<p>By studying signatures of life within extreme environments on Earth we better understand signatures of life that may be found within the geologic record on Earth as well as elsewhere in the universe. This thesis research utilized phospholipid fatty acid analysis (PLFA) in combination with carbon isotopic analysis (δ¹³C) to understand microbial carbon cycling and biosignatures within two Mars analogue environments.</p> <p>The Golden Deposit was an acid sulfate environment that was an analogue to the acid-sulfate conditions present on early Mars. Mixing of inflowing surface water and upwelling acidic groundwater on the deposit caused variations in water chemistry. Such variations resulted in differences in the microbial communities and carbon isotopic compositions within the Golden Deposit. The most acidic groundwater seep on the deposit had a distinct geochemistry, which selected for a unique microbial community and distinct carbon isotopic signatures. Variations detected within the small spatial area of the Golden Deposit indicated that biosignatures preserved on Mars might be highly variable making the unambiguous identification of past life on the planet more difficult.</p> <p>Three saline lakes of the Cariboo Plateau were used for this study and were analogues to environmental conditions present on early Earth as well as the alkaline, evaporitic conditions present on early Mars. All three lakes contained benthic microbial mat communities dominated by cyanobacteria. Photosynthetic influences by cyanobacteria resulted in an isotopic enrichment of the DIC pool. Enriched lake DIC values were preserved in precipitated carbonates (δ¹³C_carb) and the ∆¹³C_DIC-TOC(avg) was similarly preserved in ∆¹³C_carb-TOCvalues and were biosignatures of photosynthetic activity. The preservation of these biosignatures suggested that concentrations of CO₂ on early Earth did not necessarily exceed modern levels and that measuring the carbon isotopic compositions of carbon pools within saline, carbonate-rich deposits on Mars could lead to the identification of past life on the planet.</p> / Master of Science (MS)

<p>Direct VIP-nerve and indirect ICC-mediated inhibitory control of colon motility were studied using biochemical techniques. In order to define a basis for VIP action in colon, longitudinal muscle, ICC-poor circular muscle and ICC-rich layers separated by dissection were characterized by electron microscopy and subcellular fractionation using markers for synaptosomes, smooth muscle membrane and mitochondria. Synaptosmal, mitochondrial and smooth muscle enriched membrane fractions were isolated but a putative ICC membrane enriched fractions has the highest 5'nucleotidase, Mg2+ATPASE activities and density of 125 I-VIP binding followed by synaptosome and smooth muscle membranes. High VIP binding density can be used as a marker for ICC membrane.</p> <p>The study of nitric oxide production in fractions from ICC-rich and-poor preparation by measuring nitrite levels showed that mitochondrial, synaptosome and ICC-rich membrane fractions produced nitric oxide but greater levels were seen in the ICC-membrane rich fraction. Nitric oxide synthase activity on ICC membrane was constitutive and calcium-dependant. VIP increased (2-fold) nitric oxide production in ICC-rich strips which was inhibited by L-NAME, reduced by EGTA and increased by exogenous calcium. Partial inhibition of VIP-induce nitric oxide production by ω-Conotoxin GVIA was recorded in the absence (22%) and presence (32%) of L-arginine suggesting that the source of nitric oxide was in large part non-neural, from ICC.</p> <p>I conclude that direct nerve-mediated control of longitudinal and circular muscle occurs through VIP binding on membrane receptors while indirect control of circular muscle occur through VIP-nerve induced production of nitric oxide from ICC to cause smooth muscle relaxation.</p> / Doctor of Philosophy (PhD)

<p>Many people suffer from some form of low back pain during their lifetime. A well designed morbidity study of general practice in England; using data collected from over 100 general practices, showed the incidence to be 1.6% per year and the prevalence to be 17.5% (62). Severe back pain may necessitate job changes or even job loss.</p> <p>Forty-two million dollars were spent on industrial back injuries during 1974 in Ontario (86).</p> <p>Review of the literature is hampered by the fact that only in a minority of cases can a diagnosis be made with any degree of certainty based on knowledge of pathophysiological mechanisms. Perhaps even less relisble is the chance that the doctor may know some therapy which will actually be superior to the spontaneous recovery rate. The spontaneous recovery rate is estimated at 70% after three weeks of symptoms (76). Because of the lack of progress in the area of diagnosis and treatment many studies lack methodological rigor.</p> <p>This thesis attempts to examine and summarize some of the physiological, anatomical and mechanical factors that may be implicated in the etiology of low back pain and serve as a basis for rational therapy. It also reviews the encouraging advances in basic research which have taken place in the last decade.</p> <p>In an effort to determine whether proven clinical outcomes occur, with certain standard treatment regimes, a randomized clinical trial is proposed to test the effectiveness of four different programs in a two by two factorial design. All participants will receive analgesics and/or anti-inflammatory agents at the discretion of their family physician. One group will receive no further treatment. The remaining three groups will have bed rest alone, bed rest with physiotherapy, or physiotherapy alone.</p> <p>Baseline measurements will be obtained by objective methods of assessing spinal flexion, pain, and activities of daily living.</p> <p>Prior to the commencement of the trial the study population will be divided into two prognostic groups by the method center based on the decision of the family physician to give the patient either major anti-inflammatory medication or minor analgesics.</p> <p>It is expected that about 260 subjects suffering from low back pain will be identified by five groups of family physicians within a period of six months. For the patients receiving physiotherapy the same physiotherapist will visit each of the five groups two half-days a week in order to supervise the treatment. Compliance with medication will be assessed by pill counts.</p> <p>Assessment of bed rest compliance will be measured by self-reporting, reports from a friend and a home visit.</p> <p>One month after treatments begin, another family physician who is blind as to which group the patient is in will perform some of the outcome measures. Outcome results will consist of relief of pain, return to work, return to normal activities, and relapse rate up to three months.</p> / Master of Science (MS)

<p>This thesis examines psychosocial effects in a population living near the Halton Regional Landfill site in Milton, Ontario. The data described and analyzed in this research were collected between 1990 and 1995 and capture key events in the landfill site's history--site approval, construction and operation. This longitudinal study, which examines indicators of stress, reappraisal and coping at three different points in time from the same individuals, is designed to document and examine the process whereby these people have experienced and come to live with the landfill facility over time. The theoretical basis for understanding this process of appraisal, reappraisal and coping is derived from environmental stress and coping theory generally, and from the framework proposed by Lazarus and Folkman (1984) specifically. Three research objectives are addressed: (1) to document changes in psychosocial effects in Milton area residents between 1990 and 1995; (2) to explore the processes of reappraisal and coping among Milton area residents; and, (3) to evaluate the appropriateness and usefulness of the Lazarus and Folkman (1984) framework in the context of a population living proximate to a solid waste facility. These objectives are addressed through three administrations of a telephone survey instrument comprised of a combination of pre-validated scales and open- and closed-ended items to a random stratified (by distance) sample of households immediately after the landfill site was approved (1990), shortly after the site was constructed and began operation (1992-3), and two years after operation had begun (1995). Due to attrition, the baseline cohort (n = 187) was reduced to 108 by time 3, although no significant bias was introduced. Data from all three surveys were obtained for a sub-group of 87 respondents. Five constructs derived from environmental stress and coping theory guide the examination of individual responses to the landfill over time: psychological distress, perceptions of the landfill site, concerns about the landfill site, actions and neighbourhood satisfaction. A series of hypotheses, which capture expectations about how Milton area residents have experienced the introduction of the landfill site into their community over time, were generated from the constructs and guide the analysis. Overall, the results portray a community that has changed from one actively opposing plans to locate a landfill site in its midst to one that has, for the most part, resigned itself to and accepted the presence of the new facility. (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)

<p>There is evidence that a number of psychiatric disorders, particularly depression and schizophrenia, are frequently associated with lateralized disturbances of brain function, or alterations in normal patterns of lateralized function. It is also known that many such disturbances, especially in schizophrenia, involve central dopamine (DA) systems. Several studies in recent years have established that lower animals often exhibit lateralization of brain function, both on the neurochemical and behavioral level. Central DA systems appear to be particularly asymmetrical. Left/right hemispheric asymmetries in rats have been reported at the population level for a variety of DA-modulated behaviors which may variously reflect motor, sensory, spatial, or stress/arousal processes. However, the directions of reported population asymmetries can vary across studies, depending in part upon the particular processes predominantly reflected in the measured behavior, and consequently leading to difficulties in interpretation. Given the potential relevance to both normal and abnormal brain function in humans, it is of much interest to determine what paraliels exist between lateralization in humans and lower animals.</p> <p>The objectives of the present thesis were twofold. First, we sought to determine which DA-mediated behaviors exhibit left/right hemispheric asymmetries at the population level in rats, by employing specific paradigms to measure motor, sensorimotor, spatial and stress-related processes. The basic approach was to compare the effects of unilateral DA-depleting lesions (with 6-hydroxydopamine), in left or right brain structures of male rats. Behavioral and neurochemical asymmetries were also examined in nonlesioned controls. A second objective was to study the role of interhemispheric connections in the expression of behavioral asymmetries characteristic of rats with unilateral lesion-induced DA depletion. Specifically, we describe the effects of sectioning the corpus callosum in unilaterally lesioned (6-OHDA) rats, on motor and sensorimotor asymmetries.</p> <p>Regarding the first objective, hemispheric population asymmetries were not found for any of three measures of motor activation, in rats with left or right lesions of the substantia nigra. These measures included ipsiversive turning behavior in response to amphetamine, contraversive turning in response to apomorphine and spontaneous locomotor measures in activity monitors. Similarly, groups did not differ in a measure of sensory/spatial bias, namely the orientation to edges during exploration of a large openfield. The same animals did differ however, in the performance of the Morris water maze task for spatial localization, suggesting that right brain mechanisms may be preferentially involved in successful task performance. A follow-up study with the water maze paradigm, using nonlesioned rats distinguished by the preferred direction of amphetamine-induced turning (and by inference the hemisphere of greater DA activity), further supported a preferential role for right brain DAergic mechanisms in this task. An additional test of population hemispheric asymmetry which focused on stress mechanisms, compared the effects of mesocortical DA depletion (left, right or hilateral) on the development of restraint stress-induced gastric pathology. Rats depleted of DA in the right anterior midline cortex, developed significantly more severe stress pathology than did nonlesioned controls. In contrast, left or bilateral cortical DA depletion resulted in nonsignificant trends for increased pathology. All three lesion types resulted in significant and unique effects on DAergic systems in subcortical brain structures, which may have in part contributed to the asymmetric effects on development of stress pathology.</p> <p>Regarding the second objective of the study, it was found that corpus callosum section eliminated the asymmetrical orientation to openfield edges in unilaterally lesioned rats. Conversely, there was no effect of callosotomy on asymmetries in direction of turning behavior, either drug-induced or externally cued in the behavioral competition for food. Taken together with the report that callosal section potentiates lateralization of emotional expression, the findings emphasize the anatomical dissociability of these functional asymmetries, despite their mediation by DAergic systems at various levels.</p> <p>Based on these and other literature reports, it is proposed that the most fundamental processes exhibiting consistent left/right hemispheric population biases in rats, are those related to stress. The greatest degree of functional asymmetry is found in the cortex, which modulates sutcortical structures in a highly asymmetrical manner. The data extend recent suggestions that the right cortex is preferentially involved in the mediation of high arousal states (such as uncontrollable stress). Other studies have shown that activation of mesocortical DA by stress initially favors the left brain, and later predominates in the right brain as stress is prolonged. Given the evidence that cortical DA facilitates coping ability, and based on a variety of neurochemical and behavioral reports of DAergic asymmetries, it is suggested that a normal left brain DAergic dominance may exist at the population level, for both rats and humans. Such an asymmetry is proposed to confer an adaptive advantage in the rapid execution of responses to minor stressors. Finally, it is proposed that disturbances in patterns of cortical activity may lead to (psycho)pathological states which are associated with vulnerability to stress.</p> / Doctor of Philosophy (PhD)

<p>This thesis examines the sensitivity and specificity of manometry and defecography assessments; the relationship between function and symptoms; and the relationship between age and parity and patient assessments.</p> <p>The manometry assessments of 72 incontinent and 50 constipated female patients were compared to 86 healthy volunteers using discriminant function and classification analysis (DFA). The defecography assesssments of a subset of these patients, 21 incontinent and 25 constipated, were compared to 22 healthy female volunteers. These data were used to examine the factors age, parity, severity of symptoms and rectal wall morphology on function.</p> <p>The results show that the variables of total squeeze pressure and resting pressure have a sensitivity of 79% for the incontinent patients and 32% for the constipated patients. The specificity was 87%. The manometry variables resting pressure, squeeze pressures, volume to urgency were significantly different in the patient groups. Aging was a significant factor for lower resting pressures and increased parity was a significant factor for lower squeeze pressures in the patient groups.</p> <p>The sensitivity of the combination of the defecography variables, lift and strain angles and junction levdls, was 90% for the incontinent patients and 88% for the constipated patients. The specificity was 95%. The defecography variables were not significantly different in the patient groups. Rest and lift angles were significantly wider with increased age and parity.</p> <p>Neither the defecography and manometry variables nor rectal wall morphology changes were associated with varying severity of either constipation or incontinence.</p> <p>The manometry and defecography assessments are presented in graphs, which may enhance the dinical usefulness of the assessments by demonstrating the difference between patient values and healthy controls. The manometry data are also presented in an index which makes areas of specific impairment more obvious.</p> <p>DFA of the manomeby and defecography variables provides probability rates which may be useful in predicting patient outcomes. The discriminant scores from the analysis of the defecography and manometry variables can be used to develop a continuum from health to incontinence.</p> / Doctor of Philosophy (PhD)

Maternally transmitted microbes that infect host germ cells are perfectly poised to influence animal genome evolution, either directly through horizontal gene transfer or indirectly through selection for host genetic variants that control symbiont proliferation. In this dissertation, both of these scenarios are examined using the obligate, intracellular bacteria Wolbachia as a model for maternally-transmitted bacteria because of its widespread distribution across 40-50% of all arthropod species and its localization to the maternal germ line cells. In the first project, whole-genome sequencing of uninfected Chorthippus parallelus grasshoppers revealed that large regions of DNA from two different supergroups of Wolbachia had horizontally transferred into the grasshopper genome. While horizontal gene transfer of Wolbachia DNA to a eukaryotic host is common given Wolbachia?s proximity to the germ line genome, this is the first known example of two divergent Wolbachia strains contributing DNA to the same host genome. Furthermore, Wolbachia inserts were present in almost all of the grasshopper chromosomes and often differed between closely-related C. parallelus subspecies, indicating that horizontal gene transfer from Wolbachia is an unusually dynamic process in grasshoppers. In the second project, a forward genetic screen was conducted to find host genomic regions responsible for regulating an 80-fold difference in Wolbachia titers between two closely-related species of Nasonia parasitoid wasps (N. vitripennis and N. giraulti). Quantitative trait loci analyses and hybrid introgressions identified two genomic regions, one each on chromosomes 2 and 3, that act additively through a maternal effect to suppress Wolbachia titers in N. vitripennis. Thirty-three significantly differentially expressed genes are present in these regions, several of which function in pathways important for host control of intracellular bacteria including immunity, autophagy, and cell-to-cell trafficking. Additionally, staining of Nasonia ovaries with a nucleic acid dye revealed that N. vitripennis may keep Wolbachia out of developing oocytes by sequestering them in the neighboring nurse cells. Candidate genes that are overexpressed in N. vitripennis, such as trichohyalin, or those involved in Wolbachia trafficking, such as kinesin, are currently being evaluated using RNAi for their role in host regulation of Wolbachia titers and transmission.

Glycosylation is the most common post-translational modification to proteins, involving the addition of chained sugars to regulate folding, localization and intermolecular interactions. Glycosylated proteins are most heavily concentrated on the extracellular side of cellular membranes, and most secreted proteins are glycosylated. Both of these glycoprotein classes are critical for cell-cell interactions, particularly during metazoan development. The nervous system is enriched for glycoproteins, and neurons appear dependent on glycosylation in the regulation of synapse structure and function. This thesis tests glycosylation roles at the synapse by analyses of two genes at the Drosophila neuromuscular junction (NMJ); mgat1 required to produce hybrid and complex glycan branches, and pmm2 required to produce all N-linked glycosylation. Loss of either gene results in defective synaptic glycosylation, and similarly overelaborated NMJ architecture and elevated neurotransmission. Moreover, both mutant conditions cause aberrant trans-synaptic signaling that normally directs the recruitment of synaptic proteins required for synaptogenesis and neurotransmission. Thus, synaptic glycosylation strongly modulates the trans-synaptic signaling that in turn drives the recruitment synaptic proteins that mediate of structural and functional synaptogenesis. This thesis produces a new genetic model for the heritable Congenital Disorders of Glycosylation (CDG) disease state CDG1a (a.k.a. PMM2-CDG), producing an avenue for the development of treatments and therapeutic interventions.

Non-muscle myosin II (MyoII) contractility is important to the regulation of many cellular processes, including migration. The small GTPase Rho has been shown to regulate MyoII contractility, but the role of other GTPases, such as Rac, in modulating contractility is not well understood. In this study, we show that activation of Rac by the guanine nucleotide exchange factor (GEF) Asef2 increases MyoII contractility to impair cell migration on two-dimensional (2D) type I collagen. Knockdown of endogenous Rac using short hairpin RNAs (shRNAs) or treatment of cells with the Rac-specific inhibitor NSC23766 results in a significant decrease in the amount of active MyoII, as determined by serine 19 (S19) phosphorylation, and negates the Asef2-promoted increase in contractility. Furthermore, treatment of cells with blebbistatin, which inhibits MyoII activity, abolishes the Asef2-mediated effect on migration. Because three-dimensional (3D) matrices more closely mimic the physiologic environment of cells, we investigated the role of Asef2 in regulating migration and MyoII activity in 3D environments. For these studies, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that Asef2 inhibits cell migration in 3D type I collagen matrices, and treatment of cells with blebbistatin abolishes the Asef2-mediated decrease in migration. Moreover, Asef2 enhances MyoII activity as shown by increased S19 phosphorylation, and treatment with NSC23766 abolishes the Asef2-promoted increase in active MyoII. Collectively, these results indicate that Rac activation, promoted by Asef2, is critical for modulating MyoII activity and cell migration in both 2D and 3D environments.

Carbonic anhydrases (CAs) are zinc-metalloenzymes that interconvert two inorganic carbon (Ci) species, CO2 and HCO3-. In Arabidopsis thaliana, there are eight Alpha-CA genes, six Beta-CA genes, three Gamma-CA genes, and two Gamma-CA-like genes. The majority of CA research in plants has focused on finding a link between CA activity and photosynthesis rates. Since the CA genes are expressed in different plant tissues and multiple CA isoforms are distributed among various organelles of the plant cell, I hypothesize that CAs facilitate CO2 diffusion among cell compartments and maintain Ci pools for carbon-requiring reactions by interconverting CO2 and HCO3-. This thesis focuses on the Alpha-CAs and Beta-CAs of Arabidopsis and how they may affect various reactions throughout the plant. CA T-DNA insertion lines were used to determine if removing one or more CAs from Arabidopsis affects the plant growth. The Beta-ca5 single mutant and Beta-ca2Beta-ca4, Alpha-ca1Beta-ca4, and Alpha-ca2Beta-ca4 double mutants show different growth phenotypes. The Beta-ca2Beta-ca4 plants were smaller in size and chlorotic in their younger leaves under low CO2 conditions, but showed improved growth in high CO2 conditions. The growth of the Beta-ca5 single mutant was severely stunted in ambient CO2 conditions and high CO2 partially rescued wildtype growth in the Beta-ca5 plants. The Alpha-ca1Beta-ca4 and Alpha-ca2Beta-ca4 double mutants were slightly smaller than wildtype plants in low CO2 conditions. Interestingly, it seems the reduced growth of the Beta-ca5 single mutant and Beta-ca2Beta-ca4 double mutant plants was not linked to deficiencies in photosynthesis rates but rather may be required for other carbon requiring reactions. These results suggest that CAs are playing more complex roles in plants than once thought and that the various isoforms are affecting different carbon-requiring pathways.