published_or_final_version / Social Work and Social Administration / Doctoral / Doctor of Philosophy

Molecular and in vitro comparisons were performed using two isolates of Encephalitozoon hellem, one from an avian host and one from a human host, and one isolate of Encephalitozoon cuniculi from a rabbit. The molecular comparisons were performed by amplifying and sequencing the gene coding for a zinc metallo-aminopeptidase from cDNA and gDNA obtained from each of the isolates. The E. hellem sequences shared >99 % identity between each other and 70% identity with the E. cuniculi sequences. Conserved HEXXH and GXMEN motifs located within the sequences classify the protein as an aminopeptidase of the M1 family, with at least one zinc atom required for catalytic activity. In vitro growth comparisons of the isolates described above were performed under simulated "mammalian and avian conditions". The models utilized mammalian and avian cell lines and sera at incubation temperatures of 37 °C and 40 °C, respectively. Three separate experiments were performed. E. cuniculi grew best under the mammalian model and significantly better than both E. hellem isolates under this model. The E. hellem isolates were able to infect and replicate under both the mammalian and avian models, which reflects the zoonotic potential of these isolates.

An in vitro model system utilizing de-mineralized periodontium tissues was developed for investigating the molecular controls involved in the spatial deposition of minerals on collagenous tissues. Preferential mineral deposition was observed when the de-mineralized tissue sections were incubated in solutions containing a supersaturation of calcium and phosphate ions with respect to hydroxyapatite (HA). Energy dispersive X-Ray (EDX) analysis demonstrated that these minerals are likely to be octacalcium phosphate (OCP) or dicalcium phosphate dihydrate (DCPD); further characterization with a secondary technique is required to draw a more definitive conclusion. The role of collagen fibrils in mineralization was tested by removing all the non-collagenous components from the tissue sections with proteolytic enzymes and exposing them to similar mineralization conditions as the control samples. A substantially less amount of minerals were formed in these samples; this correlates well with the hypothesis in the literature that collagen fibrils alone cannot initiate mineral formation.

An in vitro model system utilizing de-mineralized periodontium tissues was developed for investigating the molecular controls involved in the spatial deposition of minerals on collagenous tissues. Preferential mineral deposition was observed when the de-mineralized tissue sections were incubated in solutions containing a supersaturation of calcium and phosphate ions with respect to hydroxyapatite (HA). Energy dispersive X-Ray (EDX) analysis demonstrated that these minerals are likely to be octacalcium phosphate (OCP) or dicalcium phosphate dihydrate (DCPD); further characterization with a secondary technique is required to draw a more definitive conclusion. The role of collagen fibrils in mineralization was tested by removing all the non-collagenous components from the tissue sections with proteolytic enzymes and exposing them to similar mineralization conditions as the control samples. A substantially less amount of minerals were formed in these samples; this correlates well with the hypothesis in the literature that collagen fibrils alone cannot initiate mineral formation.

The effects of a simulated in vitro digestion model on the viscosity of solutions of locust bean gum, guar gum, fenugreek gum, xanthan gum, gum Arabic, psyllium, flaxseed gum and soy soluble polysaccharides (SSPS) were examined in this study. All hydrocolloid solutions were formulated for low viscosity (LV), medium viscosity (MV) and high viscosity (HV), which were subsequently subjected to 3 treatments of equal volumes each. The treatments consisted of 1) H2O-dilutions, 2) acid and alkali in the absence of enzymes/bile and 3) an in vitro digestion model simulating the gastric and duodenal phases with pH changes in the presence of hydrolytic enzymes and bile salts. All hydrocolloids showed substantial reductions in viscosity, with dilutions exerting the greatest effect. Depending on the concentration, xanthan gum retained 20-50% of its initial viscosity while the other solutions were in a lower range of 1-16%, thereby showing considerable resilience to the 3 simulated conditions. / NSERC

Tissue-cultured shoots and plantlets usually have leaves with non-functional, open stomata and little epicuticular and cuticular wax, resulting in excess evapotranspiration after transplantation. Various strategies were evaluated to decrease ex vitro acclimatization difficulties for 'Silvan' blackberry, including transplanting unrooted shoots, increasing the medium agar concentration from 6 to 9 or 12 g/l and diluting the basal medium. Increased medium agar concentrations and medium dilution did not improve survival or growth. Stomatal function resumed sooner in new leaves of plantlets than shoots. High relative humidity ($>$95%) and low light intensity (90 $ mu$mol s$ sp{-1}$ m$ sp{-2}$) negatively affected stomatal closure both on acclimatizing transplants and greenhouse-grown plants. Guard cells developed on leaves in vitro were physiologically active but had apparent anatomical abnormalities that inhibited closure. A rapid clearing and staining method was developed for examination of foliar morphology using intact in vitro blackberry (Rubus sp. 'Silvan') and strawberry (Fragaria x ananassa Duch. 'Totem') plantlets and sections of greenhouse-grown 'Silvan' and 'Totem' leaves. This method involved three steps: (1) removing the chlorophyll by autoclaving in 80% ethanol; (2) dissolution of the protoplasm using 5% NaOH at 80$ sp circ$C; (3) post-alkali treatment with 75% bleach (4.5% NaClO) at room temperature for tissue-cultured plantlets and at 55$ sp circ$C for greenhouse-grown leaves. Aqueous safranin (10 mg/l) was used for staining.

The objective of this study was to develop a reliable method for intracytoplasmic sperm injection (ICSI) in bovine oocytes. Oocytes recovered from abattoir-derived ovaries were centrifuged for 5 min at 6000xg to facilitate sperm injection. Sperm were pre-treated in vitro with 5mM dithiothreitol (DTT), and diluted (approximately 1:5) with 5% polyvinylpyrrolidone (PVP) in 0.9% saline. After sperm injection, various activation procedures were compared. Initially, 3 h after activation with 5muM Ionomycin (A23187), oocytes with second polar bodies were selected and treated with 1.9mM 6-dimethylaminopurine (DMAP). The cleavage rate of sperm-injected oocytes treated with Ionomycin and DMAP was higher than with Ionomycin alone (62.1 vs. 27.3%, p < 0.05). In sham-injected control oocytes treated with Ionomycin and DMAP, the cleavage rate was approximately six times higher than that of oocytes treated with Ionomycin alone (44.3 vs. 7.4%, p < 0.001). Upon examination 16 h after ICSI, pronuclear formation was observed in 33 of 47 (70.2%) DMAP-treated oocytes. Two pronuclei were present in 18 of 33 (54.6%), while one and three pronuclei were seen in 8 of 33 (24.2%) and 7 of 33 (21.2%), respectively. In sham-injected DMAP-treated control oocytes, 6 of 15 (40.0%) had one pronucleus while 9 of 15 (60.0%) had two pronuclei. Since a single Ca2+ stimulation provided insufficient activation and DMAP treatment could result in triploidy, activation by multiple Ca2+ stimulations following ICSI was tested. Three Ca2+ stimulations were given at 30-min intervals. Pronuclear formation was observed in 16 of 41 (39.0%) oocytes at 16 h after sperm injection, with one and two pronuclei found in 4 of 16 (25.0%) and 12 of 16 (75.0%), respectively. Although one pronucleus was formed in 3 of 33 (9.1%) sham-injected control oocytes treated with three Ca2+ stimulations, two pronuclei were not seen in any of these oocytes. Due to the low rate of pronuclear formation after 5muM Ionomycin, 50muM

The use of magnesium for degradable implants can fill the need for temporary, load bearing, metallic orthopaedic implants without the risks and expense of further surgeries once the bone has healed. Mg is non toxic and biocompatible, but the corrosion rate in the body is too high. The rate will need to be moderated if these implants are to be made clinically useful. A review of common orthopaedic coatings found that the biomimetic calcium phosphate coating process meets the criteria for a good coating. This process was designed for permanent implants, and its corrosion protection properties were unknown on Mg. The research presented here evaluates and optimizes aspects of the corrosion protection of biomimetic coatings in vitro. To accurately identify the corrosion mechanisms of such coatings, the in vitro behaviour of several common simulated body fluids and buffer systems was evaluated. The deposition of biomimetic coatings on Mg was compared to Ti. The effect of common surface treatments on the deposition, composition, and ultimate corrosion protection was identified in order to understand the corrosion properties of these coatings. Following the results, the biomimetic method was modified to optimize the protection by reducing the defects. The corrosion properties of these modified coatings were assessed in vitro. The limitation of the biomimetic coatings was found to be in all cases sensitive to the defects present in the coating. While these could be minimized, they were not eliminated. This led to unfavourable corrosion properties. To solve this problem, a novel treatment was developed to give the biomimetic coatings self-healing properties. This treatment promoted local repair in the coating at the defects, greatly improving the corrosion properties. The in vitro model was increased in complexity by adding first amino acids, then proteins. The corrosion behaviour of the coatings was compared in these solutions to understand the effects of these molecules. The data gathered will help to build a better model of in vivo corrosion, and allow better prediction of the performance of biomimetic coatings for corrosion resistance.

No description available.

Botulism is a foodborne intoxication caused by ingestion of Clostridium botulinum neurotoxin (BoNT). Preliminary studies focussed on the production of polyclonal antisera against BoNT/E by immunizing a rabbit with botulinal toxoid type E. The antiserum was subsequently used to detect BoNT/E using the slot blot immunoassay where samples were applied to a slot blot filtration manifold and drawn by vacuum through a membrane. The membrane was then immunologically processed before chemiluminescent detection. However, the antisera lacked specificity and cross-reacted with closely related clostridia strains. / The specificity of the antisera was increased by adsorbing cross-reactive antibodies from whole antisera with affinity columns made with total proteins from culture supernatants of closely related clostridia. Alternatively, specific antibodies were isolated with an affinity column prepared with C. botulinum type E toxoid. / Different methods of concentrating BoNT/E in each sample prior to testing them were evaluated to increase the sensitivity of the assay. / The slot blot immunoassay was then evaluated for detection of BoNT/E in mixed cultures and in food samples. (Abstract shortened by UMI.)