D-methionine (D-met) has demonstrated otoprotection from noise-, aminoglycoside-, and cisplatin-induced hearing loss in animal studies. As a result, D-met is currently progressing through translational "bench to bedside" research. However, D-met's exact otoprotective mechanisms have not been fully elucidated. This study investigated relationships between dose- and time-dependent D-met otoprotection from noise- and aminoglycoside-induced hearing loss. Further, the study correlated protective D-met dose to endogenous antioxidant enzyme activity and lipid peroxidation. Specific aim 1 tested D-met dose response protection by auditory brainstem response (ABR) threshold shift analysis and outer hair cell (OHC) quantification. D-met doses ranging from 25-200 mg/kg/dose were administered to chinchillas every 12 hours five times each before and after steady state noise exposure totaling 10 D-met doses. Results demonstrated optimal, sub-optimal, and supra-optimal bi-phasic D-met otoprotective dose response. Optimal D-met protection from steady state noise occurred at the 50 mg/kg/dose level. OHC quantification confirmed electrophysiological assessment. Specific aim 2 measured D-met rescue protection from steady state noise exposure by ABR threshold shift analysis and OHC quantification. Five intraperitoneal (ip) D-met injections were administered every 12 hours beginning 3, 5, 7, 9, 12, 18, 24, 36, or 48 hours after steady state noise exposure. Results measured full D-met protection when administration began as late as 24 hours after noise secession. Significant partial protection was also measured for the 36 hour delay. OHC quantification confirmed electrophysiological assessment. Specific aim 3 measured D-met preloading protection from steady state noise exposure by ABR threshold shift analysis and OHC quantification. Five ip D-met injections were administered every 12 hours beginning 2, 2.5, or 3 days prior to steady state noise exposure. Results measured significant D-met protection when administration ended as early as 24 hours prior to noise exposure. OHC quantification confirmed electrophysiological assessment. Specific aim 4 tested dose-dependent D-met influence on antioxidant enzyme activity and oxidative stress in steady state noise-exposed chinchillas. One ip D-met injection, ranging from 25 to 200 mg/kg/dose, was administered every 12 hours beginning 2 days prior to steady state noise exposure for a total of 5 injections. Two hours post-noise exposure, animals were sacrificed and serum, liver, and cochleae were collected for endogenous antioxidant analysis. Glutaredoxin 2 (Grx2) was also analyzed 21 days post-noise exposure. Lower D-met doses (25 and 50 mg/kg/dose) increased superoxide dismutase and catalase activity. Glutathione reductase and glutathione peroxidase activities significantly increased with D-met doses but only at high concentrations (200 mg/kg/dose). At 21 days post-noise, Grx2 activity was significantly decreased in liver but greatly increased in the cochlea with high D-met doses (200 mg/kg/dose). The endogenous enzyme studies suggest optimal protective D-met dose determined in specific aims 1 through 3 may be secondary to increased superoxide dismutase and catalase activity which may result from D-met's free radical scavenging characteristics. Glutathione pathway activity increased only with high D-met doses that resulted in less optimal protection in specific aim 1. Thus, D-met-induced glutathione pathway enhancement may be a compensatory or saturation mechanism rather than the primary protective mechanism. Further, the extended pre-loading and rescue protection may be a result of significantly increased s-glutathionylation activity in the cochlea. Specific aim 5 tested D-met protection from impulse noise exposures. D-met dose response, rescue, and antioxidant enzyme assay protocols, similar to those in specific aims 1, 3, and 4 in steady state animals, were performed on impulse noise-exposed chinchillas. D-met provided dose- and time-dependent optimal protection from impulse noise similar to the steady-state noise studies. Optimal D-met protection was measured at the 100 mg/kg/dose level with complete rescue protection as late as 24 hours post-noise exposure. Endogenous enzyme activity measures demonstrated significant superoxide dismutase, catalase, and glutathione peroxidase activity increases which correlated with optimal D-met protective dose (100 mg/kg/dose) and catalase and superoxide dismutase activity decreases at the higher doses (200 mg/kg/dose). Specific aim 6 tested dose-dependent D-met protection from tobramycin, amikacin, kanamycin, and gentamicin aminoglycoside antibiotics. Guinea pig animal models were normalized to achieve a 30-40 dB ABR threshold shift with the lowest possible aminoglycoside dose. D-met and the aforementioned single aminoglycoside were administered for 21, 28, 23, or 14 days, respectively. ABRs were collected and assessed at baseline, 2, 4, and 6 weeks after drug administration initiation. After the 6-week ABR data collection, cochleae were collected and prepared for OHC quantification. ABR threshold shifts and OHC quantifications demonstrate significant bi-phasic D-met-induced protection from each aminoglycoside type with different D-met doses. OHC quantification confirmed electrophysiological assessment. This study identified optimal protective D-met dose for aminoglycoside- and noise- induced ototoxicity. It also identified optimal protective D-met dose timing for steady state and impulse noise-induced hearing loss. Further, this study has identified dose-dependent D-met-induced endogenous antioxidant changes and Grx2 enhancement, and therefore s-glutathionylation, as a potential mechanism for D-met protection. Thus, dose- and time-dependent D-met protection influences endogenous antioxidant activity, but exact optimal D-met protection will continue to warrant further investigation.

L to D isomerization of amino acids in a commercially prepared alkali-treated food-grade soy isolate was investigated. A soy flour product was also investigated as a control which had not been alkali treated. Methionine and valine collected from two soy flour and two soy isolate samples were coupled with L-leucine-N-carboxyanhydride. If both D- and L-forms were present this would produce diastereomeric dipeptide derivatives. The derivatives were separated by ion-exchange chromatography using a TSM Amino Acid Analyzer. Comparison of the sample methionine and valine dipeptide peaks to standard dipeptide peaks revealed the presence of L-valine, L-methionine and D-methionine in both soy flour and soy isolate samples. Methionine was substantially isomerized in both products. D-valine was not observed in either soy flour or soy isolate samples. Thus, isomerization of methionine appeared to occur at a faster rate than valine. Acid hydrolysis, toasting of soy meal, and alkaline treatment are all conditions which may be responsible for the isomerization of methionine. / Master of Science

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Nesse trabalho investigamos as propriedades vibracionais em monocristais D-Metionina, utilizando as espectroscopias Raman e Infravermelho. As medidas de espectroscopia Raman foram realizadas sob a variaÃÃo de pressÃo hidrostÃtica no intervalo de 0GPA atà 7GPa, mostraram duas transiÃÃes de fase, uma por volta 2.0GPa e outra em 2.5GPa, as quais foram detectadas no espectro Raman atravÃs das mudanÃas exibidas pelos picos associados principalmente Ãs vibraÃÃes atribuÃdas à carboxila (CO2-), amina (NH3+), , ligaÃÃes CS e CSC e grupos CH2 e CH3. Esses modos vibracionais tambÃm foram sensibilizados em baixas temperaturas, sendo que suas mudanÃas serviram como referÃncia para identificar a transiÃÃo de fase nas medidas de espalhamento infravermelho no intervalo de temperatura de 298K a 84K. Nas medidas de espalhamento Raman de 298K a 13K, a transiÃÃo foi identificada pelo surgimento do modo translacional em 91 cm-1 por volta de 84K. AlÃm disso, nesse trabalho comparamos o comportamento das mudanÃas apresentadas na rede cristalina dos monocristais de D-metionina de L-metionina sob condiÃÃes extremas de pressÃo. / In this work we have been investigated the vibrational properties of the D-methionine single crystals using the Raman and Infrared spectroscopies. The Raman spectroscopy measurements were performed under varying hydrostatic pressure from 0 GPa up to 7.0 GPa, indicated two transitions, one around 2.0 GPa and another in 2.5GPa, which were detected by changes of the vibrationals modes of the carboxilic (CO2-), amino (NH3+), CS, CSC, CH2 and CH3, respectively. These vibrationals modes also have been sensitized under low temperatures, whose changes have been helpful to identify the phase transition with the measurements of the infrared scattering from 298K up to 84K. In the measurements of the Raman scattering realized between 298K and 84K, the transition was identified by emerging of the translational mode in 91 cm-1 around 84K. Further, in this work we have been compared changes presented by crystal lattice between D-methionine and L-methionine under extremes conditions of the pressure.

MELO, Wanessa David Canedo. Transições de fase em monocristais de D-Metionina. 2012. 86 f. Tese (Doutorado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2012. / Submitted by Edvander Pires (edvanderpires@gmail.com) on 2014-11-13T20:33:27Z No. of bitstreams: 1 2012_tese_wdcmelo.pdf: 20119688 bytes, checksum: 2cea088ba0adb8d462559d2b87daaf54 (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2014-11-13T20:38:19Z (GMT) No. of bitstreams: 1 2012_tese_wdcmelo.pdf: 20119688 bytes, checksum: 2cea088ba0adb8d462559d2b87daaf54 (MD5) / Made available in DSpace on 2014-11-13T20:38:19Z (GMT). No. of bitstreams: 1 2012_tese_wdcmelo.pdf: 20119688 bytes, checksum: 2cea088ba0adb8d462559d2b87daaf54 (MD5) Previous issue date: 2012 / In this work we have been investigated the vibrational properties of the D-methionine single crystals using the Raman and Infrared spectroscopies. The Raman spectroscopy measurements were performed under varying hydrostatic pressure from 0 GPa up to 7.0 GPa, indicated two transitions, one around 2.0 GPa and another in 2.5GPa, which were detected by changes of the vibrationals modes of the carboxilic (CO2-), amino (NH3+), CS, CSC, CH2 and CH3, respectively. These vibrationals modes also have been sensitized under low temperatures, whose changes have been helpful to identify the phase transition with the measurements of the infrared scattering from 298K up to 84K. In the measurements of the Raman scattering realized between 298K and 84K, the transition was identified by emerging of the translational mode in 91 cm-1 around 84K. Further, in this work we have been compared changes presented by crystal lattice between D-methionine and L-methionine under extremes conditions of the pressure. / Nesse trabalho investigamos as propriedades vibracionais em monocristais D-Metionina, utilizando as espectroscopias Raman e Infravermelho. As medidas de espectroscopia Raman foram realizadas sob a variação de pressão hidrostática no intervalo de 0GPA até 7GPa, mostraram duas transições de fase, uma por volta 2.0GPa e outra em 2.5GPa, as quais foram detectadas no espectro Raman através das mudanças exibidas pelos picos associados principalmente às vibrações atribuídas à carboxila (CO2-), amina (NH3+), , ligações CS e CSC e grupos CH2 e CH3. Esses modos vibracionais também foram sensibilizados em baixas temperaturas, sendo que suas mudanças serviram como referência para identificar a transição de fase nas medidas de espalhamento infravermelho no intervalo de temperatura de 298K a 84K. Nas medidas de espalhamento Raman de 298K a 13K, a transição foi identificada pelo surgimento do modo translacional em 91 cm-1 por volta de 84K. Além disso, nesse trabalho comparamos o comportamento das mudanças apresentadas na rede cristalina dos monocristais de D-metionina de L-metionina sob condições extremas de pressão.

MELO, Wanessa David Canedo. Transições de fase em monocristais de D-Metionina. 2012. 86 f. Tese (Doutorado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2012. / Submitted by Edvander Pires (edvanderpires@gmail.com) on 2015-10-22T18:15:02Z No. of bitstreams: 1 2012_tese_wdcmelo.pdf: 20119688 bytes, checksum: 2cea088ba0adb8d462559d2b87daaf54 (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2015-10-22T21:31:24Z (GMT) No. of bitstreams: 1 2012_tese_wdcmelo.pdf: 20119688 bytes, checksum: 2cea088ba0adb8d462559d2b87daaf54 (MD5) / Made available in DSpace on 2015-10-22T21:31:24Z (GMT). No. of bitstreams: 1 2012_tese_wdcmelo.pdf: 20119688 bytes, checksum: 2cea088ba0adb8d462559d2b87daaf54 (MD5) Previous issue date: 2012 / In this work we have been investigated the vibrational properties of the D-methionine single crystals using the Raman and Infrared spectroscopies. The Raman spectroscopy measurements were performed under varying hydrostatic pressure from 0 GPa up to 7.0 GPa, indicated two transitions, one around 2.0 GPa and another in 2.5GPa, which were detected by changes of the vibrationals modes of the carboxilic (CO2-), amino (NH3+), CS, CSC, CH2 and CH3, respectively. These vibrationals modes also have been sensitized under low temperatures, whose changes have been helpful to identify the phase transition with the measurements of the infrared scattering from 298K up to 84K. In the measurements of the Raman scattering realized between 298K and 84K, the transition was identified by emerging of the translational mode in 91 cm-1 around 84K. Further, in this work we have been compared changes presented by crystal lattice between D-methionine and L-methionine under extremes conditions of the pressure. / Nesse trabalho investigamos as propriedades vibracionais em monocristais D-Metionina, utilizando as espectroscopias Raman e Infravermelho. As medidas de espectroscopia Raman foram realizadas sob a variação de pressão hidrostática no intervalo de 0GPA até 7GPa, mostraram duas transições de fase, uma por volta 2.0GPa e outra em 2.5GPa, as quais foram detectadas no espectro Raman através das mudanças exibidas pelos picos associados principalmente às vibrações atribuídas à carboxila (CO2-), amina (NH3+), , ligações CS e CSC e grupos CH2 e CH3. Esses modos vibracionais também foram sensibilizados em baixas temperaturas, sendo que suas mudanças serviram como referência para identificar a transição de fase nas medidas de espalhamento infravermelho no intervalo de temperatura de 298K a 84K. Nas medidas de espalhamento Raman de 298K a 13K, a transição foi identificada pelo surgimento do modo translacional em 91 cm-1 por volta de 84K. Além disso, nesse trabalho comparamos o comportamento das mudanças apresentadas na rede cristalina dos monocristais de D-metionina de L-metionina sob condições extremas de pressão.