosymbionts (FLEs) present in several ticks, we assembled the genomes of the FLEs in the hard tick Amblyomma maculatum and the soft tick Ornithodoros moubata using high-throughput sequencing. While this project was in progress, another group described the genome of an FLE in the soft tick Argus (Persicargas) arboreus. Utilizing the three genomes, we show that all FLEs evolved from a mammalian pathogen, a relationship that is converse to that of C. burnetii, which likely evolved from a tick-associated non-pathogenic ancestor. Additionally, our analyses indicate that FLEs are horizontally transferred between ticks, and due to their superior metabolic capabilities could replace ancestral endosymbionts with reduced genomes.

Trypanosomatids cause human diseases such as leishmaniasis and African trypanosomiasis. Trypanosomatids are protists from the order Trypanosomatida and include species of the genera Trypanosoma and Leishmania, which occupy a similar ecological niche. Both have digenic life-stages, alternating between an insect vector and a range of mammalian hosts. However, the strategies used to subvert the host immune system differ greatly as do the clinical outcome of infections between species. The genomes of both the host and the parasite instruct us about strategies the pathogens use to subvert the human immune system, and adaptations by the human host allowing us to better survive infections. We have applied unsupervised learning algorithms to aid visualization of amino acid sequence similarity and the potential for recombination events within Trypanosoma brucei's large repertoire of variant surface glycoproteins (VSGs). Methods developed here reveal five groups of VSGs within a single sequenced genome of T. brucei, indicating many likely recombination events occurring between VSGs of the same type, but not between those of different types. These tools and methods can be broadly applied to identify groups of non-coding regulatory sequences within other Trypanosomatid genomes. To aid in the detection, quantification, and species identification of leishmania DNA isolated from environmental or clinical specimens, we developed a set of quantitative-PCR primers and probes targeting a taxonomically and geographically broad spectrum of Leishmania species. This assay has been applied to DNA extracted from both human and canine hosts as well as the sand fly vector, demonstrating its flexibility and utility in a variety of research applications. Within the host genomes, fine mapping SNP analysis was performed to detect polymorphisms in a family study of subjects in a region of Northeast Brazil that is endemic for Leishmania infantum chagasi, the parasite causing visceral leishmaniasis. These studies identified associations between genetic loci and the development of visceral leishmaniasis, with a single polymorphism associated with an asymptomatic outcome after infection. The methods and results presented here have capitalized on the large amount of genomics data becoming available that will improve our understanding of both parasite and host genetics and their role in human disease.

Nucleic acid and protein databases such as GenBank are growing at a rate that perhaps eclipses even Moore’s Law of increase in computational power. This poses a problem for the biological sciences, which have become increasingly dependant on searching and manipulating these databases. It was once reasonably practical to perform exhaustive searches of these databases, for example using the algorithm described by Smith and Waterman, however it has been many years since this was the case. This has led to the development of a series of search algorithms, such as FASTA, BLAST and BLAT, that are each successively faster, but at similarly successive costs in terms of thoroughness. Attempts have been made to remedy this problem by devising search algorithms that are both fast and thorough. An example is CAFE, which seeks to construct a search system with a sub-linear relationship between search time and database size, and argues that this property must be present for any search system to be successful in the long term. This dissertation explores this notion by seeking to construct a search system that takes advantage of the growing redundancy in databases such as GenBank in order to reduce both the search time and the space required to store the databases and their indices, while preserving or increasing the thoroughness of the search. The result is the creation and implementation of new genomic sequence search and alignment, database compression, and index compression algorithms and systems that make progress toward resolving the problem of reducing search speed and space requirements while improving sensitivity. However, success is tempered by the need for databases with adequate local redundancy, and the computational cost of these algorithms when servicing un-batched queries.

High doses of radiation (HDR) are clearly detrimental to human health, but relatively little is known about the health consequences following exposure to low doses of radiation (LDR, <10cGy). Understanding the risks associated with LDR is of great importance to the general public due to the recent dramatic increase in diagnostic radiological imaging. While HDR clearly suppress immune function, there is evidence that LDR can be immunostimulatory. Within the organism, defining the consequences of LDR is further complicated by the impact of genetic background, particularly in systems such as the immune system for which both radiosensitivity and genetic effects are profound. We addressed the issue of genetic susceptibility to LDR using the immune system as a target system and treated the LDR response as a complex trait analyzed using a systems genetics framework. Using the BXD recombinant inbred strain mouse panel as a genetic reference population allowed us to address the radiation response within the context of natural genetic variation. Our overarching hypothesis is that, within a population, the immunological effects of LDR exposure depend in part on the individual’s baseline immunoprofile and gene expression which are ultimately dependent upon genetic background. We began by establishing the immunophenotypic variation (i.e., T:B cell ratio, CD4:CD8 ratio) within the BXD panel and used baseline spleen transcriptome profiling to identify putative candidate genes controlling these traits, specifically Acp1 and Ptprk for CD4:CD8 ratio. The same set of BXD strains was exposed to LDR (10cGy gamma radiation) to determine effects on immune function and oxidative stress. LDR significantly enhanced neutrophil phagocytosis in a manner that was independent of genetic background. In contrast, genetic background significantly impacted LDR-induced changes in spleen superoxide dismutase activity. By integrating these results with our previous analyses of BXD RI strains, we have demonstrated that baseline expression of Sod2 correlates with LDR-induced SOD activity, and baseline CD4:CD8 ratio is inversely correlated with LDR-induced neutrophil phagocytosis. In addition, spleen transcriptomic data from the BXD parental strains further highlighted the impact of genetic background on LDR responses. These data provide the groundwork for predicting LDR responses using baseline expression, immunophenotypes, and/or genotype.

The beginning of the twenty-first century has been characterized by an explosion of biological information. The avalanche of data grows daily and arises as a consequence of advances in the fields of molecular biology and genomics and proteomics. The challenge for nowadays biologist lies in the de-codification of this huge and complex data, in order to achieve a better understanding of how our genes shape who we are, how our genome evolved, and how we function. Without the annotation and data mining, the information provided by for example high throughput genomic sequencing projects is not very useful. Bioinformatics is the application of computer science and technology to the management and analysis of biological data, in an effort to address biological questions. The work presented in this thesis has focused on the use of Grid and High Performance Computing for solving computationally expensive bioinformatics tasks, where, due to the very large amount of available data and the complexity of the tasks, new solutions are required for efficient data analysis and interpretation. Three major research topics are addressed; First, the use of grids for distributing the execution of sequence based proteomic analysis, its application in optimal epitope selection and in a proteome-wide effort to map the linear epitopes in the human proteome. Second, the application of grid technology in genetic association studies, which enabled the analysis of thousand of simulated genotypes, and finally the development and application of a economic based model for grid-job scheduling and resource administration. The applications of the grid based technology developed in the present investigation, results in successfully tagging and linking chromosomes regions in Alzheimer disease, proteome-wide mapping of the linear epitopes, and the development of a Market-Based Resource Allocation in Grid for Scientific Applications. / QC 20100622

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

Sorghum (Sorghum bicolor [L.] Moench) is the world's fifth most economically important cereal crop, grown worldwide as a source of food for both humans and livestock. Sorghum is a C4 grass that is well adapted to hot and arid climes and is popular for cultivation on lands of marginal quality. Recent interest in development of biofuels from lignocellulosic biomass has drawn attention to sorghum, which can be cultivated in areas not suitable for more traditional crops, and is capable of generating plant biomass in excess of 40 tons per acre. While the quantity of biomass and low water consumption make sorghum a viable candidate for biofuels growth, the biomass composition is enriched in lignin, which is problematic for enzymatic and chemical conversion techniques. The genetic basis for stem composition was analyzed in sorghum populations using a combination of genetic, genomic, and bioinformatics techniques. Utilizing acetyl bromide extraction, the variation in stem lignin content was quantified across several sorghum cultivars, confirming that lignin content varied considerably among sorghum cultivars. Previous work identifying sorghum reduced-lignin lines has involved the monolignol biosynthetic pathway; all steps in the pathway were putatively identified in the sorghum genome using sequence analysis. A bioinformatics toolkit was constructed to allow for the development of genetic markers in sorghum populations, and a database and web portal were generated to allow users to access previously developed genetic markers. Recombinant inbred lines were analyzed for stem composition using near infrared reflectance spectroscopy (NIR) and genetic maps constructed using restriction site-linked polymorphisms, revealing 34 quantitative trail loci (QTL) for stem composition variation in a BTx642 x RTx7000 population, and six QTL for stem composition variation in an SC56 x RTx7000 population. Sequencing the genome of BTx642 and RTx7000 to a depth of ~11x using Illumina sequencing revealed approximately 1.4 million single nucleotide polymorphisms (SNPs) and 1 million SNPs, respectively. These polymorphisms can be used to identify putative amino acid changes in genes within these genotypes, and can also be used for fine mapping. Plotting the density of these SNPs revealed patterns of genetic inheritance from shared ancestral lines both between the newly sequenced genotypes and relative to the reference genotype BTx623.

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

Aquesta tesis doctoral està basada en estudiar la resposta immunològica dels peixos en models d'estrès i d'activació del sistema immune des la genòmica funcional. L'aplicació de tecnologies moleculars com el Differential Display van permetre identificar y clonar por primera vegada en orades (Sparus aurata) y en altres especies de peix, el gen enolasa. Aquest enzim glucolític s'ha plantejat per primera vegada com un bon marcador molecular per estudiar el benestar dels peixos. Per mitjà de l'ús d'una plataforma de microarrays dissenyada específicament per a salmònids, i altres metodologies biomoleculars, es va comprovar que els nivells d'enolasa eren regulats en diferents teixits y en diferents especies de peix, com també en adverses situacions per l'animal. D'altra banda, s'han estudiat diferents gens immunològics candidats a ser possibles gens per l'estudi del sistema immunològic dels peixos. Aquests gens s'han estudiat a nivell d'expressió en teixits de truites (Oncorhynchus mykiss) mitjançant PCR convencional i PCR quantitativa, i l'ús de metodologies biomoleculars i bioinformàtiques. Entre ells, destaca el factor de transcripció PU.1, un gen indispensable per el desenvolupament de l'hematopoesi. Aquest gen, s'ha clonat i caracteritzat per primera vegada en salmònids. L'expressió de PU.1 s'ha estudiat mitjançant l'ús d'hibridacions in situ en ronyó anterior y en cervell de truita. A més, l'ús de microarray en aquest dos teixits han permès fer un estudi exhaustiu i pioner a nivell de transcriptòmica en peixos. Les anàlisis del xip de microarray, ha revelat que grups de gens s'activen o s'inhibeixen com a conseqüència d'un estrès immunològic.En resum, aquesta tesis doctoral ha aplicat el desenvolupament de noves tecnologies moleculars pioneres en peixos, com el microarray, la clonació de noves seqüències gèniques i la bioinformàtica, per estudiar la genòmica funcional dels peixos en situacions d'activació dels mecanismes d'estrès i del sistema immune. / The main results of the present thesis can be integrated to a better understanding the stress and the immune responses in fish at a transcriptional level. The application of functional genomic tools, which encloses from using simple PCR analysis to more modern, sophisticate and fashionable microarray technique, allowed us to identified transcriptional regulations of certain set of genes which are enhanced or repressed under stress conditions. Our findings contribute to increase knowledge of molecular mechanism involved in coping the stress and immune responses in fish and provides a better understanding of fish physiology when fish health is threatened. Furthermore, thesis results may be interesting for aquaculture which looks for good biomolecular markers that may improve fish production and fish quality. The isolation, characterization and gene expression study with further microarray analysis of the enolase gene, allowed us to describe enolase as a possible biomolecular marker to determine fish welfare. The in situ hybridization study of the hematopoietic transcription factor PU.1, contributed to amplify the knowledge of the development of the fish immune system. Throughout this thesis, DNA sequences and mRNA expression levels of several genes studied, have contributed to enlarged genomic fish database. In summary, this thesis described from a transcriptional level, gene expression and molecular mechanisms activated or repressed when fish welfare is threatened and contributes to a better understanding of transcriptiomic mechanisms required to cope with the stress.

Using rice chromosome 1 pseudomolecule as a reference, a minimal tiling path for the euchromatic arms of sorghum chromosome 3 was constructed, in which 23 contigs contain an estimated 57.56 Mb of DNA. A total of 409 EST-STS markers and 255 genetic markers have been mapped onto the euchromatic arms providing excellent integration of the genetic and physical maps. A total of 21 contigs containing 9 ESTSTS and 35 genetic markers have been constructed across the heterochromatin block of sorghum chromosome 3 which comprise 16.57 Mb of DNA. Macrocolinearity between sorghum chromosome 3 and rice chromosome 1 was examined based on the mapped EST-STS markers. Approximately 85% of the EST-STS markers were colinear between these two homeologous chromosomes. Estimates of recombination were also determined, which indicates the existence of recombination cold and hot spots. Microcolinearity between sorghum chromosome 3 and rice chromosome 1 was examined at two different levels. In one case, overlapping sorghum BAC pools orthologous to a 5.1 Mb region of rice chromosome 1 were constructed and sequence skimmed. Alignment of the sorghum skim sequences to the TIGR rice gene models revealed ~62% colinearity between the two orthologous regions. In addition, colinearity between sorghum chromosome 3 and rice chromosome 5 was detected within this region which is likely due to the segmental homology between rice chromosome 1 and rice chromosome 5. Microcolinearity between sorghum and rice was also examined by comparing 2 fully sequenced sorghum chromosome 3 BAC clones to the orthologous region of rice chromosome 1. In this analysis, ~65% colinearity was detected for sorghum BAC 82G24 and ~59% colinearity was detected for sorghum BAC 181g10. Microcolinearity was largely confined to gene coding regions and sequences of exons displayed the highest percent identities. Small-scale gene rearrangements were also detected. Finally, RT-PCR analysis was carried out between a set of colinear and non-colinear genes from sorghum and rice to determine whether the loss of colinearity between orthologous genes resulted in a change in transcriptional regulation. No direct link between loss of colinearity and expression pattern was detected in these experiments.