Thesis (M.S.)--University of Wisconsin--Madison, 1966. / eContent provider-neutral record in process. Description based on print version record. Bibliography: l. 19-20.

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M. Tech. / Yeast such as Candida albicans are the major cause of human diseases such as genital thrush and oral thrush. Some of the genital isolates of C. albicans that were studied by Shah et al. (1991 & 1995) were found to produce the medically important immunosuppressing mycotoxin gliotoxin, which has potential important medical consequences. The biosynthesis of this mycotoxin is regulated and expressed by the presence of the gliP and gliZ genes, which were identified on the putative gene cluster of A. fumigatus. Most Candidal infections are treated using a single or a combination of antifungal agents such as amphotericin B (AmB), fluconazole, flucytosine, voriconazole, caspofungin, itraconazole, posaconazole and ketoconazole. The mode of action for these antifungal agents differs in terms of what molecule or processes are inhibited. The details of each antifungal agent and its mode of action are discussed in chapter 6 (page 54). These antifungal agents are usually recommended for the treatment of candidosis and currently the most common Candida spp. have developed resistance to these antifungal agents. The identification of Candida isolates was done using 2 different types of identification methods i.e., the chromogenic medium CHROMagar Candida and the biochemical test kit API 10 Candida. The chromogenic medium was inoculated with the Candida spp. supplied and incubated for 3 days at 37oC. The API 10C test strips were loaded with the culture suspension and incubated for 24 hours at 37oC. For the screening of gliotoxin, 2 supplemented mediums were used to cultivate the isolates that is the yeast extract sucrose (YES) and Eagles minimal essential medium (EMEM) and the isolates were grown at 37oC for 72 days. The methods that were used to identify the gliotoxin were thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) to quantify the levels of gliotoxin.

The Wharton’s Jelly (WJ) of human umbilical cord (HU) contains multipotent stem cells (WJ-MSC) which express mesenchymal markers but not hematopoietic markers. WJ-MSC are increasingly being tested for use in stem cell therapy, with intravenous delivery being the preferred route. Fetal stem cells from embryonic germ layers are present in maternal blood and can home to damaged maternal tissues. This study investigates how WJ-MSC cross the fetal and adult endothelial barriers; including the cellular and molecular mechanisms employed. WJ-MSC were isolated from HU (n=27) which were taken from normal term pregnancies after elective Caesarean section. Flow cytometry and immunofluorescence were used to check presence/absenc of mesenchymal versus haematopoietic markers. Cells were induced to become adipocytes, chondrocytes and osteocytes by using specific induction medium. Isolated WJ-MSC were added after labelling with PKH26 to confluent monolayers of isolated human umbilical vein endothelial cells (HUVEC) or commercially bought human uterine microvascular endothelial cells (HUtMEC) at a 1:5 ratio. Cell-cell interactions were monitored with real time microscopy for 24 to 40h. Fluorescence and confocal scanning microscopy, after vascular endothelial (VE) cadherin immunocytochemistry were used for detailed analysis of VE-cadherin junctional occupancy and spatio-temporal location of stem cells. Tyrosine phosphorylation status of VE-cadherin, whether at Tyr685 or Tyr731, at different time points were investigated by immunoblotting whilst levels of vascular endothelial growth factor (VEGF) in the conditional media (CM) were measured by ELISA. Three different isolates were tested, with 3 experimental repeats for all expermints. Statistical analyses were performed with ANOVA (One or Two way). Cells (>95%) from each passage were positive for the mesenchymal markers CD 29, CD 105, CD 90, CD 73 and CD 44. <2% cells showed positivity to the haematopoietic markers CD 34, HLA-DR, CD 14, CD 19 and CD 45. WJ-MSC differentiated into adipocytes, osteocytes and chondrocytes. WJ-MSC displayed exploratory behaviour for a minimum of 30 min on HUtMEC or 60 min on HUVEC with interrogation of paracellular openings before crossing rather than replacing endothelial cells. By 2h, half were found at sub-endothelial positions, with a majority reaching this within 16-22h. There was accompanying loss of junctional VE-cadherin (64.9 + 3.7 %; p<0.001 in HUVEC; 63 + 4.6%; p< 0.001 in HUtMEC) in the endothelial monolayers followed by a return at 16h and increased continuity by 22h (p<0.01 in HUVEC; p<0.001 in HUtMEC). Junctional disruptions were found close to overlying or migrating WJ-MSC. Confocal microscopy confirmed paracellular extravasation. VE-cadherin protein levels matched controls in the early hours (0-2h) and increased after 22h co-culture in both fetal and uterine endothelium. VE-cadherin showed a 2-fold increase in phosphorylation at Tyr685 from 30 min to 2h. P-Tyr731 remained unchanged, similar to untreated endothelial layers, then decreased at 2h and 22h. VEGF levels in WJ-MSC – HUtMEC co-culture supernatants was highest at 2h (88 + 3 pg/ml) and decreased by 22h, reaching negligible levels by 48h. Anti-VEGF blocked Tyr685 phosphorylation but did not affect the decrease in P-Tyr731; this was accompanied by a 25% decrease in transmigration of cells in the first two hours and a 43% decrease in total by 22h. in WJ-MSC – HUtMEC co-cultures. However, in HUVEC-WJ-MSC co-cultures, no VEGF were detected and anti-VEGF did not block Tyr685 phosphorylation and Tyr731 de-phosphorylation. WJ-MSC from term umbilical cords can be easily isolated and expanded in culture. They retain mesenchymal stem cell properties for the passages tested (up to P5) making them a valuable model for studies into mechanisms underlying extravasation. The data obtained suggest that WJ-MSC can influence expression of VE-cadherin, with perturbation during transmigration followed by upregulation and repair once the adlumenal side is reached. There was a similarity in the cellular and molecular mechanisms employed by WJ-MSC in their paracelluar migration across fetal and uterine endothelium, although VEGF may not be the key player in HUVEC interactions. For both endothelial types, WJ-MSC appear to induce phosphorylation events linked with paracellular permeability and de-phosphorylation events normally associated with leukocyte extravasation. The data from the uterine endothelial investigations suggests that fetal stem cells are able to influence paracellular junctional dynamics and strengthens the growing hypothesis that they may also play a role in re-modelling the uterine circulation for fetal advantage. The extra-embryonic WJ-MSC holds the promise of use in restoring junctional maturity and vascular repair in future therapeutic applications.

Please read the abstract in the section 00 front of this document / Dissertation (M Med Vet (Clinical Laboratory Diagnostics))--University of Pretoria, 2001. / Companion Animal Clinical Studies / unrestricted

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Electromechanical (EM) coupling in cells and the cell membrane is the product of basic electrical forces acting on the cell membrane and has been shown to produce voltage-driven membrane movements. While this type of EM coupling phenomenon has been observed in several cell types, including cultured neurons, its precise mechanism and physiological significance remain unclear. We have developed a novel platform that combines Atomic force microscopy (AFM) and patch clamp to measure voltage driven mechanical changes in the membranes of cultured HEK 293T cells. Using this technique, we have measured the mechanical effects of changes in membrane potential, determining the force and displacement at the membrane surface, as a function of voltage. Using our experimental data and basic physical principles, we developed a model of the effects of electrical forces on the membrane, which correlates highly with our observations. Importantly, our model predicts that the sum of electrical forces acting on the AFM tip and lipid bilayer will be different, suggesting that the membrane will experience a much larger tension change from EM coupling than was previously thought. The voltage driven tension is difficult to measure directly, but is predicted to act on mechanosensitive ion channels, resulting in a conductance profile that scales with the square of membrane potential. Conductance measurements exhibited a non-linear change in conductance that agreed with the predicted effects of EM coupling forces, as well as a mechanosensitive response to AFM induced tension. Our findings suggest that EM coupling could have a significant physiological role that had previously been underestimated.

The purpose of this investigation was to provide pure cultures of normal human prostatic epithelium free of fibroblasts in order to study malignant conversion by chemical carcinogens. Normal epithelial cells were needed because this was the cell type implicated in prostatic malignancies of human subjects. Unfortunately fibroblasts grew faster than epithelial cells so that cultures were always overgrown with connective tissue elements. It was considered important to find a method which would eliminate fibroblasts so that normal epithelial cells could grow out in pure culture.

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