Organellar genomes are remnants of more complex bacterial genomes reduced until reach the simplest and most efficient content. Regularly depicted as circular, these genomes can form other structures, like linear, ramified, or entangled chromosomes, or a combination of those. Nonetheless, their gene content is nearly constant throughout flowering plants based on the multiple plastid genomes (plastomes) and the comparatively few mitochondrial genomes (mitogenomes) sequenced to date. Here, I explored the evolution of the organellar genomes in orchids from a phylogenetic perspective. For this research, plastomes and mitogenomes were assembled from short pair-ended reads obtained using Illumina sequencing technology. I developed a workflow to confidently recover plastid and mitochondrial sequences, even for regions without references in databases (chapter 1). The comparison among taxa from all orchid subfamilies identified patterns of gain, loss and rearrangement of coding and non-coding DNA. Plastid and mitochondrial protein-coding genes present in all samples were used to reconstruct the phylogenetic history of orchids that was coincident in terms of topology (chapter 1). Plastomes can suffer degradation in heterotrophic species, however that is not true for mixotrophic species, as I discovered by comparing albino and green individuals of the orchid Epipactis helleborine. I found that albino plants did not suffer loss of any genes and that the sequence was almost identical to the photosynthetic plants (chapter 2). In contrast to what it is observed in angiosperm plastomes, for which the structure, content and size is conserved, plant mitogenomes are highly variable in size, which can increase by the acquisition of external DNA via horizontal gene transfer. In some orchids, the mitogenome hosts a sixteen-gene sequence transferred from a fungal mitogenome to a clade of epidendroid orchids 12-60 My ago, and has been fragmented, conserved, or fully lost since (chapter 3). Transfer RNA genes are variable in number and origin throughout orchid evolution. I identified that they had four different sources, three novel possible replacement events of the native genes with plastid-origin genes, seven tRNA remodeling events in orchids and three more in other angiosperms (chapter 4). Our comparative studies conclude that there are three main dynamics that shape the organellar genomes: gain, loss and rearrangement of genomic content. I presented examples of them in orchids (chapter 5). Additionally, I created two sets of genomic resources: one comprises eighteen new orchid mitogenomes and plastomes, and the second consists of a well-curated set of references of tRNA genes in mitogenomes discriminated by origin. These results contribute to increasing the knowledge of angiosperm organellar genomes and highlight the importance of comprehensive studies that allow the interpretation of the genomic changes in the light of the phylogenetic evolution.

The porcine antral follicle, which consists of an oocyte and surrounding follicular components, including theca, granulosa, and cumulus cells and follicular fluid, is an essential microenvironment for oocyte development and maturation. Investigating cellular and molecular events in the context of the whole follicle will aid in our understanding of interactions between the oocyte and the follicular components. The objective of this dissertation was to develop a novel bioluminescent imaging model to visualize and measure cellular and molecular events in living intact ovarian follicles in vitro. Bioluminescence imaging was employed to facilitate noninvasive, dynamic, and real-time transgene analysis in living intact follicles. The time courses of luciferase-luciferin reactions, effective plasmid DNA and D-luciferin doses and their combinations were determined as the first step toward developing a new real-time bioluminescence imaging model. In addition, the efficient nonviral gene delivery methods: cationic lipid mediated gene transfer (chemical) and electroporation (physical) for the living intact follicles were determined. For the cationic lipid mediated gene transfer method, the 1:3 DNA lipid ratio was optimal. It was also found that the optimal condition of electroporation (4 electric pulses with 100 ms duration at field strength of 100 V/cm) resulted in 15 times higher luciferase activity and increased granulosa cell viability over the cationic lipid mediated gene transfer method. Moreover, increased granulosa cell viability, increased follicular fluid progesterone content, and oocytes with expanded cumulus cells were observed in intact follicles transfected by electroporation at a field strength of 100 V/cm. Finally, bioluminescence imaging was applied to quantify functional and ligand-activated estrogen receptor (ER) activity within living intact follicles. The functional ERs were differentially activated during the different stages of the estrous cycle in the mature sow; the levels of functional ER activity in cultured granulosa cells and intact follicles in vitro were increased from late luteal phase to early follicular phase and then significantly decreased at late follicular phase. The methodology developed herein can be applicable to further our understanding of oocyte and follicle development and oocyte maturation.

Communities are defined as the ensemble of populations that interact with each other and with the environment in a specific time and location. Community ecology studies how communities assemble, what are the patterns of diversity, abundance, and composition of species, and the processes driving these patterns. It includes four basic mechanisms for the assembly of communities: dispersal, drift, selection, and speciation, with each mechanism influencing how the communities change in a different way. Dispersal, the movement of species from one geographical location to another, plays a major role in the recolonization of barren environments and the introduction of new species to established environments. Drift (i.e., random birth and death events within a community) could, theoretically, be negligible in bacterial communities where the high population densities are expected to buffer its effect. Conversely, horizontal gene transfer can be a strong selective force, as horizontally transferred genetic material is a source of functional traits that may provide selective advantages to the recipient cells, especially in environments where strong selection pressure occurs. In my Ph.D. thesis, I aim to examine these three contrasting mechanisms in controlled, simplified bacterial communities that are designed and studied through a synthetic ecology approach. I found that even at low dispersal rates, the species abundance of planktonic bacterial communities can be homogenized by migration. This homogenization can occur even when there are strong variable selection forces interacting in each environment. I also found strong evidence on the importance of stochasticity in communities. Drift can decrease the community similarity by up to 6.3%, and increases the probabilities that species become extinct, especially in the case of rare taxa. In contrast, I found that naturally competent bacteria are favored to uptake more DNA in communities that are highly productive and phylogenetically diverse. This pattern is explained by a potential higher availability of naked DNA for naturally competent bacteria, presumably because there are more cells and the predation systems are more effective. Altogether, our findings support the theory on the importance of stochastic forces and their interaction with deterministic forces on the shaping of microbial community assembly.

Yes / Several bacterial families are known to be highly abundant within the human microbiome, but their ecological roles and evolutionary histories have yet to be investigated in depth. One such family, Lachnospiraceae (phylum Firmicutes, class Clostridia) is abundant in the digestive tracts of many mammals and relatively rare elsewhere. Members of this family have been linked to obesity and protection from colon cancer in humans, mainly due to the association of many species within the group with the production of butyric acid, a substance that is important for both microbial and host epithelial cell growth. We examined the genomes of 30 Lachnospiraceae isolates to better understand the origin of butyric acid capabilities and other ecological adaptations within this group. Butyric acid production-related genes were detected in fewer than half of the examined genomes with the distribution of this function likely arising in part from lateral gene transfer (LGT). An investigation of environment-specific functional signatures indicated that human gut-associated Lachnospiraceae possess genes for endospore formation, whereas other members of this family lack key sporulation-associated genes, an observation supported by analysis of metagenomes from the human gut, oral cavity, and bovine rumen. Our analysis demonstrates that adaptation to an ecological niche and acquisition of defining functional roles within a microbiome can arise through a combination of both habitat-specific gene loss and LGT. / Canadian Institute for Health Research (grant number CMF-108026), Genome Atlantic and the Canada Research Chairs program to R.G.B.

No description available.

No description available.

No description available.

<p>Prokaryotes evolve, in part, by lateral gene transfer (LGT). This transfer of genetic material is likely important in the evolution of operons, a group of genes that are transcribed as a single mRNA. Genes that are transferred may then be integrated into genomes by homologous recombination. In this thesis, it was proposed that homologous recombination is the mechanism of integration of laterally transferred genes into operons. To investigate this proposal, a phylogenetic tree of Bacillus was inferred using DNA sequence alignments. LGT was inferred using a parsimony algorithm, and operons were inferred using OperonDB. Homologous recombination breakpoints were identified by permutation tests, <em>GENECONV</em> and maximum chi square algorithm. The results indicate that there is evidence for integration of functionally annotated genes into operons by homologous recombination. There are several laterally transferred genes that have recombination breakpoints before the start codon or after the stop codon of the genes. It was also proposed in this thesis that LGT causes an increase in the rate of evolution of genes that are neighbours of laterally transferred genes. To investigate this proposal, genes that are neighbours of laterally transferred genes in Bacillus were identified. These genes were classified as upstream or downstream genes to the LGT event. Genes that are not neighbours of laterally transferred genes were also identified as a control. Selection and the rate of evolution was studied using maximum likelihood models implemented in CodeML of PAML. Genes under positive selection were inferred using likelihood ratio tests. The results indicate that only a few neighbouring genes were under positive selection, and the rate of evolution of the neighbouring genes was slightly higher than that of the non-neighbouring genes. The high rates of evolution of the neighbouring genes are likely due to relaxed selection on the neighbouring genes.</p> / Master of Biological Science (MBioSci)

Enterococcus faecalis is a commensal bacterium, which upon acquisition of virulence factors on mobile genetic elements can cause sepsis, urinary tract infections and endocarditis. E. faecalis isolates can be multi-drug resistant and have been implicated in the dissemination of antibiotic resistance genes to other genera. Although the host range of pheromone inducible conjugative plasmids is restricted to Enterococci, they often carry transposons, which are capable of transposing into the chromosome of other genera. The plasmid pCF10 contains the antibiotic resistance gene tetM on a conjugative transposon Tn925. Tn925 is a Tn916-like plasmid and is capable of pCF10-independent conjugative transfer to multiple bacterial species at low levels. Biofilms are communities of bacteria growing within a matrix. In biofilms, bacteria are more difficult to kill because of their lower susceptibility to antibiotics. In hospital settings, biofilms can grow on medically implanted devices, catheters or even human tissue. In mixed species biofilms, antibiotic resistances are able to be transferred through horizontal gene transfer from E. faecalis to other bacterial species. In mixed species biofilms, it has been show that Tn925 can transpose into S. aureus at rates of 10-8 by Ella Massie Schuh. Using static mixed species biofilms, the transfer of tetM from E. faecalis to S. aureus was studied, hoping to better understand the underlying mechanisms. The goal of these studies was to determine if residence on pCF10 increased the transfer frequency of Tn925 in mixed species biofilms. Mixed species biofilms containing E. faecalis (pCF10) and S. aureus (pALC2073aPSM) were established and pCF10 conjugation was induced with pheromone cCF10. Transfer of Tn925 / Biomedical Sciences

Phylum Euglenozoa contsists of four groups - Euglenoidea, Kinetoplastea, Symbiontida and Diplonemea. Phototrophic euglenids, which constitute a clade, possess green plastid acquired via secondary endosymbiosis from green alga related to recent genus Pyramimonas. According to generally accepted plastid-late hypothesis, the endo- symbiosis took place after split between phototrophic euglenids and all other euglenozo- ans. Endosymbiotic event is always associated with gene transfer from endosymbiont to nuclei of host. Even if the endosymbiont is completely lost we should be able to observe enrichment of the host genome with the genes derived from endosymbiont. Some recent phylogenetic analyses uncovered genes related to green algae in trypanosomas (Kineto- plastea: Trypanosomatida). Based on this observation, authors postulated a hypothesis that the plastid was present already in common ancestor of kinetoplastids and euglenids and was lost in kinetoplastids and some euglenids including osmotrophic Rhabdomonas costata. During analysis of transcriptome of R. costata we found 63 genes, which could originated from green (24 genes) or other (49 genes) algae. In phylogenetic trees only one was robustly related to green and four were robustly related to other algae. Since the number of genes related to...