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Phenotypic and Genotypic Characterization of the Streptococci Isolated from Sputum of Adult Cystic Fibrosis Patients
No full textThe cystic fibrosis (CF) lung microbiome is a complex polymicrobial community with numerous organisms involved in disease progression. The non-pyogenic streptococci, such as the Streptococcus milleri group, have been implicated in CF disease progression and pathogenesis, but other members are less understood as there has been limited study. The objectives of this study were to characterize a collection of diverse non-pyogenic streptococci and investigate their pathogenic potential and interactions with principal CF pathogens. Genotypic analysis fully characterized 100 unique isolates from CF sputum that did not identify with known species of streptococci by partial 16S rRNA sequencing. To examine these unique isolates, I did extensive genotypic characterization with cpn60, sodA, rpoB and ddl in addition to full 16S rRNA sequencing. 48 isolates identified as novel streptococci isolates, of which 20 of the novel presented with divergent multiple 16S rRNA gene sequences. Phenotypic characterization was expanded to a larger collection of 297 non-pyogenic streptococci isolates with profiling done by several assays. I observed poor correlation between genotypic methods and there was high phenotypic heterogeneity, particularly with regards to increased β-hemolysis under anaerobic condition on human blood as compared to standard conditions. Due to chronic antibiotic use by CF patients, the assessment of the streptococci antibiotic resistome from 459 isolates was done against nine antibiotics relevant in CF treatment. High antibiotic resistance was observed against the macrolides azithromycin (56.4%) and erythromycin (51.6%). Investigation of the molecular mechanisms of macrolide resistance yielded an unexpected result in that nearly half the isolates were found to have point mutations at position 2058 or 2059 of the 23S ribosomal subunit, an uncommon molecular mechanism of resistance within the streptococci. The synergistic interactions of the oropharyngeal flora (OF) with P. aeruginosa were investigated with the construction of transposon mutant libraries in two of the synergen isolates. Mutants were obtained that displayed attenuation or up-regulation of these synergistic interactions and will be used for further study and identification. The work presented here provides the framework to elucidate the role of the diverse streptococci in CF disease progression.indefinit
Nitric oxide and enteric glia in the regulation of intestinal ion transport in health and disease
No full textBackground & Aims: Enteric glia are increasingly recognized as an important
functional component of the enteric nervous system. They have been indirectly implicated in the regulation of epithelial ion transport, which regulates homeostatic water and ion balance, through the release of the gaseous neurotransmitter nitric oxide, which has multifaceted roles in the physiological regulation and pathophysiological dysregulation of ion transport. However, the roles of enteric glia and nitric oxide in the regulation of ion transport are poorly understood in health and disease. Using nitric oxide synthase inhibition and a model of glial metabolic inhibition, the primary aims of this work were to investigate the physiological regulation of epithelial ion transport by nitric oxide and enteric glia, and how this regulation is altered during intestinal inflammation. Methods: Ion transport was measuring using full-thickness segments of mouse colon in an Ussing chamber apparatus. The release of nitric oxide from enteric glia and neurons within the myenteric plexus was assessed using amperometry and the cellular sites of nitric oxide release were investigated using nitric oxide imaging. Results: Under physiological conditions, nitric oxide release from enteric glia and neurons within the myenteric plexus regulates the nicotinically-mediated biphasic stimulation of epithelial ion transport in a concentration-dependent manner, while nitric oxide and enteric glia are not involved in the regulation of electrically-evoked stimulation of ion transport. Following intestinal inflammation, ion transport is hyporesponsive to stimulation, as the biphasic nicotinic response is reduced, and the response to electrically-evoked stimulation is absent. The inhibition of nitric oxide synthase or
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the metabolic inhibition of enteric glia further inhibits the nicotically-mediated response, and restores the electrically-evoked secretory response. Conclusions: This work demonstrates novel functional roles for nitric oxide released from enteric glia in the nicotinically-mediated physiological regulation and electrically-evoked pathophysiological dysregulation of intestinal ion transport. Additionally, a previously unappreciated role for the myenteric plexus in the regulation of nicotinically-mediated ion transport under physiological conditions is described. The data presented in this thesis highlight and extend the important roles of nitric oxide and enteric glia within the myenteric plexus in gastrointestinal health and disease.indefinit
Characterization of Salicylates as Novel Catalytic Inhibitors of Human DNA Topoisomerase II Alpha
No full textTopoisomerase II (topo II) is a ubiquitous enzyme required for the maintenance of DNA topology. Due to its essential nature, many pharmaceuticals have been found to or developed to target the enzyme for both laboratory and clinical uses. Topo II poisons, such as doxorubicin and etoposide, are commonly used in cancer chemotherapy regimens and are cytotoxic due to their ability to covalently trap topo II on cleaved DNA, leading to the accumulation of DNA double-stranded breaks. In contrast, compounds that inhibit topo II catalytic activity without generating DNA double-stranded breaks are classified as catalytic inhibitors and impair enzyme function at a number of stages in the catalytic cycle, including DNA binding, DNA cleavage, ATP hydrolysis, and enzyme dissociation. Some catalytic inhibitors have been found to counter the DNA damaging effects of topo II poisons. This thesis describes work identifying salicylate as a novel catalytic inhibitor of topo II. It demonstrates that pretreatment of cells with salicylate, prior to treatment with topo II poisons, attenuates poison-induced DNA damage signaling mediated by the ATM protein kinase. This is associated with a concomitant loss of poison-induced DNA double- stranded breaks and consequently decreased cytotoxicity following treatment with topo II poisons. This work also demonstrates that salicylate does not block topo II-DNA binding, or trap the enzyme as a closed clamp on DNA. Rather, salicylate blocks DNA cleavage and enzyme-mediated ATP hydrolysis is impaired in a non-competitive manner. Furthermore, salicylate-mediated inhibition of topo II catalytic activity is selective for the alpha isoform of topo II. In investigating the structural determinants modulating potency of topo II inhibition, many compounds with structural similarities to salicylate, including the salicylate-based pharmaceuticals sulfasalazine and diflunisal, were found to also act as catalytic inhibitors of topo II. It was determined that potency is determined by modifications at the 2’-position that are electronegative and capable of donating a hydrogen bond, but that these effects are further influenced by substitutions at the 5’-position. Considered together, the data presented in this thesis establish a new role for a long-established drug.indefinit
On Deformity: Bodies in Contemporary Canadian Fiction
No full textThis dissertation ponders how deformity acts as an index of resistance to the conventional family saga; it challenges the authority of the genre, which perpetuates
conformity to affirm the existence of a national identity. I open with a history of the trope of deformity and a theory on its applicability to questions of the nation in Canadian fiction. Bonnie Burnard’s A Good House begins the literary analysis and considers how Daphne’s asymmetrical face exemplifies the novel’s overarching deformation of the domestic realist text. Ann-Marie MacDonald’s Fall on Your Knees rereads Victorian conventions to demonstrate the perversity of power and the purity of individuality as the characters contest hegemonic cultural projects. The giants and runts that make up the Hervé family in D. Y. Béchard’s Vandal Love rewrite the traditional roman de la terre and exhibit how the trope of deformity underscores an indefinable Canadian identity – one that possesses roots only by seeking to disavow those roots. Anna’s body in Susan Swan’s The Biggest Modern Woman of the World resists all acts of colonization, including historical proofs, and performs a parody that reverses the ideal of the female
body as a trope for the mother nation. Lastly, the stories of Fielding and Smallwood in A Colony of Unrequited Dreams and The Custodian of Paradise destabilize the formulation of a collective unconscious based on historical writings. Their symbolic deformities reveal how Newfoundland’s status as a nation is both fact (as historically represented by Smallwood) and fiction (as imagined by Fielding).
The deformed bodies in these texts are non-compliant disruptions of national
discourses that enable national identity through exclusive ideological frameworks; they destabilize centralized myths of the nation frequently employed by supporters of the classical canon to reaffirm a sense of cultural existence. Though they criticize the
superficiality of kinship, they nonetheless highlight a still prevalent need for roots,
physical and historical, as they reconstitute a more flexible sense of community.Indefinit
The Role of Sodium-Calcium Exchanger in the Regulation of Endothelial Function
No full textAppropriate release of nitric oxide (NO) is critical for normal physiological functioning of the cardiovascular system. Although a rise in intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells (ECs) is thought to play an important role in the coordination of NO release, the molecular mechanism underlying this influx is poorly understood. The work presented here outlines the molecular mechanisms responsible for regulating endothelial [Ca2+]i and its implication to NO production and release, and involved two major areas of study. Firstly, we identified the presence of a signaling complex comprised of stromal interaction molecule 1 (STIM1), transient receptor potential protein (canonical subtype) 1 (TRPC1), and the Na+/Ca2+ exchanger 1 (NCX1) in cultured ECs, which may be an important component of the Ca2+ influx pathway necessary for the activation of endothelial NO synthase (eNOS) during agonist stimulation. Secondly, recruitment of the NCX in reverse mode was shown to play an important role in flow-mediated dilation and the corresponding phosphorylation of serine-1177 of eNOS (S1177-eNOS). This is the first study to identify S1177-eNOS phosphorylation in response to flow in small (< 300 μm), pressurized, myogenic rat cerebral arteries. These findings are significant because the potential involvement of a TRP-NCX signaling complex in ECs has been an unresolved issue for some time. Therefore the work of this thesis provides unique insight into the molecular mechanisms that contribute to the regulation of endothelial [Ca2+]i.indefinit
Stress, sex and synapses: an exploration of stress-associated male/female differences in electrophysiological properties of corticotropin-releasing hormone neurons of the paraventricular nucleus of the hypothalamus
No full textActivation of the neuroendocrine stress response in response to challenge is coordinated by corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN). The stress responsiveness of the brain in general and this system in particular, is determined, in part, by intrinsic factors including genetic background, age and sex. There is little information on whether the sex of an animal, prior to increases in sex hormones during adolescence, impacts cellular and synaptic mechanisms responsible for regulating CRH neurons. I characterized the differences in synaptic transmission and postsynaptic firing characteristics in identified CRH neurons from p. 21-30 male and female mice. I noted a longer delay to first spike in female mice and tested the hypothesis that this was a consequence of the actions of circulating glucocorticoids. The data in this thesis demonstrate how intrinsic sex differences and stress can exert control over postsynaptic CRH neuron activity.indefinit
Cartilage boundary lubrication and rheology of proteoglycan 4 + hyaluronan solutions and synovial fluid
No full textSynovial fluid (SF) is the viscous fluid present within articular joints that contributes to load bearing and lubrication functions. Proteoglycan 4 (PRG4) and hyaluronan (HA) in SF contribute synergistically to cartilage boundary lubrication. However, changes in SF PRG4 and HA content with osteoarthritis (OA) and associated effects on cartilage boundary lubricating function are not fully understood. Furthermore, the effects of PRG4+HA interaction on solution viscosity have not been thoroughly characterized.
The objectives of this thesis were to 1) investigate the relationship between PRG4 and HA composition and boundary lubricating function of normal and OA SF, and 2) to investigate how the concentration and structure of PRG4 contributes to interactions with itself and HA, and subsequently the boundary lubricating and rheological properties of SF.
Novel and previously characterized biochemical and biomechanical methods were used to evaluate boundary lubricant composition and lubricating ability of SF. While not all OA SF samples had low PRG4, samples that had low PRG4 concentration and decreased HA molecular weight (MW) demonstrated decreased cartilage boundary lubricating ability in vitro, which could be restored by addition of PRG4. SF aspirated after a flare reaction to intra-articular injection that had low PRG4 and an approximately normal HA MW distribution demonstrated normal cartilage boundary lubricating ability. In purified solutions of PRG4 and HA, decreased PRG4 or decreased high MW HA limited cartilage boundary lubricating ability. PRG4 and recombinant human PRG4 increased the viscosity of HA solutions at low concentrations, but decreased the viscosity of high concentration HA solutions. The intra- and inter-molecular disulfide bonded structure of PRG4 was observed to be important for its contributions to both PRG4+HA cartilage boundary lubricating ability and PRG4+HA solution viscosity.
These results demonstrate that alterations in both PRG4 and HA content in SF may have negative effects on SF cartilage boundary lubricating and rheological function, and are consistent with a non-covalent, crowding mechanism of interaction. They suggest that maintaining PRG4 and HA content in SF during injury and disease, through the development of new PRG4±HA biotherapeutic treatments, may be able to both protect cartilage from degeneration and restore SF viscosity in vivo.indefinit
Sunmine: Evaluating The Potential Of Grid-connected Solar Photovoltaics With Energy Storage
No full textGlobal warming mitigation is translating into actions worldwide. Canada targets higher levels of clean energy sources in its electricity mix. Solar photovoltaics is the fastest growing renewable electricity source and energy storage is positioned as the enabling technology to increase its contribution integrated with the grid by providing reliable and greenhouse gas free electricity when needed. This project consolidates knowledge from multiple sources and applies it to SunMine, the largest solar photovoltaics plant in British Columbia. The goal is to
maximize revenue at the lowest cost by investigating the economics of adding battery storage to allow photovoltaic electricity to be sold when the price is higher. While batteries used for a single service do not result in a net-economic benefit, this project unveils the roadmap to enhance its value proposition through strong collaboration between utility, plant owner, solar and energy storage suppliers towards the Canadian electricity market of the future
Megaliths to Medicine Wheels: Boulder Structures in Archaeology
No full textProceedings of the 11th Annual Chacmool Archaeology Conferenc
Analysis of an Electricity Market Restructuring Reform: The Case of Mexico
No full textIn 2013, Mexico started reforming its electricity sector, expecting improvements in the performance of the industry and increases in consumers’ welfare. The 2012-2018 administration introduced competition to their generation segment and created a Wholesale Electricity Market through a series of constitutional amendments. It aimed to attract private capital to modernize the sector and improve the quality and reliability of energy supply in the country. It also sought to decrease the high electric costs and encourage the incorporation of clean energy in the sector. However, consumer prices have not reflected the improvements in competition in the market. This framework has allowed the current government to propose measures that are opposed to the competitive model. The purpose of this project is to analyze the implemented policies and their consequences, as well as the implications of keep increasing market competitions or returning to a state-owned utility monopoly. Paul Joskow developed some key principles for reforming processes to create efficient electricity markets. These principles can be used as a “standard model” for jurisdictions restructuring their power sector. The model can be helpful to analyze any country’s restructuring electricity reforms. This project compares them to Mexico’s measures to identify barriers to competition and other sources of inefficiencies in the Mexican electricity market. According to the literature review, the model based on Joskow’s principles for a successful liberalizing reform mainly consists of: i) Privatization of state-owned utilities, ii) Vertical separation of competitive segments, iii) Designation of a single Independent System Operator (ISO), iv) Promotion of efficient access to the transmission network, v) Creation of voluntary public wholesale spot energy and operating reserve market institutions, vi) Development of active demand-side institutions and vii) Creation of independent regulatory agencies. For a reform to be successful, it also needs a strong political commitment to it. Furthermore, nonstable market rules and regulatory imperfections deter potential investments in new generating capacity. Mexico complied with the vertical and horizontal separation of the state utility, the designation of an ISO, the creation of the institutions for a wholesale market and the creation of independent regulatory agencies. Nevertheless, the Mexican restructuring process did not privatize its state-owned utility. That decision makes it harder to incentivize performance improvements and the utility can be used to pursue political agendas. Also, Mexico is still in an early stage in the development of demand-side management institutions, limiting the efficiency of the market to incorporate demand responses. Moreover, prices have not decreased due to the increasing congestion in various links of the national transmission network. Additionally, with the change in administration, there is currently weak political support for the reform and the market rules are unstable, discouraging investment. The legal separation of CFE was pulled back and two temporarily suspended agreements may allow for exclusionary behaviour in the access to transmission. These actions impose further barriers to competition