For many years man has been searching for a cure for lime-induced chlorosis. This disease is prevalent throughout the entire western United States and is characterized by a yellowing of the leaves, destruction of the chlorophyll, and eventual death of the plant. It has brought devastation of fruit orchards in Utah, costing fruit producers millions of dollars. Hardly any county in Utah is free from this destructive nutritional disease.

The spread of the pulmonary disease among humans is a very rapid process and it stands as the third highest killer in the United States of America. Computed Tomography (CT) scanning allows us to obtain detailed images of the pulmonary anatomy including the airways. The complexity of the tree makes the process of manual segmentation tedious, time-consuming, and variant across individuals. The resultant airway segmentation, whether arrived at manually or through the aid of computers, can then be used to measure airway geometry, study airway reactivity, and guide surgical interventions. The thesis addresses these problems and suggests a fully automated technique for segmenting the airway tree in three-dimensional (3-D) micro-CT images of the thorax of an ex-vivo mouse. This novel technique is a several step approach consisting of: 1. The feature calculation of individual voxels of the micro-CT image, 2. Selection of the best features for classification (obtained from 1), 3. KNN-classification of voxels by the best selected features (from 2) and 4. Region growing segmentation of the KNN classified probability image. KNN classification algorithm has been used for the classification of the voxels of the image (into airway and non-airway voxels) based on the image features, the results of which have then been processed using the region growing segmentation algorithm to obtain the final set of results for segmentation. The segmented airway of the ex-vivo mouse lung volume can be analyzed using a commercial software package to obtain the measurements.

The purpose of this research project was to develop and characterise matrix type microspheres prepared from Eudragit® L100, containing insulin as model peptide drug as well as an absorption enhancer, N-trimethyl chitosan chloride (TMC), to improve intestinal absorption via the paracellular route. Insulin loaded microspheres were prepared using a single water in oil emulsification/evaporation method in accordance with a fractional factorial design (23) and subsequently characterised in terms of morphology as well as internal structure. Also, insulin and TMC loading were determined using a high pressure liquid chromatography analysis (HPLC) and colorimetric assay, respectively. Scanning electron microscopic characterisation revealed that most microsphere formulations showed a spherical shape and smooth surface with a sponge-like internal structure as well as relatively good homogeneity in terms of size distribution. Insulin loading ranged from 27.9 ± 14.25 – 52.4 ± 2.72% between the different formulations. TMC loading was lower than for insulin and ranged from 29.1 ± 3.3 - 37.7 ± 2.3% between the different formulations. The pronounced difference in insulin and TMC loading between the microsphere formulations is probably the result of the multitude parameters involved as well as the complex physicochemical processes which govern emulsification/solvent evaporation. Based on the microsphere characterisation results, two formulations were selected (i.e. B and F) for further characterisation (i.e. particle size distribution, dissolution behaviour, and enteric nature) and for in vitro evaluation of insulin transport across excised Fischer (FSR) rat intestinal tissue using a Sweetana-Grass diffusion chamber. Particle size analysis by means of laser light diffraction of the two selected microsphere formulations revealed that the mean particle size (based on volume) ranged from 135.7 ± 41.05 to 157.3 ± 31.74 m. Dissolution results for microsphere Formulations B and F revealed that both insulin and TMC were released from the microsphere formulations in an alkaline environment (pH 7.4). The mean dissolution time (MDT) for insulin ranged from 34.5 ± 4.01 to 42.6 ± 9.06 min, while the MDT for TMC ranged from 1.2 ± 1.73 to 6.8 ± 6.42 min. Statistical analysis revealed no significant differences in the MDT of either insulin or TMC (p-value > 0.05) between the two formulations, although the difference between insulin and TMC of each formulation was significant (p-value < 0.05). Microsphere formulations B and F released 36.92 and 48.21% of their total drug content over a period of 1 h in 0.1 M HCl. Microsphere Formulation B showed 8.3 ± 0.52% and formulation F 8.9 ± 2.26% transport of the initial insulin dose after a period of 120 min across excised rat intestinal tissue. The increase in insulin transport by the microsphere formulations compared to that of the control group (i.e. insulin alone) correlated well with the decrease in transepithelial electrical resistance (TEER) caused by the microsphere formulations. The transport of insulin from Formulations B and F represented transport enhancement ratios of 10.67 and 9.68, respectively. Insulin loaded EudragitL100 microspheres containing TMC were successfully prepared by emulsification/solvent evaporation that demonstrated promising potential to serve as oral drug delivery systems for insulin. The microspheres exhibited improved insulin permeability across intestinal epithelial tissue; however, its enteric properties should be improved and clinical effectiveness need to be confirmed by future in vivo studies. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

The purpose of this research project was to develop and characterise matrix type microspheres prepared from Eudragit® L100, containing insulin as model peptide drug as well as an absorption enhancer, N-trimethyl chitosan chloride (TMC), to improve intestinal absorption via the paracellular route. Insulin loaded microspheres were prepared using a single water in oil emulsification/evaporation method in accordance with a fractional factorial design (23) and subsequently characterised in terms of morphology as well as internal structure. Also, insulin and TMC loading were determined using a high pressure liquid chromatography analysis (HPLC) and colorimetric assay, respectively. Scanning electron microscopic characterisation revealed that most microsphere formulations showed a spherical shape and smooth surface with a sponge-like internal structure as well as relatively good homogeneity in terms of size distribution. Insulin loading ranged from 27.9 ± 14.25 – 52.4 ± 2.72% between the different formulations. TMC loading was lower than for insulin and ranged from 29.1 ± 3.3 - 37.7 ± 2.3% between the different formulations. The pronounced difference in insulin and TMC loading between the microsphere formulations is probably the result of the multitude parameters involved as well as the complex physicochemical processes which govern emulsification/solvent evaporation. Based on the microsphere characterisation results, two formulations were selected (i.e. B and F) for further characterisation (i.e. particle size distribution, dissolution behaviour, and enteric nature) and for in vitro evaluation of insulin transport across excised Fischer (FSR) rat intestinal tissue using a Sweetana-Grass diffusion chamber. Particle size analysis by means of laser light diffraction of the two selected microsphere formulations revealed that the mean particle size (based on volume) ranged from 135.7 ± 41.05 to 157.3 ± 31.74 m. Dissolution results for microsphere Formulations B and F revealed that both insulin and TMC were released from the microsphere formulations in an alkaline environment (pH 7.4). The mean dissolution time (MDT) for insulin ranged from 34.5 ± 4.01 to 42.6 ± 9.06 min, while the MDT for TMC ranged from 1.2 ± 1.73 to 6.8 ± 6.42 min. Statistical analysis revealed no significant differences in the MDT of either insulin or TMC (p-value > 0.05) between the two formulations, although the difference between insulin and TMC of each formulation was significant (p-value < 0.05). Microsphere formulations B and F released 36.92 and 48.21% of their total drug content over a period of 1 h in 0.1 M HCl. Microsphere Formulation B showed 8.3 ± 0.52% and formulation F 8.9 ± 2.26% transport of the initial insulin dose after a period of 120 min across excised rat intestinal tissue. The increase in insulin transport by the microsphere formulations compared to that of the control group (i.e. insulin alone) correlated well with the decrease in transepithelial electrical resistance (TEER) caused by the microsphere formulations. The transport of insulin from Formulations B and F represented transport enhancement ratios of 10.67 and 9.68, respectively. Insulin loaded EudragitL100 microspheres containing TMC were successfully prepared by emulsification/solvent evaporation that demonstrated promising potential to serve as oral drug delivery systems for insulin. The microspheres exhibited improved insulin permeability across intestinal epithelial tissue; however, its enteric properties should be improved and clinical effectiveness need to be confirmed by future in vivo studies. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

The purpose of the present study was to examine the effects of external laryngeal vibration on voice function. The current study was based on a recent pilot study using silicone vocal folds that demonstrated a decrease in phonation threshold pressure (PTP; cmH2O) when an external oscillation was applied to the vocal folds. Using a within-subjects experimental design, a custom external oscillatory device was fitted to the posterior portion of 12 excised pig larynges using a traditional benchtop phonation setup. For each larynx, phonation was elicited during 30 repeated trials, including 15 with and 15 without external oscillation. During the phonation trials, aerodynamic measures were collected. The outcome measure for this study was PTP, which has been established in the literature as being correlated with physiologic and self-perceived vocal effort. Furthermore, PTP is used routinely as an aerodynamic indicator of voice function, vocal efficiency, and the nature and severity of voice disorders. Although the aim was to quantify either positive (i.e., PTP decrease) or negative (i.e., PTP increase) effects of external oscillation on PTP, it was hypothesized that external oscillation would result in a reduction in average PTP values. The results of the study indicate that application of an external oscillatory device results in significantly lower PTP. These findings have important clinical implications for PTP signal acquisition and the potential use of external oscillation as a therapeutic tool to improve voice function.

Animal models are used extensively in voice research to study aspects of phonation, including physiology, kinematics, structure, and histology. Animals such as dog, cow, pig, sheep, deer, monkey, ferret, and rabbit have been used in voice research, with pig being one of the most common models. It is thought that the pig larynx is highly similar to the human larynx and one of the best models used in animal translational research. As with any model, however, the pig larynx does have some limitations. Perhaps a limitation most important to the rationale of this investigation is that pigs are difficult animals to study in vivo. Maintenance for a pig is challenging due to its large size and the variability of phonation use in the animal. Therefore, viable and practical alternatives are needed for in vivo voice research. The current study collected preliminary normative data from an alternate animal model, the rabbit, which has been used more recently in studies to model human phonation. The rabbit model was chosen due to its histological similarities to humans, in vivo phonation patterns, size, and practicality. The rabbit represents a more practical model for some longitudinal designs, as well as ex vivo phonatory models with aerodynamic measures as the primary variables. The current study involved a comparison of two aerodynamic measures, specifically phonation threshold pressure (PTP) and phonation threshold flow (PTF) between two groups, pig and rabbit larynges. The purpose of this study was to determine normative aerodynamic values for rabbits and to compare these with normative values for pigs during excised larynx benchtop phonation. Each group consisted of 15 larynges that were finely dissected to reveal the true vocal folds. Each larynx was then connected to a pseudolung and humidified air was passed through it. Fifteen phonation trials were elicited and the results averaged for each larynx. The results indicated that PTP and PTF were significantly different between the two groups, with PTP and PTF being lower for the rabbit group. Additionally, PTP values for rabbits were closer than pigs to the typical human value; however, some methodological challenges to rabbit benchtop models, including size and structural integrity, also exist. But the results from this study indicate that rabbits should be considered a viable option for voice research that would be more feasible with a small animal option that translates well to humans than a large animal option.

This thesis examined the effects of combination inhaled corticosteroids (ICs) on the stability of six aerodynamic measures of phonation utilizing a traditional benchtop model with leporine larynges. The motivation for this study was based on the increase of voice disorders associated with IC use in recent years. The aerodynamic measures examined were phonation threshold pressure (PTP), phonation threshold flow (PTF), onset resistance, sustained pressure, sustained flow, and sustained resistance. Leporine larynges were selected as the model for this study due to histological similarities between leporine and human vocal folds that make them ideal for translational research. Rabbits were either exposed to saline solution or ICs for 8 weeks before being sacrificed. After being sacrificed, larynges were excised and dissected. After dissection, the larynges were mounted on a benchtop, the aerodynamic data were gathered, and stability over multiple phonation trials was calculated. The results indicate that the variation between individual rabbits across the measures did not differ significantly. However, after controlling for trial, the average variation of the groups across all trials did differ significantly. PTP and sustained pressure were more variable for the inhaler group, while PTF, sustained flow, onset resistance, and sustained resistance were more variable for the control group. These results suggest that some level of variability in aerodynamic measures both within and between subjects is to be expected when using the leporine benchtop model. Furthermore, while IC exposure does not seem to impact within-subject variability, it does influence between-subjects variability.

The purpose of this thesis is to examine the effects of inhaled corticosteroid drugs (ICs) on the voice due to their frequent use in treating an increasing prevalence of asthma disorders. As part of a larger five-year study, the focus of this thesis is specifically on whether 8 weeks of in vivo exposure to ICs will cause changes in the sustained subglottal pressure, sustained airflow, and visual-perceptual ratings of edema and erythema in excised rabbit larynges. Researchers administered either ICs or a control nebulized isotonic saline solution to 22 rabbits in vivo, sacrificed them, and harvested their larynges for benchtop research. While ensuring proper tissue preservation, researchers then finely dissected the larynges to expose the true vocal folds and run phonation trials. Dependent variables included continuous acoustic signals (Hz), subglottal pressure (cm H2O), and airflow (L/min) data for 15 phonation trials per rabbit larynx. Researchers also collected still image photographs at this time and subsequently normalized them for use in the visual-perceptual portion of this thesis. For visual-perceptual ratings, raters used a 0-3 equal appearing interval scale to rate aspects of edema and erythema on left and right vocal fold and arytenoid tissues. Results indicate that, when compared to control larynges exposed to nebulized isotonic saline, experimental larynges treated with ICs require significantly higher subglottal pressure to maintain phonation, p < .05. Mean sustained phonation for experimental larynges is 11.24 cm H2O compared to 8.92 cm H2O for that of control larynges. Phonation trials for experimental larynges have significantly higher sustained airflow with a mean of 0.09 L/min compared to 0.07 L/min for that of control larynges, p < .05. Surprisingly, experimental larynges have higher average fundamental frequencies with less variability (mean: 519 Hz, standard deviation: 66 Hz) than that of control larynges (mean: 446 Hz, standard deviation: 130 Hz). On visual-perceptual ratings, experimental larynges have significantly higher severity ratings on all eight items rated, p < .0001 - p = .0305. Based on these results, it is concluded that ICs cause significant damage to rabbit vocal folds, as evidenced by higher sustained pressure, higher airflow, and higher severity ratings for experimental versus control larynges. The dependent variables in this thesis are novel in benchtop model research and demonstrate a unique perspective on this research question. Thus, this thesis informs future phonation, benchtop, and visual-perceptual research.

Combination corticosteroid inhalers are the primary treatment option for long-term pulmonary disorders including asthma, persistent bronchitis, and chronic obstructive pulmonary disease. Common side effects of these medications are xerostomia in the mouth and throat, hoarseness, and soreness in the oropharynx. Research indicates that a large percentage of the inhaler particles are deposited onto laryngeal tissue, leaving an alteration of laryngeal mucosal properties. As the first stage in a long-term project, this thesis addresses the need for baseline phonatory data that will lay groundwork for quantifying inhaler-induced phonatory changes. Excised larynx research is a powerful tool for assessing aerodynamic alterations that accompany laryngeal pathology. Porcine (pig) larynges are a traditional species employed in voice disorder research, though leporine (rabbit) larynges are an emerging species that lends itself to histologic vocal fold studies as they have the most similar vocal fold cover structure to humans compared to any other animal to date. The purpose of this study was to examine the measurement stability of six aerodynamic parameters in a traditional excised larynx benchtop model. Specifically, the current author assessed measurement stability of leporine larynges compared to porcine larynges with the following aerodynamic metrics: phonation onset pressure (PTP; cmH2O), phonation onset flow (PTF; L/m), sustained pressure (cmH2O), sustained flow (L/m), onset laryngeal resistance (cmH2O/L/m), and sustained laryngeal resistance (cmH2O/L/m). A total of 30 larynges—15 leporine and 15 porcine—were mounted on a benchtop setup; phonation was sampled over 15 trials for each larynx. Measurement stability for the above six tokens was examined using coefficient of variation (%) analyses. Leporine larynges demonstrated significantly less variation across all six aerodynamic parameters when compared to porcine larynges. The leporine PTP values were most stable as compared to leporine and porcine pressure and airflow values. Leporine airflow values were also more stable than porcine PTP and PTF values. These results indicate that leporine larynges might be a preferred excised larynx specimen for certain benchtop phonation studies. These findings are important for establishing expected measurement variability in porcine and leporine larynges, particularly when translating benchtop research to laryngeal pathology.

The purpose of this thesis is to investigate the reversibility of the adverse effects of combination inhaled corticosteroids (ICs) on vocal fold health as part of a five-year study. This pilot study tested the hypothesis that the adverse effects of ICs may be induced and then reversed, using a benchtop model and rabbit larynges measuring sustained subglottic pressure and airflow. Eighteen rabbits were assigned randomly to 5 subgroups, including baseline, induction experimental, induction control, reversibility experimental, and reversibility control. Baseline rabbits did not receive any treatment. Both experimental groups, induction and reversibility, were administered Advair until visual-perceptual ratings were noted. Their paired control groups were administered nebulized saline in the same dosage levels. Induction groups were immediately sacrificed, while reversibility groups entered a withdrawal phase until visual-perceptual ratings showed a return to baseline. Larynges were dissected and mounted on a benchtop model for phonation trials. Dependent variables included sustained phonatory pressure (cm/H2O) and sustained phonatory airflow (L/min). The results of phonation trials indicate that sustained pressure and flow were higher for both induction and reversibility experimental groups when compared to baseline and control groups. The reversibility experimental group had lower sustained pressure and flow than the induction experimental group. These results indicate a reduction of signs after a period of rest. These findings suggest that voice symptoms may be reversible to a degree after combination IC use. These are important preliminary data that support future studies with larger sample sizes to confirm findings.