acase@tulane.edu

acase@tulane.edu

acase@tulane.edu

acase@tulane.edu

In this dissertation, a novel optical video microscopic technique was employed to visually study: coalescence or noncoalescence of microscopic n-hexadecane oil drops; hetero-aggregation among microscopic oil drops and two types of polystyrene latex particles; and selective aggregation among the latex particles. Using two micropipettes, two microscopic oil drops (one at each tip) were produced and released inside the microcapillary. Stationary contact coalescence was studied by carefully bringing the two drops into apparent contact and measuring the time elapsed between apparent contact and coalescence. This is the coalescence time, $t\sb{c}.$ Spontaneous coalescence was observed at highly acidic pH's. Rapid coalescence, although not spontaneous, was also observed at highly basic pH's. At pH's close to 7.0, coalescence could not be accomplished even by forcing the drops against one another in a head-on collision. Collision coalescence was qualitatively studied. For stationary contact coalescence, the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory has been used to theoretically explain the experimental observations. To test the general applicability of the DLVO theory, the principal parameters viz. the pH and ionic strength, were changed independently i.e., (i) changing the pH at constant ionic strength, and (ii) changing ionic strength at constant pH of the interacting medium. The total interaction energy curves together with previously reported measurements of critical thickness of rupture of aqueous thin films was used to predict the coalescence and noncoalescence of microscopic oil drops during interaction in a surfactant-free aqueous medium. Results indicate that coalescence occurs by rupture of the thin film at a finite thickness in the critical thickness range In the DLVO equations the boundary electrostatic potential $(\Psi\sb{d})$ for the oil drop surface was assumed equal to its zeta potential. Measurements of electrophoretic mobility and drop size distributions for n-hexadecane oil-in-water were made to determine the zeta potential of the drop surface as a function of pH. From the data, it was noticed that the surface potentials and $\kappa a$ values fell into a range for which there was no simple procedure readily available in literature for converting the mobility data to zeta potential. Therefore, a graphical procedure involving the modified Booth equation was incorporated into a computer program for calculating the zeta potential from mobility thereby eliminating the use of graphs. This program is applicable for all potentials and $\kappa a$ values and greatly eases the effort in determining zeta potentials This dissertation examines visual evidence of reversibility or irreversibility of hetero-aggregation, selective aggregation and non-aggregation among microscopic n-hexadecane oil drops and two types of surfactant-free polystyrene latex particles in terms of the sphere-sphere DLVO interaction model. Visual observations showed that drop-particle interactions were distinctly different from particle-particle interactions and that partial wetting of particle surfaces by oil appears to be a key factor in the irreversibility of drop-particle hetero-aggregation. (Abstract shortened by UMI.) / acase@tulane.edu

acase@tulane.edu

acase@tulane.edu

The microRNA (miRNA) pathway regulates differentiation and proliferation in a variety of organisms. Here we analyzed the function of the miRNA pathway in regulating differentiation and proliferation of human multipotent stromal cells from bone marrow (hMSCs) Differentiation of hMSCs into osteoblasts and adipocytes was inhibited using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA. Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were upregulated during osteogenic differentiation, 20 during adipogenic differentiation of which 11 were commonly upregulated in both osteogenic and adipogenic differentiation. In silico models predicted that 5 of the upregulated miRNAs targeted leukemia inhibitory factor (LIF) expression. The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF We then assessed the role of the miRNA pathway in hMSC proliferation using Drosha and Dicer knockdown hMSCs. hMSCs with reduced Drosha expression had a significantly reduced proliferation rate, while hMSCs with reduced Dicer expression displayed a proliferation rate similar to untransduced cells. Cell cycle analysis identified that unlike Dicer knockdown, Drosha knockdown hMSCs contained an increased number of G1 phase cells, with a reduced level of cells in S phase, compared to controls. ELISAs of hMSCs revealed decreased levels of pRB and stable levels of total RB with Drosha knockdown. Two key regulators of the G1/S phase transition, cyclin dependent kinase inhibitor 2A (p16) and cyclin dependent kinase inhibitor 2B (p15), were increased in Drosha knockdown cells but not in Dicer knockdown. Transcripts of 28S and 18S rRNA were significantly reduced in Drosha knockdown hMSCs, with no change in rRNA levels in Dicer knockdown hMSCs. 45S pre-rRNA transcripts were not significantly different in either knockdown model. The above results indicate that Drosha modifies hMSC proliferation through a miRNA independent mechanism, potentially by regulating rRNA processing / acase@tulane.edu

Two distinct paraldehyde-fuchsin (PAF)-positive loci were characterized in the scolex of the rat tapeworm, Hymenolepis diminuta. The first represents an unicellular endocrine gland. The cell body lies in the distal scolex and proximal neck region of the worm. Long, tendrillar cell processes extend apically into the musculature of the suckers. The secretory product is PAF-positive in worms of all ages and is packaged in spherical, electron dense, membrane bound granules. Secretion apparently occurs by exocytosis into the extracellular matrix surrounding the fibrillar portions of the acetabular musculature. A function as a long-acting modulator of muscle activity was suggested. The second PAF-positive loci is located in the apical portion of the rostellum, and represents 12-15 flask-shaped tegumentary cytons which support the rostellar tegument. The cells initially become PAF-positive at about 3 days postinfection, concurrent with the appearance of large, ovoid granules in the cyton cytoplasm. By 14 days postinfection, fuchsinophilia extends throughout the cytons and surface tegumental covering, as do the large, ovoid granules. Subsequently, however, the fuchsinophilia diminishes and finally disappears, although the large, ovoid granules persist throughout the life of the worm. The function of the glands has not been ascertained. Although previous authors had postulated a role in the regulation of either growth or maturation, this hypothesis was based on the assumption that the glands were neurosecretory. Such is not the case; the glands are a modified portion of the tegument. The granules sequestered in the rostellar tegument are apparently secreted, with limiting membrane intact. This secretory mechanism is apparently unique and not in keeping with theories on the function of the tegumental inclusions of the strobila. Experiments in which worms were removed from a rat host subsequent to the loss of fuchsinophilia, destrobilated and surgically reimplanted in a new rat host indicate that the cycle of fuchsinophilia can be reactivated. In vitro experiments failed however, to distinguish between a reactivation caused by destrobilization or by the trauma of the experimental procedure. Histochemical and cytochemical studies revealed that the granules within the rostellar tegument contain a diastase-stable, protein/neutral carbohydrate-rich material. Lipids and alkaline and acid phosphatase were not detected in the tegument. Autoradiographic studies revealed an apical translocation of granules, but at a slower rate than in the strobilar tegument. In striking contrast to the strobilar tegument, the rostellar tegument apparently does not incorporate ('3)H-galactose. The function of the rostellar glands is apparently secretion although the role of the secretory product and the significance of the fuchsinophilia associated with these products remains unclear / acase@tulane.edu

acase@tulane.edu