<p>Asthma is a disease characterized by airway hyperresponsiveness (AH) to various spasmogens. Ozone-inhalation causes transient AH of human and canine airway smooth muscle (ASM) and has therefore been used as a model of AH. The mechanism(s) underlying ozone-induced or asthma-related AH are not completely understood, but there is a growing body of evidence that there is a causal relationship between airway inflammation and AH.</p> <p>In these studies, the mechanisms regulating canine bronchial smooth muscle (3rd to 5th order) activity were investigated using intracellular microelectrodes (to monitor electrical activity) and muscle baths (to monitor mechanical activity). The effects of ozone-inhalation and inflammatory mediators on regulation of ASM tone were also investigated.</p> <p>Excitation of ASM (membrane depolarization and contraction) was elicited by activation of muscarinic receptors or thromboxane receptors; the former were found to be of the M₃-subtype. The data indicated that pharmacomechanical (rather than electromechanical) coupling was involved.</p> <p>Inhibition of ASM was found to be initiated by activation of β-adrenoceptors; these were found to be predominantly of the β₁-subtype, though there was evidence that some β₂-adrenoceptors were also involved. There was evidence of physiological antagonism between cholinergic receptor-mediated excitation and adrenoceptor-mediated inhibition due to reciprocal effects on adenylate cyclase.</p> <p>Release of acetylcholine from the cholinergic nerve endings was antagonized by activation of muscarinic receptors (M₁-subtype) or adrenoceptors (both β₁- and β₂- subtypes), and potentiated by activation of thromboxane receptors; these receptor populations were presumed to be located on the nerve endings. There was indirect evidence that neurotransmitter-release was also antagonized by a cyclo-oxygenase metabolite of arachidonic acid (presumably PGE₂).</p> <p>Ozone-inhalation significantly enhanced contractions in response to electric stimulation, but did not significantly after those to exogenously-added cholinergic agonist or to KCl. This AH was prevented by indomethacin (cyclo-oxygenase inhibitor) or by L-670,596 (thromoboxane receptor antagonist). It was concluded that the mechanism underlying ozone-induced AH involved decreased prejunctional inhibition (mediated by PGE₂) and/or increased prejunctional excitation (mediated by TxA₂).</p> / Doctor of Philosophy (PhD)

<p>The proliferative behaviour of human lung fibroblasts in vitro was examined. Fibroblasts derived from the lung of patients with idiopathic pulmonary fibrosis proliferated faster compared to fibroblasts from control lung tissue. An examination of clonally-derived fibroblast lines showed a substantial degree of fibroblast heterogeneity which followed a normal distribution, and also the existence of a significantly greater number of fast-growing clones in the panels of clones derived from primary fibroblast lines established from fibrotic tissue. Heterogeneity with respect to the expression of collagen genes was also documented.</p> <p>The effect of an acute challenge of peripheral blood monocyte and/or alveolar macrophage supernatants on fibroblast proliferation was examined. Supernatants from peripheral blood monocytes and alveolar macrophages stimulated with lipopolysacharide elicited a dose-dependent inhibition of fibroblast proliferation, likely induced by interleukin 1. This effect could be prevented by pre-treating the fibroblasts with indomethacin and reconstituted by adding exogenous prostaglandin E$\sb2$, thus indicating the involvement of the prostaglandin E$\sb2$ pathway of the fibroblast. Exogenous prostaglandin E$\sb2$ directly caused an inhibition of fibroblast proliferation, and fibroblasts derived from fibrotic tissues were shown to be hyporesponsive to this mediator. Supernatants from unstimulated alveolar macrophages obtained from rats which had been given intratracheal bleomycin caused a similar effect on the proliferation of rat lung fibroblasts.</p> <p>Primary lines of fibroblasts chronically exposed to peripheral blood monocyte supernatants became hyporesponsive to both these supernatants and prostaglandin E$\sb2$, and released greater amounts of prostaglandin E$\sb2$, upon rechallenge compared to unexposed fibroblasts. An examination of clonally-derived lines showed marked heterogeneity in the responsiveness of individual clones to peripheral blood monocyte supernatants and prostaglandin E$\sb2$ as well as a change in the clonal distribution after chronic exposure to peripheral blood monocyte supernatants. Chronic exposure of sensitive clones to peripheral blood monocyte supernatants did not alter their level of responsiveness.</p> / Doctor of Philosophy (PhD)

<p>Endothelial cells contain a high density of vasoactive intestinal peptide (VIP) receptors. Since VIP is a potent vasodilator, activation of these receptors may lead to the release of endothelium-dependent relaxing factor (EDRF). Using patch-clamp techniques it was demonstrated that VIP modulates plasma membrane K$\sp+$ channels in bovine pulmonary artery endothelial cells. VIP inhibited activity of inwardly rectifying K$\sp+$ channels (I$\sb{\rm Kin}$) and activated opening of the Ca$\sp{2+}$-dependent K$\sp+$ channels (K$\sb{\rm Ca}$). Activation of K$\sb{\rm Ca}$ channels tends to hyperpolarize the cell membrane. This transient hyperpolarization increases the driving force for influx of extracellular Ca$\sp{2+}$ through nonselective cation channels. This way VIP may contribute to Ca$\sp{2+}$ influx necessary for the potential EDRF production.</p> <p>Although VIP activates adenylate cyclase and production of cyclic AMP (cAMP) in many cell types, this study provides evidence allowing us to exclude cAMP as a second messenger for VIP action in endothelial cells. Direct evidence comes from the measurements of cAMP level in bovine pulmonary artery endothelial cells stimulated with VIP and isoproterenol. Isoproterenol increased cAMP level, whereas VIP did not, or even decreased cAMP production in some cases. Indirect evidence comes from the results of patch-clamp experiments. VIP strongly suppressed I$\sb{\rm Kin}$ channel activity, whereas the inhibitory effect of isoproterenol on this channel was very weak (about 20% of the VIP effect). cAMP elevation may reduce the activity of I$\sb{\rm Kin}$ channel, but is not a mediator for the inhibitory effect of VIP on these channels. In addition, VIP was still able to inhibit I$\sb{\rm Kin}$ channel activity in outside-out patches, where the second messenger system is not operating. This effect was mediated by a G protein that most likely couples directly to the channel. This unidentified G protein revealed GTP-$\gamma$-S and cholera toxin sensitivity and resistance to pertussis toxin.</p> <p>Since elevated intracellular Ca$\sp{2+}$ is a trigger for EDRF production, it has been demonstrated that modulation of the calcium fluxes at the level of internal stores did influence an influx of extracellular Ca$\sp{2+}$. Cyclopiazonic acid (CPA), an inhibitor of SR/ER Ca$\sp{2+}$ pump, (i) induced K$\sb{\rm Ca}$ currents, presumably as a consequence of the spontaneous leakage of Ca$\sp{2+}$ from internal stores, (ii) reduced the I$\sb{\rm Kin}$ currents, and (iii) enhanced influx of extracellular Ca$\sp{2+}$ through nonselective cation channels. Moreover, CPA activated K$\sb{\rm Ca}$ currents in endothelial cells by a mechanism independent of 1,4,5-triphosphate (IP$\sb3$). An expected consequence of this action of CPA may be an influx of extracellular Ca$\sp{2+}$. In parallel studies, it has been demonstrated that CPA relaxed rat aorta in endothelium-dependent manner, suggesting CPA-induced release of EDRF. Ca$\sp{2+}$ enters the endothelial cell through nonselective cation channels, which can be activated by an agonist or a mechanical stimulus (e.g. stretch). It is still unclear whether these nonselective cation channels in bovine pulmonary artery endothelial cells are controlled by free intracellular Ca$\sp{2+}$, empty Ca$\sp{2+}$ ER stores or an agonist.</p> / Doctor of Philosophy (PhD)

<p>Significant differences in the cardiovascular mortality rate exist between Canadians of South Asian, Chinese and European origin. This thesis represents a six year effort to detennine the prevalence of cardiovascular disease, the burden of atherosclerosis, and the prevalence of their major determinants among three ethnic groups in Canada. In addition to the study of the relationship of the "classical" cardiovascular risk factors to disease outcomes, the contribution to disease outcomes of selected "emerging" risk factors (e.g. markers of thrombosis, socio-economic, dietary, and psychosocial stress factors) was studied.</p> <p>The major findings reported in this thesis include the confirmation that South Asians in Canada have the greatest prevalence, Europeans have an intermediate prevalence, and the Chinese have the lowest prevalence of cardiovascular disease. However, Europeans have significantly more atherosclerosis compared to the South Asians and Chinese. Yet, for any given amount of atherosclerosis, South Asians suffer more cardiovascular events. This may reflect an increased propensity of South Asians to develop arterial thrombosis.</p> <p>Major differences in the daily calorie consumption, and in the sources of calories exist between the ethnic groups. Diet was measured using food frequency questionnaires developed for each ethnic group, which were found to reliably classify individuals into high or low consumers for a given macronutrient. This classification facilitates the study of diet-disease relationships.</p> <p>Psychosocial stress was measured in a valid and reliable way using a single questionnaire created for use by all participants. Discrimination in the workplace is a challenge to measure, and while it was reported more often among South Asians and Chinese, its reproducibility was relatively low. Markers of social stability (e.g. income and employment) were associated with psychosocial stress factors, neurohormones, atherosclerosis and cardiovascular disease. This demonstrates the powerful influence that "social factors" have on the health of free-living Canadians.</p> / Doctor of Philosophy (PhD)

Previous studies suggested that teniposide is a strong clastogen, and that the DNA breakage effect of this drug is mediated by the nuclear enzyme topoisomerase II. Ripley et al found evidence for a correspondence between sites of acridine-induced frameshift mutations in bacteriophage T4 and sites of in Vitro DNA cleavage by T4 topoisomerase II. To identify the sequence specificity of teniposide-induced deletion and insertion mutations in mammalian cells, the CHO-D422 cell line, which is hemizygous at the aprt locus, was employed in this study. Sixty-eight teniposide-induced and 42 spontaneous aprt mutants were analyzed at the DNA sequence level. Compared with the spectrum of spontaneous mutations in which two thirds of the mutations are base substitutions, two thirds of teniposide-induced mutations are deletions and insertions of different sizes. Significant site correspondence between teniposide-induced deletion/insertion mutations and in vitro DNA double strand cleavages by purified mammalian topoisomerase II was also found in this study, which suggests that the majority of teniposide-induced deletions and insertions observed in this study were generated at the sites of topoisomerase II mediated DNA double strand breaks in the cells. However, considering the positions of the double cleavage sites in the mutation sequences, no consistent pattern was found which could suggest a unified mechanism of DNA double strand break repair. Three models are proposed to try to explain the possible events occurring in the cells following teniposide exposure which resulted in observed deletion and insertion mutations.

Summary: 1. The type of animal blood used in preparing blood agar plates did not significantly affect the demonstration of hemolysis by any streptococcal species except for S.faecalis. S.faecalis more consistently displayed an alpha reaction on sheep blood agar while expressing a beta reaction on horse and rabbit blood agars. 2. A recommended scheme for identifying streptococci, adaptable to most any clinical laboratory, was developed and is outlined in Table 13. 3. S.agalactiae (group B) was the most frequent beta hemolytic streptococcal isolant. Significantly, it was the only species over-all to be recovered from every clinical source. The CAMP test proved to be the best method for the presumptive identification of the group B streptococci. 4. The alpha streptococci were isolated in substantial numbers from a variety of infectious situations suggesting that their role in infection may be greater than previously believed. 5. S.avium accounted for a greater percentage of group D isolations than previously reported in other studies of a similar nature. 6. The bile-esculin test was supported as the best nonserological method for distinguishing the group D streptococci. Arginine hydrolysis was shown to be the best method for distinguishing between the enterococci and the nonenterococci. The test commonly used for this purpose, growth in 6.5% NaCl, resulted in the misidentification of S.avium as an enterococcal species. 7. Urinary tract isolants from every division (notably S.agalactiae, S.mutans. and S.faecalis) exhibited patterns of overall greater antibiotic resistance than those strains isolated from other sources. 8. Among the group D species, S.faecalis displayed the greatest antibiotic resistance followed by S.avium, S.faecium, and S.bovis. S.avium, a nonenterococcus. showed greater drug resistance than S.faecium, an enterococcus. S.bovis was the least resistant with susceptibility patterns more closely related to the alpha streptococci.

Innate immunity depends on pattern recognition receptors, which recognize pathogen associated molecular patterns (PAMPS), such as Toll-like receptor 4 (TLR4), which detects the gram-negative bacterial toxin, lipopolysaccharide. Engagement of TLR4 by LPS sets off a cascade ending in the activation of pro-inflammatory cytokines and interferon-β (IFN-β) which alerts the host to the infection. However, these responses can be mal-adaptive, especially in the context of bacterial sepsis, where a "cytokine storm" results in death of the host. Pharmacological modulation of these responses may therefore be a promising treatment modality. Inhibition of classic pro-inflammatory cytokines such as IL-1β and TNF-α has been the (largely unfruitful) focus of much research. However it has recently emerged that mice with defects in type I IFN signaling are also substantially resistant to challenge with endotoxin. We therefore wish to investigate pharmacological inhibition of IFN signaling as a potential means to control sepsis. We analyzed the effects of Trichostatin A (TSA) and Suberoylanilide hydroxamic acid (SAHA) (both broad spectrum HDAC inhibitors) and ST-2-92 (HDAC6 specific inhibitor) on IFN regulation and endotoxic shock. We created an in vivo mouse model for this treatment with TSA and SAHA (which are well tolerated in mouse and human) to look for possible alteration in the survival rate following endotoxin challenge. We as well as others found that treatment with SAHA (50mg/kg) significantly improves survival rate. We also characterized in-vitro modulation of IFN responses through SAHA by mouse DNA microarray. We noticed a decreased expression of many innate immune regulated genes in the SAHA and LPS treated condition compared to the LPS treatment alone. Additionally we observed a decrease in protein levels of IFN-β IL-1β, IL-6, IL-12p40, RANTES and TNF-α in cell culture supernatants treated with SAHA or ST-2-92 and LPS compared to LPS only treatment. These results show the ability of broad spectrum HDACi through SAHA to increase mouse survival following LPS challenge as well as modulate the induction of innate immune responsive genes in vitro. Furthermore we have shown that HDAC specific inhibition through ST-2-92 can decrease pro-inflammatory transcript as well as protein levels.

In the United States, blood product availability is dependent entirely on donations from volunteer blood donors. Current trends in blood collection and utilization raise concerns about the ability to meet future demands for blood products. At a time of high demand and deferrals, the Armed Services Blood Program has been unable to meet its requirements for blood and consequently needs to purchase blood from civilian agencies to meet the dual demands of the military community at home, as well as those deployed around the world. This creates a need to better understand the military blood donor in an effort to recruit and retain those relative few who are willing and eligible to donate. The purpose of this survey-based research is to characterize the demographics of the military blood donor and to understand, through descriptive and regression analysis, the relationship between a donor’s attitude, subjective norms, and perceived behavioral control and their intention to donate blood again in the next six months. The study uses the framework of the theory of planned behavior to design the survey and evaluate relationships between the theoretical constructs. Descriptive analysis results of the sample demographics describe the typical respondent as White, young, male, and a junior service member. Results of the multivariate regression analysis showed that a respondent’s attitude toward blood donation and perceived behavioral control over donating blood were positively related to their intention to donate again in the next six months. However, contrary to the theory, there was no statistically significant relationship between the influence of subjective norms and intention to donate again. This study is the first to apply a theoretical framework to identify those factors that influence a military blood donor to donate blood. Further, it has taken steps to provide a clear description of the typical military blood donor. Future experimental research can now be designed with the aim of developing efficient and effective blood donor recruiting and retention campaigns. Specifically, the understanding of the demographics of the population allows targeted interventions to underrepresented groups, and theoretical research will further guide interventions that target those important motivational factors influencing blood donation.

Enamel demineralization in the form of white spot lesions (WSLs) around fixed orthodontic appliances is a persistent problem in patients with poor oral hygiene.These lesions can form rapidly within 4 weeks of bracket placement.The purpose of this in-vitro study was to investigate the fluoride recharging capability ofa commercially available orthodontic primer used to minimize the development of WSLs in patients. The three groups tested were: OpalSeal (n=20, Ultradent, South Jordan, UT), ProSeal (n=20, Reliance, Itasca, IL) and Transbond XT (control, n=10, 3M Unitek, Monrovia, CA). The samples(5mmin diameter x 1mm in thickness) weresuspended individually in vials filled with 10mL of deionized water usinga fishing line. The baseline fluoride ion release from all of the samples was measured after two weeks of changing the solution every other day. The samples were then randomly divided into two groups, toothbrush or gel. The samples in the toothbrush group were brushed for one minute every day for 7d, with fluoride containing toothpaste (Colgate-Palmolive Company, New York, NY) and placed in a new solution after each brushing. After 7d of brushing the fluoride ion release was measured. The samples in the gel group were immersed in 10mL of acidulated phosphoric fluoride gel (APF) for one minute, following manufacturer’s instructions, and then placed in a new vial with 10mL of deionized water. At the end of 24hrs fluoride ion release measurementswere made and the samples were placed individually in a new solution. The solution was changed weekly in the gel group over six weeks to simulate the typical length of time between two orthodontic appointments. A final fluoride ion release measurement was taken of all the discs in the gel group 6 weeks after the fluoride gel treatment. The results of repeated-measures analysis indicated that there were no significant differences between the groups at baseline and after 7d of toothbrushing time points. Opal Seal exhibited a significant increase in fluoride uptake (1.0ppm) after 24hrs of fluoride gel exposure but these levels gradually decreasedover 6 weeks (0.04ppm). Pro Seal and Transbond showed no significant fluoride release after the gel or toothpaste applications. The fluoride-containing primer, Opal Seal, had the ability to be recharged with fluoride ions from APF gel. However, the amount of fluoride released from recharged discs decreased gradually over a 6 weeks of time.

Epigenetics is the study of heritable gene expression due to alterations in the DNA structure other than the underlying DNA sequence. DNA methylation is one of the three types of epigenetic modifications found in the eukaryotic system. It involves the incorporation of a methyl group at the 5-position of cytosine residues in the DNA. DNA methylation is associated with several notorious disorders and diseases including Fragile X Syndrome, neurodegenerative disease (Parkinson’s, Alzhiemer, etc), diabetes and cancer. Cytosine methylation of mitochondrial DNA (mtDNA) was first demonstrated several decades ago but the mechanism of generating cytosine modification and its functional importance remain elusive. Our laboratory recently demonstrated that the enzyme involved in cytosine modification of mtDNA is a novel mitochondrial isoform of DNA Methyltransferase 1, mtDNMT1. This protein is encoded in the nucleus and targeted to the mitochondria via a N-terminal targeting sequence. Bioinformatic analysis of the DNMT1 coding sequence showed a consensus NRF1 binding site that coincidently overlaps a p53 binding site within the promoter region, previously shown by this group to repress DNMT1 expression. Previous studies in the Taylor laboratory showed that mtDNMT protein expression was regulated by the transcription factor NRF1 as well as its coactivator PGC1α. PGC1α and NRF1 stimulate a large body of genes that are involved in mitochondrial biogenesis and cellular respiration in response to environmental stress. Considering the previous findings in our laboratory regarding mtDNMT1 regulation and the importance of PGC1α and NRF1 in oxidative homeostasis, we asked whether there is a mitochondrial epigenetic component in the cell’s response to cellular stress and whether up-regulation of mtDNMT1 might be part of the general response to this stress. To investigate the relationship between mtDNA methylation and oxidative homeostasis we examined the regulation of mtDNMT1 by transcription factors that respond to oxidative stress. Conditions that induced oxidative stress were applied to HCT 116 and SH-SY5Y cell lines and the protein expression of DNMT1 was observed. Ethanol and hypoxia- induced oxidative stress were observed to increase to protein level of mtDNMT1 while total DNMT1 level either remained constant or decreased. The protein level of PGC1α and NRF1 remained low in HCT 116 cells exposed to hypoxic stress, despite elevated mtDNMT1 protein level. ChIP analysis of HCT 116 cells exposed to hypoxic stress demonstrated that NRF1 and PGC1α are not regulating the transcription of DNMT1i in the mitochondria. However, we observed that p53 dissociated from the DNMT1 promoter upon hypoxic stress, indicating that the up-regulation of mtDNMT1 is through the relief of p53 suppression. The findings of this investigation proved that mtDNMT1 is receptive to oxidative stress through the regulation by p53 and suggested that mitochondrial epigenetics may be playing an integral role in the cellular stress response toward hypoxia.