Background: Relatively little is known about the genomic basis and evolution of wood- feeding in beetles. We undertook genome sequencing and annotation, gene expression assays, studies of plant cell wall degrading enzymes, and other functional and comparative studies of the Asian longhorned beetle, Anoplophora glabripennis, a globally significant invasive species capable of inflicting severe feeding damage on many important tree species. Complementary studies of genes encoding enzymes involved in digestion of woody plant tissues or detoxification of plant allelochemicals were undertaken with the genomes of 14 additional insects, including the newly sequenced emerald ash borer and bull-headed dung beetle. Results: The Asian longhorned beetle genome encodes a uniquely diverse arsenal of enzymes that can degrade the main polysaccharide networks in plant cell walls, detoxify plant allelochemicals, and otherwise facilitate feeding on woody plants. It has the metabolic plasticity needed to feed on diverse plant species, contributing to its highly invasive nature. Large expansions of chemosensory genes involved in the reception of pheromones and plant kairomones are consistent with the complexity of chemical cues it uses to find host plants and mates. Conclusions: Amplification and functional divergence of genes associated with specialized feeding on plants, including genes originally obtained via horizontal gene transfer from fungi and bacteria, contributed to the addition, expansion, and enhancement of the metabolic repertoire of the Asian longhorned beetle, certain other phytophagous beetles, and to a lesser degree, other phytophagous insects. Our results thus begin to establish a genomic basis for the evolutionary success of beetles on plants.

Over the last few decades, phylogenetics has emerged as a very promising field, facilitating a comparative framework to explain the genetic relationships among all the living organisms on earth. These genetic relationships are typically represented by a bifurcating phylogenetic tree — the tree of life. Reconstructing a phylogenetic tree is one of the central tasks in evolutionary biology. The different evolutionary processes, such as gene duplications, gene losses, speciation, and lateral gene transfer events, make the phylogeny reconstruction task more difficult. However, with the rapid developments in sequencing technologies and availability of genome-scale sequencing data, give us the opportunity to understand these evolutionary processes in a more informed manner, and ultimately, enable us to reconstruct genes and species phylogenies more accurately. This thesis is an attempt to provide computational methods for phylogenetic inference and give tools to conduct genome-scale comparative evolutionary studies, such as detecting homologous sequences and inferring gene families. In the first project, we present FastPhylo as a software package containing fast tools for reconstructing distance-based phylogenies. It implements the previously published efficient algorithms for estimating a distance matrix from the input sequences and reconstructing an un-rooted Neighbour Joining tree from a given distance matrix. Results on simulated datasets reveal that FastPhylo can handles hundred of thousands of sequences in a minimum time and memory efficient manner. The easy to use, well-defined interfaces, and the modular structure of FastPhylo allows it to be used in very large Bioinformatic pipelines. In the second project, we present a synteny-aware gene homology method, called GenFamClust (GFC) that uses gene content and gene order conservation to detect homology. Results on simulated and biological datasets suggest that local synteny information combined with the sequence similarity improves the detection of homologs. In the third project, we introduce a novel phylogeny-based clustering method, PhyloGenClust, which partitions a very large gene family into smaller subfamilies. ROC (receiver operating characteristics) analysis on synthetic datasets show that PhyloGenClust identify subfamilies more accurately. PhyloGenClust can be used as a middle tier clustering method between raw clustering methods, such as sequence similarity methods, and more sophisticated Bayesian-based phylogeny methods. Finally, we introduce a novel probabilistic Bayesian method based on the DLTRS model, to sample reconciliations of a gene tree inside a species tree. The method uses MCMC framework to integrate LGTs, gene duplications, gene losses and sequence evolution under a relaxed molecular clock for substitution rates. The proposed sampling method estimates the posterior distribution of gene trees and provides the temporal information of LGT events over the lineages of a species tree. Analysis on simulated datasets reveal that our method performs well in identifying the true temporal estimates of LGT events. We applied our method to the genome-wide gene families for mollicutes and cyanobacteria, which gave an interesting insight into the potential LGTs highways. / <p>QC 20161010</p>

Due to recent advances in molecular biology and genomic research, numerous diseases have been given their genetic identities for which gene therapy may be a possible prescription. Gradually, the development of viral and non-viral vectors to translocate genes has become a bottleneck. For non-viral vectors, polyethylenimine (PEI) is considered as a potential vector candidate for gene delivery because of its ability to compact DNA and its intrinsic pH buffering capacity. PEI and its derivates have been widely tested in both in-vitro and in-vivo gene transfection experiments. The progress is limited due to the lack of a better understanding of the intracellular mechanism. So far, their cytotoxicity is relatively high and gene transfection efficiency is low. This study was designed to modify PEI and optimize its cytotoxicity and gene transfection efficiency. / During the complexes formation, both LLS and zeta-potential were used to follow the process. The results showed that most of anionic DNA are complexed by cationic PEI-based polymers when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches &sim;3, but the gene transfection reaches the highest efficiency when N:P &sim;10. When N:P > 3, there exist two population of PEI chains in the solution mixture: bound to DNA and free in the solution. The bound PEI chains condense and protect DNA. Our current study confirms that it is those free PEI chains that play a vital role in promoting the gene transfection. Our preliminary data shows that the promotion mainly occurs in the intracellular space. The detailed mechanism is still lacking at this moment. Nevertheless, our finding leads to a totally different way in the development of non-viral vectors. / Further, we grafted PEI with polyethylene glycol (PEG), respectively via a reductive disulfide -S-S- and a non-degradable -C-C- bond to form two copolymer vectors. A comparative study shows that the polyplexes formed between the two copolymers and DNA are more stable than that formed between unmodified PEI and DNA under the physiological condition, presumably because the grated PEG chains form a protective hydrophilic shell on the PEI/DNA polyplexes. However, PEGylation reduces the internalization of the copolymer/DNA polyplexes in in-vitro experiments. For the two copolymer vectors, PEG-SS-PEI is 2-8 times more effective than its counterpart (PEG-CC-PEI) in the gene transfection, presumably due to the cleavage of the grafted PEG chains inside the reductive cytosol, which promotes the release and translocation of DNA. Our results demonstrate that using the disulfide as a linker is a promising approach to overcome the PEGylation dilemma in the development of low cytotoxic and high efficient non-viral polymeric vectors. / It has been known that short PEI chains are less toxic, but long chains are more effective in gene transfection. Therefore, we decide to use the disulfide bond (-S-S-) to extend short PEI chains to increase efficiency and also utilize the reductive cytosol environment to cleave such extended PEI chains to reduce their cytotoxicity inside the cell. Laser light scattering (LLS) was used to in-situ monitor the linking reaction between short PEI chains (M w = 2000 g/mol) and dithiobis(succinimidyl propionate) (DSP). The molar mass and crosslinking degree of the extended PEI chains was controlled by either the amounts or the adding rate of DSP. A comparative study of two linked PEI samples (PEI-7K-L and PEI-400K-L, respectively with M w = 6.5 x 103 and 3.8 x 10 5 g/mol) reveals that cytotoxicity and gene transfection efficiency of such extended PEI chains are related to the chain length and structure. Namely, PEI-7K-L with an extended chain structure is less cytotoxic and 2--10 times more effective in the gene transfection than the "golden standard" (PEI25K) and the widely used commercial vector, Lipofectamine 2000RTM. Comparatively, PEI-400K-L with a spherical microgel structure is ineffective in spite of its non-toxicity. Our study clearly demonstrates that a proper control of the chain length and structure is important. / by Deng, Rui. / Adviser: Chi Wu. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Philosophiae Doctor - PhD / Plants are continuously exposed to varying conditions in their environment, to which they have to adapt by manipulating various cellular processes. Environmental (abiotic) and pathogen (biotic) stress are challenges against which plants have to defend themselves. Many plant responses to stress stimuli are a result of cellular processes that can be divided into three sequential steps; namely signal perception, signal transduction m1d execution of a response. Stress signal perception is, in most of these cases, facilitated by cell surface or intracellular receptors that act to recognize molecules presented to the cell. In several cases, hormones are synthesized in response to stress signals and in turn these hormones are perceived by cellular receptors that trigger signal transduction cascades. Propagation of signal transduction cascades is a complex process that results from activation of various signaling molecules within the cell. Second messengers like calcium (Ca2+) and guanosine 3', 5'-cyclic monophosphate (cGMP) play a vital role in mediating many signal transduction processes. The result of these signal transduction cascades is, in most instances, expression of genes that contribute to the plant's ability to cope with the challenges presented to it. Plant natriuretic peptides (PNPs) are novel plant hormones that regulate water and salt homeostasis via cGMP-dependent signaling pathways that involve deployment of Ca2+. The aim of this study is to partially characterize a PNP and a guanylyl cyclase, both from Arabidopsis thaliana. Guanylyl cyclases synthesize cGMP from the hydrolysis of guanosine 5' -triphosphate (GTP) in the cell. The study also aims to investigate the effect of drought and salinity on cGMP levels in plants, using sorbitol to mimic the osmolarity/dehydration effect of drought and NaCl as a source of salinity stress and thus link NaCl and sorbitol responses to both AtPNP-A and cGMP up-regulation.

Yue, Yanan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 62-63). / Abstract also in Chinese. / ABSTRACT (Chinese) --- p.i / ABSTRACT --- p.iii / CONTENT --- p.v / ACKNOWLEDGMENT --- p.vii / ABBREVIATIONS --- p.viii / Chapter CHAPTER 1 --- Introduction and Background / Chapter 1.1 --- Methods of Gene Delivery --- p.1 / Chapter 1.1.1 --- Viral Delivery Systems --- p.2 / Chapter 1.1.2 --- Non-Viral Delivery Systems --- p.3 / Chapter 1.2 --- The Gene-delivery Problems --- p.7 / Chapter 1.2.1 --- Extracellular Barriers --- p.8 / Chapter 1.2.2 --- Intracellular Barriers --- p.10 / Chapter 1.3 --- Polymer-Mediated Systems for Gene Delivery --- p.13 / Chapter 1.3.1 --- Polyethylenimine (PEI)-Based Vectors --- p.13 / Chapter 1.3.2 --- Cyclodextrin-Based Vectors --- p.15 / Chapter 1.4 --- Objective and Main Achievements --- p.16 / Chapter 1.5 --- References --- p.18 / Chapter CHAPTER 2 --- Effect of Free Polyethylenimine-Mediated Polycations on Gene Delivery: Fundamentals and Vital Factors / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Experimental Section --- p.25 / Chapter 2.3 --- Results and Discussions --- p.29 / Chapter 2.3.1 --- Fundamentals --- p.29 / Chapter 2.3.2 --- Vital Factors for the Efficacy of Free Chains --- p.37 / Chapter 2.4 --- Conclusions --- p.42 / Chapter 2.5 --- References --- p.42 / Chapter CHAPTER 3 --- Effect of Free Polyethylenimine-Mediated Polycations on Gene Delivery: Mechanistic Study / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Experimental Sections --- p.46 / Chapter 3.3 --- Results and Discussion / Chapter 3.3.1 --- Potential Effect of Free PEI Chains on Cellular Uptake --- p.49 / Chapter 3.3.2 --- Potential Effect of Free PEI Chains on Endolysosomal Release --- p.51 / Chapter 3.3.3 --- Exploration on Proton Sponge Hypothesis --- p.53 / Chapter 3.3.4 --- Interactions of PEI-based Polycations and Phospholipid Membranes --- p.55 / Chapter 3.4 --- Conclusions --- p.61 / Chapter 3.5 --- References --- p.62

The ubiquitous nature of antibiotic resistance and antibiotic resistance genes (ARGs) among environmental pathogens from a variety of wastewater effluents, suggests that the aquatic environment, and specifically alternative wastewater treatment systems, may act as reservoirs for drug resistant bacteria and ARGs, thereby contributing to the widespread dissemination of antibiotic resistance. More research is necessary to contribute to our understanding of the occurrence, fate, and mobility of antibiotic resistance and ARGs among bacterial indicators of faecal contamination as well as pathogenic bacteria within Canadian wastewater treatment systems. The primary objective of this research was to determine the prevalence, fate, and potential transfer of bacterial resistance and ARGs among selected environmental pathogens exposed to alternative wastewater treatment systems, while considering the impact of treatment strategies on the expression of antibiotic resistance. A detailed analysis was initially conducted with respect to the characterization and quantification of microbial populations (including antibiotic resistant bacteria) in a variety of treatment systems and waste effluent sources. Traditional culture-based screening techniques in combination with molecular characterization (through colony or multiplex PCR), and molecular quantification using real-time quantitative PCR were utilized in order to help establish a preliminary environmental assessment of selected pathogens (Escherichia coli, Enterococcus spp., Salmonella spp.) and ARGs (tetA, blaSHV, & ampC) within a variety of wastewater treatment systems (lab-scale mesocosms, constructed wetland, constructed lagoon system, and pilot-scale biological nutrient removal (BNR) system). Overall, the level of multiple antibiotic resistance (MAR) among culturable indicator (E. coli & Enterococcus spp.) and environmental bacteria was high (reaching 100% in several instances) within different types of wastewater treatment systems and effluent sources (poultry waste effluent, municipal wastewater, aquaculture wastewater). Common antibiotic resistance profiles among E. coli isolates included simultaneous resistance to between three and five antimicrobials, whereas common MAR profiles among Enterococcus spp. isolates showed resistance to ten or more antibiotics. Real time quantitative PCR was used to determine the concentration of three bacterial pathogens; E. coli, Enterococcus faecalis, and Salmonella spp., and three ARGs; tetA, ampC, and blaSHV, within a variety of wastewater samples. Based on the results, it was concluded that high concentrations of ARGs were present in the treated effluent (10⁴- 10⁶ target gene copies/100 mL), regardless of system type (i.e. constructed lagoon, pilot-scale BNR, or constructed wetland), which may ultimately serve as a potential route for entry of ARGs and antibiotic resistant bacteria into the natural environment. Water is considered an important medium for transfer of resistance genes and resistant bacteria to the broader environment. Few studies have examined the transferability via conjugation of ARGs in E. coli and Salmonella spp. isolated from wastewater. Identification of three resistance determinants (tetA, strA, strB) conferring resistance to tetracycline and streptomycin was performed on selected multi-drug resistant Salmonella spp. and E. coli isolates. The potential for transfer of tetracycline and streptomycin resistance genes was demonstrated through broth conjugation experiments using multi-drug resistant Salmonella spp. and E. coli isolates as donors, and E. coli K12 as the recipient. Conjugation was successfully observed in 75% (9/12) of donor isolates, occurring in both Salmonella spp. and E. coli isolates. Six strains (50%) were capable of transferring their tetA, strA, and strB genes to the recipient strain, resulting in 58.5% (38/65) of total transconjugant strains acquiring all three resistance determinants. The results confirm the role of environmental bacteria (isolated from wastewater treatment utilities) as a reservoir of antibiotic resistance and ARGs, containing mobile genetic elements, which are capable of disseminating and transferring ARGs. As concerns about water quality and environmental contamination by human and agricultural effluents have increased, it has become increasingly more important to consider the prevalence and transferability of ARGs to opportunistic and human pathogens. As observed in this research, the ubiquitous nature of multi-drug resistant bacteria in water and wastewater effluents, the presence of diverse ARGs of human and veterinary health significance, as well as the transfer of resistance determinants through conjugative plasmids to recipient bacteria, suggests that environmental exposure through contact or consumption with contaminated water is probable. However, a lack of critical information still exists regarding the movement of resistance genes within and between microbial populations in the environment. In addition, the extent of human exposure to ARGs and antibiotic resistant bacteria is still not well understood, and future studies on human exposure to these resistant contaminants are necessary.

Brachyspira species are bacteria that colonize the intestines of some mammalian and avian species with different degrees of pathogenicity. Brachyspira intermedia is a mild pig and bird pathogen with an unknown genomic sequence. In this project, we completed the genome of Brachyspira intermedia PWS/AT and did a comparative genomic analysis between B. intermedia PWS/AT and the already completed genomes of B. hyodysenteriae WA1, B. murdochii 56-150T and B. pilosicoli 95/1000. A table containing 15 classes of unique and shared genes was developed and analyzed in order to gain a better understanding of species-specific traits and clues behind the different degree of pathogenicity. Our result shows that genes are overall poorly annotated and further studies are of great importance for understanding different and shared properties. The largest number of unique features was found in B. intermedia and B. murdochii. B. hyodysenteriae and B. pilosicoli has most likely developed independently towards different biological niches and B. pilosicoli has undergone a major reductive evolution. One plasmid and six prophages were found in B. intermedia, where two of the phages appear to be capable of horizontal gene transfer. Further genome sequencing of more strains will probably increase the understanding of species-specific traits even more.

The efficiency of retrovirus-mediated gene transfer can be dramatically enhanced by inducing flocculation of viruses. Addition of oppositely charged polymers to virus stocks resulted in the formation of virus-polymer complexes. The complexes specifically incorporated virus particles and only few other proteins, were not cytotoxic, did not reduce the stability of the viruses, and were large enough to sediment, delivering the viruses to the cells more rapidly than by simple diffusion. Increases in the rate of transport of viruses correlated with increases in the rate of transduction, as the polymers did not affect the efficiency of post-binding steps of transduction. The formation of virus-polymer complexes also permitted concentrating viruses and purifying the stocks from inhibitors of transduction. Pelleting of the complexes followed by resuspension of the pellet in a reduced volume of fresh cell culture medium resulted in substantial enhancement of transduction. Purified virus stocks could be used in smaller quantities than unprocessed stocks to achieve a given level of gene transfer and reduced uncertainties about the relationship between the amount of virus used and the number of genes transferred. When using high concentrations of purified viruses, the efficiency of gene transfer was dependent on the number of envelope proteins displayed on the surface of each virus particle. Viruses with a low number of envelope proteins transduced cells more efficiently than did viruses with a high number of envelope proteins, and allowed more integrations of the transgene per target cell. In contrast, when the number of envelope proteins per virus particle was high, transduction appeared to be limited by a reduction in availability of functional receptors for viruses pseudotyped with the same envelope. Taken together, this novel method for processing retrovirus stocks and a better understanding of major limitations of transduction should simplify efforts to predict the outcome of retrovirus transduction protocols and should help to increase the likelihood that human gene therapy protocols will succeed.

The angiosperm order Malpighiales includes \(\sim 16,000\) species and constitutes up to 40% of the understory tree diversity in tropical rain forests. Despite remarkable progress in angiosperm phylogenetics during the last 20 years, relationships within Malpighiales have remained poorly resolved, possibly due to its rapid rise during the mid-Cretaceous. Using phylogenomic approaches, including analyses of 82 plastid genes from 58 species, we identified 12 new clades in Malpighiales and substantially increased resolution along the backbone (Chapter 1). This greatly improved phylogeny revealed a dynamic history of shifts in net species’ diversification rates across Malpighiales, with bursts of diversification noted in the Barbados cherries (Malpighiaceae), cocas (Erythroxylaceae), and passion flowers (Passifloraceae). We also found that commonly used a priori approaches for partitioning data in similar large-scale analyses, by gene or by codon position, performed poorly relative to the use of partitions identified a posteriori using a Bayesian mixture model. Another aspect of my thesis focused on investigating horizontal gene transfer (HGT) in Malpighiales. Recent studies have suggested that plant genomes have undergone potentially rampant HGT. Parasitic plants have provided the strongest evidence of HGT, which appears to be facilitated by the intimate physical association between the parasites and their hosts. Using phylogenomic approaches, we analyzed the nuclear transcriptome (Chapter 2) and mitochondrial genome (Chapter 3) of the holoparasite Rafflesiaceae, which represents an enigmatic subclade of Malpighiales. Our analyses show that several dozen actively transcribed nuclear genes, and as many as 34–47% of its mitochondrial gene sequences, show evidence of HGT depending on the species. Some of these HGTs appear to have maintained synteny with their donor and recipient lineages suggesting that vertically inherited genes have likely been displaced via homologous recombination, as is common in bacteria. Finally, our results establish for the first time that although the magnitude of HGT involving nuclear genes is appreciable in these parasitic plants, HGT involving mitochondrial genes is substantially higher. Moreover, the elevated rate of unidirectional host-to-parasite gene transfer raises the possibility that HGTs may provide a fitness benefit to Rafflesiaceae for maintaining these genes.

Lateral gene transfer (LGT) is an important factor contributing to the evolution of prokaryotic genomes. The Aquificae are a hyperthermophilic bacterial group whose genes show affiliations to many other lineages, including the hyperthermophilic Thermotogae, the Proteobacteria, and the Archaea. Here I outline these scenarios and consider the fit of the available data, including two recently sequenced genomes from members of the Aquificae, to different sets of predictions. Evidence from phylogenetic profiles and trees suggests that the ?-Proteobacteria have the strongest affinities with the three Aquificae analyzed. However, this phylogenetic signal is by no means the dominant one, with the Archaea, many lineages of thermophilic bacteria, and members of genus Clostridium and class ?-Proteobacteria also showing strong connections to the Aquificae. The phylogenetic affiliations of different functional subsystems showed strong biases: as observed previously, most but not all genes implicated in the core translational apparatus tended to group Aquificae with Thermotogae, while a wide range of metabolic systems strongly supported the Aquificae - ?-Proteobacteria link. Given the breadth of support for this latter relationship, a scenario of ?-proteobacterial ancestry coupled with frequent exchange among thermophilic lineages is a plausible explanation for the emergence of the Aquificae.