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.

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.

An excised animal larynx model has been used in many studies to better understand the physiological and anatomical properties of the human larynx. One difference between an ex vivo model and an in vivo model is that ion loss occurs postmortem. To compensate for this in the excised model, researchers most commonly use a preservation method that includes completely submerging the specimen in isotonic saline (0.9% Na+Cl-) and then flash freezing it in liquid nitrogen. The flash freezing method allows researchers to maintain the integrity of the structures while also being able to gather specimens as they become available. Not enough research has been done to understand the effects of a preservation method on the outcomes of the study. Additionally, no common method has been established for preservation across studies to ensure that results are not being influenced by this variable. This prospective, mixed experimental design study includes three groups, a control group and two experimental groups. The control group consisted of 10 bench-mounted porcine larynges that were soaked in isotonic saline and flash frozen with liquid nitrogen. Prior to the experiment, the frozen larynges were thawed overnight before trials. The other two groups consisted of 10 bench-mounted porcine larynges each; these larynges were soaked in either isotonic saline or Ringers solution, a balanced fluid used in vivo to counteract dehydration. Larynges from these two groups were kept fresh and stored in a refrigerator overnight before trials. On the day of experimentation, each larynx was mounted on a bench top setup including three micropositioners to stabilize, adduct, and elongate the vocal folds. All the larynges were connected to a pseudolung via the trachea and humidified air was passed through to the vocal folds until phonation was achieved. Phonatory trials consisted of brief phonation followed by 5-minute desiccation intervals until phonation was no longer achieved. Phonation threshold flow (PTF), defined as the flow observed at the onset of phonation, was observed during each phonation trial; and flow values were compared within and between groups. Statistically significant differences were found between the Ringers group and the fresh saline group as well as between the Ringers group and the frozen saline group, indicating that PTF is influenced by the larynx preservation method.

Dissertation submitted in fulfilment of the requirements for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2012. / Apoptosis, or programmed cell death, is a process which is pivotal in eliminating damaged, infected, or unwanted cells from the body. It has been studied in numerous types of cell lines ranging from normal to infected cell lines, and there have been a wide range of studies on apoptosis in laryngeal cancer because this type of cancer has become one of the most common types of head or neck cancer due to the high incidence of alcohol consumption, tobacco smoking and chewing of betel quid amongst populations. Laryngeal cancer is usually treated with radiotherapy or is surgically removed, but due to the loss of the function of the larynx after surgery, it has been suggested that alternative strategies or ways of treating laryngeal cancer are required. This has prompted the use of, and research in the field of, plant medicine to combat laryngeal cancer. Plant medicine has been used for centuries by the Chinese, Indian and Arabian population in Uhani, Ayurveda and Siddha as a form of replacing conventional medicine with complementary and alternative medicine, these include many plants from the family Asphodelaceae, which have become marketable commodities owing to their medicinal values and traditional uses. Amongst these plants, the genus Bulbine has been used as a form of natural medicine in rural Africa and they are also exploited for their aloe vera properties as well as their possession of phytochemical compounds such as isoflavanoids, nor-lignans, naphthalene derivatives, anthracene and poly prenylated flavonoids. There has been a compelling amount of literature on the traditional uses of the Bulbine spp. because these are linked to the Bulbine spp. having secondary metabolites such as pyroles, chromones, coumarins, bianthraceane, benzene as well as alkaloids. However, for Bulbine natalensis and B. frutescens, the plants of interest in this study, the location of anticancer compounds in them are the only amounts of information available. It has been reported, traditionally, that B. natalensis possesses the anticancer potential in the roots, while the anticancer potential for B. frutescens is in the leaves. However, this requires scientific clarification. Therefore, this study was conducted to assess programmed cell death or apoptosis by analysing the responses of the human laryngeal carcinoma cell line (HEp-2) to crude aqueous and organic (50% and 100% ethanol) fractions of B. natalensis and B. frutescens. In order to have achieved this, the HEp-2 cell line was exposed to the above mentioned fractions at three different final concentrations (20, 2 and 1μg/ml) and assessed for cytotoxicity using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay as an indicator of cell death after fraction utilisation (3 days) for 5 and 8 days. The differences in the potency of the Bubline spp. fractions were confirmed using the non-parametric ANOVA test. Thereafter, selected fractions were screened for apoptotic potential using reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the expression of bax and caspase-3 biomarkers, which are the biomarkers that participate in mitochondrial, endoplasmic reticulum and receptor-ligand mechanism of apoptosis. The fractions of B. frutescens were selected relative to those of B. natalensis for the RT-PCR procedure (read section 3.4.1. for the selection procedure) and links between the cytotoxicity and gene expression results were analysed. It was found that the B. natalensis fractions had a much greater cytotoxic effect on the HEp-2 cell line compared to fractions of B. frutescens by the fifth day of the MTT assay. On the eight day of incubation, there was an increase in HEp-2 cell line proliferation by the fractions of both plant species administered. The fractions selected for bax and caspase-3 gene expression analysis for B. natalensis were the: 20 μg/ml root and corm aqueous fractions, 20 μg/ml leaf and corm 100% ethanol fractions, 20 μ g/ml corm 50% ethanol fraction, 2 μg/ml root aqueous fraction, 2 μg/ml leaf 100% ethanol fraction and the corm 1 μg/ml aqueous and 50% ethanol fractions. The fractions that were compared to B. natalensis were the 20 μg/ml root and leaf aqueous and 100% ethanol fractions respectively, the 2 μg/ml root aqueous fraction and the 2 μg/ml leaf 100% ethanol fraction. It was found from RT-PCR analysis that all of the B. natalensis fractions tested induced expression of caspase-3, which indicated that those fractions were capable of inducing apoptosis in laryngeal carcinoma in vitro, since caspase-3 is the molecular indicator of apoptosis. The aqueous B. frutescens root fraction, did not induce expression of caspase-3 gene, although it caused expression of bax. This implied that the root aqueous B. frutescens fraction, may be involved in some other form of cell death, other than apoptosis. It was also found that there was variability in the response of the HEp-2 cell line to the Bulbine spp. fractions because of the variation in bax expression among fractions of different concentration. It was difficult, from this study, to classify fractions into categories for their mechanism of action, because not all of the fractions that caused the expression of capase-3, induced bax gene expression. Hence, proper conclusions were unable to be made, more so, because all the mechanisms of apoptosis mentioned, involve bax gene activation in order to proceed to completion. Therefore for those Bulbine spp. fractions to which the HEp-2 cell line exhibited a variable response to, it was postulated that cell death or apoptosis occurred through some other unknown mechanism. Overall, the cytotoxicity result didn’t correlate to the gene expression results because fractions that promoted HEp-2 cell line growth by day five, expressed apoptotic markers, which highlighted the sensitivity and accuracy of the cells-to-cDNATM II kit for detecting a few possibly apoptosed cells. This was confirmed by the fact that the HEp-2 cell line used in the MTT cytotoxicity assay and gene expression study had the same passage number and were viable, the latter being achieved because the MTT assay only measures the cytotoxicity of compounds once they have been taken up by viable cells – measuring mitochondrial activities expressed as absorbances. Therefore, the deduction that HEp-2 cell death may be due to bax/caspase-3 expression was valid because the mRNA was isolated from viable HEp-2 cells that had been killed by Bulbine spp. fractions of different polarity. Furthermore, the lack of correlation between the cytotoxicity and gene expression results indicated the amount of HEp-2 cell line proliferation by the fraction out-competes those that died, thereby producing a negative cytotoxicity result. There was a relationship between the traditional information about the anticancer potential for B. natalensis and B. frutescens. For example, the aqueous root fractions of B. natalensis were found to be non-toxic to the HEp-2 cell line, but did express caspase-3, which indicated the possibility of apoptosis. Similarly, the 100% ethanol leaf B. frutescens fractions were non-toxic to the HEp-2 cell line, but were able to induce apoptosis as well. This emphasised that the MTT cytotoxicity assay should be compared with other methods of measuring cytotoxicity when performing studies like this, because although literature has emphasised many advantages of using the MTT cytotoxicity assay in apoptotic studies, this study proved otherwise. When identical HEp-2 cells were treated with the same extract, only some cells were killed (apoptosis) whereas others proliferated. This was because although the cells were identical phenotypically, they were all probably at different phases of the cell cycle resulting in the HEp-2 cells responding variably to the same fraction at different concentrations. It was also found that the responses were concentration independent. For example, the 1 μg/ml B. natalensis corm fraction exhibited the highest toxicity of the three concentrations administered. The lowest cytotoxicity was achieved for the 20 μg/ml fraction – showing a proliferative effect on the HEp-2 cell line. Similarly, the 2 μg/ml aqueous B. natalensis leaf fraction induced the highest cytotoxicity level in the HEp-2 cell line followed by the 1 μg/ml and then the 20 μg/ml fractions. Apart from the genetic variation in identical HEp-2 cells; this indicated that the HEp-2 cell line was selective to particular fractions of the Bulbine spp. for utilisation. Concentration independence and HEp-2 cell preferential selection has been reported in many other studies involving plant fractions/extracts and natural products. This study demonstrated that although all the tested B. natalensis fractions were capable of inducing HEp-2 cell death possibly via. apoptosis (caspase-3 induction), a lack of any link between apoptosis and the cytotoxicity results (hence the 20 μg/ml corm fraction had a negative cytotoxicity but expressed both apoptotic markers), indicated the need for phytochemical screening of both Bulbine spp. in future, to determine the compounds that are responsible for the cytotoxicity and gene expression result outcomes of both Bulbine spp. fractions. Furthermore, procaspase genes also have to be analysed since genes are expressed to form procaspases, which then form active caspases. Although normal cells also express caspase-3 genes during apoptosis, this study focused exclusively on the effect of Bulbine natalensis and B. frutescens fractions (selected relative to the cytotoxicity results of B. natalensis) on the HEp-2 cell line (read cell culture and cytotoxicity discussion for selection of HEp-2 cell line). The validity of this study is confirmed by similar experimental designs that assayed the cytotoxicity of plant-derived or natural compounds on cancer cell lines only, and the detection of apoptosis through caspase- 3 induction and other unrelated methods. This is the first study to report the induction of apoptosis in cancer cell lines by Bulbine spp. fractions using cytotoxicity and the expression of bax and caspase-3 apoptotic markers. It provides insight into the interaction between the HEp-2 cell line and the aqueous and organic fractions of B. natalensis and B. frutescens by analyzing links between cytotoxicity and bax and caspase-3 gene expression; which could probably contribute to drug design with selected Bulbine spp. fractions. Further investigations are required in future, to confirm the possible drug targets of the studied Bulbine spp. fractions in an attempt of assaying their therapeutic importance. / National Research Foundation

An investigation was conducted to determine if laryngeal valving economy, as reflected in measures of laryngeal airway resistance during vowel production, varies across adulthood. Toward this end, sixty healthy men were studied, ten from each of six age groups--25, 35, 45, 55, 65, and 75 years (± 2 years). Results showed that there are age-related differences in laryngeal airway resistance during vowel production, and that these differences are characterized by a lower mean resistance in 75-year-old men than in younger men of the ages studied. This finding provides insight into mechanisms regarding the impact of age on laryngeal function and has important clinical implications for the evaluation and management of men with voice disorders.

The purpose of this study was to compare major theories of laryngeal vibration. The basic hypothesis of the study was that the differences and similarities between the major theories of laryngeal vibration could be made evident and clear through a comparative study. It was assumed that there are two or more theories of laryngeal vibration and that all the major theories of laryngeal vibration from 1945 to the present have been described in written form in English.

Introdução: Diversas afecções laríngeas exigem o uso de enxertos para correção de insuficiências anatômicas ou de múltipla aplicação de fármacos para controle da doença. Alguns bioimplantes, especialmente aqueles com propriedades de liberação controlada de drogas, podem ser úteis no tratamento dessas afecções laríngeas, como a papilomatose laríngea. O poli ácido lático-co-glicólico (PLGA), tem sido bastante testado em aplicações intraoculares e topicamente na cavidade nasal, com resultados bastante promissores. No entanto, um implante de PLGA nunca foi avaliado quanto à sua biocompatibilidade em tecidos laríngeos. Objetivo: Avaliar as alterações histológicas decorrentes da aplicação de PLGA em laringes de coelhos. Materiais e Métodos: Grupos de sete a oito coelhos foram sacrificados em diferentes tempos pós-manipulação cirúrgica (1, 2, 4 e 8 semanas), comparando o lado implantado com PLGA e o lado sham-operado. Os achados histopatológicos foram comparados entre os dois grupos em cada tempo cirúrgico, assim como foi avaliada a diferença comparada a laringes não manipuladas. Resultados: A implantação de PLGA promoveu maior infiltração de células inflamatórias, indução de fibrose e neovascularização até a 4ª semana pós-operatória avaliada. No entanto, após oito semanas de evolução, as hemilaringes implantadas com PLGA se apresentaram semelhantes às laringes sham-operadas, ou mesmo não manipuladas cirurgicamente, com retorno quase que completo às condições basais. Por outro lado, não houve diferença da quantidade de depósito de colágeno tipo I e III nas hemilaringes implantadas com PLGA, em nenhum dos tempos avaliados. Conclusão: O implante de PLGA parece ser seguro para aplicações intralaríngeas, produzindo reações inflamatórias nas quatro primeiras semanas, mas que são reversíveis em oito semanas. / Introduction: Several laryngeal affections require the use of grafts to correct anatomical insufficiencies or multiple application of drugs to control the disease. Some bioimplants, especially those with controlled drug release properties, may be useful in the treatment of such laryngeal conditions, such as laryngeal papillomatosis. Poly (lactic acid-co-glycolic acid), PLGA, has been widely tested in intraocular and nasal cavity applications, with very promising results. However, a PLGA implant has never been assessed for its biocompatibility in laryngeal tissues. Aim: To evaluate the histological changes resulting from the application of PLGA in rabbit larynx. Materials and Methods: Groups of 7-8 rabbits were sacrificed at different postsurgical times (1, 2, 4, and 8 weeks), comparing the implanted side with PLGA and the sham-operated side. Histopathological findings were compared between the two groups at each surgical time, as was the difference compared to the non-surgically manipulated larynx. Results: PLGA implantation promoted a higher infiltration of inflammatory cells, induction of fibrosis and neovascularization until the 4th postoperative week evaluated. However, after 8 weeks of evolution, PLGA implanted hemilarynx were similar to sham-operated or even non-surgically manipulated larynx, with a complete return to baseline conditions. On the other hand, there was no difference in the amount of collagen deposition type I and III in the hemilarynx implanted with PLGA, in none of the evaluated times. Conclusion: The PLGA implant appears to be safe for intralaryngeal applications, producing inflammatory reactions in the first 4 weeks, but which are reversible within 8 weeks.

Thesis. 1975. Ph.D. cn--Massachusetts Institute of Technology. Dept. of Electrical Engineering and Computer Science. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING. Vita. / Bibliography: leaves 293-303. / Ph.D.cn

Objetivo: Verificar se pacientes com disfunção velofaríngea marginal modificam a resistência laríngea como uma estratégia para alcançar o fechamento velofaríngeo completo. Modelo: Análise prospectiva. Local de execução: Laboratório de Fisiologia do Hospital de Reabilitação de Anomalias Craniofaciais - USP (HRAC-USP). Participantes: 19 pacientes com fissura de palato operada, de ambos os sexos com idade entre 12 e 47 anos, apresentando fechamento velofaríngeo marginal (grupo M), e 19 indivíduos sem fissura (grupo C), de ambos os sexos com idade entre 14 e 35 anos. Variáveis: Resistência laríngea (R), pressão aérea intra-oral (Po) e fluxo oro-nasal (V), obtidos por meio de avaliação aerodinâmica utilizando-se o sistema PERCI-SARS, durante a produção da sílaba /pa/, com e sem a oclusão das narinas. Resultados: O valor médio de R, Po e V, no grupo com fechamento velofaríngeo marginal foi de, respectivamente, 4,8±10,8cmH2O/l/seg, 4,8±1,4cmH2O, 144,8±34,0ml/s sem a oclusão das narinas (Ms); de 4,0±14,3cmH2O/l/seg, 4,8±1,1cmH2O, 150,9±38,7ml/s com a oclusão das narinas (Mc) e de 9,2±13,4cmH2O/l/seg, 4,8±0,8cmH2O, 133,9±50,2ml/s no grupo controle (C). Não houve diferença estatisticamente significante (p<0,05) entre os valores médios de R, Po e V dos grupos estudados. Conclusão: Esses resultados mostraram que os pacientes com fechamento velofaríngeo marginal estudados não modificaram a resistência laríngea como uma estratégia para melhorar a ressonância de fala. / Objective: To investigate whether patients with marginal velopharyngeal dysfunction modify the laryngeal resistance as a strategy to achieve complete velopharyngeal closure. Design: Prospective analysis. Setting: Laboratory of Physiology at the Hospital for Rehabilitation of Craniofacial Anomalies - USP (HRAC-USP). Participants: 19 patients with repaired cleft palate, of both genders, aged 12 to 47 years, presenting marginal velopharyngeal closure (M group), and 19 individuals without clefts (C group), of both genders, aged 14 to 35 years. Variables: Laryngeal resistance (R), intraoral air pressure (Po) and oronasal airflow (V), obtained by aerodynamic evaluation by the PERCI-SARS system, during production of the syllable /pa/, with and without nostril occlusion. Results: The mean R, Po and V values for the group with marginal velopharyngeal closure was, respectively, 34.8±10.8cmH2O/l/sec, 4.8±1.4cmH2O, 144.8±34.0ml/s without nostril occlusion (Ms); 34.0±14.3cmH2O/l/sec, 4.8±1.1cmH2O, 150.9±38,7ml/s with nostril occlusion (Mc); the values observed for the control group (C) were 39.2±13,4cmH2O/l/sec, 4.8±0.8cmH2O, 133.9±50.2ml/s. There was no statistically significant difference (p<0.05) among the mean R, Po and V values of the study groups. Conclusion: These results demonstrate that the patients with marginal velopharyngeal closure investigated did not modify the laryngeal resistance as a strategy to improve speech resonance.

No description available.