abstract: Measuring molecular interaction with membrane proteins is critical for understanding cellular functions, validating biomarkers and screening drugs. Despite the importance, developing such a capability has been a difficult challenge, especially for small molecules binding to membrane proteins in their native cellular environment. The current mainstream practice is to isolate membrane proteins from the cell membranes, which is difficult and often lead to the loss of their native structures and functions. In this thesis, novel detection methods for in situ quantification of molecular interactions with membrane proteins are described. First, a label-free surface plasmon resonance imaging (SPRi) platform is developed for the in situ detection of the molecular interactions between membrane protein drug target and its specific antibody drug molecule on cell surface. With this method, the binding kinetics of the drug-target interaction is quantified for drug evaluation and the receptor density on the cell surface is also determined. Second, a label-free mechanically amplification detection method coupled with a microfluidic device is developed for the detection of both large and small molecules on single cells. Using this method, four major types of transmembrane proteins, including glycoproteins, ion channels, G-protein coupled receptors (GPCRs) and tyrosine kinase receptors on single whole cells are studied with their specific drug molecules. The basic principle of this method is established by developing a thermodynamic model to express the binding-induced nanometer-scale cellular deformation in terms of membrane protein density and cellular mechanical properties. Experiments are carried out to validate the model. Last, by tracking the cell membrane edge deformation, molecular binding induced downstream event – granule exocytosis is measured with a dual-optical imaging system. Using this method, the single granule exocytosis events in single cells are monitored and the temporal-spatial distribution of the granule fusion-induced cell membrane deformation are mapped. Different patterns of granule release are resolved, including multiple release events occurring close in time and position. The label-free cell membrane deformation tracking method was validated with the simultaneous fluorescence recording. And the simultaneous cell membrane deformation detection and fluorescence recording allow the study of the propagation of the granule release-induced membrane deformation along cell surfaces. / Dissertation/Thesis / Doctoral Dissertation Electrical Engineering 2018

Zhang, Xinran. / Thesis submitted in: December 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 80-87). / Abstracts in English and Chinese. / TITLE PAGE --- p.i / ABSTRACT --- p.ii / 中文摘要 --- p.v / ACKNOWLEDGEMENTS --- p.vii / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xiv / ABBREVIATIONS --- p.xv / Chapter CHAPTER 1. --- Introduction / Chapter 1.1. --- Rationale of the Study --- p.1 / Chapter 1.2. --- Current technologies for formulating long-acting parenteral protein deliver system --- p.3 / Chapter 1.2.1. --- Chemical Modification --- p.3 / Chapter 1.2.2. --- Sustained-release formulation --- p.4 / Chapter 1.2.2.1. --- Phase separation method --- p.4 / Chapter 1.2.2.2. --- Solvent evaporation/extraction method --- p.5 / Chapter 1.2.2.3. --- Spray drying method --- p.6 / Chapter 1.2.2.4. --- Causes for protein instability --- p.6 / Chapter 1.2.2.4.1. --- Water/organic solvent interface --- p.6 / Chapter 1.2.2.4.2. --- Lyophilization --- p.8 / Chapter 1.2.2.4.3. --- Polymer --- p.11 / Chapter 1.2.2.4.4. --- Stabilizing additive --- p.13 / Chapter 1.3. --- Aqueous-aqueous emulsion technology --- p.17 / Chapter 1.3.1. --- Background --- p.17 / Chapter 1.3.2. --- Basic Principle --- p.17 / Chapter 1.3.3. --- Phase diagram --- p.18 / Chapter 1.3.4. --- Formation of aqueous-aqueous emulsion --- p.19 / Chapter 1.3.4.1. --- Introduction of a water-soluble charged polymer as stabilizer --- p.19 / Chapter 1.3.4.2. --- Freezing-induced phase separation --- p.20 / Chapter 1.3.5. --- General Protocol --- p.21 / Chapter 1.3.5.1. --- Introduction of a water-soluble charged polymeric stabilizer --- p.22 / Chapter 1.3.5.2. --- Freezing-induced phase separation --- p.22 / Chapter 1.3.6. --- Merits and limitations of the aqueous-aqueous emulsion technology --- p.23 / Chapter 1.3.7. --- Protein selection for the sustained release formulation --- p.25 / Chapter 1.4. --- Aims and scope of study --- p.26 / Chapter "CHAPTER 2," --- Materials and Methods / Chapter 2.1. --- Materials --- p.28 / Chapter 2.1.1. --- Proteins --- p.28 / Chapter 2.1.2. --- Polymers --- p.28 / Chapter 2.1.3. --- Media for TF-1 Cell Culture --- p.28 / Chapter 2.1.4. --- Chemicals and Solvents for Cell Proliferation Assay --- p.29 / Chapter 2.1.5. --- Other Chemicals and Solvents --- p.29 / Chapter 2.1.6. --- Materials for Cell Culture --- p.29 / Chapter 2.1.7. --- Materials for Reagent Kits --- p.30 / Chapter 2.2. --- Methods --- p.30 / Chapter 2.2.1. --- Determination of the Partition Coefficients of Proteins Between PEG and Dextran --- p.30 / Chapter 2.2.2. --- Preparation of Glassy Particles --- p.31 / Chapter 2.2.2.1. --- Standard Stable Aqueous-aqueous Emulsion Method --- p.31 / Chapter 2.2.2.2. --- Freezing-induced Phase Separation --- p.32 / Chapter 2.2.3. --- Preparation of Protein-loaded and Blank Microspheres Using S-o-w Solvent Extraction Technique --- p.32 / Chapter 2.2.4. --- Optical Microscopy and Scanning Electron Microscopy --- p.33 / Chapter 2.2.5. --- Determination of Protein Loading --- p.34 / Chapter 2.2.5.1. --- Within Dextran Particles --- p.34 / Chapter 2.2.5.2. --- Within PLGA microspheres --- p.34 / Chapter 2.2.6. --- Evaluation of Protein Structural Integrity and Bioactivity in Dextran Particles and PGLA Microspheres --- p.35 / Chapter 2.2.7. --- In vitro Release Study --- p.36 / Chapter 2.2.8. --- RhIFN Stability Determination under Simulated In Vitro Release Conditions --- p.37 / Chapter 2.2.8.1. --- In the Absence of PLGA --- p.37 / Chapter 2.2.8.2. --- In the Presence of PLGA --- p.37 / Chapter 2.2.9. --- MicroBCÁёØ Protein Assay --- p.38 / Chapter 2.2.10. --- Size Exclusion Chromatography (SEC) - High Performance Liquid Chromatography (HPLC) --- p.38 / Chapter 2.2.11. --- ELISA --- p.39 / Chapter 2.2.12. --- Bioactivity Assay --- p.40 / Chapter 2.2.12.1. --- RhIFN --- p.40 / Chapter 2.2.12.2. --- RhGM-CSF --- p.41 / Chapter CHAPTER 3. --- Results and Discussions / Chapter 3.1. --- Sustained-release RhIFN Formulation --- p.45 / Chapter 3.1.1. --- Partition Coefficient of RhIFN --- p.45 / Chapter 3.1.2. --- Formulation Based on the Standard Aqueous-aqueous Emulsion (SA-AE) Method With Sodium Alginate as Stabilizer --- p.45 / Chapter 3.1.2.1. --- Surface Morphology --- p.45 / Chapter 3.1.2.2. --- Formulation Characterization --- p.46 / Chapter 3.1.2.3. --- In Vitro Release of RhIFN from PLGA Microsheres --- p.54 / Chapter 3.1.3. --- Formulation Based on the Freezing-induced Phase Separation (FIPS) Technique without Sodium Alginate --- p.56 / Chapter 3.1.3.1. --- Formulation Characterization --- p.56 / Chapter 3.1.3.2. --- In Vitro Release of RhIFN from PGLA Microsphees --- p.59 / Chapter 3.2. --- RhIFN Stability Assessment under Simulated In Vitro Release Conditions --- p.63 / Chapter 3.2.1. --- In the Absence of PLGA --- p.63 / Chapter 3.2.2. --- In the Presence of PLGA --- p.65 / Chapter 3.3. --- Sustained-release RhGM-CSF Formulation --- p.68 / Chapter 3.3.1. --- Partition Coefficient Determination of RhGM-CSF Between PEG and Dextran --- p.68 / Chapter 3.3.2. --- Formulation Based on Freezing-induced Phase Separation --- p.68 / Chapter 3.3.2.1. --- Validation of MTT Assay Conditions --- p.69 / Chapter 3.3.2.2. --- Formulation Characterization --- p.71 / Chapter 3.3.2.3. --- In Vitro Release of RhGM-CSF from PLGA Microspheres --- p.75 / Chapter CHAPTER 4. --- Conclusion and Future Studies / Chapter 4.1. --- Conclusion --- p.78 / Chapter 4.2. --- Future Studies --- p.79 / References --- p.80

Este estudo visa o desenvolvimento de microemulsões que, após captação de água do tecido subcutâneo, transformar-se-ão em gel nanoestruturado de fase hexagonal para liberação sustentada de naltrexona e tratamento do alcoolismo. A microemulsão selecionada, composta por monooleína, tricaprilina, propilenoglicol e água (ME-MO) resultou na liberação in vitro de 31 % de naltrexona em 96 h. Após sua administração subcutânea, foi observada formação do gel em 48 h, o qual persistiu por mais de 30 dias in vivo, promovendo liberação prolongada do marcador fluorescente Alexa flúor. A eficácia da formulação foi avaliada em modelo de preferência condicionada por lugar induzida por etanol; ME-MO com 5 e 10% de naltrexona foi comparada à solução de naltrexona diária. Não observou-se diferença significativa entre a solução de naltrexona e ME-MO 5%, enquanto que ME-MO 10% diferiu destas, e antagonizou a preferência condicionada por lugar. Esses resultados demonstram o potencial de ME-MO como uma plataforma para liberação prolongada de fármacos no tratamento de dependência química. / This study focuses on the development of microemulsions that after in vivo water uptake of the subcutaneous tissue will turn into liquid-crystalline gels for sustained release of naltrexone, used in the treatment of alcoholism. Three microemulsions based on monoolein and tricapryline (ME-MO), vitamin E TPGS and propylene glycol, TPGS and Span were selected. The latter resulted is faster drug release (65% in 96 h). Based on the ability of the gel formed to withstand dilution, ME-MO was selected for in vivo studies. After subcutaneous administration, hexagonal phase formation was observed in 48 h and its persistence for more than 30 days in those animals. The efficacy of the formulation was assessed using conditioned preference place model. The animals were divided into four groups: Saline (control); Naltrexone solution (1 mg / kg) daily for 8 days (30 min before ethanol administration), and ME-MO with 5% or 10% naltrexone (single administration). The results suggest that ME-MO 10% antagonized the preference induced by ethanol.

Esse trabalho apresenta resultados de modificações estruturais, caracterizações e aplicações de derivados de quitosana como carreadores de fármacos. Derivados anfifílicos de quitosana, contendo grupos hidrofílicos e grupos hidrofóbicos, foram caracterizados por técnicas de espectroscopia de ressonância magnética nuclear (RMN1H), espectroscopia na região do infravermelho (IV), espectroscopia na região do UV-Vis, técnicas termoanalíticas (termogravimetria (TGA), termogravimetria derivada (DTG), análise térmica diferencial (DTA) e calorimetria exploratória diferencial (DSC)), fluorescência no estado estacionário, espalhamento dinâmico de luz (DLS) e microscopia eletrônica de transmissão (MET). Os resultados de caracterização mostraram que as sínteses propostas foram realizadas com sucesso. A determinação da concentração de agregação crítica (CAC) e os estudos de DLS e MET confirmam que os derivados se auto-organizam em solução aquosa formando agregados com diâmetros variando entre 230 a 500 nm. Esses valores, associados aos potenciais zeta obtidos (+14,1 mV a +44,8 mV), demonstram que os agregados são estáveis em solução, característica fundamental para aplicação no transporte de fármacos. A capacidade de encapsulamento do fármaco quercetina por esses derivados foi avaliada por estudos de incorporação utilizando a espectroscopia UV-Vis. Os resultados obtidos demonstraram que o comportamento dos derivados depende de parâmetros como o grau de hidrofilicidade e grupo hidrofílico, grau de hidrofobicidade e pH do meio de encapsulamento, possibilitando controlar a quantidade de fármaco contida nos carreadores. A atividade biológica dos agregados formados pelos derivados de quitosana foi testada em células de adenocarcinoma de mama (MCF-7) e os resultados indicaram baixa toxicidade dos carreadores, além de potencialização do efeito terapêutico do fármaco. Estudos de microscopia confocal de varredura a laser evidenciaram que agregados marcados com proteína verde fluorescente com afinidade por quitosana (CAP-sfGFP) foram internalizados pelas células MCF-7. Resultados de hemocompatibilidade indicaram que os polímeros apresentam baixa destruição de glóbulos vermelhos do sangue e liberação de hemoglobina. Portanto, esses derivados possuem características adequadas para aplicação no transporte e liberação controlada de fármacos. / This thesis presents results of structural modifications, characterizations and applications of chitosan derivatives as drug carriers. Amphiphilic derivatives of chitosan containing hydrophilic and hydrophobic groups were characterized by nuclear magnetic resonance spectroscopy (RMN1H), infrared spectroscopy (IV), uv-vis spectroscopy, thermoanalytical techniques (thermogravimetry (TGA), derivative thermogravimetry (DTG), differential thermal analysis (DTA) and differential exploratory calorimetry (DSC)), fluorescence, dynamic light scattering (DLS) and transmission electron microscopy (MET). The characterization results showed that the proposed syntheses were successfully performed. The critical aggregation concentration (CAC), DLS and MET studies confirmed that the derivatives self-assembled in aqueous solution forming aggregates with diameters ranging from 230 to 500 nm. These values, associated with zeta potentials (+14.1 mV to +44.8 mV), demonstrate that the aggregates were stable in solution. The ability to encapsulate quercetin by these derivatives was assessed by incorporation studies using UV-Vis spectroscopy. The results showed that the behavior of the derivatives depends on parameters such as the degree of hydrophilicity and hydrophilic groups, degree of hydrophobicity and pH of the encapsulation medium. The biological activity of the aggregates formed by the chitosan derivatives was tested in breast cancer cells (MCF-7) and the results indicated low toxicity of the carriers, in addition to improving the therapeutic effect of the drug. Confocal laser scanning microscopy studies showed that aggregates stained with chitosan-affinity protein (CAP-sfGFP) were internalized by MCF-7 cells. Hemolysis assays showed good hemocompatibility of chitosan derivatives. Therefore, the derivatives have suitable characteristics for application as drug delivery systems.

Foram realizados 2 experimentos no Departamento de Zootecnia da ESALQ/USP com o intuito de se avaliar o desempenho de tourinhos Nelore em confinamento. No experimento 1 foi avaliada a substituição da uréia (U) convencional por farelo de soja (FS) ou uréia de liberação lenta (ULL) (Optigen®) em rações de bovinos em terminação. Foram utilizados 100 tourinhos da raça Nelore (389 kg), distribuídos em 20 baias por 90 dias. As rações continham 8% de feno de Tifton (% da MS) e 65 a 69% de polpa cítrica no concentrado. Os 5 tratamentos experimentais foram assim designados (%MS): FS - ração com 5% de farelo de soja + 0,9% de uréia; U - ração com 1,7% de uréia; ULL 0,5 - ração com 0,5% de uréia de liberação lenta + 1,2% de uréia; ULL 1,0 - ração com 1,0% de uréia de liberação lenta + 0,8% de uréia; ULL1,5 - ração com 1,5% de uréia de liberação lenta + 0,3% de uréia. A ingestão de MS, o ganho de peso diário, a eficiência alimentar, o rendimento de carcaça, a espessura de gordura subcutânea e a área de olho de lombo não foram afetados pelas fontes de N testadas (P>0,05). Em conclusão, o desempenho de tourinhos Nelore em terminação alimentados com rações ricas em polpa cítrica, não é melhorado com a substituição da uréia convencional por farelo de soja ou uréia de liberação lenta na ração. No experimento 2 foram utilizados 192 animais (403 kg), distribuídos em 32 baias por 99 dias com objetivo de comparar rações contendo milho duro moído fino (M), laminado (L), ensilado (SGU) ou floculado (F), com 2 níveis de inclusão de bagaço de cana-deaçúcar: 12 ou 20% da MS da ração. Não houve interação entre processamentos do grão de milho e os níveis de forragem para nenhum parâmetro avaliado (P>0,05). A IMS foi maior (P<0,05) para o tratamento L comparado como os demais. O GPD foi maior (P<0,05) para os tratamentos F e SGU e a EA foi maior (P<0,05) para os tratamentos F, SGU e M comparados com L e para o tratamento F comparado com M. O rendimento de carcaça (RC) foi maior (P<0,05) para os tratamentos F e M. Os maiores valores (P<0,05) de energia líquida de manutenção e de ganho de peso das rações foram obtidos com os tratamentos F e SGU. A digestibilidade total do amido (DTA) foi maior nas rações contendo milho floculado, intermediária com milho moído fino e com milho ensilado e menor com milho laminado (P<0,05). A IMS foi menor (P<0,05) para os tratamentos contendo rações com 12% bagaço de cana-de-açúcar na MS da ração. O GPD, a EA, e o RC foram maiores nos animais dos tratamentos com 12% de bagaço de cana-de-açúcar na MS da ração (P<0,05). Os valores de energia líquida de manutenção e de ganho de peso das rações foram superiores (P<0,05) para os tratamentos com 12% de bagaço de cana-de-açúcar na MS. A DTA não foi alterada (P<0,05) em função do nível de inclusão do bagaço de cana-de-açúcar nas rações. Com base nos resultados conclui-se que o milho floculado e ensilado úmido são superiores, o milho moído é intermediário e o laminado inferior como fontes energéticas para tourinhos Nelore em terminação. Rações com 12% de bagaço de cana-de-açúcar contêm maior densidade energética e permitem melhor desempenho de tourinhos Nelore na fase de terminação comparadas com rações contendo 20% de bagaço de cana-de-açúcar. / Two trials were conducted at the Animal Sciences Department of the University of São Paulo Piracicaba-SP to evaluate the performance of finishing Nellore bulls. In trial 1 soybean meal (FS) and slow release urea (SRU) (Optigen®) replaced urea (U) on diets high in citrus pulp for finishing cattle. One hundred Nellore bulls (389 kg) were grouped in 20 pens for 90 days. Diets contained (%DM) 8% Tifton-85 grass hay and 92% concentrate (65 to 69% citrus pulp). The 5 treatment diets were isonitrogenous as follow (%DM): FS 5% soybean meal + 0.9% urea; U 1.7% urea; SRU 0.5 0.5% slow release urea + 1.2% urea; SRU 1.0 1.0% slow release urea + 0.8% urea; SRU 1.5 1.5% slow release urea + 0.3% urea. Animals were grouped in blocks according to initial BW. DMI, ADG, ADG/DMI, and carcass traits were not affected by N sources (P>0.05). In conclusion, on high citrus pulp diets, replacing soybean meal or slow release urea for urea does not improve performance of finishing Nelore bulls. In trial 2 192 finishing Nellore bulls (403 kg) grouped in 32 pens were fed for 99 days to compare diets containing fine ground, dry rolled, high moisture or steam flaked flint corn and two levels (12 or 20% on DM) of sugar cane bagasse. There was no interaction between corn processing methods and diet forage levels (P>0.05). DMI was higher (P<0.05) for dry rolled corn compared to the other 3 processing methods. ADG was higher (P<0.05) for steam flaked and high moisture corn than for ground or rolled corn. Feed efficiency (ADG/DMI) was higher (P<0.05) for steam flaked corn than for fine ground or dry rolled corn, and higher (P<0.05) for high moisture and ground corn than for dry rolled corn. Dressing was higher (P<0.05) for steam flaked and ground corn than for high moisture and dry rolled corn. The highest (P<0.05) diet energy values were observed for steam flaked and high moisture corn. Total tract starch digestibility was highest for steam flaked, intermediate for high moisture and ground corn and lowest for dry rolled corn (P<0.05). DMI was lower and ADG, feed efficiency, dressing and diet energy values were higher for cattle fed 12% than 20% forage diets (P<0.05). Forage level had no effect on diet starch digestibility (P>0.05). In conclusion, steam flaked and high moisture corn are highest, ground corn is intermediate and dry rolled corn is lowest in energy for finishing Nellore bulls. Performance of finishing Nellore bulls is improved with 12% sugar cane bagasse forage diets compared to 20% forage diets.

This thesis investigates the feasibility of loading silicone hydrogel (SiHy) contact lenses with two different hydrophobic therapeutics, latanoprost and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), for treatment of glaucoma and hyperemia respectively. The two methods of loading were 1) thermal loading in an aqueous medium and 2) convective loading in a solution of n-propanol. Dailies Total1® lenses prepared in this manner were tested for their loading and their release into artificial tears. Continuous release over 1-4 days at therapeutic levels is achievable from thermal loading of DMPC, convective loading of DMPC, and convective loading of latanoprost. The DMPC loading processes can be naturally integrated into standard manufacturing lines for Dailies Total1®. Both DMPC and latanoprost release at rates proportional to the amount loaded into a contact lens. Latanoprost loads into a contact lens strictly proportionally to the loading concentration and the time of loading. The convective loading step represents a significant improvement on both the time of loading (reduced from days to minutes) and the loading capacity of silicone hydrogel contact lenses. This thesis also compares the loading and release of latanoprost in the convective loading procedure using the SiHy contact lenses of Acuvue Advance® (Johnson & Johnson Vision Care, Jacksonville, FL) , Air Optix® (Alcon, Copenhagen, Denmark), Biofinity® (CooperVision), PureVision® (Bausch & Lomb), and Dailies Total1® (Alcon), and the polyHEMA lens, SofLens 38® (Bausch & Lomb), finding that silicone hydrogels load an order of magnitude more drug than the polyHEMA lens and release into artificial tears for an order of magnitude longer. Overall, these experiments provide a quantitative understanding of the dynamics of loading and release for both DMPC and latanoprost.

Improved cochlear implant (CI) designs and surgical techniques have allowed CI patients to retain acoustic hearing in the implanted ear post-operatively. These EAS (electric-acoustic stimulation) CI users listen with a combination of acoustic and electric hearing in the same ear. While electric hearing alone improves speech recognition in quiet, preserved acoustic hearing allows EAS CI users to outperform traditional CI users in speech recognition in noise and demonstrate “speech masking release,” an improvement in speech recognition in temporally fluctuating noise relative to steady noise. Masking release is arguably an ecologically valid metric, as listeners often attend to target speech embedded in fluctuating competing speech. Improved speech recognition outcomes have been attributed to the spectral and temporal resolution provided by acoustic hearing. However, the relationship between spectral and temporal resolution and outcomes in EAS CI users is not clear. This study evaluated speech masking release, spectral ripple density discrimination thresholds, and fundamental frequency difference limens (f0DLs) in EAS CI users. Both the ripple and f0DL tasks are thought to measure underlying spectral resolution and temporal fine structure. EAS CI subjects underwent testing in three listening modes: acoustic-only, electric-only, and acoustic+electric. Comparisons across listening modes allowed the benefit provided by acoustic hearing to be quantified. It was hypothesized that speech masking release, spectral ripple density discrimination thresholds, and f0DLs would be poorest with electric-only hearing and would improve in the acoustic-only and acoustic+electric listening modes. This would reflect the benefit of preserved acoustic hearing. It was also hypothesized that speech masking release would correlate with spectral ripple density discrimination thresholds and f0DLs, reflecting the roles of spectral and temporal fine structure cues. Lastly, it was hypothesized that EAS CI users with more residual hearing (lower audiometric thresholds) would perform better on all three tasks. Speech masking release was evaluated using a 12-alternative-forced-choice (AFC) spondee recognition in noise task. The noise was a two-talker and a ten-talker babble presented at -5 dB SNR, and masking release was quantified as the difference in spondee recognition in two-talker babble relative to ten-talker babble. Spectral ripple density discrimination thresholds were assessed in a 3-AFC task using a broadband stimulus that contained spectral peaks and valleys logarithmically spaced on the frequency axis. The spacing between spectral peaks (ripple density) was varied to determine the threshold at which listeners could no longer resolve the individual spectral peaks. F0DLs were assessed via a 3-AFC task using a broadband harmonic complex with a baseline f0 = 110 Hz. The f0 of the test intervals was varied to determine the smallest change in f0 that the listener could detect. Results showed that performance in all three measures was poorest when EAS CI users were tested using electric-hearing only, with significant improvements when tested in the acoustic-only and acoustic+electric listening modes. F0DLs, but not spectral ripple density discrimination thresholds or audiometric thresholds, significantly correlated with speech masking release. Speech masking release also significantly correlated with open-set AzBio sentence recognition in noise scores obtained from clinical records. Results indicated that preservation of residual acoustic hearing allows for speech masking release, likely due to access to temporal fine structure cues provided by residual hearing. The significant correlation between speech masking release and sentence recognition in noise indicates that the ability to extract target speech embedded in temporally fluctuating competing speech is important for speech recognition in noise. Funded by National Institutes of Health/National Institutes on Deafness and Other Communication Disorders (NIH/NIDCD) P50 DC000242, American Speech-Language-Hearing Foundation Student Research Grant, and American Academy of Audiology Student Investigator Research Grant.

Sustainable landscaping includes utilization of plants requiring few inputs. We chose four species showing potential for use in arid landscapes: Purshia mexicana, Shepherdia argentea, Shepherdia rotundifolia, and Alnus maritima. We sowed seeds of S. rotundifolia, S. argentea and P. mexicana in three substrates with various water-holding properties due to differing amounts of organic matter (OM). S. rotundifolia germination was maximized in a calcined clay (66.2%) containing no OM and had low germination (12.7 - 21.8%) in the other substrates. S. argentea germination (42.3 to 53.7%) was similar in all substrates. Poor seed quality of P. mexicana resulted in inconclusive results. Our results suggest that germination of some species is enhanced by substrates with excellent drainage properties. We also investigated effects of different rates of controlled-release fertilizer (CRF) on symbiotic nodule formation in seaside alder. We found that lower than prescribed rates of CRF enhanced nodulation without compromising nitrogen status.

La nanotechnologie possède la potentialité d’améliorer la sécurité, les procédés, l’emballage alimentaire et le concept d’ingrédient fonctionnel. La nano-encapsulation d’agents actifs, est un concept innovant permettant de protéger les agents actifs d’une dégradation éventuelle pendant le procédé de fabrication de l’aliment et son stockage. Le principal objectif de ce travail est de développer et d’optimiser une méthode de marquage fluorescent afin d’effectuer des études de transfert dans différents films de bio-polymères et dans l’aliment et de nano-encapsuler la nisine. La nano-encapsulation de la nisine dans différents nano-liposomes par micro-fluidisation (CCDS) est une technique innovante pour la fabrication de nano-systèmes de re-largage. L’incorporation de nisine sous forme de nano-émulsion est une méthode efficace de contrôle des flores pathogènes sans altérer les caractéristiques des films d’HPMC. La nisine Z a été marquée par un composé fluorescent, et a une masse moléculaire de 3713,3. Des études en microscopie confocale ont permis de démontrer que l’interaction de la nisine avec les membranes bactériennes se situait au niveau de site de division de la cellule. L’HPMC, le chitosane, le caséinate et l’acide poly-lactique agissent comme des réservoirs et libèrent progressivement la nisine afin d’obtenir un effet inhibiteur durable. Le choix du biopolymère affecte la biodispinibilité du composé à la surface et à l’intérieur de l’aliment. La prochaine révolution concernant la sécurité alimentaire par l’emballage mettra en avant le dernier concept technologique « 3-BIOs » qui se réfère aux notions Bioactif - Biodégradable - Bionanocomposite / Food nanotechnology has the potential to improve food safety and bio-security, food processing, food packaging and functional ingredients. Nano-encapsulation of active agents is an innovative concept to protect them against possible denaturation during processing and storage. The overall objective of the present work was to optimize and develop fluorescent labeling and encapsulation of nisin for molecular transfer study in different packaging based on biopolymers and in the food. Nanoencapsulation of nisin in different nanoliposomes by using continuous cell disruption system (CCDS) has provided an innovative method for nano-delivery systems fabrication. Incorporation of nisin in nano-emulsion form (encapsulated and free) can possibly be an effective approach to control pathogen without compromising the basic physico-chemical attributes of composite HPMC coatings. The fluorescently labeled nisin Z prepared had a molecular weight of 3717.3 Da. Confocal microscopic studies demonstrated the interaction of nisin with the bacterial membranes at the cell-division sites as possible mechanism of action against food borne pathogen. HPMC, CTS, SC and PLA packaging bio-membranes act as a reservoir and progressively release nisin to sustain a constant inhibitory effect. Choice of biopolymer is significant in providing requisite bioavailability of antimicrobial compounds at exterior surface and inside the food system. Based on the present study results, the emerging revolution concerning food safety through packaging possibly will rely on « 3-BIOs » blend with nanotechnology, which refers to Bioactive, Biodegradable and Bio-nanocomposite

The successful completion of this work is dedicated first to my grandparents for having always shown their unwavering love and encouragement in my journeys (most of which they kindly and politely only pretended to understand) and for having also served as life-long role models who upheld an unparalleled work ethic. To many whom I consider to be my chosen family, especially Ann Williams and Brittany Sears, who kept me laughing, but more importantly, kept my crazy train from derailing during these tumultuous years. To Wayne Price and Tom Jackman, who fostered the success of my career and are the epitome of patience and kindness. To DeAngelis, for the many hours of laughter, conversations, and adventuresome treks that further kindled my knowledge, love, and respect for Florida’s ecology. To family in Alabama who have either helped shape my brazen character or made this education possible. To Taego, the one to whom I am bound through so many of the stories that begin with, “Remember when…?” and who is often so kind and thoughtful though he still holds tightly to the stereotype of the selfish youngest sibling. Finally, to Fen for being my smiling, bright blue-eyed, spunky kid who has been on this journey with me from the get-go; for keeping me from getting too big for my britches; for your intrinsic fire that burns for equality, fairness, and friendship; and for inspiring me to be the best example of a mother that I can possibly be.