(運動・神経生理)

ABSTRACT In this dissertation, several adaptive control design schemes for a class of nonlinear systems are proposed. The first topic of the research is concerned with self-tuning PID controller design. The main problem of designing PID controller is how to determine the values of three control gains, i.e., proportional gain , integral gain , and derivative gain . We attempt to use the technique of adaptive control based on the Lyapunov approach to design the PID controller for some class of partially known nonlinear systems. Three PID control gains are adjusted on-line such that better output performance can be achieved. The stability of the closed-loop PID control systems is analyzed and guaranteed by introducing a supervisory control and a modified adaptation law with projection. Second, two kinds of adaptive neural control systems including the direct and indirect neural controls are considered by using simple single auto-tuning neuron. We will first propose a novel neuron called auto-tuning neuron and use it to take place of the roles of the traditional neural networks used in the direct and indirect adaptive neural control systems. This can greatly reduce the computational time and network complexities due to the simple configuration of the auto-tuning neuron. It is also easy for hardware implementation. Third, based on the idea borrowed from natural evolution, genetic algorithm can search for optimal or near-optimal solutions for an optimization problem over the search domain. An optimization technique of real-coded genetic algorithm is used to design the PID controller by minimizing the performance index of integrated absolute error. The improvements of our results over that using other methods are also illustrated. In the last part of each section, some computer simulation results will also be provided to illustrate our proposed methods.

A fast method for gathering large-scale data sets through the serial sectioning of brain tissue is described. These data sets are retrieved using knife-edge scanning microscopy, a new technique developed in the Brain Networks Laboratory at Texas A&M University. This technique allows the imaging of tissue as it is cut by an ultramicrotome. In this thesis the development of a knife-edge scanner is discussed as well as the scanning techniques used to retrieve high-resolution data sets. Problems in knife-edge scanning microscopy, such as illumination, knife chatter, and focusing are discussed. Techniques are also shown to reduce these problems so that serial sections of tissue can be sampled at resolutions that are high enough to allow reconstruction of neurons at the cellular level.

Mutations of the á1A subunit of P/Q-type voltage-gated calcium channels are responsible for several inherited disorders affecting humans, including familial hemiplegic migraine, episodic ataxia type 2 and spinocerebellar ataxia type 6. These disorders include phenotypes such as a progressive cerebellar atrophy and ataxia. The leaner mouse also carries a mutation in the alpha(1A) subunit of P/Q-type voltage-gated calcium channels, which results in a severe cerebellar atrophy and ataxia. The leaner mutation causes reduced calcium ion influx upon activation of P/Q-type voltage-gated calcium channels. This disrupts calcium homeostasis and leads to a loss of cerebellar neurons, including cerebellar Purkinje cells. Because of its similarities with human P/Qtype voltage-gated calcium channel mutations, leaner mouse has served as a model for these disorders to aid our understanding of calcium channel function and neurodegeneration associated with calcium channel dysfunction. The aims of this dissertation were: (1) to precisely define the timing and spatial pattern of leaner Purkinje cell death and (2) to assess the role of caspases and specifically of caspase 3 in directing leaner Purkinje cell death. We used the mechanism independent marker for cell death Fluoro-Jade and demonstrated the leaner Purkinje cell death begins around postnatal day 25 and peaks at postnatal day 40 to 50. Based on this temporal pattern of Purkinje cell death we then investigated the role of caspases in leaner Purkinje cell death. These studies showed that caspase 3 is specifically activated in dying leaner cerebellar Purkinje cells. In addition, in vitro inhibition of caspase 3 activity partially rescued leaner Purkinje cells. Further investigation revealed that caspase 3 activation may be working together with or in response to macroautophagy. This study also indicated a potential role for mitochondrial signaling, demonstrated by the loss of mitochondrial membrane potential in leaner cerebellar Purkinje cells. However, our study revealed that if the loss of mitochondrial membrane potential is associated with leaner Purkinje cell death, this process is not mediated by the mitochondrial protein cytochrome C.

Modern neuron models are large, complex entities. The ability to better simulate these complex models has been iven by the development of ever more powerful and cheap computers. The capacity to manage and understand the models has lagged behind improvements in simulation ability almost from the inception of neuron modeling. Despite the computing power currently available, more powerful simulation platforms and strategies are needed to cope with current and next generation neuron models. This thesis develops methodologies aimed at better characterizing motoneuron models. The hypothesis presented is that relationships between model outputs in addition to the relationships between model inputs (parameters) and outputs (behaviors) provide a characteristic description of the model that describes the model in a more useful way than just model behaviors. This description can be used to compare a model to different implementations of the same motoneuron and to experiment data. Data mining and data reduction techniques were used to process the data. Principal component analysis was used to indicate a significant, consistent reduction in dimensionality in an intermediate, mechanistic layer between model inputs and outputs. This layer represents the non-linear relationships between input and output, implying that if the non-linear relationships of a model were better understood and accessible, a model could be manipulated by varying the mechanism layer members, or rather the model parameters that primarily affect a mechanism layer member. Hierarchical cluster analysis showed similarity between sensitivity analyses data from models with random parameter sets. A main cluster represented the main region of model behavior with outlying clusters representing non-physiological behavior. This indicates that sensitivity analysis data is a good candidate for a model signature. The results demonstrate the usefulness of cluster analysis in identifying the similarities between data used as a model characterization metric or model signature. Its application is also valuable in identifying the main region of useful activity of a model, thus helping to identify a potential 'average' parameter set. Furthermore, factor analysis also proves functional in identifying members of the mechanism layer as well as the degree to which model outputs are affected by these members.

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Serotonergic neurons modulate a wide range of behaviors and functions, from mood and aggression to vital autonomic processes like heart rate, respiratory dynamics, and body temperature. We hypothesize that this broad scope reflects the collective actions of many functionally and molecularly distinct subtypes of serotonergic neurons, each with specialized roles in different neural processes. Supporting this idea are examples of heterogeneity among serotonergic neurons with respect to developmental origin, biophysical properties, and molecular expression; yet deciphering the functional and behavioral relevance of these differences has been challenging. In order to better understand serotonergic system organization, we have developed and applied a set of mouse genetic tools to subdivide serotonergic neurons into groups based on molecular criteria, and then to query these subtypes for differences with respect to biophysical properties, hodology, gene expression, and whole animal function. We applied these tools in a stage-wise fashion, from neural system en masse, as reference, and then to specific serotonergic neuron subtypes. From this, we have established that serotonergic neurons play key roles in at least two life-sustaining reflexes - the respiratory chemoreflex (breathing modulation to keep tissue PCO2/pH within physiological limits) and body temperature regulation. We found that chemoreflex modulation, but not body temperature regulation, maps to a specific serotonergic neuron subtype - that subtype with a developmental history of Egr2 gene expression. Further, in brain slice preparations, we found that this subtype is chemosensitive, increasing firing rate in response to conditions of hypercapnic acidosis. Thus, in vivo, Egr2-serotonergic neurons likely transduce chemosensory information into action potential firing to increase respiratory drive and ultimately breathing. Further, we found that Egr2-serotonergic neurons project selectively to respiratory nuclei involved in PCO2/pH sensory signal transduction, but not primary respiratory motor nuclei. This indicates that the serotonergic system has distinct sensory and motor divisions - another unexpected finding. In summary, these results establish a previously unappreciated functional modularity and organization to the serotonergic system, and open up potential for tailored function-specific therapeutic strategies, for example here as relates to disorders of respiratory homeostasis or thermoregulation.

<p>The recent discovery that fibroblasts can be reprogrammed directly to functional neurons with lentivirus has reinvigorated the belief that autologous human cell therapies against many neurodegenerative diseases may be achievable in the near future. To increase the clinical translatability of this approach, we have developed a technique to perform this direct conversion without the use of virus. We transfected fibroblasts with plasmids condensed into non-viral nanoparticulate carriers with a bioerodible peptidomimetic polymer, pCBA-ABOL. Gene delivery with pCBA-ABOL was exceptionally effective and nontoxic, producing high transfection efficiencies and enabling serial dosing of plasmid cocktails. We delivered plasmids encoding neural lineage-instructive transcription factors to primary mouse embryonic fibroblasts (PMEFs), eliciting: drastic morphological changes, activation of endogenous neuronal transcription factor expression, neuronal promoter activity, and the appearance of neuronal proteins. Serial dosing of pCBA-ABOL complexes produced increasingly higher yields of these non-virally induced neurons (NiNs). The majority of NiNs fired action potentials under patch clamp. This is the first description of a method capable of directly inducing functional neuronal cells from fibroblasts without the use of virus.</p><p>We then moved on to further increase the yield of NiN generation, in an effort to produce a sufficient quantity of neurons for cell therapies. Informed by neurodevelopmental cues and by precedents set by the induced pluripotent stem cell (iPSC) field, we performed non-viral neuronal reprogramming in the presence of soluble microenvironmental factors that either inhibited GSK-3beta; and SMAD signaling, or induced chronic membrane depolarization. Chronic depolarization doubled the number of cells expressing neuronal markers produced with glutamatergic as well as with dopaminergic transcription factor cocktails. Inhibition of GSK-3beta; and SMAD signaling similarly doubled the yield of glutamatergic NiNs, and enabled induction of neuronal markers and morphological transformation in human fibroblasts.</p><p>In addition to soluble microenvironmental factors, the physical microenvironment can also strongly influence various cellular phenotypes. This list includes many phenotypes related to endocytosis - the transit mechanism of nanoparticulate gene carriers entering cells during non-viral transfection. As such, we set out to determine if patterned physical substrate topography could be used to increase non-viral transfection. We employed a high-throughput screen of micropitted substrate topographies, and indeed found that larger, denser micropits could support significantly higher transfection efficiencies in fibroblasts, compared to smooth substrates. The same topographies produced higher reverse transfection efficiencies, and greater siRNA-mediated knockdown of a reporter gene. The control of transfection with substrate topography is a new design parameter that could find broad application in in vitro non-viral reprogramming strategies, as well as in the intelligent design of nucleic acid-eluting scaffolds in vivo. </p><p>Encouraged by our success with direct neuronal reprogramming, and armed with a greater understanding of some microenvironmental mediators thereof, we attempted to produce non-virally-induced oligodendroglial progenitor cells (NiOPCs), which has been historically challenging for other investigators. We discovered the fibroblastic intracellular microenvironment is a significant barrier to the function of Olig2 - a master regulator of OPC phenotype - in direct reprogramming. Fibroblasts do not express Olig2 chaperones which are normally expressed in OPCs, causing Olig2 to become sequestered in the cytoplasm of transfected PMEFs. We relieved this barrier through fusion of a strong nuclear localization sequence (NLS) to Olig2, which repartitioned Olig2-NLS from the cytoplasm to the nucleus in transfected fibroblasts. The use of Olig2-NLS in iOPC reprogramming cocktails resulted in a drastic improvement in the yield of OPC-specific marker expression. The improvement associated with Olig2-NLS was insufficient to elicit significant myelin protein expression with the non-viral system, but the yield of virally-induced oligodendrocyte-like cells (iOLs) was improved dramatically. Further enhancements will be required to generate fully-reprogrammed NiOPCs, but the increased efficiency of viral iOPC generation is immediately useful for disease modeling and potentially in cell replacement therapies if human cells can be converted for the first time using this technique. During direct reprogramming, CNS-specific transcription factors are delivered to foreign intracellular contexts as a rule, which may reduce their ability to function effectively; we have shown this can be a critical yet under-appreciated determinant of the success or failure of a direct reprogramming system.</p><p>Taken together, the technological and intellectual advancements we describe herein represent significant improvements to non-viral transfection and reprogramming systems. These techniques can find broad appeal to the many researchers and clinicians deploying these systems. More specifically, we present significant steps towards realization of the dream of safe and effective autologous cell therapies against devastating and currently-intractable neurodegenerative diseases.</p> / Dissertation

Throughout neuronal development, bouts of spontaneous electrical activity are critical for the proper wiring of neuronal connections. Alterations in firing activity can affect growth cones, which tip developing and regenerating neurites and are responsible for the integration of extracellular guidance cues into pathfinding behaviors. While growing evidence implicates gaseous signaling molecules, nitric oxide (NO) and carbon monoxide (CO), as modulators of neuronal firing activity, less is understood about how they affect growth cone motility. Therefore, in this dissertation, I focus on how NO and CO affect electrical activity of developing and regenerating neurons and how these effects translate into changes at the growth cone level. The specific goals of this dissertation were to investigate 1) the neuron-type-specific effects of NO on growth cone motility; 2) the role of CO in the regulation of neuronal firing activity and excitability; and 3) the role CO plays in the regulation of growth cone motility. Using the well-established developmental model, Helisoma trivolvis, neurons were isolated in single-cell culture allowing for the maximal control over environmental conditions for the direct characterization of NO and CO. In the study of NO, differences in B5 and B19 growth cone responses to NO were due to neuron-type-specific differences in action potential duration. Moreover, the non-responsive B19 growth cones could be made responsive to NO treatment upon the pharmacological broadening of its action potentials. While NO has been found to increase firing activity, the study of CO revealed that CO had the opposite effect on electrical activity, silencing spontaneous firing activity and decreasing neuronal excitability. The study of CO on growth cone motility showed that CO increased growth cone filopodial length through a soluble guanylyl cyclase/protein kinase G/ryanodine receptor mediated pathway without inducing robust increases in growth cone calcium concentration. Taken together, this dissertation reveals new insight into how NO and CO regulate electrical activity and growth cone motility, providing evidence for these gases as important signaling messengers during for the development and regeneration of nervous system.

No description available.