No description available.

OBJETIVO GENERAL: Determinar las alteraciones histomorfométricas de la capa granular de la corteza órbitofrontal de Rattus norvegicus, a nivel de las neuronas granulares y glías de la serie astrocítica sometidas a administración crónica de bebidas alcohólicas adulteradas. MATERIAL Y MÉTODOS: Estudio experimental, prospectivo, comparativo y longitudinal. Se utilizaron cuarenta y cinco ratas de laboratorio Rattus norvegicus, variedad albina, obtenidos de los bioterios del CENAN y trasladados al bioterio de la Facultad de Medicina Humana de la UNMSM. Fueron divididos en 3 grupos de trabajo: el grupo control (Grupo O) y los grupos experimentales (Grupo A y Grupo B), el Grupo A recibió la bebida alcohólica denominada “Tumbaloco” (mezcla de pisco, anisado y cognac adulterado) y el Grupo B la bebida alcohólica denominada “Penal” (mezcla de pisco, anisado y cognac adulterado más cerveza). Se enviaron dichas bebidas alcohólicas al Centro de Información, Control Toxicológico y Apoyo a la Gestión Ambiental (CICOTOX) para el análisis cuantitativo de etanol y metanol por el método de espectrofotometría de absorción atómica. Se les administró de manera crónica, por un tiempo de 12 semanas, bebidas alcohólicas adulteradas al grupo experimental y agua al grupo control mediante el uso de una sonda orogástrica. Luego de las 12 semanas se procedió al sacrificio del animal mediante la decapitación, se procedió a la extracción del encéfalo murino en un tiempo no mayor de 4 minutos luego del cual fue colocado en una solución de formol neutro al 10%. Se hizo un corte coronal del encéfalo a nivel de la corteza prefrontal, una parte del tejido fue incluido en parafina para la realización de láminas histológicas con la coloración Hematoxilina & Eosina e inmunohistoquímica con GFAP (marcador específico para astrocitos) la otra parte del tejido fue llevada a congelación para la elaboración de láminas histológicas con la coloración Argéntica del Río Ortega impregnación simple. Dichas láminas histológicas fueron fotografiadas y posteriormente analizadas mediante el procesador de imágenes JMicrovision 1.2.5. RESULTADOS: No existió diferencia estadísticamente significativa en el peso de los animales al inicio y al final del experimento. El área determinada por las neuronas necróticas de la capa granular fue menor en los grupos que recibieron bebidas alcohólicas adulteradas a predominio del grupo que recibió la bebida “Penal”. El mayor porcentaje de neuronas necróticas se observó en el grupo que recibió bebidas alcohólicas adulteradas más cerveza el cual fue estadísticamente superior que el grupo control, el porcentaje de neuronas necróticas en el grupo que recibió sólo bebidas alcohólicas adulteradas también fue estadísticamente superior que el grupo control pero menor que el grupo que recibió bebidas alcohólicas adulteradas más cerveza. Se observó gliosis de la serie astrocítica en los grupos que recibieron bebidas alcohólicas adulteradas también a predominio del grupo que recibió la bebida “Penal”. CONCLUSIONES: El consumo crónico de bebidas alcohólicas adulteradas produce una disminución de las neuronas granulares y un aumento de las glías de la serie astrocítica a nivel de la capa granular de la corteza órbitofrontal. Las alteraciones fueron mayores en el grupo que recibió la bebida alcohólica “Penal”, mezcla de 3 bebidas alcohólicas adulteradas más cerveza.

Autosomal dominant adult onset neuronal ceroid lipofuscinoses (ANCL/CLN4) is a rare neurodegenerative disorder caused by mutations in the human gene DNAJC5 which encodes cysteine string protein alpha (CSPα). ANCL is characterized by the appearance of aberrant lysosomal storage material in the post-mortem brains of patients, who usually die from widespread neuronal loss within 10 years from the onset of symptoms. CSPα is a neuroprotective co-chaperone specifically localized to synaptic vesicles (SVs) and is evolutionarily conserved in all animals. CSPα forms a chaperone complex with HSC70 to properly fold a limited number of synaptic proteins. Complete loss of CSP leads to neurodegeneration and reduced lifespans in flies and mice. However, the mechanism of degeneration induced by ANCL mutations is currently unknown and there are no available animal models to study the dysfunctional proteins in situ. In this thesis, I describe the generation and subsequent characterization of the first animal model of ANCL, using the fruit fly Drosophila melanogaster. First, I show that human CSPα (hCSPα) is conserved functionally from humans to flies. Wildtype hCSPα expressed in flies localizes properly to SVs and is able to rescue lifespan defects in CSP null mutant flies. Overexpression of hCSPα proteins with the ANCL causing L115R and L116Δ mutations recapitulates numerous phenotypes consistent with human disease pathology. This includes the appearance of high molecular weight (HMW) SDS-resistant aggregates on western blots, accumulation of aberrant osmophilic membrane structures observed via electron microscopy, and a dose-dependent reduction in adult viability. Mutant hCSPα is mislocalized from SVs to enlarged abnormal endosomes, which accumulate in neuronal axons and somata. These endosomes strongly co-localize with the endosomal sorting required for transport (ESCRT) complex protein HRS, contain large amounts of ubiquitinated proteins, and lack markers of lysosomal maturation. This suggests that the ANCL causing mutations may cause disruptions in endo-lysosomal trafficking via an ESCRT related mechanism. To probe the genetic nature of the mutant alleles I expressed the mutant hCSPα transgenes with various doses of endogenous Drosophila CSP (dCSP). I show that loss of dCSP suppresses toxicity, as well as the aberrant endosomal accumulations and HMW aggregates induced by overexpression of mutant hCSPα. Additionally, expression of a combination of the wildtype and mutant hCSPα showed a super-additive effect on viability and HMW aggregates. This suggests that the disease-causing mutations may act as hypermorphic gain of function alleles, contrary to existing models, which suggest a dominant-negative mechanism. I also performed an F1 candidate screen for genetic modifiers of toxicity, using a robust and easy-to-score adult eye morphology and pigmentation phenotype. Using this approach, I discovered several strong interactors, both enhancers and suppressors, including member of the ESCRT trafficking pathway and other known CSP-interacting proteins. Of particular interest was the CSP co-chaperone Hsc70, which had several loss of function alleles among the strongest observed suppressors. Loss of Hsc70 also greatly reduces toxicity and endosomal accumulations of overexpressed mutant hCSPα but interestingly does not have a significant effect on the levels of HMW CSPα aggregates. This further supports the model that ANCL mutations act as hypermorphs, with a toxic mechanism involving CSP’s endogenous interactions with HSC70. Finally, I discuss the implications of these findings in relation to previous studies of the ANCL causing mutations and endogenous CSPα/HSC70 function and propose a novel mechanistic disease model. This model postulates that mutant CSP is properly trafficked to synapses but, after a brief lifespan as a properly functioning HSC70 co-chaperone, is then ubiquitinated and localized onto endosomes. Ubiquitinated mutant CSP is then clustered by HRS but is unable to mature properly through an ESCRT dependent degradation pathway. These endosomes are retrogradely trafficked through the axon to the soma where they fuse, accumulate, and persist, eventually leading to cellular toxicity via an unknown mechanism. The hypermorphic nature of the mutants can be explained by the novel observation that normal endogenous CSP also traffics through a retrograde ESCRT dependent pathway, where it intersects and co-accumulates with mutant CSP, potentially contributing to toxicity.

No description available.

Antibodies directed against AMPAR and GABA_BR subunits have been implicated in forms of limbic encephalitis (LE), a disease characterised by memory loss and seizures. Patients with LE show clinical improvement with immunomodulatory treatment, suggesting that the associated antibodies are pathogenic. To explore further the AMPAR and GABA_BR antibodies, an in house cell based assay (CBA) was established for screening and potential pathogenicity was explored using a series of in vitro experiments. Human embryonic kidney (HEK) cells transfected with AMPAR and GABA_BR subunits and primary neuronal cultures were used to detect antibodies in patient sera and CSF. In total, 15/1361 (1.1%) AMPAR antibody positive samples and 24/1438 (1.7%) GABABR antibody positive samples were identified. The predominant antibody subclass for AMPAR and GABA_BR antibodies was shown to be IgG1. Interestingly, on transfected cells, only AMPAR antibodies showed complement deposition, and therefore had the potential to activate the classical pathway of the complement cascade. Application of IgG purified from AMPAR antibody positive patients, but not GABA_BR antibody positive patients caused a down regulation of the receptor from the cell surface of transfected HEK cells and primary hippocampal cultures. Electrophysiological analysis showed changes in Up state duration and spike rate in the entorhinal cortex following application of purified GABA_BR antibody IgG on brain slices. These findings suggest that GABABR antibodies are having a direct short term effect on GABA_BRs, and by extension cortical networks. Finally, we attempted to study whether or not viral infection could be a trigger for antibody production to known and novel antigen targets in a cohort of Japanese encephalitis viral (JEV) samples. A JEV cohort of 44 children was screened for antibodies to neuronal surface proteins by CBA. Twenty-seven percent of patients had antibodies to known neuronal surface antigens, with NMDAR and CASPR2 as the most common antigen targets. Interestingly, screening the cohort on primary neuronal cultures revealed that 65.9% of children with JEV have neuronal surface antibodies. The identification of the novel antigen target was attempted using immunoprecipitation and mass spectrometry techniques. In total, 4 neuronal surface proteins were identified that could be potential targets in JEV patients. In summary, antibodies directed against previously described antigenic targets were explored further for pathogenic potential, and a cohort of patients with a viral infection with potential novel antigen targets was investigated.

The Endocannabinoid system has been found in the gastrointestinal tract, where it plays an important role in gut under both physiological and pathological conditions. Although the major effects of cannabinoids in the gut are mediated through effects on enteric neurons, the role of cannabinoids in the enteric nervous system is poorly understood. In the present study, we have used the primary cultures of myenteric ganglia and a newly developed fetal enteric neuronal cell line to identify whether the endocannabinoid, anandamide, affects ganglionic and neuronal survival and the pathways involved. Anandamide had a biphasic effect on ganglionic survival, increasing survival at low concentrations (1nM-0.1uM) and decreasing survival at high concentrations (1-10uM). Maximal survival (68% increase in number of ganglia surviving) occurred at 0.1uM and the ED50 was 3nM. This effect on promoting survival was inhibited by the CB1 antagonist AM251 (1uM) and by AraC (10uM), but not the CB2 antagonist AM630 (1uM). AM630 (1uM) significantly blocked the decreased survival induced by high concentration anandamide (10uM). The enteric glia was involved in anandamide-induced ganglion survival. Anandamide had no effect on the number of neurons/ganglion in the presence of enteric glia, but decreased the number of neurons/ganglion by 15-20% in absence of enteric glia. This effect was partially reversed by CB1 antagonist, AM251 (1uM) (20%-145% at 1nM-10uM) and by CB2 antagonist AM630 (1uM) (40%-185% at 1nM-10uM). In the fetal enteric neural cell line (IM-FEN), anandamide decreased enteric neuronal survival in a concentration-dependent manner at both 39 and 33 degree (11-45% and 10-22%decrease in survival at 1nM-10uM, respectively). Coculture of astrocytes with the enteric neuronal cells was not able to reverse anandamide-mediated neuronal death. Immunocytochemistry and western blot confirmed that the presence of both CB1 and CB2 receptors in enteric neurons (primary cultures and IM-FEN) and glia (primary cultures). In addition, the PLC-beta inhibitor U73122 (1uM) inhibited anandamide induced ganglia survival significantly. Anandamide also induced increased expression of phospho-P44/42MAPK (13-48% at 1nM-10uM) and phospho-AKT (1-28% at 1 nM-10uM) in IM-FEN. We conclude that anandamide has a differential effect on survival of enteric ganglia and neurons. It promotes ganglionic and neuronal survival by CB1 receptors in the presence of glia and this involves the PLC-beta pathway. Conversely, anandamide promotes neuron death in absence of glia as a result of effects on both the MAPK and PI-3K/AKT pathways. Since the endocannabinoid system is upregulated in inflammatory bowel diseases, these effects may play a role in the pathogenesis of the response to inflammation as well as the recovery and reinnervation of the gut following the acute phase of inflammation. The further significance of this work could contribute to developing new therapeutic methods for treatment of inflammatory bowel disease and related symptoms in clinic practice.

En esta investigación se propone una metodológica alternativa a la metodología de Box y Jenkins, donde se podrá evidenciar el modelamiento de series temporales no lineales, mediante el enfoque paramétrico y el enfoque No paramétrico. En el enfoque paramétrico me inclinaré por la extensión de los métodos de Box y Jenkins, es decir, los modelos ARCH, GARCH, TGARCH entre otros, para el modelado de series temporales no lineales, en la cual obtendré los pronósticos del año 2012 para la serie temporal:  Número de peruanos retornantes según año de regreso mediante medio de transporte aéreo. En el enfoque No paramétrico presentaré el método de la red neural de retropropagación resiliente para el modelado de series temporales no lineales, en la cual obtendré los pronósticos del año 2012 para la serie temporal:  Número de peruanos retornantes según año de regreso mediante medio de transporte aéreo. La serie de tiempo estudiada para esta investigación presenta un cambio estructural durante los años del 2000-2003, lo que induce a la no linealidad de la serie. La estimación de los dos enfoques serán comparados y se elegirá el enfoque que otorgue mejores pronósticos, la cual escogeré mediante indicadores de validación como por ejemplo el MAD (Desviación Media Residual) y SSE (Suma de los Cuadrados de los Residuos). / In this research an alternative to Box and Jenkins methodology, where you can demonstrate the modeling of nonlinear time series, using parametric and nonparametric approach is proposed methodological approach. In the parametric approach, we prefer the extension of the methods of Box and Jenkins, ie ARCH, GARCH, TGARCH models among others, for modeling nonlinear time series, in which we obtain forecasts for 2012 for the series time:  Number of returnees back Peruvians by year by means of air transport. In the parametric approach not present the method of the Resilient Backpropagation Neural Network for modeling nonlinear time series, in which we obtain forecasts of 2012 to the time series:  Number of returnees back Peruvians by year by means of air transport. The time series studied for this research presents a structural change during the years of 2000-2003, which leads to the nonlinearity of the series. The estimation of the two approaches will be compared and approach that gives better predictions will be chosen, which will choose validation using indicators such as MAD (mean deviation residual) and SSE (sum of the squares of the waste)

No description available.

Experimentos têm revelado que redes de neurônios, tanto in vitro como in vivo, mantêm atividade descrita por avalanches e se organizam em um estado crítico no qual essas avalanches são distribuídas de acordo com leis de potência. Mostramos no presente trabalho que um modelo de rede de elementos excitáveis com sinapses dinâ- micas é capaz de exibir criticalidade auto-organizada para ampla região do espaço de parâmetros. Nossos resultados estão de acordo com outros estudos que indicam que a depressão sináptica de curto prazo constitui mecanismo suciente para produzir criticalidade em avalanches neuronais. No entanto, segundo diversos pesquisadores, embora o ajuste de parâmetros seja grosso para que haja criticalidade no modelo, é mais preciso dizer que o sistema não apresenta criticalidade auto-organizada genu ína, mas sim quasi-criticalidade auto-organizada, como os demais modelos não conservativos presentes na literatura. / Experiments have shown that neuronal networks, both in vitro and in vivo, maintain activity described by avalanches and they are organized into a critical state in which these avalanches are distributed according to power laws. We have demonstrated that a model based on a network of excitable elements with dynamical synapses is able to exhibit self-organized criticality for a wide range of the parameter\'s space. Our results are consistent with other studies that suggest short-term synaptic depression is enough to produce criticality in neuronal avalanches. However, according to several researchers, in spite of the tuning to be gross to ensure that there is criticality in the model, it is more accurate do not say that the system presents genuine self-organized criticality, but self-organized quasi-criticality as the other non-conservative models in the literature.

O protocolo conhecido como Dynamic Clamp consiste em utilizar um computador para introduzir condutâncias artificiais em um neurônio biológico. O modo como estas condutâncias dependem da voltagem da membrana ou do tempo são modelado por equações diferenciais que são integradas em tempo real por um computador conectado ao neurônio biológico. Resumidamente, o computador tem acesso ao potencial de membrana dos neurônios através de eletrodos intracelulares conectados a conversores analógico-digitais (ADCs), calcula as correntes a serem injetadas nos neurônios e produz os sinais de saída através de conversores digitalanalógicos (DACs) que produzem a injeção das correntes nos eletrodos intracelulares. De um certo modo, o Dynamic Clamp utiliza os neurônios como simuladores, permitindo investigar a importância de um tipo de condutância para a atividade elétrica de um neurônio, assim como determinar o efeito produzido pelas sinapses em uma rede, combinando o controle e flexibilidade de uma simulação no computador com a acurácia e o realismo de um experimento em eletrofisiologia. Descrevemos a implementação de um protocolo de Dynamic Clamp utilizando um computador pessoal tipo IBM-PC que permitiu contornar 3 das principais limitações que apresentam alguns dos programas de Dynamic Clamp comerciais/gratuitos disponíveis atualmente: (a) Garantia de que o sistema roda em tempo real - nossa implementação é baseada em um programa de Dynamic Clamp que roda em uma plataforma Linux Real-Time que além de controlar os experimentos em tempo real consiste em software livre com codigo fonte aberto e que pode ser instalado gratuitamente; (b) Não necessita de hardware de aquisição de dados dedicado para eletrofisiologia - utilizamos uma placa ADC/DAC comercial comum marca National Instruments modelo PCIMIO16E4. Com o driver COMEDI instalado para placas de aquisição de dados Linux, a maioria das placas ADC/DAC tipo PCI disponíveis no mercado podem ser utilizadas em implementações futuras; (c) Aumentar o número de neurônios que podem ser conectados simultaneamente - desenvolvemos um circuito demultiplex analógico que permite controlar até 8 neurônios biológicos/artificiais a partir das duas saídas analógicas que as placas DAC comerciais possuem e ainda atingir frequências de atualização da corrente de até 3 kHz (para 8 correntes de saída). Apresentamos os resultados de diversos testes que fizemos usando o programa adaptado e o circuito demultiplex para produzir sinapses em tempo real e conectar diversos neurônios artificiais em pequenas redes. Também mostramos alguns resultados preliminares obtidos com a primeira implementação de um modelo de neurônio estocástico tipo Hodgkin-Huxley em um programa de Dynamic Clamp. / The Dynamic Clamp protocol consists in using a computer to introduce artificial conductances in a biological neuron. The voltage- and time-dependency of each conductance is modeled by differential equations integrated in real-time by the computer connected to the biological neurons. In short, the computer executes a 3-phase cycle in which the membrane potential of the neurons is measured by intracellular electrodes and digitized by analog-to-digital converters (ADCs), the currents are calculated based in the digitized membrane potentials and current signals are generated by digital-to-analog converters (DACs). These currents are actually injected in the neurons by other intracellular electrodes. In some extent the Dynamic Clamp uses the neurons as simulators, allowing one to investigate the role of a specific conductance in the intrinsic activity of a neuron as well as to look for the effects of a synapse in the behavior of a small network. The Dynamic Clamp combines the control and flexibility of a computer simulation with the reality of an experiment in electrophysiology. We describe an implementation of a Dynamic Clamp protocol that allowed us to surmount 3 of the main drawbacks present in some commercial/freely available Dynamic Clamp programs: (a) Runs in real time - our implementation is based in a program that runs in a Real-Time Linux platform. This operating system not only ensures the experiments will be controlled in real time but also consists in open source software that can be freely downloaded and installed; (b) No need of special electrophysiology acquisition hardware - we used a commercial ADC/DAC acquisition board model PCI-MIO16E4 from National Instruments. With the COMEDI Linux package driver that is used most of the PCI commercial ADC/DAC boards can be used in future implementations with no change needed in the program itself. (c) We can connect more than two neurons with artificial synapses - we developed an analog demultiplex circuit that allowed us to control simultaneously up to 8 biological/artificial neurons from the two analog outputs available in most of the commercial ADC/DAC boards and we could still reach current update rates of about 3 kHz (for 8 current outputs enabled). We present the results of several tests we performed using the program adapted to control the analog demultiplex to establish synapses and to connect several artificial neurons in small neural networks. Preliminary results from the first implementation of a stochastic whole cell Hodgkin-Huxley model neuron in a real time Dynamic Clamp program are also shown.