Bacterial transformation is defined as a mechanism of genetic exchange whereby a population of bacteria can obtain genetic informa-tion as a result of cellular uptake and integration of extracellular deoxyribonucleic acid released from other bacteria by natural or in-duced lysis. In order for a transformable strain of bacteria to take up DNA and to undergo transformation, the cells must be in a physiolog-ical state called competence. A cell is referred to as competent if it has the ability to bind extracellular DNA irreversibly and subsequently to integrate and express the DNA.

Introduction: Acute ketone monoester (KE) supplementation affects exercise responses but there are equivocal data regarding the effects on exercise efficiency. We examined the effect of ketone monoester ingestion on exercise efficiency during cycling and probed further the influence of supplement dose and exercise intensity. This study was registered prior to data collection at ClinicalTrials.org (NCT05665855). Methods: Twenty-eight trained adults were recruited [16 males, 12 females; peak oxygen uptake (V̇O2peak): 59±11 ml·kg-1·min-1]. Participants completed three experimental trials in a randomized, crossover, and double-blinded manner, each separated by ~1 week. Participants ingested either a 0.3 (KE-LO) or 0.6 g/kg (KE-HI) body mass dose of KE or a flavour-matched placebo (PLAC) ~30 min prior to exercise. The incremental cycling protocol involved a 3-minute warm-up, three 5-minute stages at 75%, 100%, and 125% of individual ventilatory threshold, and a ramp increase to volitional exhaustion. Expired gases and heart rate were measured continually during exercise. Results: Venous blood [ß-hydroxybutyrate], the major circulating ketone body, was higher in both KE conditions compared to PLAC and also different between conditions (3.0±1.1 and 2.3±0.6 vs 0.2±0.1 mM; all p<0.05). There were no differences in submaximal exercise V̇O2, exercise economy, gross efficiency, or delta efficiency between conditions (all p>0.05). Submaximal exercise heart rate and ventilation were higher in both KE conditions compared to PLAC (141±11 and 141±12 vs 137±12 beats/min; 63±14 and 62±13 vs 60±13 L/min, respectively; all p<0.05). Peak power output at V̇O2peak was lower in KE-HI compared to both KE-LO and PLAC (329±60 vs 339±62 and 341±61 W; both p<0.05). Conclusion: KE supplementation did not alter exercise efficiency during submaximal cycling. KE ingestion increased cardiorespiratory stress during submaximal exercise and the higher dose reduced peak aerobic power output. Future studies should investigate the mechanisms by which KE ingestion alters exercise responses. / Thesis / Master of Science (MSc) / Endurance exercise performance is determined by many variables including the efficiency of the individual. This can be measured during cycling by calculating the ratio of oxygen uptake relative to power output. Ketone supplements have been suggested to alter exercise efficiency. We investigated this issue by having trained adults complete an incremental cycling protocol on three occasions. Before exercise the participants ingested either a small or large dose of a ketone supplement or a taste-matched placebo drink. Exercise efficiency was not different between the conditions but ventilation rate and heart rate were higher during the ketone supplemented trials compared to the placebo. The power output that the participants could achieve at maximal exercise was reduced in the high dose ketone condition. Our study does not support the use of ketone supplements as a strategy to enhance endurance exercise performance. Future studies should investigate the mechanisms by which ketones affect exercise responses.

Sprint interval training (SIT) increases peak oxygen uptake (VO2peak) but the mechanistic basis is unclear. The Fick principle broadly attributes increases in VO2peak to changes in peak cardiac output (Qpeak) and/or peak arteriovenous oxygen difference (peak a-vO2diff). The main purpose of this thesis was to investigate the role of Qpeak, measured non-invasively using inert gas rebreathing (IGR), on SIT-induced changes in VO2peak. It also considered the time course of these responses and the influence of biological sex. The SIT protocol involved 3 x 20-s “all out” sprints performed within a 10-min session of low-intensity cycling. Study 1 measured Qpeak after 2, 6, and 12 weeks of SIT and found it was increased after 12 weeks and associated with the change in VO2peak. Peak a-vO2diff, estimated based on the Fick equation (peak a-vO2diff = VO2peak/Qpeak), was also increased after SIT and associated with the change in VO2peak. Study 2 found that a novel constant-load protocol elicited Qpeak values that were non-inferior to an established step protocol, within a margin of 0.5 L/min. Both protocols elicited VO2 values at Qpeak that were similar to VO2peak. The constant load protocol had similar day-to-day repeatability as the VO2peak test (typical error = 6.6 and 6.4%, respectively). Study 3 investigated an exploratory finding from Study 1 that suggested Qpeak was increased in male but not female participants. The design was similar, but Study 3 employed suggested best practices for making sex-based comparisons. Contrary to our hypothesis, Qpeak was unchanged after 12 weeks of SIT and there was no sex-based difference. Like Study 1, peak a-vO2diff was increased and correlated with VO2peak. This thesis advances knowledge regarding the influence of SIT on Qpeak determined non-invasively and highlights the need for more mechanistic work to comprehensively assess the basis for the increase in VO2peak. / Thesis / Candidate in Philosophy / Sprint interval training (SIT) is a form of exercise that involves brief bursts of near-maximal to “all out” efforts separated by short recovery periods. The method improves cardiorespiratory fitness — an important health marker that is quantified as the highest amount of oxygen used by the body during strenuous exercise (VO2peak) — but the mechanisms are not well understood. This thesis examined the effect of SIT on peak cardiac output (Qpeak), which is the highest rate of blood pumped by the heart each minute, and the relationship to changes in VO2peak. Qpeak was measured non-invasively by having participants breathe an inert gas mixture. Two separate 12-week training studies confirmed that SIT increased VO2peak but yielded conflicting results regarding the role of Qpeak. The findings also suggest that the capacity of skeletal muscles to extract oxygen is increased after SIT. Biological sex does not appear to influence SIT-induced changes in Qpeak or VO2peak.

Monocytes are professional antigen presenting cells (APC). They serve as precursors of macrophages and dendritic cells (DC). We have used cytopathic (cp) and non-cytopathic (ncp) Bovine Viral Diarrhea Viruses (BVDV) to determine the genes and proteins expression levels in bovine monocytes. Four specific aims were accomplished in this study. The first aim was to assess the baseline expression of the proteins involved in professional antigen presentation in bovine monocytes. The results showed that the differential detergent fractionation (DDF) approach can provide interpretable and meaningful functional information in bovine monocytes. The second aim was to evaluate the role of in vitro cp and ncp BVDV infection in the expression of the selected bovine genes involved in professional antigen presentation. The results showed that both BVDV could escape innate immune responses by modulating toll-like receptor (TLR) gene expression, followed by pro-inflammatory, type I interferon (IFN), Th1/Th2 type cytokine genes expression, and decreasing the expression levels of CD80/CD86 in professional APC. The third objective was to determine how the two biotypes affect selective antigen uptake, receptor-mediated endocytosis and non-selective uptake, macropinocytosis in bovine monocytes. The results indicated that bovine monocytes use macropinocytosis for a bulklow uptake of soluble antigens. The final aim was to characterize protein profiles in peripheral blood monocytes infected with cp BVDV isolate in vitro. Comparative profiling of the membrane and cytosolic proteins related to professional antigen presentation were assessed. The results showed that 47 bovine proteins, involved in immune function of professional APC have been significantly altered after cp BVDV infection. Overall, we hypothesize that by modulating expression levels of multiple proteins and genes related to immune responses BVDV could significantly compromise immune defense mechanisms resulting in uncontrolled immune activation or suppression.

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<p>Pap-Pit is a low affinity phosphate transporter found in <em>S. meliloti</em> and many other microorganisms. Pit is the transporter and Pap is the Pit accessory protein. Pap has been shown to be required for the function of Pap-Pit system in <em>S. meliloti</em>. In this study, <em>pap-pit</em> or <em>pit</em> alone from three species of bacteria have been expressed <em>in trans</em> in the <em>E. coli</em> Pi uptake mutants to check their ability to complement the Pi uptake deficiency of the hosts. A visualization tag, SNAP-tag, has been fused to <em>S. meliloti</em> Pap to help determine the subcellular localization of Pap. Here we show that there is an optimal level of Pap-Pit in the cells, and Pap appears to modulate this level to optimize the function of the system. We also demonstrate that Pap is probably localized intracellularly along the cell membrane. In addition, a <em>S. meliloti pap-pit </em>deletion strain has been prepared and to be used as the background strain for site-directed mutagenesis in Pap. The highly conserved surface amino acids in Pap have been identified to be the candidates for the site-directed mutagenesis.</p> / Master of Science (MSc)

<p> Gene-targeted studies in mice have shown that the murine scavenger receptor class B type I (mSR-BI) is atheroprotective and plays a key role in the clearance of high density lipoprotein (HDL) cholesterol by the liver. We focused on the analysis of human SR-BI (hSR-BI) and the role of its C-terminal cytoplasmic tail on its localization, lipid uptake activity, and regulation in hepatocytes both in vitro and in vivo. Full length hSRBI and hSR-BI lacking its C-terminal cytoplasmic tail (hSR-BI-DM) localized to vesiclelike structures in the cytoplasm, to juxtanuclear regions and to the cell surface in HepG2 cells. Similar cytoplasmic punctate distribution was observed in transfected human and mouse aortic endothelial cells. </p> <p> In HepG2 cells both hSR-BI and hSR-BI-DM mediated HDL-lipid uptake; however, the truncation mutant displayed only half ofthe activity, suggesting that removal ofthe C-terminal cytoplasmic tail reduced but did not eliminate SR-BI's activity. In HepG2 cells treated with the PKC inhibitor, calphostin C, hSR-BI or hSR-BI-DM mediated HDL-lipid uptake was decreased by 40 and 50%, respectively, indicating that this activity is regulated by PKC. </p> <p> In order to determine the effects of hSR-BI and hSR-BI-DM in vivo, we set out to generate transgenic mice with hepatic overexpression ofeach protein using a bipartite expression system requiring driver and responder transgenes. Mice expressing the responder transgenes, PTREhSR-BI and PTREhSR-BI-DM, as well as a reporter transgene (PTRdacZ), driven by the same bi-directional promoter, were generated and mated to mice with a liver-specific driver trans gene, PMuptTA. The mice were analyzed and showed the presence of a reporter protein, ~-galactosidase, in their livers, but not in other tissues tested. Total and HDL cholesterol levels were not altered in PMuPtTA I PrREhSRBI or PMuptTA I PrREhSR-BI-DM transgenic mice. Further characterization ofthe double transgenic mice revealed that hSR-BI m.RNA transcripts were detected in the livers of PMuPtTA I PrREhSR-BI mice, but not in those ofPMuPtTA I PrREhSR-BI-DM mice. However, neither PMuptTA I PrREhSR-BI nor PMuptTA I PrREhSR-BI-DM mice showed increased expression of SR-BI in their livers. </p> / Thesis / Master of Science (MSc)