L'objectif de cette thèse est d’étudier temporellement des phénomènes évolutifs à l'aide de la tomographie d'émission monophotonique (TEMP). La première partie de cette thèse traite le problème du mouvement respiratoire dans les images TEMP de souris. Nous présentons ici une méthode permettant de détecter ce mouvement respiratoire dans les images TEMP 4D, d'extraire un signal respiratoire intrinsèque, et de déterminer la phase du cycle respiratoire sans mouvement la plus large possible. Les données enregistrées durant ces phases sans mouvement sont alors utilisées pour reconstruire une seule image TEMP 3D sans artefact de mouvement par acquisition. Les images ainsi reconstruites présentent un bon compromis en terme de statistiques et de précision des mesures par rapport aux images TEMP 3D de base et TEMP 4D. Dans la deuxième partie, nous étudions la cinétique d'incorporation de l'iode dans l'estomac de souris à partir d'images TEMP 4D. Afin de comprendre le rôle biologique de cette accumulation dans l'estomac, nous avons modélisé le phénomène par une approche d'analyse compartimentale avec un modèle simplifiée à deux compartiments (paroi et cavité stomacale) et une entrée (sang). Les courbes temps-activité (TAC) de chaque compartiment sont déduites des observations et une première estimation des paramètres a été obtenue. / The aim of this thesis is to investigate temporally evolving phenomena with the use of single photon emission computed tomography (SPECT).The first part of this thesis addresses the problem of respiratory motion in SPECT images of mice. The presented method permits us to detect the respiratory motion in 4D SPECT images, to extract an intrinsic respiratory signal and to determine the widest possible phase of the respiratory cycle without movement. The data recorded during these motionless phases are then used to reconstruct a single 3D SPECT image without motion artefacts per acquisition. Reconstructed motionless SPECT images present a good compromise in terms of statistics and accuracy of the measurements with respect to basic 3D SPECT and 4D SPECT images. In the second part, we study the iodide uptake kinetics in the stomach 99mTc-pertechnetate biodistribution with the of mice through the study of use of 4D SPECT images. To understand the biological role of the iodide accumulation in the stomach, we modelled the phenomenon with a compartmental analysis approach using a simplified two-compartment (stomach wall and cavity) model with one input (blood). Time activity curves (TAC) of each compartment are deduced from observations and a first estimation of the parameters was obtained.

O presente trabalho possui como metas o estudo, simulação, identificação de parâmetros e comparação estatística de modelos compartimentais utilizados em tomografia por emissão de pósitrons (PET). Para tanto, propõe-se utilizar a metodologia de equações de sensibilidade e o método de Levenberg-Marquardt para a tarefa de estimação de parâmetros característicos das equações diferenciais descritoras dos referidos sistemas. Para comparação entre modelos, foi empregado o critério de informação de Akaike. São consideradas três estruturas compartimentais compostas, respectivamente, por dois compartimentos e duas constantes características, três compartimentos e quatro constantes características e quatro compartimentos e seis constantes características. Os dados considerados neste texto foram sintetizados preocupando-se em reunir as principais características de um exame de tomografia real, tais como tipo e nível de ruído e morfologia de função de excitação do sistema. Para tanto, foram utilizados exames de pacientes do setor de Medicina Nuclear do Instituto do Coração da Faculdade de Medicina da Universidade de São Paulo. Aplicando-se a metodologia proposta em três níveis de ruído (baixo, médio e alto), obteve-se concordância do melhor modelo em graus forte e considerável (com índices de Kappa iguais a 0.95, 0.93 e 0.63, respectivamente). Observou-se que, com elevado nível de ruído e modelos mais complexos (quatro compartimentos), a classificação se deteriora devido ao pequeno número de dados para a decisão. Foram desenvolvidos programas e uma interface gráfica que podem ser utilizadas na investigação, elaboração, simulação e identificação de parâmetros de modelos compartimentais para apoio e análise de diagnósticos clínicos e práticas científicas. / This work has as goals the study, simulation, parameter identification and statistical comparison of compartmental models used in positron emission tomography (PET). We propose to use the methodology of sensitivity equations and the method of Levenberg-Marquardt for the task of estimating the characteristic parameters of the differential equations describing such systems. For model comparison, Akaikes information criterion is applied. We have considered three compartmental structures represented, respectively, by two compartments and two characteristic constants, three compartments and four characteristic constants and four compartments and six characteristics constants. The data considered in this work were synthesized taking into account key features of a real tomography exam, such as type and level of noise and morphology of the input function of the system. To this end, we used tests of patients in the sector of Nuclear Medicine of the Heart Institute of the Faculty of Medicine, University of São Paulo. Applying the proposed methodology with three noise levels (low, medium and high), we obtained agreement of the best model with strong and considerable degrees (with Kappa indexes equal to 0.95, 0.93 and 0.63, respectively). It was observed that, with high noise level and more complex models (four compartments), the classification is deteriorated due to lack of data for the decision. Programs have been developed and a graphical interface that can be used in research, development, simulation and parameter identification of compartmental models, supporting analysis of clinical diagnostics and scientific practices.

Modelos matemáticos de motoneurônios têm sido desenvolvidos para auxiliar na compreensão dos fenômenos que envolvem o sistema neuromuscular. Entretanto, a maioria dos modelos já desenvolvidos baseou-se na premissa de que a árvore dendrítica tem um comportamento passivo, o que ocorre em animais anestesiados, mas pode não ocorrer durante o comportamento motor normal de um animal intacto. Experimentos com animais descerebrados, em que as vias monoaminérgicas encontravam-se ativas, mostraram que os motoneurônios podem apresentar comportamentos mais complexos decorrentes da presença de condutâncias iônicas voltagem-dependentes que se situam nos dendritos e são responsáveis pela gênese de uma corrente de entrada persistente. Nesse sentido, um primeiro objetivo deste trabalho foi o de desenvolver novos modelos matemáticos de motoneurônios de diferentes tipos (i.e. dos tipos S, FR e FF), computacionalmente eficientes e contendo em seus compartimentos dendríticos uma condutância de cálcio do tipo L, de forma que os fenômenos de biestabilidade, potencial platô e amplificação da corrente sináptica efetiva possam ser gerados. Um segundo objetivo foi o de verificar como a presença da condutância iônica ativa no dendrito influencia o comportamento motoneuronal quando o mesmo está sujeito a entradas sinápticas de diferentes tipos. Os novos modelos foram parametrizados baseando-se em dados da literatura experimental para motoneurônios de gatos descerebrados e validados segundo os protocolos experimentais básicos que permitem caracterizar cada tipo de modelo como sendo totalmente ou parcialmente biestável. As entradas sinápticas foram simuladas por processos pontuais de Poisson e os trens de potenciais de ação dos motoneurônios foram analisados. Uma modulação senoidal da intensidade do processo pontual foi usada para estimar as respostas em frequência de cada modelo. Observou-se que, funcionalmente, a presença da condutância iônica dendrítica pode favorecer a ação do motoneurônio durante tarefas posturais, pois, uma vez ativada, a corrente de entrada persistente eleva a excitabilidade motoneuronal tornando os disparos mais regulares, além de prover uma alta sensibilidade dos modelos a entradas sinápticas de baixa frequência, correspondentes às oscilações observadas durante a manutenção da postura ereta quieta. / Mathematical models of motoneurons have been developed as an aid to the understanding of phenomena involving the neuromuscular system, but most of these models have been based on the hypothesis of a passive dendritic tree. This holds for anesthetized animals but not necessarily during normal motor behavior of the intact animal. Experiments with decerebrate animals in which the monoaminergic tracts were maintained intact have shown that more complex behaviors may emerge in motoneurons due to dendritic voltage-gated ionic conductances, which are responsible for a persistent inward current. Therefore, the first aim of this work was to develop computationally-efficient new motoneuron models of different types (i.e. type S, FR and FF) that include a dendritic L-type calcium conductance so that bistability, plateau potential and enhancement of effective synaptic current may be generated. The second aim of this research was to evaluate the effects of the active dendritic ionic conductance on the input-output mapping of presynaptic to postsynaptic spike trains. The new models were parameterized based on data reported in experimental literature on the decerebrate cat preparation, and they were validated using appropriate protocols for either fully or partially bistable dynamics. The synaptic inputs were simulated by Poisson point processes and the output spike trains were analyzed. Sinusoidal modulation of the point process intensity was used for the estimation of each models frequency response. The results suggested that an active dendritic ionic conductance in motoneurons has a functional role during postural tasks, because, when activated, the persistent inward current enhances the motoneuronal excitability, reducing the variability of interspike intervals, and focusing the sensitivity of the models to low frequency inputs that correspond to the low-frequency oscillations that typically occur during quiet standing posture.

O presente trabalho possui como metas o estudo, simulação, identificação de parâmetros e comparação estatística de modelos compartimentais utilizados em tomografia por emissão de pósitrons (PET). Para tanto, propõe-se utilizar a metodologia de equações de sensibilidade e o método de Levenberg-Marquardt para a tarefa de estimação de parâmetros característicos das equações diferenciais descritoras dos referidos sistemas. Para comparação entre modelos, foi empregado o critério de informação de Akaike. São consideradas três estruturas compartimentais compostas, respectivamente, por dois compartimentos e duas constantes características, três compartimentos e quatro constantes características e quatro compartimentos e seis constantes características. Os dados considerados neste texto foram sintetizados preocupando-se em reunir as principais características de um exame de tomografia real, tais como tipo e nível de ruído e morfologia de função de excitação do sistema. Para tanto, foram utilizados exames de pacientes do setor de Medicina Nuclear do Instituto do Coração da Faculdade de Medicina da Universidade de São Paulo. Aplicando-se a metodologia proposta em três níveis de ruído (baixo, médio e alto), obteve-se concordância do melhor modelo em graus forte e considerável (com índices de Kappa iguais a 0.95, 0.93 e 0.63, respectivamente). Observou-se que, com elevado nível de ruído e modelos mais complexos (quatro compartimentos), a classificação se deteriora devido ao pequeno número de dados para a decisão. Foram desenvolvidos programas e uma interface gráfica que podem ser utilizadas na investigação, elaboração, simulação e identificação de parâmetros de modelos compartimentais para apoio e análise de diagnósticos clínicos e práticas científicas. / This work has as goals the study, simulation, parameter identification and statistical comparison of compartmental models used in positron emission tomography (PET). We propose to use the methodology of sensitivity equations and the method of Levenberg-Marquardt for the task of estimating the characteristic parameters of the differential equations describing such systems. For model comparison, Akaikes information criterion is applied. We have considered three compartmental structures represented, respectively, by two compartments and two characteristic constants, three compartments and four characteristic constants and four compartments and six characteristics constants. The data considered in this work were synthesized taking into account key features of a real tomography exam, such as type and level of noise and morphology of the input function of the system. To this end, we used tests of patients in the sector of Nuclear Medicine of the Heart Institute of the Faculty of Medicine, University of São Paulo. Applying the proposed methodology with three noise levels (low, medium and high), we obtained agreement of the best model with strong and considerable degrees (with Kappa indexes equal to 0.95, 0.93 and 0.63, respectively). It was observed that, with high noise level and more complex models (four compartments), the classification is deteriorated due to lack of data for the decision. Programs have been developed and a graphical interface that can be used in research, development, simulation and parameter identification of compartmental models, supporting analysis of clinical diagnostics and scientific practices.

Modelos matemáticos de motoneurônios têm sido desenvolvidos para auxiliar na compreensão dos fenômenos que envolvem o sistema neuromuscular. Entretanto, a maioria dos modelos já desenvolvidos baseou-se na premissa de que a árvore dendrítica tem um comportamento passivo, o que ocorre em animais anestesiados, mas pode não ocorrer durante o comportamento motor normal de um animal intacto. Experimentos com animais descerebrados, em que as vias monoaminérgicas encontravam-se ativas, mostraram que os motoneurônios podem apresentar comportamentos mais complexos decorrentes da presença de condutâncias iônicas voltagem-dependentes que se situam nos dendritos e são responsáveis pela gênese de uma corrente de entrada persistente. Nesse sentido, um primeiro objetivo deste trabalho foi o de desenvolver novos modelos matemáticos de motoneurônios de diferentes tipos (i.e. dos tipos S, FR e FF), computacionalmente eficientes e contendo em seus compartimentos dendríticos uma condutância de cálcio do tipo L, de forma que os fenômenos de biestabilidade, potencial platô e amplificação da corrente sináptica efetiva possam ser gerados. Um segundo objetivo foi o de verificar como a presença da condutância iônica ativa no dendrito influencia o comportamento motoneuronal quando o mesmo está sujeito a entradas sinápticas de diferentes tipos. Os novos modelos foram parametrizados baseando-se em dados da literatura experimental para motoneurônios de gatos descerebrados e validados segundo os protocolos experimentais básicos que permitem caracterizar cada tipo de modelo como sendo totalmente ou parcialmente biestável. As entradas sinápticas foram simuladas por processos pontuais de Poisson e os trens de potenciais de ação dos motoneurônios foram analisados. Uma modulação senoidal da intensidade do processo pontual foi usada para estimar as respostas em frequência de cada modelo. Observou-se que, funcionalmente, a presença da condutância iônica dendrítica pode favorecer a ação do motoneurônio durante tarefas posturais, pois, uma vez ativada, a corrente de entrada persistente eleva a excitabilidade motoneuronal tornando os disparos mais regulares, além de prover uma alta sensibilidade dos modelos a entradas sinápticas de baixa frequência, correspondentes às oscilações observadas durante a manutenção da postura ereta quieta. / Mathematical models of motoneurons have been developed as an aid to the understanding of phenomena involving the neuromuscular system, but most of these models have been based on the hypothesis of a passive dendritic tree. This holds for anesthetized animals but not necessarily during normal motor behavior of the intact animal. Experiments with decerebrate animals in which the monoaminergic tracts were maintained intact have shown that more complex behaviors may emerge in motoneurons due to dendritic voltage-gated ionic conductances, which are responsible for a persistent inward current. Therefore, the first aim of this work was to develop computationally-efficient new motoneuron models of different types (i.e. type S, FR and FF) that include a dendritic L-type calcium conductance so that bistability, plateau potential and enhancement of effective synaptic current may be generated. The second aim of this research was to evaluate the effects of the active dendritic ionic conductance on the input-output mapping of presynaptic to postsynaptic spike trains. The new models were parameterized based on data reported in experimental literature on the decerebrate cat preparation, and they were validated using appropriate protocols for either fully or partially bistable dynamics. The synaptic inputs were simulated by Poisson point processes and the output spike trains were analyzed. Sinusoidal modulation of the point process intensity was used for the estimation of each models frequency response. The results suggested that an active dendritic ionic conductance in motoneurons has a functional role during postural tasks, because, when activated, the persistent inward current enhances the motoneuronal excitability, reducing the variability of interspike intervals, and focusing the sensitivity of the models to low frequency inputs that correspond to the low-frequency oscillations that typically occur during quiet standing posture.

A oxcarbazepina (OXC) é indicada como terapia adjuvante ou monoterapia no tratamento de crises epilépticas parciais ou crises tônico-clônicas generalizadas em adultos e crianças. A OXC sofre rápida eliminação pré-sistêmica com formação do metabólito ativo 10-hidroxicarbazepina (MHD), o qual possui como enantiômeros o R-(-)- e o S-(+)-MHD. A OXC e o MHD são substratos da glicoproteína-P (P-gp), que pode ser inibida pelo verapamil. O presente estudo avalia a influência do verapamil na farmacocinética e perfusão cerebral da OXC e dos enantiômeros do MHD em voluntários sadios. Os voluntários sadios (n=12) receberam em uma ocasião doses de 300 mg/12h de OXC e em outra ocasião doses de 300 mg/12h de OXC associadas com 80 mg/8h de verapamil. As amostras de sangue foram coletadas no estado de equilíbrio durante 12 horas e a avaliação da perfusão cerebral realizada utilizando a tomografia computadorizada por emissão de fóton único (SPECT) antes do início do tratamento e nos tempos 4, 6 ou 12h após a administração da OXC. As concentrações plasmáticas total e livre da OXC e dos enantiômeros do MHD foram avaliadas por LC-MS/MS. A análise farmacocinética não compartimental foi realizada com o programa WinNonlin e a farmacocinética populacional foi desenvolvida utilizando a modelagem não-linear de efeitos mistos com o programa NONMEM. Os limites de quantificação obtidos foram de 12,5 ng OXC/mL de plasma e 31,25 ng de cada enantiômero MHD/mL de plasma para a análise da concentração total, enquanto foi de 4,0 ng de OXC/mL de plasma e de 20,0 ng de cada enantiômero do MHD/mL de plasma para a determinação da concentração livre. Os coeficientes de variação obtidos nos estudos de precisão e a porcentagem de inexatidão inter e intra-ensaios foram inferiores a 15%, assegurando a reprodutibilidade e repetibilidade dos resultados. A análise farmacocinética não compartimental da OXC em monoterapia resultou nos seguintes parâmetros: concentração plasmática máxima (Cmax) de 1,35 ?g/mL como valor total e 0,32 ?g/mL como concentração livre em 1,0 h, área sob a curva concentração plasmática versus tempo (AUC0-12) de 3,98 ?g.h/mL e meia-vida de eliminação de 2,45 h, volume de distribuição aparente (Vss/F) de 352,17 L e clearance aparente (CLss/F) de 75,58 L/h. A disposição cinética do MHD é enantiosseletiva, com observação de maior proporção para o enantiômero S-(+)-MHD em relação ao R-(-)-MHD (razão AUC0-12 S-(+)/R-(-) de 4,26). A fração livre avaliada no tmax da OXC foi 0,26 para a OXC, 0,42 para o R-(-)-MHD e 0,38 para o S- (+)-MHD, mostrando enantiosseletividade na ligação às proteínas plasmáticas do MHD. O tratamento com o verapamil reduziu o tempo médio de residência (MRT) (4,71 vs 3,83 h) e Cmax como concentração livre (0,32 vs 0,53 ?g/mL) da OXC e aumentou os valores para ambos os enantiômeros do MHD de Cmax como valor total (2,60 vs 3,27 ?g/mL para o R-(-)- e 11,05 vs 11,94 ?g/mL para o S-(+)-MHD), Cmax como concentração livre (3,11 vs 4,14 ?g/mL para o S-(+)-MHD), Cmédia (2,11 vs 2,42 ?g/mL para o R-(-)- e 8,10 vs 9,07 ?g/mL para o S-(+)-MHD) e AUC0-12 (25,36 vs 29,06 ?g.h/mL para o R-(-)- e 97,19 vs 111,37 ?g.h/mL para o S-(+)-MHD). A ii farmacocinética populacional da OXC foi melhor descrita por modelo de dois compartimentos com eliminação de primeira ordem e com um conjunto de três compartimentos de trânsito para descrever o perfil de absorção da OXC. A disposição de ambos os enantiômeros do MHD foi caracterizada por modelo de um compartimento. Os valores de CLss/F estimados na monoterapia foram de 84,9 L/h para a OXC e de 2,0 L/h para ambos enantiômeros do MHD, enquanto os valores de Vss/F foram de 587 L para a OXC, 23,6 L para o R-(-)-MHD e 31,7 L para o S-(+)- MHD. Concluindo, a associação do verapamil aumentou a biodisponibilidade da OXC em 12% (farmacocinética populacional) e aumentou os valores de AUC de ambos os enantiômeros do metabólito MHD (farmacocinética não compartimental), o que está provavelmente relacionado com a inibição da P-gp no trato intestinal. A associação do verapamil aumentou as concentrações cerebrais preditas de ambos os enantiômeros do MHD em maior extensão do que aquelas observadas no plasma. As mudanças no fluxo sanguíneo cerebral (SPECTs realizados 6h após a administração da OXC) associadas à coadministração de verapamil provavelmente foram causadas pelo aumento dos níveis cerebrais de ambos os enantiômeros do MHD. A confirmação dessa observação requer um braço experimental adicional com SPECTs realizados também após a administração do verapamil em monoterapia. / Oxcarbazepine (OXC) is indicated as adjunctive therapy or monotherapy for the treatment of partial or generalized tonic-clonic seizures in adults and children. OXC undergoes rapid pre-systemic reduction with formation of the active metabolite 10- hydroxycarbazepine (MHD), which has the enantiomers R-(-)- and S-(+)-MHD. OXC and MHD are substrates of P-glycoprotein (P-gp), which can be inhibited by verapamil. The present study evaluates the influence of verapamil on the pharmacokinetics and cerebral perfusion of OXC and the MHD enantiomers in healthy volunteers. The healthy volunteers (n=12) received on one occasion doses of 300 mg/12h OXC and on another occasion they received doses of 300 mg/12h OXC associated with 80 mg/8h of verapamil. Blood samples were collected at steady state for 12 hours and the assessment of cerebral perfusion was performed using a single-photon emission computed tomography (SPECT) before the beginning of treatment and at times 4, 6 or 12 hours after OXC administration. The total and free plasma concentrations of OXC and MHD enantiomers were assessed by LC-MS/MS. The non-compartmental pharmacokinetics analysis was performed using the WinNonlin program, and population pharmacokinetics was developed using nonlinear mixed effects modelling with NONMEM.The limits of quantification obtained were 12.5 ng/mL plasma for OXC and 31.25 ng of each MHD enantiomer/mL plasma for total concentration analysis, while it was 4.0 ng OXC/mL plasma and 20.0 ng of each MHD enantiomer/mL plasma for the free concentration determination. The coefficients of variation obtained in studies of accuracy and the percentage of inaccuracy inter and intra-assay were less than 15%, ensuring the result reproducibility and repeatability. The non-compartmental pharmacokinetic analysis of OXC in monotherapy treatment, resulted in the following parameters: maximum plasma concentration (Cmax) of 1.35 ?g/mL as total concentration and 0.32 mg/mL as free concentration in 1.0 h, area under the plasma concentration vs time curve (AUC0-12) was 3.98 ?g.h/mL, half-life of 2.45 h, apparent volume of distribution (Vss/F) of 352.17 L and the apparent clearance (CLSS/F) of 75.58 L/h. The MHD kinetic disposition is enantioselective, with observation of a greater proportion of the S-(+)-MHD enantiomer compared to R-(-)-MHD (ratio AUC0-12 S-(+)/R-(-) of 4.26). The free fraction measured in the tmax of OXC was 0.26 for OXC, 0.42 for R-(-)-MHD and 0.38 for S-(+)-MHD, showing enantioselectivity in the plasma protein binding of MHD. Verapamil treatment reduced the mean residence time (MRT) (4.71 vs 3.83 h) and Cmax (0.26 vs 0.31 ?g/mL) as free concentration for OXC and increased the both MHD enantiomers values of Cmax (2.60 vs 3.27 ?g/mL for R-(-)- and 11.94 vs 11.05 ?g/mL for S-(+)-MHD) as total concentration, Cmax (3.11 vs 4,14 ?g/mL for S- (+)-MHD) as free concentration, Cavg (2.11 vs 2.42 ?g/mL for R-(-)- and 8.10 vs 9.07 ?g/mL for S-(+)-MHD) and AUC0-12 (25.36 vs 29.06 ?g.h/mL for R-(-)- and 97.19 vs 111.37 ?g.h/mL for S-(+)-MHD). The population pharmacokinetics of oxcarbazepine was best described by a two-compartment model with first-order elimination and a iv set of three transit compartments to describe the absorption profile of the parent compound. The disposition of both MHD enantiomers was characterised by onecompartment model. The CLss/F estimates in monotherapy were 84.9 L/h for OXC and 2.0 L/h for both MHD enantiomers, whereas the values of Vss/F were 587 L for OXC, 23.6 L for R-(-)-MHD and 31.7 L for S-(+)-MHD. In conclusion, verapamil coadministration increased the OXC bioavailability in 12% (population pharmacokinetics) and increased the AUC of both metabolite MHD enantiomers (non-compartmental pharmacokinetics), which is probably related to the inhibition of P-gp in the intestinal tract. Verapamil co-administration increased the predicted brain concentrations of both MHD enantiomers in a greater extent than those observed in plasma. Changes in cerebral blood flow (SPECTs performed 6h after administration of OXC) associated with co-administration of verapamil were probably caused by an increase in brain levels of both MHD enantiomers. Confirmation of this observation requires additional experimental arm with SPECTs also performed after administration of verapamil in monotherapy.

Potassium (K+) is an essential nutrient and the most abundant cation in plant cells. Plants possess two transport systems for K+ acquisition: a high-affinity system (HATS), operating at external K+ concentrations ([K+]ext) below 1 mM, and showing reduced transport activity in the presence of ammonium (NH4+); and, a low-affinity system (LATS), operating at [K+]ext above 1 mM, that is not affected by NH4+. K+ transport and compartmentation were investigated in barley (Hordeum vulgare L.) and rice (Oryza sativa L.) using the non-invasive technique of compartmental analysis by tracer efflux (CATE), to simultaneously determine unidirectional membrane fluxes, ion concentrations, and exchange characteristics in subcellular compartments. These studies revealed striking differences in unidirectional K+ fluxes between HATS and LATS. It was found that flux measurements, using traditional direct influx (DI) protocols, accurately represented HATS influx, but underestimated LATS influx by as much as seven-fold. In both barley and rice, LATS K+ fluxes were found to undergo rapid, futile cycling, with the ratio of efflux:influx 3 to 5 times greater, and the cytosolic exchange rate 2 to 3 times faster than under HATS. Based upon plasma-membrane electrical potential measurements, efflux was found to be active under LATS conditions. LATS-mediated conditions for K+ were found to provide relief from NH4+ toxicity in barley by immediately reducing NH4+ influx by more than 50%, and significantly reducing NH4+ futile cycling. Employing the K+ channel inhibitors cesium, lanthanum, and tetraethylammonium, NH4+ was shown to have both K+-sensitive and –insensitive influx pathways at high [NH4+]ext. Based on current models of flux energetics, the combined uptake of K+ and NH4+ was found to utilize 60% of root oxygen consumption. Barley and rice both showed signs of NH4+ toxicity at low [K+]ext, but rice recovered at much lower [K+]ext, suggesting a crucial role of K+ in the NH4+-tolerance of rice. These experiments address fundamental aspects of K+ fluxes, and help provide a physiological framework for future studies of K+ transport and mineral nutrition.

Potassium (K+) is an essential nutrient and the most abundant cation in plant cells. Plants possess two transport systems for K+ acquisition: a high-affinity system (HATS), operating at external K+ concentrations ([K+]ext) below 1 mM, and showing reduced transport activity in the presence of ammonium (NH4+); and, a low-affinity system (LATS), operating at [K+]ext above 1 mM, that is not affected by NH4+. K+ transport and compartmentation were investigated in barley (Hordeum vulgare L.) and rice (Oryza sativa L.) using the non-invasive technique of compartmental analysis by tracer efflux (CATE), to simultaneously determine unidirectional membrane fluxes, ion concentrations, and exchange characteristics in subcellular compartments. These studies revealed striking differences in unidirectional K+ fluxes between HATS and LATS. It was found that flux measurements, using traditional direct influx (DI) protocols, accurately represented HATS influx, but underestimated LATS influx by as much as seven-fold. In both barley and rice, LATS K+ fluxes were found to undergo rapid, futile cycling, with the ratio of efflux:influx 3 to 5 times greater, and the cytosolic exchange rate 2 to 3 times faster than under HATS. Based upon plasma-membrane electrical potential measurements, efflux was found to be active under LATS conditions. LATS-mediated conditions for K+ were found to provide relief from NH4+ toxicity in barley by immediately reducing NH4+ influx by more than 50%, and significantly reducing NH4+ futile cycling. Employing the K+ channel inhibitors cesium, lanthanum, and tetraethylammonium, NH4+ was shown to have both K+-sensitive and –insensitive influx pathways at high [NH4+]ext. Based on current models of flux energetics, the combined uptake of K+ and NH4+ was found to utilize 60% of root oxygen consumption. Barley and rice both showed signs of NH4+ toxicity at low [K+]ext, but rice recovered at much lower [K+]ext, suggesting a crucial role of K+ in the NH4+-tolerance of rice. These experiments address fundamental aspects of K+ fluxes, and help provide a physiological framework for future studies of K+ transport and mineral nutrition.

In light of the complex and highly uncertain nature of dynamical systems requiring controls, it is not surprising that reliable system models for many high performance engineering and life science applications are unavailable. In the face of such high levels of system uncertainty, robust controllers may unnecessarily sacrifice system performance whereas adaptive controllers are clearly appropriate since they can tolerate far greater system uncertainty levels to improve system performance. In this dissertation, we develop a Lyapunov-based direct adaptive and neural adaptive control framework that addresses parametric uncertainty, unstructured uncertainty, disturbance rejection, amplitude and rate saturation constraints, and digital implementation issues. Specifically, we consider the following research topics: direct adaptive control for nonlinear uncertain systems with exogenous disturbances; robust adaptive control for nonlinear uncertain systems; adaptive control for nonlinear uncertain systems with actuator amplitude and rate saturation constraints; adaptive reduced-order dynamic compensation for nonlinear uncertain systems; direct adaptive control for nonlinear matrix second-order dynamical systems with state-dependent uncertainty; adaptive control for nonnegative and compartmental dynamical systems with applications to general anesthesia; direct adaptive control of nonnegative and compartmental dynamical systems with time delay; adaptive control for nonlinear nonnegative and compartmental dynamical systems with applications to clinical pharmacology; neural network adaptive control for nonlinear nonnegative dynamical systems; passivity-based neural network adaptive output feedback control for nonlinear nonnegative dynamical systems; neural network adaptive dynamic output feedback control for nonlinear nonnegative systems using tapped delay memory units; Lyapunov-based adaptive control framework for discrete-time nonlinear systems with exogenous disturbances; direct discrete-time adaptive control with guaranteed parameter error convergence; and hybrid adaptive control for nonlinear uncertain impulsive dynamical systems.

Isoelectric trapping (IET) has become an accepted preparative-scale electrophoretic separation technique. However, there are still a number of shortcomings that limit its utility. The performance of the current preparative-scale IET systems is limited by the serial arrangement of the separation compartments, the difficulties in the selection of the appropriate buffering membranes, the effect of Joule heating that may alter separation selectivity and a lack of methods for the determination of the true, operational pH value inside the buffering membranes. In order to bolster the current membrane pH determination methods which rely on the separation of complex ampholytic mixtures, a fluorescent carrier ampholyte mixture was synthesized. The use of a fluorescent mixture allows for a reduced load of carrier ampholytes, thereby reducing a possible source of error in the pH determinations. A mixture of carrier ampholytes tagged with an alkoxypyrenetrisulfonate fluorophore was shown to have suitable fluorescence and ampholytic properties and used to accurately determine the pH of high pH buffering membranes under actual IET conditions. In a more elegant solution to the difficulties associated with pH determinations, a method utilizing commercial UV-transparent carrier ampholytes as the ampholyte mixture to be separated was developed. By using commercial carrier ampholytes and eliminating the need to synthesize, purify, and blend fluorescently tagged ampholytes, the new method greatly simplified the determination of the operational pH value of the buffering membranes. In order to address the remaining limitations, a new system has been developed that relies on (i) parallel arrangement of the electrodes and the collection compartments, (ii) a directionally-controlled convection system for the delivery of analytes, (iii) short anode-to-cathode distances, (iv) short intermembrane distances, and (v) an external cooling system. This system has been tested in four operational modes and used for the separation of small molecule ampholytic mixtures, for the separation of protein isoforms, and direct purification of a target pI marker from a crude reaction mixture.