Thesis (Ph. D.)--Oregon State University, 2006. / Printout. Includes bibliographical references. Also available on the World Wide Web.

"The C. albicans community is currently laying the foundation of understanding how this human pathogen causes infection. C. albicans infections represent a major medical and economic burden for today’s society with an estimated 400,000 blood stream infections worldwide and direct costs exceeding 1$ billion dollar a year in the U.S. alone. Although finding the biological causes of this disease seemed to be beyond our reach in the past, various aspects of the infection have been recently unveiled including its pathology, immunology, histology, and epidemiology. Here we explored the genetic components of this disease by studying the complex host-pathogen dynamics through a series of in vivo, ex vivo and in vitro experiments. By using a pathogen unbiased reverse genetic approach and a host gene candidate strategy we uncovered some of the genes and pathways that are important for pathogenicity and immunity. In particular we explored the complex host-pathogen dynamics using a C. albicans - C. elegans model system and identified four novel putative virulence factors. We focused on Zcf15, a C. albicans transcription factor that has been poorly characterized in the literature and that plays an important role in the pathogen’s ability to resist host generated reactive oxygen species (ROS). By leveraging the power of RNASeq and ChIP-Seq we identified Zcf15 transcriptional targets and DNA binding sites. These studies suggest that Zcf15 plays a critical role in carbon metabolism and that it exerts its ability to protect the pathogen from ROS by controlling the expression of thiol- peroxidases and other detoxifying enzymes. We also showed here that in C. elegans, the host’s ability to counteract the infection relies on the MAPK pathway, evidence that mirrors what has been found by others in mammals and that emphasizes the usefulness of studying C. albicans infections in smaller genetically traceable organisms like C. elegans. The nematode model is also shown here to be a powerful tool not only to study the genetic bases that drive infection and immunity but also to identify new compounds that can be used for therapeutic intervention. This model was instrumental in identifying filastatin, a small molecule that was subsequently found by our collaborators to be capable of reducing virulence in mammals. The antifungal properties of filastatin are currently undertaking further preclinical testing. Overall this thesis shed light on the complex mechanisms of C. albicans pathogenicity and host immunity and identified novel virulence determinants that can be used by the larger community for further biological studies or even drug development. "

This study examines the effect of ecophysiology on growth of cells and production of enzymes and secondary metabolites produced by the fungi Aspergillus niger (lysozyme) and a Phoma sp. (squalestatin S1). The effect of interactions of water activity (aw) (0.99-0.90), temperature (20, 30 and 35°C) and modifying aw solute (glycerol, NaCl) on growth and sporulation of a wild type strain of Aspergillus niger (W) and two genetically engineered lysozyme producing strains (L11, B1) was examined for the first time. Maximum growth rates were achieved for both strains (L11 and B1) under moderate aw levels. Optimum conditions for growth of strain L11 were estimated by means of contour plot surfaces and found to be 0.965 aw with glycerol as a solute at 35ºC (10.5 mm day-1). A model combining the effect of aw and temperature on growth of strains of Aspergillus niger, and comparison with data on food spoilage moulds in the literature was developed. The growth of two strains of A. niger, as a function of temperature (25-30oC) and aw (0.90-0.99) was developed. The estimation of the minimum aw (awmin) and optimal aw (awopt) levels were in accordance with data in the literature for a range of other Aspergillus and related species, regardless of the solutes used for aw modification. A central composition design was used to describe the effects of water activity (aw, 0.98, 0.97 and 0.96), inoculum size (2.7x105, 2.7x104 and 2.7x103 spores ml-1), and three autoclaving procedure (A = all components autoclaved together, B = medium autoclaved + maltose filtered and, C = medium autoclaved + maltose & soya milk filtered) on the production of lysozyme by two genetically-engineered strains of Aspergillus niger (B1 and L11) in a liquid culture fermentation. Although both strains produced similar lysozyme concentrations (15 mg l-1), different production patterns were found under the experimental conditions. However, strain B1 produced relatively higher amounts of lysozyme under water stress (0.96 aw) with all the substrates autoclaved together. Subsequently, a central composition design was used to investigate: different immobilized polymer types (alginate and pectate), polymer concentration (2 and 4% (w/v)), inoculum support ratios (1:2 and 1:4) and gel-inducing agent concentration (CaCl2, 2 and 3.5% (w/v)) on lysozyme production. Overall immobilization in Ca-pectate resulted in higher lysozyme production compared to immobilization in Ca-alginate. Similar effects were observed when the polymer concentration was reduced. A 13 fold higher lysozyme production was achieved with Ca-pectate in comparison to Ca-alginate (20-23 and 0.5-1.7 mg l-1 respectively). Polymer modifications also significantly affected the final pH and aw of the immobilized cell fermentation. The aw factor is a very significant parameter in the immobilization design. A combined statistical methodology of orthogonal design L27(313) and surface response methodology was applied to optimize the composition and concentration of a liquid fermentation medium for the production of squalestatin S1 by a Phoma species. Confirmatory experiments of the optimal medium composition produced average concentrations of 434 mg l 1 in five days fermentation at 25oC. This represented an improvement over 60% of the maximum concentration achieved in the initial experiment and a two-fold higher productivity in comparison with reported productivities of S1 in liquid fermentations with different fungal species. Different liquid height and column diameter (HL/Hr) ratios 3.7, 7.4 and 11.4 were studied in a bubble column (Dr=0.07 m) with a porous plate gas distributor, to find the effect on the gas hold up, power consumption (PG/VL) and volumetric mass transfer coefficient, kLa performance, under different superficial gas velocities calculated from the liquid properties and flow rates (2, 4, 6 and 8 l min-1) and temperatures (15, 25 and 30oC). Two kLa models were proposed based on the geometrical ratio (HL/Dr) and superficial gas velocity (m s-1) (R2=0.951), and power consumption (PG/VL) (R2=0.950). A free cell fermentation was performed in the bubble column, ratio (HL/Dr)= 3.7 and superficial gas velocity U= 0.120 m s-1, at 25oC. The S1 production reached a level of 420 mg l 1. The bioreactor scale up succeeded in maintaining the high S1 concentration obtained in our previous work 434 mg l 1 in Erlenmeyer flasks but in a shorter time. A Plackett-Burman design was used to improve the S1 produced by different immobilized designs. The immobilized cell fermentation design considered: polymerization with alginate and polygalacturonate and copolymerization, polymer concentration (alginate 3, 3.5 and 5 % w/v and pectate 4, 6 and 8 % w/v), 0.98, 0.96 and 0.94 aw levels, inoculum levels of 10, 20 and 30 % wt. v/v, gel-inducer (CaCl2) 3, 4 and 5 % w/v, gel-reinforce agent 0, 0.75 and 1.5 g l-1, air flow 4, 6 and 8 l min-1. Production of S1 reached levels of 883 mg l-1 which represent a 34 % improvement over the 660 mg l 1 produced in a stirred tank bioreactor (STR) with a free cell fermentation.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 8 uppsatser.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1940. / Typescript. Includes abstract and vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 50-53).

This study examines the effect of ecophysiology on growth of cells and production of enzymes and secondary metabolites produced by the fungi Aspergillus niger (lysozyme) and a Phoma sp. (squalestatin S1). The effect of interactions of water activity (aw) (0.99-0.90), temperature (20, 30 and 35°C) and modifying aw solute (glycerol, NaCl) on growth and sporulation of a wild type strain of Aspergillus niger (W) and two genetically engineered lysozyme producing strains (L11, B1) was examined for the first time. Maximum growth rates were achieved for both strains (L11 and B1) under moderate aw levels. Optimum conditions for growth of strain L11 were estimated by means of contour plot surfaces and found to be 0.965 aw with glycerol as a solute at 35ºC (10.5 mm day-1). A model combining the effect of aw and temperature on growth of strains of Aspergillus niger, and comparison with data on food spoilage moulds in the literature was developed. The growth of two strains of A. niger, as a function of temperature (25-30oC) and aw (0.90-0.99) was developed. The estimation of the minimum aw (awmin) and optimal aw (awopt) levels were in accordance with data in the literature for a range of other Aspergillus and related species, regardless of the solutes used for aw modification. A central composition design was used to describe the effects of water activity (aw, 0.98, 0.97 and 0.96), inoculum size (2.7x105, 2.7x104 and 2.7x103 spores ml-1), and three autoclaving procedure (A = all components autoclaved together, B = medium autoclaved + maltose filtered and, C = medium autoclaved + maltose & soya milk filtered) on the production of lysozyme by two genetically-engineered strains of Aspergillus niger (B1 and L11) in a liquid culture fermentation. Although both strains produced similar lysozyme concentrations (15 mg l-1), different production patterns were found under the experimental conditions. However, strain B1 produced relatively higher amounts of lysozyme under water stress (0.96 aw) with all the substrates autoclaved together. Subsequently, a central composition design was used to investigate: different immobilized polymer types (alginate and pectate), polymer concentration (2 and 4% (w/v)), inoculum support ratios (1:2 and 1:4) and gel-inducing agent concentration (CaCl2, 2 and 3.5% (w/v)) on lysozyme production. Overall immobilization in Ca-pectate resulted in higher lysozyme production compared to immobilization in Ca-alginate. Similar effects were observed when the polymer concentration was reduced. A 13 fold higher lysozyme production was achieved with Ca-pectate in comparison to Ca-alginate (20-23 and 0.5-1.7 mg l-1 respectively). Polymer modifications also significantly affected the final pH and aw of the immobilized cell fermentation. The aw factor is a very significant parameter in the immobilization design. A combined statistical methodology of orthogonal design L27(313) and surface response methodology was applied to optimize the composition and concentration of a liquid fermentation medium for the production of squalestatin S1 by a Phoma species. Confirmatory experiments of the optimal medium composition produced average concentrations of 434 mg l 1 in five days fermentation at 25oC. This represented an improvement over 60% of the maximum concentration achieved in the initial experiment and a two-fold higher productivity in comparison with reported productivities of S1 in liquid fermentations with different fungal species. Different liquid height and column diameter (HL/Hr) ratios 3.7, 7.4 and 11.4 were studied in a bubble column (Dr=0.07 m) with a porous plate gas distributor, to find the effect on the gas hold up, power consumption (PG/VL) and volumetric mass transfer coefficient, kLa performance, under different superficial gas velocities calculated from the liquid properties and flow rates (2, 4, 6 and 8 l min-1) and temperatures (15, 25 and 30oC). Two kLa models were proposed based on the geometrical ratio (HL/Dr) and superficial gas velocity (m s-1) (R2=0.951), and power consumption (PG/VL) (R2=0.950). A free cell fermentation was performed in the bubble column, ratio (HL/Dr)= 3.7 and superficial gas velocity U= 0.120 m s-1, at 25oC. The S1 production reached a level of 420 mg l 1. The bioreactor scale up succeeded in maintaining the high S1 concentration obtained in our previous work 434 mg l 1 in Erlenmeyer flasks but in a shorter time. A Plackett-Burman design was used to improve the S1 produced by different immobilized designs. The immobilized cell fermentation design considered: polymerization with alginate and polygalacturonate and copolymerization, polymer concentration (alginate 3, 3.5 and 5 % w/v and pectate 4, 6 and 8 % w/v), 0.98, 0.96 and 0.94 aw levels, inoculum levels of 10, 20 and 30 % wt. v/v, gel-inducer (CaCl2) 3, 4 and 5 % w/v, gel-reinforce agent 0, 0.75 and 1.5 g l-1, air flow 4, 6 and 8 l min-1. Production of S1 reached levels of 883 mg l-1 which represent a 34 % improvement over the 660 mg l 1 produced in a stirred tank bioreactor (STR) with a free cell fermentation.

No description available.

BACKGROUND: Fungal corneal ulcer is one of the major causes of visual impairment worldwide. Treatment of fungal corneal ulcer mainly depends on anti-fungal agents. In the current study, we developed an integrated combination therapy of cryotherapy and anti-fungal agents to facilitate effective treatment of fungal corneal ulcer. METHODS: Rabbit models of cornea infection were established using a combined method of intrastromal injection and keratoplasty. After treatment with cryotherapy and anti-fungal agents, scanning electron microscopy, transmission electron microscopy, and confocal microscopy were conducted to observe changes in microstructure in the rabbits. Periodic acid Schiff A and hematoxylin and eosin staining were used for detection of histological changes. RESULTS: Continuous scanning electron microscopy and transmission electron microscopy observations showed that cryothermal treatment inhibited growth of fungal mycelium by destroying fungal cellular structures. Typical cryotherapy was effective in curing fungal corneal ulcer. Different fungi showed different susceptibilities to treatment. The curative effect of Candida albicans was the best, while that of Aspergillus fumigates was the worst. CONCLUSIONS: Our study provides a novel method of a combination of cryotherapy and anti-fungal agents for treatment of fungal corneal ulcer. This treatment could help facilitate the practice of fungal keratitis treatment in the future.

Thesis (Ph. D.)--West Virginia University, 1998. / Title from document title page. "December 11, 1998." Document formatted into pages; contains ix, 94 p. : ill. (some col.) Vita. Includes abstract. Includes bibliographical references.

published_or_final_version / Botany / Master / Master of Science