No description available.

The pathological mechanisms of variant Creutzfeldt-Jakob disease (vCJD) in the human brain remain poorly understood. Gene expression data may provide insight into the molecular mechanisms involved. This requires analysis of human postmortem brain tissue however; the variability in RNA preparations from human brain material is a concern. A method for the isolation of RNA from vCJD brains which minimized infectivity and reduced Proteinase K resistant prion protein levels to undetectable by biochemical assay was developed. RNA preparations were made from sample of the frontal parasagital cortex, sub-frontal cortex and cerebellum of 78 human autopsy cases; 21 vCJD, 26 other neurological disease (OND) and 31 nonneurological disease (NND). Suitable RNA metrics for these human brain RNA preparations were evaluated and the intra- and inter-case variability of RNA preparations was determined. There was marked intra- and inter-case variability in RNA integrity number (RIN), A260:280 absorbance ratio and RNA yield. In particular, RIN and A260:280 showed little variation intra-patient, although RNA yield was more variable. The effects of postmortem interval, tissue pH, age at death, gender, freeze-thaw cycles (including storage method and temperature) and agonal state were investigated; none of these parameters correlated with the marked variability observed. Parameters for matching vCJD and OND/NND cases were considered and RNA from three age and gender matched comparison groups, each containing one OND, one NND and one vCJD case, were used for gene expression analysis. Data was generated using Superarray GEArray® Focused DNA Microarray and analysed using the GEArray Expression Analysis Suite and Significance Analysis of Microarray software. A comparison between matched vCJD and NND control cases identified 26 up-regulated and 16 down-regulated genes, showing >1.5-fold change with a false discovery rate of 9%. The modulated genes were involved in cell signaling, cell death, cholesterol and lipid metabolism. Involvement of these pathways is consistent with findings in other transmissible spongiform encephalopathy studies.

Ziel der vorliegenden Arbeit war es einerseits zu untersuchen, ob unter den Bedingungen eines regulären Schlachtbetriebs die stumpfe Schuss-Schlagbetäubung eine den tierschutzrechtlichen Anforderungen entsprechende Alternative zur Bolzenschussbetäubung darstellt. Andererseits sollte überprüft werden, ob es zu einer nachweisbaren Embolisierung von ZNS-Gewebe in die Lungengefäße, das Herzlumen oder das Herzspitzengewebe kommt.

The dorsal vagal nucleus (DMNX) and nucleus ambiguus (NA) are two anatomically distinct regions of the medulla oblongata of the brainstem involved with the control of the heart on a beat to beat basis. The vagus nerve has parasympathetic cell bodies located in the DMNX and NA. The presence of the disease associated prion (PrPD) in the DMNX and NA is used in the post mortem diagnosis of transmissible spongiform encephalopathies (TSEs) in animals. It has been shown that PrPD alters the neuronal discharge properties of infected tissue (Barrow, Holmgren et al.1999; Collinge, Whittington et al. 1994). I wished to investigate whether a change in heart rate variability (HRV) influenced by the presence of PrPD deposits in brainstem areas of animals and people incubating TSEs would be detectable. Recordings from control and infected sheep, cattle and humans, consisting of three hundred-second samples of electrocardiogram (ECG) were collected from species specific healthy controls and subjects incubating TSE disease. Data were digitised at a sampling frequency of 1kHz and were translated and analysed using standard software (CED Spike2 ; IBM SPSS). Artefacts and missed beats were corrected based upon screening by eye. ECG R-wave timings were obtained in order to determine variability in the R-R intervals. An instantaneous tachogram was constructed from which power spectra were calculated. Power spectral analysis along with simpler time domain estimates of HRV, such as RMSSD, were employed to investigate differences between control and infected animals. In addition R wave variability within each breath was utilized to examine the vagal control of the heart in relation to breathing and thus investigate a change in function of the specific neurological areas of the brainstem used as diagnostic criteria for such diseases. It was found there were significant differences (p<0.05) in the HRV of infected sheep, cattle and humans incubating TSE disease compared to control samples. Repeated non-invasive longitudinal tests may provide a means to screen animals and humans for the presence of disease associated prions and may give applications in the objective assessments of putative therapeutics in addition to identifying TSE disease at a preclinical stage.

No description available.

Ziel der vorliegenden Arbeit war es einerseits zu untersuchen, ob unter den Bedingungen eines regulären Schlachtbetriebs die stumpfe Schuss-Schlagbetäubung eine den tierschutzrechtlichen Anforderungen entsprechende Alternative zur Bolzenschussbetäubung darstellt. Andererseits sollte überprüft werden, ob es zu einer nachweisbaren Embolisierung von ZNS-Gewebe in die Lungengefäße, das Herzlumen oder das Herzspitzengewebe kommt.

Príons são patógenos infecciosos que causam um grupo de doenças neurodegenerativas fatais. A proteína normal, PrP celular, denominada PrPC, é convertida em PrPSc, isoforma anormal e patogênica de PrP, através de um processo no qual uma porção de -hélice da estrutura é reenovelada em folhas . A conversão de PrPC em PrPSc ocorre por um mecanismo auto-catalítico. Para um melhor entendimento do mecanismo de propagação dos príons, têm sido propostos vários modelos matemáticos. Nesse trabalho, estudamos o tempo de incubação de algumas doenças causadas por príons: Encefalopatia Espongiforme Bovina (BSE), ou mal da vaca louca; doença variante de Creutzfeldt-Jakob (vCJD), que afeta humanos, através da exposição ao agente de BSE; e Scrapie murina, uma infecção priônica experimental em camundongos. A distribuição de probabilidades da duração do período de incubação foi suposta ser lognormal, modelo este extensamente aceito em doenças infecciosas. Os objetivos desse trabalho foram esclarecer aspectos obscuros sobre a cinética de replicação priônica e o mecanismo de toxicidade das doenças priônicas, através de comparação dos resultados de simulações computacionais com os perfis de distribuição de tempos de incubação de BSE, vCJD e Scrapie murina. Foram realizadas simulações computacionais, utilizando o Método Monte Carlo Dinâmico (MCD) e o modelo Difusão Limitada à Agregação. Primeiramente, estudamos o modelo de Eigen (1996), através de simulações computacionais usando o MCD, para verificar quais termos são importantes para a cinética priônica. De posse desse resultado, partimos então para o estudo sobre a toxicidade das doenças priônicas, usando o modelo DLA e o método MCD: considerando que PrPC se converte em PrPSc quando existe contato (auto-catálise); e PrPCs são livres e podem se movimentar por uma rede, enquanto PrPScs, ou agregados de PrPScs são fixos. Confirmamos a suspeita de Eigen de que o termo mais importante nas equações de cinética priônica é o termo de Michaelis-Menten, ou termo auto-catalítico. Os resultados obtidos através das simulações MCD e modelo DLA foram comparados com os perfis de distribuições de tempos dessas doenças (BSE, vCJD e Scrapie murina). Conseguimos o ajuste de diferentes perfis de distribuição de tempos de incubação para algumas doenças priônicas, lognormal para BSE e vCJD, e lognormal com segundo pico para Scrapie murina. A auto-catálise é o mecanismo mais importante na cinética priônica, a conversão espontânea de PrPC em PrPSc pode ser negligenciada. A partir do modelo DLA, fica reforçada a hipótese de que para BSE e vCJD, doenças priônicas de ocorrência natural, a toxicidade é causada, principalmente, pela formação das placas amilóides. Para Scrapie murina, uma infecção experimentalmente induzida, a toxicidade é, possivelmente, causada por dois mecanismos: formação das placas amilóides e depleção de PrPC. Apenas com a mudança dos parâmetros iniciais e finais, conseguimos ajustar as distribuições de tempos de incubação das três doenças priônicas estudadas, apesar de o modelo ser bastante simples. A lognormalidade, de acordo com o modelo, é resultado do processo difusivo. As concentrações de PrPC devem ser baixas, menores que 1% e o número de PrPScs deve ser menor que 10 para que a lognormalidade ocorra sem a depleção de PrPC. / Prions are infectious agents responsible for a group of fatal neurodegenerative disorders. A pathogenic isoform of the prion protein (PrPSc) generated by a posttranslational process involving the conversion of alpha helices into beta sheets of the normal cellular prion protein (PrPC) is believed to be the main component of these infectious agents. The conversion of a normal PrPC into an abnormal isoform PrPSc, kinetically follows through an autocatalytic process. For better understanding of this kind of abnormal protein propagation, many analytical models have been proposed. Thus, we studied, using the Monte Carlo method, the distribution of the incubation periods in some of these neurodegenerative disorders, such as: bovine spongiform encephalopathy well known as mad cow disease (BSE), Variant Creutzfeldt Jakob disease (vCJD) and murine scrapie, an experimental murine prionic disease. The distribution of the incubation times of these diseases were considered lognormal. The aim of this study was to investigate some aspects of toxicity and replication of the prionic diseases, by comparing the results of computational simulations with the incubation times of BSE, vCJD and murine scrapie, previously established. Computational simulations, using a Dynamical Monte Carlo method (DMC) and the diffusion limited aggregation model (DLA), were worked out. At first, we evaluate the Eigen model through computational simulations using the DMC to verify the essential parameters in the kinetic of the prionic diseases. Following the results, we studied the toxicity of the prionic diseases using the DMC and the DLA model; by considering that PrPC converting in PrPSc just when exists contact (autocatalysis) and free PrPCs are allowed to diffuse randomly to their nearest neighbour sites in a square lattice, while isolated PrPScs or aggregate of PrPScs are fixed. Confirming the Eigen suspicion, the most important parameter in the equation of the prionic kinetic is the Michaelis Menten term (or the autocatalytic term). The results obtained through simulations using DMC and DLA model were compared with the time distribution profiles of the prionic diseases already established (BSE, vCJD and murine Scrapie). We get the fitting in different profiles of the distribution of the incubation periods (lognormal to BSE and vCJD and lognormal with a second peak to murine scrapie). It is concluded that autocatalysis is an essential mechanism for the prionic kinetics and the spontaneous conversion of PrPC in PrPSc can be neglected. Starting from the DLA model, is reinforced that the hypothesis for BSE and vCJD, prionic diseases of natural occurrence, the toxicity is caused, mainly, by the formation of amyloid plaques. For Scrapie murina, an experimentally induced infection, the toxicity is, possibly, caused by two mechanisms: formation of amyloid plaques and depletion of PrPC. Just with the change of the initial and final parameters, we fitted all studied prionic diseases, in spite of the model to be quite simple. The lognormality from the model, is resulting of a diffusive process. Concentrations of PrPC should be low, smaller than 1% and the number of PrPScs should be smaller than 10 for the lognormality take place without the depletion of PrPC.

Príons são patógenos infecciosos que causam um grupo de doenças neurodegenerativas fatais. A proteína normal, PrP celular, denominada PrPC, é convertida em PrPSc, isoforma anormal e patogênica de PrP, através de um processo no qual uma porção de -hélice da estrutura é reenovelada em folhas . A conversão de PrPC em PrPSc ocorre por um mecanismo auto-catalítico. Para um melhor entendimento do mecanismo de propagação dos príons, têm sido propostos vários modelos matemáticos. Nesse trabalho, estudamos o tempo de incubação de algumas doenças causadas por príons: Encefalopatia Espongiforme Bovina (BSE), ou mal da vaca louca; doença variante de Creutzfeldt-Jakob (vCJD), que afeta humanos, através da exposição ao agente de BSE; e Scrapie murina, uma infecção priônica experimental em camundongos. A distribuição de probabilidades da duração do período de incubação foi suposta ser lognormal, modelo este extensamente aceito em doenças infecciosas. Os objetivos desse trabalho foram esclarecer aspectos obscuros sobre a cinética de replicação priônica e o mecanismo de toxicidade das doenças priônicas, através de comparação dos resultados de simulações computacionais com os perfis de distribuição de tempos de incubação de BSE, vCJD e Scrapie murina. Foram realizadas simulações computacionais, utilizando o Método Monte Carlo Dinâmico (MCD) e o modelo Difusão Limitada à Agregação. Primeiramente, estudamos o modelo de Eigen (1996), através de simulações computacionais usando o MCD, para verificar quais termos são importantes para a cinética priônica. De posse desse resultado, partimos então para o estudo sobre a toxicidade das doenças priônicas, usando o modelo DLA e o método MCD: considerando que PrPC se converte em PrPSc quando existe contato (auto-catálise); e PrPCs são livres e podem se movimentar por uma rede, enquanto PrPScs, ou agregados de PrPScs são fixos. Confirmamos a suspeita de Eigen de que o termo mais importante nas equações de cinética priônica é o termo de Michaelis-Menten, ou termo auto-catalítico. Os resultados obtidos através das simulações MCD e modelo DLA foram comparados com os perfis de distribuições de tempos dessas doenças (BSE, vCJD e Scrapie murina). Conseguimos o ajuste de diferentes perfis de distribuição de tempos de incubação para algumas doenças priônicas, lognormal para BSE e vCJD, e lognormal com segundo pico para Scrapie murina. A auto-catálise é o mecanismo mais importante na cinética priônica, a conversão espontânea de PrPC em PrPSc pode ser negligenciada. A partir do modelo DLA, fica reforçada a hipótese de que para BSE e vCJD, doenças priônicas de ocorrência natural, a toxicidade é causada, principalmente, pela formação das placas amilóides. Para Scrapie murina, uma infecção experimentalmente induzida, a toxicidade é, possivelmente, causada por dois mecanismos: formação das placas amilóides e depleção de PrPC. Apenas com a mudança dos parâmetros iniciais e finais, conseguimos ajustar as distribuições de tempos de incubação das três doenças priônicas estudadas, apesar de o modelo ser bastante simples. A lognormalidade, de acordo com o modelo, é resultado do processo difusivo. As concentrações de PrPC devem ser baixas, menores que 1% e o número de PrPScs deve ser menor que 10 para que a lognormalidade ocorra sem a depleção de PrPC. / Prions are infectious agents responsible for a group of fatal neurodegenerative disorders. A pathogenic isoform of the prion protein (PrPSc) generated by a posttranslational process involving the conversion of alpha helices into beta sheets of the normal cellular prion protein (PrPC) is believed to be the main component of these infectious agents. The conversion of a normal PrPC into an abnormal isoform PrPSc, kinetically follows through an autocatalytic process. For better understanding of this kind of abnormal protein propagation, many analytical models have been proposed. Thus, we studied, using the Monte Carlo method, the distribution of the incubation periods in some of these neurodegenerative disorders, such as: bovine spongiform encephalopathy well known as mad cow disease (BSE), Variant Creutzfeldt Jakob disease (vCJD) and murine scrapie, an experimental murine prionic disease. The distribution of the incubation times of these diseases were considered lognormal. The aim of this study was to investigate some aspects of toxicity and replication of the prionic diseases, by comparing the results of computational simulations with the incubation times of BSE, vCJD and murine scrapie, previously established. Computational simulations, using a Dynamical Monte Carlo method (DMC) and the diffusion limited aggregation model (DLA), were worked out. At first, we evaluate the Eigen model through computational simulations using the DMC to verify the essential parameters in the kinetic of the prionic diseases. Following the results, we studied the toxicity of the prionic diseases using the DMC and the DLA model; by considering that PrPC converting in PrPSc just when exists contact (autocatalysis) and free PrPCs are allowed to diffuse randomly to their nearest neighbour sites in a square lattice, while isolated PrPScs or aggregate of PrPScs are fixed. Confirming the Eigen suspicion, the most important parameter in the equation of the prionic kinetic is the Michaelis Menten term (or the autocatalytic term). The results obtained through simulations using DMC and DLA model were compared with the time distribution profiles of the prionic diseases already established (BSE, vCJD and murine Scrapie). We get the fitting in different profiles of the distribution of the incubation periods (lognormal to BSE and vCJD and lognormal with a second peak to murine scrapie). It is concluded that autocatalysis is an essential mechanism for the prionic kinetics and the spontaneous conversion of PrPC in PrPSc can be neglected. Starting from the DLA model, is reinforced that the hypothesis for BSE and vCJD, prionic diseases of natural occurrence, the toxicity is caused, mainly, by the formation of amyloid plaques. For Scrapie murina, an experimentally induced infection, the toxicity is, possibly, caused by two mechanisms: formation of amyloid plaques and depletion of PrPC. Just with the change of the initial and final parameters, we fitted all studied prionic diseases, in spite of the model to be quite simple. The lognormality from the model, is resulting of a diffusive process. Concentrations of PrPC should be low, smaller than 1% and the number of PrPScs should be smaller than 10 for the lognormality take place without the depletion of PrPC.