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Pseudomonas aeruginosa bacterial biofilms are the leading cause of mortality among cystic fibrosis (CF) patients. Biofilms contain bacteria attached to a surface and encased in a protective matrix. Since bacteria within a biofilm are less susceptible to antibiotics, a new approach is to use dispersion compounds that cause the biofilms to release free-swimming bacteria. Our approach has focused on combining nutrient dispersion compounds with antibiotics to increase eradication of bacteria within biofilms. This approach takes advantage of the enhanced susceptibility of free-swimming bacteria to antibiotics, compared to bacteria within biofilms. Ultimately, this research will guide the development of an aerosol therapy containing both antibiotic and dispersion compounds to treat bacterial biofilm infections. To study the effect of antibiotic and dispersion compound treatments on biofilm eradication, a high-throughput screening assay was used to assess the effect on young Pseudomonas aeruginosa biofilms. In addition, a Lab-Tek chambered coverglass system imaged via confocal microscopy was used to assess the effect on mature Pseudomonas aeruginosa biofilms. Seven antibiotics (amikacin disulfate, tobramycin sulfate, colistin sulfate, colistin methanesulfonate (CMS), polymyxinB sulfate, erythromycin, and ciprofloxacin hydrochloride) were tested alone or in combination with four nutrient dispersion compounds (sodium citrate, succinic acid, xylitol, and glutamic acid) to assess the level of eradication of bacteria within biofilms. For young biofilms, 15 of 24 combinations significantly eliminated more live bacteria within the biofilms (measured in colony forming units per milliliter) compared to antibiotics alone. In the more mature biofilm system, only 3 out of 26 combinations resulted in a higher percentage of live biofilm bacteria being eliminated compared to antibiotics alone, showing the importance of biofilm age in the effectiveness of these potential combination therapies. To aid in confocal microscopic analysis of biofilms, an automated quantification program called STAINIFICATION was developed. This new program can be used to simultaneously investigate connected-biofilm bacteria, unconnected bacteria (dispersed bacteria), the biofilm protective matrix, and a growth surface upon which bacteria are grown in confocal images. The program contains novel algorithms for the assessment of bacterial viability and for the quantification of bacteria grown on uneven surfaces, such as tissue. The utility of the viability assessments were demonstrated with confocal images of Pseudomonas aeruginosa biofilms. The utility of the uneven surface algorithms were demonstrated with confocal images of Staphylococcus aureus biofilms grown on cultured human airway epithelial cells and Neisseria gonorrhoeae biofilms grown on transformed cervical epithelial cells. Finally, a proof-of-concept study demonstrated that dry powder aerosols containing both antibiotic and nutrient dispersion compounds could be developed with properties optimized for efficient deposition in the lungs. A design of experiments study showed that solution concentration was the most significant parameter affecting aerosol yield, particle size, and in vitro deposition profiles. Collectively this work demonstrated that bacterial dispersion from biofilms can enhance antibiotic susceptibility and can be better quantified using the new STAINIFICATION software. Formulation of dispersion compounds and antibiotics into a dry powder aerosol could enable more effective treatment of biofilm infections in the lungs.

L'étude des propriétés luminescentes de nanoparticules permet d'accéder à des informations sur les mécanismes élémentaires liés à cette luminescence. À l'instar de ce qui a été fait pour les semiconducteurs (effets de confinement quantique par exemple), nous souhaitons étudier l'influence de paramètres tels que la taille ou la composition de nanoparticules isolantes sur leur luminescence. Pour cela il fallait créer un outil polyvalent capable d'exciter efficacement ces particules, d'en effectuer des images luminescentes et enfin d'en faire la spectroscopie. Le microscope confocal achromatique élaboré dans le cadre de mon travail de thèse et hébergé au sein de Nanoptec est à même de remplir ces objectifs : longueur d'onde d'excitation accordable allant de l'UV dur (210 nm) à l'IR (près de 1 μm), résolution spatiale de l'ordre du μm (permet l'étude de particules assez espacées), aspect confocal permettant d'isoler spatialement la luminescence de l'objet étudié, système de détection capable d'isoler spectralement cette luminescence... Cet outil a permis des collaborations diverses avec des équipes au sein et hors du laboratoire, comme la cartographie spatiale de la répartition de dopants dans des fibres laser, l'évaluation des inhomogénéités lumineuses au sein de matériaux céramiques, la mesure de dispersion spatiale de nanoparticules dans des plastiques... Le microscope confocal achromatique nous sert également à étudier plus fondamentalement les effets de la puissance d'excitation sur les propriétés luminescentes de nanoparticules de tailles et de compositions diverses.

<p>This thesis considers the effect of manufacturing conditions and polymer ratio on water permeability and morphology of free-films. A novel spray method for producing ethyl cellulose (EC) and hydroxypropyl cellulose (HPC) free-films was developed where several process parameters was controlled. The process was optimised by pre-spraying solvent until the system reached a steady-state temperature. This minimised the variation of outlet air temperature to < 2.5 °C. Coating time was approximately 4 minutes excluding drying.</p><p>Free-films were produced using 94 wt% solvent (95 %-ethanol) and 6 wt% polymer. The amount of HPC in the films was varied (wt% HPC defined as HPC/(HPC+EC)*100). Films with 30-40-50-57 wt% HPC were studied. Phase diagrams was constructed to study the phase transformation of polymer mixtures. Results show that all polymer mixtures with HPC content above 30 wt% were phase separated prior to film manufacturing. Temperature had an effect on the polymer phase transformation. In the phase diagram, the 2-phase area was larger for temperatures above 40 °C.</p><p>The investigated manufacturing conditions were outlet air temperature (°C) and spray rate (g/min). Outlet air temperature was controlled by adjusting the inlet air temperature. The films were characterized by measuring water permeability (m²/s). Cross section structure of the films was analyzed with confocal laser scanning microscopy (CLSM). FITC-HPC was added for enhanced contrast between the domains.</p><p>Higher outlet air temperature gave higher water permeability of the film whereas higher spray rate gave lower water permeability. The outlet air temperature had an impact on evaporation rate. The evaporation rate together with spray rate affected the solidification and hence the structure of the film. Images show that longer solidification time smeared the domains into larger domains. Lower water permeability was caused by less connectivity between the pores.</p><p>In conclusion, experiments show that water permeability of EC/HPC free-films was highly dependent on the manufacturing conditions.</p><p></p>

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Aspergillus nidulans is a filamentous fungus whose cells have highly polarized growth. This requires many genes including hypA, which affects hyphal morphogenesis by promoting tip cell growth and suppressing growth in basal regions. The hypA locus was cloned previously by complementing the hypA1 phenotype. The hypA orthologues in Saccharomyces cerevisiae and Schizosaccharomyces pombe are essential, whereas hypA deletion strains in Aspergillus nidulans are viable but morphologically abnormal. The A. nidulans hypA locus has two temperature sensitive alleles, hypA1 and hypA6. These alleles were generated independently, but using the same mutagen, and were identified and characterized by different groups. Although the hypA1 strain has been shown to sporulate at restrictive temperature, the hypA6 strain was described as having a restrictive arrest phenotype, which suggested it was a lethal defect and was at odds with the report that hypA was dispensable for growth. To resolve this discrepancy, my study compared the hypA1 and hypA6 strains using morphometry, and compared their genetic lesions. I show that the hypA1 and hypA6 phenotypes are indistinguishable and mutation events in their coding regions are identical. Both hypA1 and hypA6 strains were able to sporulate at restrictive temperature as shown by scanning electron microscopy. Their cell morphologies were indistinguishable after growth under restrictive conditions, as were their repolarization kinetics when restrictive-grown cells were shifted to permissive temperature. Sequencing the hypA locus from a hypA6 strain, and comparing it to a hypA1 strain showed that they had identical lesions (G329R), which is consistent with the morphometric analysis. Unexpectedly, the hypA1 and hypA6 strains shared additional non-conservative amino acid substitutions, K885F and E932K, with their wildtype parent A28 compared to the strain used for complementing hypA1. The repair of these two amino acid changes can not rescue hypA1 or hypA6 phenotype at 42C. The S. cerevisiae homologue of hypA protein is TRS120p, which is involved in endomembrane trafficking. FM4-64 endomembrane staining of hypA1 and hypA6 strains showed they had aberrant endomembrane arrays at restrictive versus permissive temperature, which recovered the wildtype pattern after a return to permissive conditions. This is consistent with the similarities between the hypA and TRS120 sequences. The disparity between the published descriptions of the hypA1 and hypA6 restrictive phenotypes has been resolved, and the allele renamed hypA1/A6.

Aspergillus nidulans is a filamentous fungus whose cells have highly polarized growth. This requires many genes including hypA, which affects hyphal morphogenesis by promoting tip cell growth and suppressing growth in basal regions. The hypA locus was cloned previously by complementing the hypA1 phenotype. The hypA orthologues in Saccharomyces cerevisiae and Schizosaccharomyces pombe are essential, whereas hypA deletion strains in Aspergillus nidulans are viable but morphologically abnormal. The A. nidulans hypA locus has two temperature sensitive alleles, hypA1 and hypA6. These alleles were generated independently, but using the same mutagen, and were identified and characterized by different groups. Although the hypA1 strain has been shown to sporulate at restrictive temperature, the hypA6 strain was described as having a restrictive arrest phenotype, which suggested it was a lethal defect and was at odds with the report that hypA was dispensable for growth. To resolve this discrepancy, my study compared the hypA1 and hypA6 strains using morphometry, and compared their genetic lesions. I show that the hypA1 and hypA6 phenotypes are indistinguishable and mutation events in their coding regions are identical. Both hypA1 and hypA6 strains were able to sporulate at restrictive temperature as shown by scanning electron microscopy. Their cell morphologies were indistinguishable after growth under restrictive conditions, as were their repolarization kinetics when restrictive-grown cells were shifted to permissive temperature. Sequencing the hypA locus from a hypA6 strain, and comparing it to a hypA1 strain showed that they had identical lesions (G329R), which is consistent with the morphometric analysis. Unexpectedly, the hypA1 and hypA6 strains shared additional non-conservative amino acid substitutions, K885F and E932K, with their wildtype parent A28 compared to the strain used for complementing hypA1. The repair of these two amino acid changes can not rescue hypA1 or hypA6 phenotype at 42C. The S. cerevisiae homologue of hypA protein is TRS120p, which is involved in endomembrane trafficking. FM4-64 endomembrane staining of hypA1 and hypA6 strains showed they had aberrant endomembrane arrays at restrictive versus permissive temperature, which recovered the wildtype pattern after a return to permissive conditions. This is consistent with the similarities between the hypA and TRS120 sequences. The disparity between the published descriptions of the hypA1 and hypA6 restrictive phenotypes has been resolved, and the allele renamed hypA1/A6.

Abstract Both in the closed and open eye state the superior limbus is covered by the upper lid. This region is of physiological interest and clinical importance because in chronic hypoxia, neovascularization of the cornea commonly occurs here. The limbal region in general is additionally of importance as the stem cells which are the source of the new corneal cells are located in the epithelium of the limbus and these are vital for normal functioning and are affected under certain adverse conditions. Purpose: In this experiment I examined corneal morphology in the limbal area and in particular under the upper lid in order to primarily examine the variation in the corneal limbal epithelial and total thickness as well as epithelial and endothelial cell density. Methods: I measured 30 eyes OD/OS (chosen randomly) of thirty healthy subjects aged from 18 to 55 years in the first study and twelve participants in the second study, with refractive error ≤ ±4 D and astigmatism ≤ 2 D. The thickness and cell density of five positions: superior, inferior, temporal, nasal limbal and central cornea was determined with optical coherence tomography (OCT) and confocal microscopy. At least three scans of each position were taken in both studies with OCT. At least 40 of 100 adjacent sagittal scans of each image were measured using OCT software program. In the confocal study, image J software was used to determine cell densities. Results: The epithelial and corneal limbal thickness were significantly thicker than the epithelial and central corneal thickness (p<0.05). The limbal, inferior cornea is thinner than the three other positions and the temporal region of the cornea is the thickest both in epithelial and total cornea. Epithelial cell density was significantly lower in the superior cornea than the four other positions. There was no significant difference in the endothelial cell density. Conclusions: Using OCT with high resolution and cross-sectional imaging capability and confocal microscope with high magnification, I found that the limbal cornea is significantly thicker than the central cornea both in total and in epithelial thickness. In the limbus, one might expect the superior cornea (under the lid) to be thickest (because of the expected hypoxia) whereas I found the temporal cornea was thickest. The epithelial cell density was lower in the superior cornea but there was no significant difference in cell densities in the endothelium. Further morphological investigation is of interest.

Advances in computational modelling offer unprecedented potential for obtaining insights into the mechanics of cell-cell interactions. With the aid of such models, cell-level phenomena such as cell sorting and tissue self-organization are now being understood in terms of forces generated by specific sub-cellular structural components. Three-dimensional systems can behave differently from two-dimensional ones and since models cannot be validated without corresponding data, it is crucial to build accurate three-dimensional models of real cell aggregates. The lack of automated methods to determine which cell outlines in successive images of a confocal stack or time-lapse image set belong to the same cell is an important unsolved problem in the reconstruction process. This thesis addresses this problem through a contour grouping algorithm (CGA) designed to lead to unsupervised three-dimensional reconstructions of biological cells. The CGA associates contours obtained from fluorescently-labeled cell membranes in individual confocal slices using concepts from the fields of machine learning and combinatorics. The feature extraction step results in a set of association metrics. The algorithm then uses a probabilistic grouping step and a greedy-cost optimization step to produce grouped sets of contours. Groupings are representative of imaged cells and are manually evaluated for accuracy. The CGA presented here is able to produce accuracies greater than 96% when properly tuned. Parameter studies show that the algorithm is robust. That is, acceptable results are obtained under moderately varied probabilistic constraints and reasonable cost weightings. Image properties – such as slicing distance, image quality – affect the results. Sources of error are identified and enhancements based on fuzzy-logic and other optimization methods are considered. The successful grouping of cell contours, as realized here, is an important step toward the development of realistic, three-dimensional, cell-based finite element models.

Purpose: Hyper-reflective superficial cells were an unexpected finding while examining the corneal epithelium using confocal microscopy (CM), during an MSc thesis conducted in 2006 at the University of Waterloo, Canada. The author1 suggested that the appearance of these hyper-reflective cells could be associated with solution induced corneal staining (SICS) that was also observed in those participants who had manifested these hyper-reflective cells. However, this hypothesis has not been reported in the literature. This thesis aimed to investigate variables that could possibly predict the appearance of hyper-reflective superficial cells. These investigated variables were the effect of: contact lenses, contact lens solutions, lens/solution combinations, long-term use of certain contact lenses and solutions, age, dry eye symptom, topical anaesthetics and sodium fluorescein. In addition to this, the normal superficial epithelium of controls was defined. Methods: CM images of the superficial epithelium were obtained during the various experiments from: 32 non-contact lens wearing participants, 18 post-menopausal participants symptomatic of dry eye and 18 post-menopausal age-matched asymptomatic women and 147 adapted soft contact lens wearers. For one experiment CM was performed with the contact lens in situ, making the use of a topical anaesthetic unnecessary. Superficial cellular appearance of CM images was graded using a custom grading scale. Hyper-reflective cells were counted. Corneal staining was assessed using sodium fluorescein. Results: Results obtained during the various experiments revealed that hyper-reflective cells predominately appeared with the use of a specific lens/solution combination. Also, the number of hyper-reflective cells peaked after two hours of lens wear. It was also shown that when hyper-reflective cells occurred during an experiment, not every participant who was exposed to that specific lens/solution combination manifested hyper-reflective cells. Also, a great deal of inter-subject variability in observed numbers of hyper-reflective cells was noted. Conclusion: In conclusion, this thesis established that the hyper-reflective cells that were observed by Harvey were reproducible and may co-occur with corneal staining induced by a specific lens/solution interaction