Algunos óxidos electroactivos pueden ser conductores iónicos mixtos (conductividad iónica-electrónica) con una química de oxido-reducción que involucra procesos con reacciones de intercalación/desintercalación en estructuras abiertas o con vacantes. Estos materiales pueden presentar propiedades físicas relacionadas con los cambios en los estados de oxidación de los elementos involucrados en la estructura de óxido. Algunos de estos óxidos muestran comportamientos biocompatibles, lo que los convierte en materiales ideales para ser usados como electrodos en sistemas biológicos si se combina esta biocompatibilidad con su electroactividad. En el presente trabajo de investigación han sido obtenidas capas finas de TiO2, IrOx y un óxido mixto de los dos componentes anteriores para ser aplicados como substratos para crecimiento neuronal. Una vez sintetizadas, las capas de los tres materiales en estudio fueron caracterizadas estructuralmente mediante GIXRD, Reflectometría de Rayos X, XPS, XAS, Espectroscopía Raman y ATR. La microestructura fue evaluada mediante las técnicas de AFM, SEM y la medida del ángulo de contacto. Posteriormente se realizó una caracterización electroquímica y una evaluación de las propiedades de transporte mediante CV, IS y ECQM y por último se evaluó el posible uso de estos materiales como substratos para el crecimiento neuronal mediante cultivos de células neuronales obtenidas del córtex cerebral de embriones de rata E14 y E18 Wistar. Las capas de TiO2 fueron obtenidas por sol - gel y depositadas por spin coating en substratos de Indium Tin Oxide y cuarzo y se trataron térmicamente a temperaturas de 250oC, 350oC y 450oC en el caso de las muestras depositadas sobre ITO y 350oC, 450oC, 500oC y 600oC en el caso de las muestras depositadas sobre cuarzo. Los estudios de XPS revelan que TiO2 obtenido a 450oC o a largos tiempos de tratamiento térmico contiene cantidades pequeñas, pero significativas en la superficie, de Na+ proveniente del vidrio puede afectar algunas aplicaciones. Las muestras tratadas a 250oc y 350oC son amorfas mientras que las tratadas a 450oC presentaron una estructura anatasa con una microestructura nanoestructurada y homogénea. Las capas de TiO2 obtenidas fueron aislantes y no pasivan el paso de corriente de un substrato conductor y su ventana de acción electroquímica es de -0.7 V a 0.5 V. Los cultivos neuronales muestran que hay crecimiento neuronal sobre las capas obtenidas de TiO2 aunque con inhibición del desarrollo de dendritas que parece estar motivado por la química del material más que por su microestructura. Las capas de IrOx fueron obtenidas por deposición electroquímica a corriente constante (35 μA/cm2) y a potencial dinámico (0.55V, 10mV/s) con 17, 25, 50 y 100 ciclos. La estructura resultante fue amorfa o quasi-amorfa con una estructura local tipo rutilo que permite la inserción de H+ o K+ y con grados de homogeneidad variable. La caracterización electroquímica y eléctrica muestran que el IrOx obtenido es un conductor electro-iónico (mixto) con capacidad de sufrir cambios en el estado de oxidación del Ir, e intercalación simultánea de protones en forma hidratada, con facilidad. Los resultados obtenidos por ECQM muestran que el proceso de formación electroquímica del IrOx consume 4.2 e-/Ir y por tanto involucra un proceso adicional a la oxidación del Ir(III) precursor. Los cultivos celulares sobre IrOx lo muestran como el mejor substrato probado con diferencias significativas con respecto al TiO2. El crecimiento de dendritas no parece estar inhibido ya que la supervivencia neural es muy alta, equivalente a la referencia. Las capas del óxido mixto (Ir-Ti)Ox fueron obtenidas por spin coating en substratos de Pt/cuarzo partiendo de disoluciones de Ti (donde el Ti está en forma de isopropóxido) e Ir (donde el Ir está como cloruro). Las capas depositadas se trataron térmicamente a temperaturas de 600oCy 650oC. Los resultados de GIXRD muestran que las capas del óxido mixto son más cristalinas que los óxidos simples con tres fases presentes: TiO2 anatasa y rutilo e IrOx rutilo. Además diferencias en la intensidad de los picos con la variación del ángulo de incidencia hacen pensar que el material puede ser un compuesto estratificado con IrOx localizado en mayor proporción en la superficie. Las capas presentan una microestructura homogénea menos rugosa y más hidrofílica que en el caso de los óxidos simples. Los cultivos neuronales muestran una buena adhesión de las neuronas a las capas del óxido mixto y es evidente que la supervivencia celular mejora incrementando la cantidad de iridio. Al evaluar los tres materiales en términos de supervivencia y desarrollo neuronal se puede afirmar que el iridio tiene un papel determinante en la aplicación de dichos materiales como posibles substratos para crecimiento celular y electroestimulación. Además las capas de IrOx se muestran como la mejor opción para ser utilizadas como soporte neuronal. / Some electroactive oxide ionic conductors can be mixed ionic conductors (ionic-electronic conductivity) with an oxidation-reduction chemistry involving intercalation/deintercalation processes in open structures or with vacant. These materials may have physical properties related with changes in oxidation states of the elements involved in the oxide structure. Some of these oxides show biocompatible behaviours, which makes them ideal materials to be uses as electrodes in biological systems when combined with its electroactivity and biocompatibility. In this research work thin films of TiO2, IrOX and a mixed oxides of the two previous components have been obtained to be applied as substrates for neuronal growth. Once synthesized, the layers of the three materials under study were structurally characterized by GIXRD, X-ray reflectometry, XPS, XAS, Raman spectroscopy and ATR. The microstructure was evaluated using the techniques of AFM, SEM and contact angle measurement. Subsequently an electrochemical characterization and evaluation of the transport properties by CV, IS and ECQM was done. Finally the possible use of these materials as substrates for neuronal growth was evaluated by neuronal cells cultures of the cerebral cortex of rat embryos Whistar E14 and E18. TiO2 layers were obtained by sol - gel and deposited by spin coating on Indium Tin Oxide and quartz substrates; the films were heat treated at temperatures of 250oC, 350oC and 450oC in the case of samples deposited on ITO and 350oC, 450oC, 500oC and 600oC in the case of samples deposited on quartz. XPS studies reveal that TiO2 films treated at 450oC or with long heat treatment times contains small amounts, but significant on the surface, of Na ions from the glass substrate that may affect some applications. The samples treated at 350oC 250oc and are amorphous while those treated at 450oC showed an anatase structure with a nanostructured and homogeneous microstructure. TiO2 layers obtained do not passive current flow across a conductor substrate and its electrochemical action window is from -0.7 V to 0.5 V Vs Pt. The neuronal cell cultures show that there is neuronal growth on the TiO2 layers although an inhibition in the development of dendrites appears motivated by chemistry of the material rather than microstructure. IrOX layers were obtained by electrochemical deposition at constant current (35μA/cm2) and dynamic potential (0.55V, 10mV / s) with 17, 25, 50 and 100 cycles. The resulting structure was amorphous or quasi-amorphous with a rutile local structure that allows the insertion of H+ or K+, with variable degrees of homogeneity. The electrochemical and electrical characterization show that the IrOX obtained is an electro-ionic conductor (mixed) with the capacity to undergo changes in oxidation state of Ir, and simultaneous intercalation of protons in hydrated form. ECQM results show that the process of electrochemical formation of the IrOX films consumes 4.2 e-/Ir and therefore involves an additional oxidation process of the Ir (III) precursor. Cell cultures on IrOX show this material as the best substrate tested with significant differences respect to the TiO2. The growth of dendrites does not seem to be inhibited and the neural survival is very high, equivalent to the reference. The layers of mixed oxide (Ir-Ti)Ox were obtained by spin coating on substrates of Pt / quartz from solutions of Ti (where Ti is in the isopropoxide form) and Ir (where Ir is as a chloride). The layers deposited were treated at temperatures of 650oC and 600oC. GIXRD results show that the mixed oxide coatings are more crystalline than the simple oxides with three phases: anatase and rutile TiO2 and IrO2 rutile. Besides differences in the intensity of the peaks with the variation of the incidence angle suggest that the material can be laminated with the IrOX located mostly at the surface. The layers have a homogeneous microstructure, less rough and more hydrophilic than in the case of the simple oxides. The neuronal cell cultures show a good adhesion of the neurons on the mixed oxide layers and it is clear that cell survival improves by increasing the iridium amount. When the tree materials are evaluated in terms of neuronal survival and development can be said that the iridium has an important role in the implementation of these materials as potential substrates for cell growth and electrostimulation. Furthermore IrOX layers are shown as the best option to be used as neuronal support.

This dissertation studies two problems related to geometric representation of neuroanatomical data: (i) spatial representation and organization of individual neurons, and (ii) reconstruction of three-dimensional neuroanatomical regions from sparse two-dimensional drawings. This work has been motivated by nearby development of new technology, Knife-Edge Scanning Microscopy (KESM), that images a whole mouse brain at cellular level in less than a month. A method is introduced to represent neuronal data observed in the mammalian brain at the cellular level using geometric primitives and spatial indexing. A data representation scheme is defined that captures the geometry of individual neurons using traditional geometric primitives, points and cross-sectional areas along a trajectory. This representation captures inferred synapses as directed links between primitives and spatially indexes observed neurons based on the locations of their cell bodies. This method provides a set of rules for acquisition, representation, and indexing of KESMgenerated data. Neuroanatomical data observed at the gross level provides the underlying regional framework for neuronal circuits. Accumulated expert knowledge on neuroanatomical organization is usually given as a series of sparse two-dimensional contours. A data structure and an algorithm are described to reconstruct separating surfaces among multiple regions from these sparse cross-sectional contours. A topology graph is defined for each region that describes the topological skeleton of the region’s boundary surface and that shows between which contours the surface patches should be generated. A graph-directed triangulation algorithm is provided to reconstruct surface patches between contours. This graph-directed triangulation algorithm combined together with a piecewise parametric curve fitting technique ensures that abutting or shared surface patches are precisely coincident. This method overcomes limitations in i) traditional surfaces-from-contours algorithms that assume binary, not multiple, regionalization of space, and in ii) few existing separating surfaces algorithms that assume conversion of input into a regular volumetric grid, which is not possible with sparse inter-planar resolution.

Purification, chemistry and application are three very important aspects of current research on carbon nanotubes (CNTs). In the dissertation, the purification of nitric acid treated single-walled carbon nanotubes (SWNTs), the dissolution and dichlorocarbene addition of SWNTs, and the effects of chemically functionalized CNTs on neuronal growth are discussed.The nitric acid treated SWNTs were purified by chemical treatment, cross-flow filtration, and centrifugation methods. The effects of nitric acid treatment on the SWNTs and the efficiency of different purification methods was evaluated by the measurement of purify of SWNTs via solution phase NIR. Nitric acid reflux followed with controlled pH centrifugation can produce SWNTs with high purity. This purification mechanism was explained by the relationship of the concentration of the acidic sites on SWNTs and the zeta potential of SWNTs.The dissolution of SWNTs was achieved via chemical functionalization of SWNTs with octadecylamine (ODA). Dichlorocarbene addition to the sidewall of both ODA functionalized and as-prepared SWNTs was investigated. ODA functionalized HiPco-SWNTs were found to have the highest functionality of dichlorocarbene. Vis-NIR spectra of the dichlorocarbene functionalized SWNTs showed a significant decrease in the interband transitions of the semiconducting SWNTs, which indicated that the chemical functionalization of the sidewall of SWNTs changes the electronic properties of SWNTs. Far-IR spectra of the dichlorocarbene functionalized SWNTs showed a dramatic decrease in the electronic transitions at the Fermi level of metallic SWNTs, which was opposite to the effect of ionic doping by bromine. This difference in the far-IR spectroscopy can be used to distinguish covalent chemical functionalization and ionic doping effects of SWNTs.Chemically functionalized multi-walled carbon nanotubes (MWNTs) were applied as substrates for neuronal growth. By manipulating the charge carried by functionalized MWNTs we are able to control the outgrowth and branching pattern of neuronal processes. Chemically functionalized water soluble SWNTs graft copolymers were used in the modulation of outgrowth of neuronal processes. The graft copolymers were prepared by the functionalization of SWNTs with poly-m-aminobenzene sulphonic acid and poly-ethylene glycol. These functionalized water soluble SWNTs were able to increase the length of selected neuronal processes after their addition to the culturing medium.

Programmed cell death (PCD) is a highly evolved mechanism of cellular suicide that is aberrantly activated following neural injury. Two fundamental PCD signaling pathways termed the extrinsic (caspase-8-mediated) and intrinsic (caspase-9-mediated) pathways, have been described. While each pathway is initiated by distinct cellular stimuli, both pathways culminate in the activation of downstream executioner caspases. Previous efforts to isolate the in vivo contribution of each pathway have been hindered by the embryonic lethality of casp8 and casp9 null mice. In the present study, I overcame this obstacle to directly assess the contribution of each pathway following two well-characterized forms of acute neural injury; excitotoxic destruction of CA1 pyramidal neurons, and the loss of motor neurons following facial nerve transection. To determine the role of caspase-8, I constructed several lines of mice in which caspase-8 was conditionally ablated within the relevant neuronal populations. The results obtained from these animals definitively demonstrate that caspase-8 is not required by either motor neurons or CA1 pyramidal neurons to undergo PCD following injury. Therefore, these findings have provided the first direct experimental evidence to counter the widely held dogma of caspase-8 as the central effector of death receptor-mediated signaling within neurons. With respect to the intrinsic pathway, several lines of evidence suggest that the apoptosome predominantly regulates the death of motor neurons. I tested this hypothesis by performing facial axotomies in mice containing a point mutation introduced (“knocked in”) into the genomic locus of cytochrome c which abolishes its ability to activate the intrinsic pathway. Homozygous cytochrome c knock-in mice displayed a significant enhancement in motor neuron survival in comparison to control littermates following injury. However, the level of motor neuron protection differed from that previously reported in mice either overexpressing anti-apoptotic or lacking pro-apoptotic members of the Bcl-2 family. Therefore, the results of this study directly demonstrate the influence of the apoptosome on injury-induced neuronal PCD isolated from upstream Bcl-2 family-mediated effects. In addition, my results have provided the first evidence that activation of the apoptosome is required for the release of apoptosis inducing factor (AIF) from the mitochondria of injured motor neurons in vivo.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 6 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes bibliographical references (leaves 126-136). Also available online.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / "August 1997." Typescript. Vita. Includes bibliographical references (leaves 144-151). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves : 144-151). Also available on the Internet.

Myelin is essential for normal nervous system conduction as well as providing metabolic support for the ensheathed axon and has been implicated to influence axon calibre (diameter of the axon body) growth. In demyelinating diseases, the disruption of these functions causes axon degeneration resulting in neurological impairment. The neurons that are myelinated in the CNS and the axon-oligodendrocyte (axon- OL) interactions that might regulate axon calibre and myelination during myelinated axon formation are still mostly unknown, preventing a deeper understanding of CNS development and repair. This doctoral thesis identifies a specific subset of interneurons that are myelinated and investigates the axon-oligodendrocyte interactions during axon calibre growth and initial myelination. In the zebrafish spinal cord, Commisural Primary Ascending interneurons (CoPA), Circumferential Descending interneurons (CiD) and reticulospinal neurons are amongst the first to be myelinated, whereas Commisural Bifurcating Longitudinal interneurons (CoBL) and Circumferential Ascending interneuron (CiA) are not myelinated during early developmental stages. Of the myelinated neurons, axon calibre of reticulo spinal neurons is increased in time with myelin ensheathment, while the axon calibre of CoPA and CiD interneurons is not increased with the onset of myelination. In order to investigate whether there might be a causative relationship between axon calibre increase and myelin ensheathment, the majority of oligodendrocytes were eliminated by olig2 morpholino knockdown. In the absence of oligodendrocytes, the axon calibre of reticulospinal neurons was normal, demonstrating that axon calibre growth is independent of axon-OL interactions and myelin ensheathment. In order to further investigate which aspects of myelinated axon formation might be regulated by axon-OL interactions, axonal activity was reduced through inhibition of synaptic vesicle release by global expression of Tetanus-toxin (TetTx). TetTx treated zebrafish showed a 40% decrease of myelinated axons in the spinal cord. Interestingly, only 10% of this reduction was caused by a decrease in oligodendrocyte number in the spinal cord. Single cell analysis of individual oligodendrocytes revealed a 30% reduction of myelin sheaths per oligodendrocyte in TetTx treated animals, indicating a positive correlation between synaptic vesicle release and the extent of myelination. Timelapse analysis of the myelinating behaviour of individual oligodendrocytes revealed that the decrease in myelin sheaths per cell in the absence of synaptic vesicle release results from a reduction in the initial formation of sheaths rather than an increased retraction of myelin sheaths. Furthermore, individual myelin sheaths formed by the same oligodendrocyte exhibit a dynamic range of different growth rates in control animals, which was reduced to a more uniform, slow growth of myelin sheaths in the absence of synaptic vesicle release. This suggests that local axon-OL interactions can regulate the dynamic myelin sheath growth through synaptic vesicle release. The analyses in this doctoral thesis identifies a subset of the neurons that are myelinated during the onset of myelination in the zebrafish spinal cord, demonstrates that axon caliber growth of these neurons is independent of myelin ensheathment and that axon-OL interactions mediated by synaptic vesicle release can regulate the extent of myelination and influence the dynamic myelinating behavior of oligodendrocytes in vivo. These findings begin to elucidate the axon-OL interactions underlying myelinated axon formation during CNS development, from which future studies might derive neuro-regenerative treatments for demyelinating diseases.

O fenômeno da potenciação de longa duração (LTP) é amplamente aceito como correlato celular da formação de memórias. A tarefa de Reconhecimento de Objetos (RO) é particularmente útil para estudar as memórias declarativas em roedores, porque se baseia em sua preferência inata por objetos novos sobre objetos familiares. Neste estudo, camundongos com eletrodos implantados nas vias colaterais de Schaffer hipocampais foram treinados na tarefa de RO. Registros de campo de potenciais excitatórios pós-sinápticos evocados na sinapse CA3-CA1 foram obtidos durante a sessão de treino ou a diferentes momentos após o seu término. Um fenômeno de potenciação sináptica “LTP-like” foi observado 6 horas após o treino. Uma sessão de teste foi conduzida 24 horas após o treino, na presença de um objeto novo e de um objeto familiar. Uma facilitação sináptica foi observada enquanto os animais exploravam os objetos, independentemente do tipo de objeto (novo ou familiar). Um curto período de depotenciação foi observado logo após o teste, seguido de uma fase tardia de potenciação sináptica. Demonstrou-se, portanto, que a consolidação da memória de RO é acompanhada por uma potenciação sináptica transitória na sinapse CA3-CA1, enquanto que a reconsolidação desta memória desencadeia uma fase curta de depotenciação - que poderia ser a responsável pela vulnerabilidade característica da memória – que é seguida por uma fase tardia de potenciação sináptica.