Espécies muito próximas da família Calliphoridae apresentam hábitos alimentares muito distintos, como alimentação em tecido de um hospedeiro vivo (parasitismo obrigatório) e alimentação em matéria orgânica em decomposição (necro-saprofagia). As origens evolu-tivas do hábito de parasitismo nesta família ainda são desconhecidas. No entanto, o que a torna ideal para o estudo da evolução do hábito alimentar é o aparecimento do parasitis-mo obrigatório em pelo menos três ocasiões independentes em sua história. Neste traba-lho, foram utilizados métodos para entender a evolução do parasitismo obrigatório assim como os genes envolvidos em três diferentes hábitos alimentares. O primeiro passo foi a inferência do hábito ancestral de Calliphoridae. Com o mapeamento de caracteres na filo-genia da família, o hábito ancestral mais provável seria a necro-saprofagia e o parasitismo obrigatório teria surgido posteriormente (independentemente do parasitismo facultati-vo). Ensaios de escolha com fêmeas permitiram a classificação precisa das espécies quan-to ao hábito alimentar. Ensaios com as larvas mostraram que tanto espécies necro-saprófagas como parasitas facultativas se alimentam tanto de carne em decomposição como carne fresca. Por outro lado, a espécie parasita obrigatória Co. hominivorax, apresentou um comportamento aversivo pela carne em decomposição e se desenvolveu somente na carne fresca. Esses resultados permitiram a formulação da hipótese de que o parasitismo tenha surgido a partir de uma mudança da atração das fêmeas pelos substra-tos para oviposição, seguida da especialização da larva parasita. A busca dos genes envol-vidos nos diferentes hábitos foi realizada por meio da análise de expressão gênica dife-rencial em dados de RNA-seq gerados para seis califorídeos. No total, foram encontrados 230 potenciais genes candidatos para investigação futura. Além disso, o padrão geral observado indicou que variações tanto nas regiões regulatórias como codificadoras, sofrem a ação predominante de seleção purificadora / Closely related species of the Calliphoridae family have contrasting feeding habits, such as feeding on living tissues of a host (obligate parasitism) and feeding on decaying organ-ic matter (necro-saprophagy). The evolutionary origins of parasitism in Calliphoridae are still unknown. However, what makes this family ideal for the study of the evolution of feeding habits is the appearance of obligate parasitism in at least three independent oc-casions. In this study, we used methods to understand the evolution of parasitism, as well as the genes involved in in three different feeding habits. First, we inferred the ancestral habit of Calliphoridae. By using stochastic character mapping along the phylogeny of the family, the most likely ancestral habit was revealed as necro-saprophagy. Obligate para-sitism could have evolved later (with an independent evolution of the facultative parasit-ism). Two-choice essays with females allowed the precise classification of the species regarding their feeding habits. Essays with larvae showed that both necro-saprophagous and facultative parasites feed on decaying flesh and fresh meat. On the other hand, the obligate parasite, Co. hominivorax, showed an aversive behavior to decaying meat and able to develop only in fresh meat. These results led to the formulation of the hy-pothesis that parasitism arose from a shift in the attraction of the female attraction to new oviposition sites, followed by the specialization of the parasitic larvae. The search for the genes involved in the different feeding habits was performed through an analysis of differential gene expression using RNA-seq data generated for six califorids. Within a da-taset containing more than 1000 candidate genes, 230 genes potential candidate genes were found for future research. In addition, the general pattern observed indicated that both regulatory and coding regions have predominantly undergone the action of purifying selection

Neste estudo foi implementado um pipeline bioinformático para processar e analisar dados de RNA-Seq total e fita-específico gerados em nosso laboratório a partir de amostras pareadas de tumor e tecido adjacente não tumoral de 14 pacientes com o objetivo de catalogar com alta-resolução a composição e alterações no transcritoma no PDAC incluindo genes codificadores e não codificadores de proteína. / In the present work, we applied a bioinformatic pipeline to process and analyse data from total RNA-seq strand-oriented generated in our laboratory from matched samples of tumor and non-tumor adjacent pancreatic tissue from 14 patients with the goal of generate a high resolution catalog of the composition and the alterations in the transcriptome of PDAC, including protein coding and non coding genes.

Herbicide resistance is increasing in incidence among weed populations and poses a threat to food security. In Sunflower County, MS, a population of junglerice was identified with resistance to four herbicides, fenoxaprop-P-ethyl, imazamox, quinclorac, and propanil, each representing a different mechanism of action. The target site of fenoxaprop-P-ethyl, acetyl coenzyme A carboxylase (ACCase), was investigated. The ACCase contained none of the known resistance-conferring point mutations and an enzyme assay revealed no difference in response to increasing levels of fenoxaprop-P-ethyl between the resistant biotype and a sensitive biotype, indicating that the ACCase enzyme in the resistant biotype was sensitive to the herbicide. Whole-plant dose response assays in the presence and absence of cytochrome P450 and glutathione-S-transferase (GST) inhibitors did not increase efficacy of fenoxaprop-P-ethyl in the resistant biotype. However, when malathion, a cytochrome P450 inhibitor, was applied with imazamox or quinclorac, a reduction in resistance was observed in the resistant biotype, suggesting that a cytochrome P450 was important to the resistance mechanism for these two herbicides. RNA was isolated from the resistant and sensitive biotypes before and one hour after imazamox treatment for RNA-seq analysis. The reads from all samples were pooled to assemble the first E. colona leaf transcriptome. Differential gene expression analysis comparing untreated and treated samples for both biotypes revealed that several stress response genes were upregulated following herbicide exposure. A time course examining six of these genes showed that expression peaked between 4 and 12 hours and then dropped to untreated levels by 48 hours. Comparison of untreated resistant and sensitive plants revealed that a kinase and GST were significantly upregulated in the resistant biotype and an F-box protein was significantly downregulated. SNP analysis of cytochrome P450 sequences identified several nonsynonymous point mutations of interest including two transcripts that had premature stop codons in the sensitive but not the resistant biotype. These transcripts and their products should be the subject of future studies to determine if and how they are involved in resistance.

Vision is a crucial aspect of life for humans and animals. Impaired vision can lead to reduced quality of life along with other complications. Cataracts are a leading cause of impaired vision and blindness worldwide. Cataracts develop as a process of aging, although several environmental and lifestyle factors increase the risk of this disease. The toxic metal cadmium (Cd) has been associated with cataract formation and other ocular diseases such as macular degeneration. Cadmium exposure experiments were conducted to investigate potential pathways or mechanisms by which Cd may contribute to cataract formation and ocular disease. Zebra fish larvae (72, 96, and 120 hours post fertilization), adult zebra sh (6-month male, 10-month male, and 10-month female) and the B3 human lens epithelial (HLE) cell line were acutely exposed to varying concentrations of Cd. Transcriptomic changes relative to control (0 μM Cd) were determined using microarray analysis for zebra sh larvae and RNA sequencing (RNA-Seq) for adult zebra sh and HLE cells. Gene Ontology (GO) enrichment analysis for the zebra sh larvae exposure (50 μM Cd for 4 or 8 hours) enriched the "retina development in camera-type eye" term, and genes involved in enrichment (dnmt1, ccna2, fen1, mcm3 and slbp) were down-regulated. Gene set enrichment analysis (GSEA) for the 10-month male zebra sh exposure (50 μM Cd for 4 hours) enriched the "embryonic eye morphogenesis" gene set and signi ficant genes involved in enrichment (tcf7l1a, pitx2, fzd8a, sfrp5, lmx1bb, mfap2, six3b, lum, phactr4b, and foxc1a) were down-regulated. GSEA for the 10-month female zebra sh (50 μM Cd for 4 hours) enriched the "photoreceptor cell differentiation" gene set and signi cant genes involved in enrichment (odc1, thrb, and ush2a) were up-regulated. GO enrichment analysis for up-regulated genes in the HLE cell exposure (10 μM Cd for 4 hours) enriched the terms "eye development" (22 genes) and "lens development in camera-type eye" (CITED2, SKIL, CRYAB, SLC7A11, TGFB2, EPHA2, BCAR3, WNT5B, and BMP4). These results show cadmium is capable of altering transcription of eye-related genes in both zebra sh and human models, which may contribute to the formation of ocular disease. Many of these genes are involved in lens and retina development, yet they are also associated with diseases in these eye structures. Future studies could assess the consequences of altered transcription of these genes which could help elucidate the mechanisms of these changes and the overall effect of cadmium exposure on ocular disease. Ultimately, our study characterized the regulation of eye-related genes in response to Cd exposure and provided valuable knowledge laying the foundation for identi fication of the molecular mechanisms contributing to ocular diseases. / Thesis / Master of Science (MSc) / The eye is a sphere-like organ which is important for visualizing your surroundings. It is composed of many different structures such as the cornea, lens and retina. Many eye diseases have been characterized by abnormalities in eye structures; for example, a cataract occurs when the lens becomes cloudy and unable to focus light while macular degeneration is defined by progressive deterioration of the retinal macula region. While these diseases can occur through the natural aging process, certain environmental factors can increase risk. Exposure to cadmium, a toxic heavy metal which causes negative effects in animals, has shown to be associated with eye disease like cataracts and macular degeneration. In order to expand on this knowledge, we exposed both zebrafish and human lens cells to cadmium. By utilizing different experimental methods such as microarray analysis and RNA sequencing, eye-related genes which were affected by cadmium were revealed. Identifying the relationship between eye diseases, cadmium and gene expression will help identify the mechanism by which cadmium contributes to eye disease formation.

Optimizing the production of useful macromolecules from transgenic microorganisms is crucial to biopharmaceutical companies. Improving bacterial growth and replication depends largely on the efficiency of translation, which is rate-limited by initiation. Among the most important interactions between the mRNA translation initiation region (TIR) and the translation machinery is the association between the Shine-Dalgarno (SD) sequence in the TIR and the complementary anti-SD (aSD) sequence which is located within a short unstructured segment that includes the 3’ terminus (3’ TAIL) of the mature 16S rRNA. However, the mature 3’ TAIL has been poorly characterized in the majority of bacteria, rendering optimal SD/aSD pairing unclear in these species. In light of this, we established a novel strategy to characterize the mature 3’ TAILs of bacterial species that leverages the availability of publically stored RNA sequencing (RNA-Seq) data. In chapter 2, we devised a RNA-Seq-based approach to successfully recover the experimentally verified 3’ TAIL in E. coli (5’-CCUCCUUA-3’) and resolve inconsistencies surrounding the identity of the 3’ TAIL in Bacillus subtilis. In chapter 3 we improve the method introduced in chapter 2 to clearly and more reliably define the 3’ TAIL termini for 13 bacterial species with available protein abundance data. Our results reveal considerable heterogeneity in the termini of 3’ TAILs among closely related species and that sites downstream of the canonical CCUCC aSD motif are more important to initiation than previously believed. My research contributes to advance our understanding in microbial translation efficiency in two significant ways: 1) providing an RNA-Seq-based approach to characterize rRNA transcripts, and 2) elucidating optimal recognition between protein-coding genes and the rRNA translation machinery.

The malaria pre-erythrocytic stages have been identified as an ideal therapeutic target, but complex in vitro models for Plasmodium vivax and Plasmodium falciparum lack the efficiency needed for rapid screening and evaluation of new vaccines and drugs, especially targeting the P. vivax hypnozoite. To address this challenge, we employed a multi-parameter approach using “omics’” to identify pre-erythrocytic targets and biomarkers, guide phenotypic therapeutic screening, and study parasite functionality with innovative bioassays using highcontent screening. Herein, we discuss three novel bioassays formatted in 384-well plate systems with utilization of commercially-available materials and application of high-content imaging for rapid bio-image analysis. To refine functional assessment of pre-erythrocytic targets in early infection phases, we developed a real-time, ‘live’ sporozoite motility assay and a live sporozoite hepatocyte cell traversal assay to examine chemotherapeutic and immunoprophylactic interventions in biologically relevant environments. Furthermore, our 384-well primary hepatocyte culture system and methodology maintains stable hepatocyte physiology of cryopreserved primary human hepatocytes in addition to primary non-human primate hepatocytes for greater than 30 days, thus ideal for robust liver parasite development following infection with P. vivax, P. falciparum or P. cynomolgi sporozoites. We report antimalarial drug and vaccine studies performed in all bioassays with identification of novel anti-LS inhibition mechanisms. Additionally, this research discusses the discovery of potential sporozoite and liver stage targets identified through transcriptomic profiling of freshly isolated P. vivax and P. cynomolgi sporozoites using a candid approach of recapitulating the pivotal transition period from mosquito to human through microenvironment reconstruction and exposure to biological stimuli. We further characterize sporozoite invasive phenotypes through the application of the bioassays. Together, these novel functional assays enable us to rapidly evaluate potential preerythrocytic therapeutic candidates and analyze complex Plasmodium sporozoite phenotypes.

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.

Gamma-aminobutyric acid type B (GABAB) receptor is a G protein coupled receptor (GPCR) that mediates decreased neural activity. It has two subunits, GABAB1 and GABAB2. Previous complementary DNA (cDNA) microarray data showed strong GABAB1 signals from human prefrontal cortex using an intron 4 region probe, and these studies indicated that novel intron 4 containing GABAB1 splicing variants exist. We cloned GABAB1k, l, m, and n including mouse GABAB1j. Expression of these variants are much lower than other major known splicing variants, but GABAB1k, l, m, and n levels are similar across brain tissues. GABAB1l and GABAB1m impair GABAB receptor induced function. To better define GABAB1 splicing in alcoholic brains, whole transcriptome shortgun sequencing (RNA-seq) experiments were proposed. Due to the complexity of GABAB1 splicing, we used gene specific libraries as well as whole transcriptome libraries to maximize GABAB1 specific splicing junction search. The splicing junction search data found that GABAB1 gene is 2 to 3 times longer than the previous known gene length. Extremely low expression at 5’ end exons was found, and GABAB1 exons were grouped based on expression levels. Chronic alcohol altered exon/intron expression and splicing junctions more than overall gene expression. Decreased exon expression at a GABA binding site, a transmembrane domain (TM), and a microRNA (miRNA) binding site may diminish the normal GABAB1 transcript population and compromise signal transduction following chronic alcohol exposure. This may explain why GABAB receptor agonists have therapeutic benefit in treating alcoholism. During the sequence mapping, read pile-ups and gaps were found from whole transcriptome libraries in known exons. These may prevent single nucleotide polymorphism (SNP) and splicing junction identification and gene expression calculations. Sequence analysis found sequence biases from their mapped reads. The major sequence biases were from RNaseIII RNA fragmentation and T4 polynucleotide kinase (T4PNK) reaction. Heat fragmentation and OptiKinase treatment removed the read pile-ups and gaps including the sequence biases. The identification of RNaseIII target sequences can be incorporated into methods of miRNA gene prediction. These data showed the complexity of GABAB1 receptor splicing and the perturbation of splicing by chronic alcohol abuse demonstrate the power of RNA-seq to provide new insight into gene expression and the role of GABAB receptors in alcoholism. In addition, many other important brain genes may have unexplored splicing variants which will be important for alcoholism and other psychiatric diseases. Also, new RNA-seq library constructions improved the quality of gene expression studies. / text

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.