Scrapie é uma doença neurodegenerativa infecciosa que afeta ovinos e caprinos, o qual está relacionada a uma alteração conformacional da proteína priônica, que leva a deposição e agregação da proteína no sistema nervoso central. A predisposição a infecção pelo agente príon está associado a polimorfismos de nucleotídeos únicos no gene da proteína priônica. Os principais polimorfismos relacionados à infecção estão presentes nos códons 136, 154 e 171, sendo o genótipo VRQ o mais suscetível e o ARR o genótipo mais resistente. O presente estudo teve como objetivo identificar os polimorfismos de nucleotídeos únicos em ovinos das raças Suffolk, Dorper e Santa Inês, provenientes de surtos de scrapie clássico e de rebanhos livre de scrapie, os quais os proprietários estavam dispostos a colaborar com o projeto. O primeiro trabalho analisou polimorfismos de nucleotídeos únicos em 15 códons do gene da proteína priônica em um rebanho Suffolk afetado com scrapie clássico no Brasil. Dos 15 códons analisados, 3 apresentaram polimorfismos (136, 143 e 171). O códon 171 apresentou o maior número de polimorfismos, os quais foram encontradas todas as formas alélicas. Quando avaliado os grupos de risco, cerca de 96% do rebanho pertenceu aos grupos 1 a 3 (risco muito baxi a moderado). O segundo trabalho relatou o desenvolvimento da técnica de PCR em tempo real, baseado em sondas TaqMan, para a identificação de polimorfismos nos códons 136, 154 e 171 e sua aplicabilidade em rebanhos brasileiros. Um total de 142 amostras foram analisadas por PCR em tempo real. Ao comparar os resultados do PCR em tempo real com o sequenciamento, 100% das amostras foram idênticas. Para o códon 136, a maioria dos ovinos apresentou o genótipo AA. Para o códon 154, o genótipo RR foi o mais frequente, e para o códon 171, os genótipos mais frequentes foram QQ e QR. O terceiro trabalho descreve a caracterização de três surtos de scrapie clássico em ovinos da raça Dorper, em diferentes regiões do sul do Brasil. Os surtos ocorreram nos anos de 2011 a 2013, sendo que no segundo e no terceiro foram identificados ovinos do primeiro caso. Além disso, foi analisada a associação de scrapie com a genotipagem do gene da proteína priônica em ovinos presentes nos três rebanhos. No total, 22 ovinos foram positivos no teste de imuno-histoquímica, sendo que 4 deles apresentaram sinais clínicos da doença. Em todos os estudos, presentes as três raças analisadas, foi possível evidenciar a presença de ovinos, na sua maioria, geneticamente suscetíveis a infecção, pois a maioria pertenceu ao grupo de risco 3, considerado moderado. / Scrapie is an infectious neurodegenerative disease affecting sheep and goats. This is related to an altered conformational of the prion protein (PrPSc) that leads to the deposition and aggregation of protein in the central nervous system. The predisposition to prion infection agent is associated with single nucleotide polymorphisms in the prion protein gene. The mostly polymorphisms related with infection are present in codons 136, 154 and 171, being more susceptible genotype VRQ and ARR more resistant genotype. This study aimed to identify single nucleotide polymorphisms in sheep breeds Suffolk, Dorper and Santa Ines, from outbreaks of classical scrapie flocks and free scrapie flocks, which the owners were willing to collaborate with the project. The first article analyzed single nucleotide polymorphisms in 15 codons of the prion protein gene in a Suffolk sheep affected with classical scrapie in Brazil. Of the 15 codons analyzed, 3 showed polymorphisms (136, 143 and 171). Codon 171 showed the greatest number of polymorphisms, which were found all allelic forms. When assessed risk groups, about 96% of the herd belonged to groups 1 to 3 (very baxi to moderate risk). The second article reported the development of PCR real time based on TaqMan probes for the identification of polymorphisms in codons 136, 154 and 171 and their applicability in Brazilian herds. A total of 142 samples were analyzed by real-time PCR. When comparing the results of real time PCR with the sequencing of the samples were 100% identical. For codon 136, the majority of the sheep had the AA genotype. For codon 154, the RR genotype was the most frequent, and the codon 171, the most common genotypes were QQ and QR. The third article describes the characterization of three outbreaks of classical scrapie in Dorper sheep, in different regions of southern Brazil. The outbreaks occurred in the years 2011-2013, and the second and third were identified sheep of the first case. Furthermore, it was analyzed its association with genotyping prion protein gene in sheep present the three herds. In total, 22 sheep were positive in immunohistochemical testing, and 4 of them showed clinical signs of disease. In all studies, presents three races analyzed, it was possible to demonstrate the presence of sheep, mostly genetically susceptible to infection because the majority belonged to the risk group 3, considered moderate.

Scrapie é uma doença neurodegenerativa infecciosa que afeta ovinos e caprinos, o qual está relacionada a uma alteração conformacional da proteína priônica, que leva a deposição e agregação da proteína no sistema nervoso central. A predisposição a infecção pelo agente príon está associado a polimorfismos de nucleotídeos únicos no gene da proteína priônica. Os principais polimorfismos relacionados à infecção estão presentes nos códons 136, 154 e 171, sendo o genótipo VRQ o mais suscetível e o ARR o genótipo mais resistente. O presente estudo teve como objetivo identificar os polimorfismos de nucleotídeos únicos em ovinos das raças Suffolk, Dorper e Santa Inês, provenientes de surtos de scrapie clássico e de rebanhos livre de scrapie, os quais os proprietários estavam dispostos a colaborar com o projeto. O primeiro trabalho analisou polimorfismos de nucleotídeos únicos em 15 códons do gene da proteína priônica em um rebanho Suffolk afetado com scrapie clássico no Brasil. Dos 15 códons analisados, 3 apresentaram polimorfismos (136, 143 e 171). O códon 171 apresentou o maior número de polimorfismos, os quais foram encontradas todas as formas alélicas. Quando avaliado os grupos de risco, cerca de 96% do rebanho pertenceu aos grupos 1 a 3 (risco muito baxi a moderado). O segundo trabalho relatou o desenvolvimento da técnica de PCR em tempo real, baseado em sondas TaqMan, para a identificação de polimorfismos nos códons 136, 154 e 171 e sua aplicabilidade em rebanhos brasileiros. Um total de 142 amostras foram analisadas por PCR em tempo real. Ao comparar os resultados do PCR em tempo real com o sequenciamento, 100% das amostras foram idênticas. Para o códon 136, a maioria dos ovinos apresentou o genótipo AA. Para o códon 154, o genótipo RR foi o mais frequente, e para o códon 171, os genótipos mais frequentes foram QQ e QR. O terceiro trabalho descreve a caracterização de três surtos de scrapie clássico em ovinos da raça Dorper, em diferentes regiões do sul do Brasil. Os surtos ocorreram nos anos de 2011 a 2013, sendo que no segundo e no terceiro foram identificados ovinos do primeiro caso. Além disso, foi analisada a associação de scrapie com a genotipagem do gene da proteína priônica em ovinos presentes nos três rebanhos. No total, 22 ovinos foram positivos no teste de imuno-histoquímica, sendo que 4 deles apresentaram sinais clínicos da doença. Em todos os estudos, presentes as três raças analisadas, foi possível evidenciar a presença de ovinos, na sua maioria, geneticamente suscetíveis a infecção, pois a maioria pertenceu ao grupo de risco 3, considerado moderado. / Scrapie is an infectious neurodegenerative disease affecting sheep and goats. This is related to an altered conformational of the prion protein (PrPSc) that leads to the deposition and aggregation of protein in the central nervous system. The predisposition to prion infection agent is associated with single nucleotide polymorphisms in the prion protein gene. The mostly polymorphisms related with infection are present in codons 136, 154 and 171, being more susceptible genotype VRQ and ARR more resistant genotype. This study aimed to identify single nucleotide polymorphisms in sheep breeds Suffolk, Dorper and Santa Ines, from outbreaks of classical scrapie flocks and free scrapie flocks, which the owners were willing to collaborate with the project. The first article analyzed single nucleotide polymorphisms in 15 codons of the prion protein gene in a Suffolk sheep affected with classical scrapie in Brazil. Of the 15 codons analyzed, 3 showed polymorphisms (136, 143 and 171). Codon 171 showed the greatest number of polymorphisms, which were found all allelic forms. When assessed risk groups, about 96% of the herd belonged to groups 1 to 3 (very baxi to moderate risk). The second article reported the development of PCR real time based on TaqMan probes for the identification of polymorphisms in codons 136, 154 and 171 and their applicability in Brazilian herds. A total of 142 samples were analyzed by real-time PCR. When comparing the results of real time PCR with the sequencing of the samples were 100% identical. For codon 136, the majority of the sheep had the AA genotype. For codon 154, the RR genotype was the most frequent, and the codon 171, the most common genotypes were QQ and QR. The third article describes the characterization of three outbreaks of classical scrapie in Dorper sheep, in different regions of southern Brazil. The outbreaks occurred in the years 2011-2013, and the second and third were identified sheep of the first case. Furthermore, it was analyzed its association with genotyping prion protein gene in sheep present the three herds. In total, 22 sheep were positive in immunohistochemical testing, and 4 of them showed clinical signs of disease. In all studies, presents three races analyzed, it was possible to demonstrate the presence of sheep, mostly genetically susceptible to infection because the majority belonged to the risk group 3, considered moderate.

The cellular prion protein (PrP) has been shown to act as a receptor for soluble oligomers of amyloid-beta (Aβ), an ~4 kDa amyloidogenic peptide that is found in neuritic plaques that are a pathological hallmark of Alzheimer’s disease (AD). Oligomeric forms of the Aβ peptide are thought to be synaptotoxic, and have been shown to produce PrP-dependent dendritic spine loss, suppression of long term potentiation (LTP), and behavioral changes in mouse models of AD. However, the specific molecular interactions between PrP and Aβ have not been fully characterized. In this work, we conducted a robust examination of the kinetic processes leading to Aβ fibril formation, and present evidence that PrP significantly inhibits Aβ polymerization. Using established mathematical models of polymerization kinetics, we show that inhibition is based on binding between PrP and the ends of Aβ filaments, an interaction that blocks elongation. To support these results, we conducted multiple binding assays to show that PrP binds to monomers of Aβ with low affinity, oligomers with intermediate affinity, and to fibrils with high affinity. These results extend upon previous studies, which have focused only on the interaction between oligomeric Aβ and PrP. To better understand the molecular interactions required for binding and inhibition of polymerization, we performed assays with a series of PrP deletion mutants, which revealed that low-affinity binding to Aβ monomer is dependent on the presence of the C-terminal domain of PrP. This domain is also required for Aβ polymerization inhibition. Based on our results, we propose a model in which the unstructured N-terminal domain of PrP binds to the ends of Aβ fibrils, while the C-terminal domain interrupts the docking of new monomers to fibril ends, in part through competing for similar binding sites. This study provides an important contribution to our understanding of the PrP-Aβ interaction that leads to synaptoxicity.

Memoria para optar al Título Profesional de Médico Veterinario. / El Scrapie es una enfermedad neurodegenerativa y fatal que afecta a pequeños rumiantes como ovinos, caprinos y muflones. El causante de esta enfermedad es una partícula proteinácea infecciosa llamada prión, que se origina por un cambio conformacional de una proteína priónica celular del hospedero (PrPC). Los programas de vigilancia consideran a la Histopatología como método diagnóstico y a la Inmunohistoquímica (IHQ) como método confirmatorio para el Scrapie. Esta memoria fue desarrollada en el laboratorio de la Unidad de Patología del Servicio Agrícola y Ganadero de Lo Aguirre. Se utilizaron 50 muestras de óbex de caprinos mayores de 2 años, sin importar raza ni sexo, provenientes de mataderos de la IV Región de Chile. De cada muestra de óbex se obtuvieron cortes seriados con el propósito de destinar uno a la tinción de Hematoxilina y Eosina (H/E), y un corte homólogo a la técnica de Inmunohistoquímica. Así en la tinción H/E se comprobó la aptitud de la muestra evaluada a partir de la observación de la integridad de núcleos nerviosos de presentación bilateral. En total se trabajó con 50 muestras que fueron sometidas a los métodos tradicionales de histopatología para la tinción de H/E. De éstas, 50 cortes resultaron aptos para aplicar la técnica de Inmunohistoquímica. De los cortes sometidos a IHQ ninguno presentó el precipitado granular rojo característico de la inmunorreacción por la presencia de priones, por lo cual se determinó que todas las muestras de los caprinos estudiados fueron negativas, al igual que los controles negativos de óbex. Por su parte, los controles positivos de óbex siempre presentaron el precipitado granular rojo indicativo de una inmunorreacción positiva, tal como lo había indicado el Centro de Referencia para ese tipo de controles. De esta manera, este trabajo colaboró con la vigilancia pasiva anual que realizó el Servicio Agrícola y Ganadero durante el año 2015, con respecto a la detección de priones en la especie caprina en un determinado grupo de animales de nuestro país / Scrapie is a neurodegenerative and fatal disease, affecting small ruminants such as sheep, goats and mouflons. The cause of these diseases is an infectious proteinaceous particle called prion, which originates from a conformational change of a prion cellular protein of the host (PrPC). Surveillance programs consider histopathology as the diagnostic method and Immunohistochemistry (IHC) as a confirmatory method for Scrapie. This study was developed in the Laboratory of Pathology Unit of the Agricultural and Livestock Service of Lo Aguirre. Fifty obex samples of goats, older than 2 years, regardless of race or sex, were used from slaughterhouses in IV Region of Chile. Serial sections were obtained from each obex sample in order to assign one slice to the Hematoxylin and Eosin staining (H/E) and its homologous slice to the Immunohistochemistry technique. Thus, in the H/E staining the suitability of the slices were evaluated, from the observation of the integrity of the following nerve nuclei, with bilateral presentation: of the solitary tract, parasympathetic of the vagus nerve, of the spinal tract of the trigeminal nerve and motor of the hypoglossus. In total, 50 slices were performed, which were subjected to the traditional methods of histopathology and H/E staining. Of these, 50 were able to apply the technique of Immunohistochemistry (IHC). Of the samples submitted to IHC, none of them presented the red granular precipitate characteristic of the immunoreaction due to the presence of prions, whereby it was determined that all samples of the goats studied were to be classified as negative, as were the negative controls of Obex. On the other hand, positive obex controls always presented the red granular precipitate indicative of a positive immunoreaction, as indicated by the Reference Center for such controls. In this way, this work collaborated with the annual passive surveillance carried out by the Agricultural and Livestock Service during the year 2015, regarding the detection of prions in the goat species in a determined group of animals of our country

The aim of the diploma thesis was to find out how the frequency of the prion protein genotypes affects the production and reproductive characteristics of sheep in the Czech Republic. We also used graphs to evaluate changes in the frequency of allele of prion protein genotypes from 2003 to present in ten selected breeds of sheep genotyped within the breeding program for scrapie resistance in the Czech Republic. We also dealt with administrative interventions in the genotyping process by the state and recognized breeders' associations. In the next part we evaluated the changes in the frequency of the allele in relation to the development of the sheep properties in the Czech Republic. The results of the research were processed by appropriate mathematical and statistical methods. We have found that the sheep production properties are influenced by the allele frequencies of the prion protein genotypes, and that administrative interventions have contributed to changes in the frequencies of the risk groups in some breeds. Further, we have discovered that most of the highest values of utility properties are associated with the risk group R1.

The misfolding of native, cellular prion protein (PrPc) to a conformationally altered pathogenic isoform, designated scrapie PrPsc, is the main molecular process involved in the pathogenesis of prion diseases. Prion diseases are marked by the accumulation of conformationally modified forms of cellular prion protein. An N-terminal portion of the prion protein, PrP (106-128), is a 23-residue peptide fragment and is characterized by an amphipathic structure with two domains: a hydrophilic N-terminal domain and a hydrophobic C-terminal domain. In this study, the aggregation characteristics of the PrP (106-128) peptide were investigated using a combination of biophysical approaches. We investigated the effect of different factors including concentrations, pH, and metal ions, on the aggregation of the peptide. Our results demonstrated that the peptide steadily aggregates at concentrations higher than 25 M. The aggregation propensity and fibril formation is higher at pH 7.4 and pH 8.1, and the aggregation is inhibited at pH lower than 6. Furthermore, our results indicate that the Cu2+ has much less effect on the peptide amyloidogenesis, while Zn2+ has a significant influence on the PrP (106-128) amyloidogenesis. We further presented a systematic analysis of the impact of phospholipid liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-racglycerol) (POPG) in the absence or presence of cholesterol, on the amyloidogenesis of PrP (106-128). The results showed that POPC vesicles does not significantly influence the aggregation kinetics of the peptide. However, the anionic lipid POPG delays the aggregation in a concentration-dependent manner, whereas the addition of POPG with the cholesterol shows fast kinetics of fibrillization, thus reducing the lag time of the aggregation kinetics. We also monitored the effect of cholesterol and its derivatives including cholesterol-SO4 and DC-cholesterol on PrP (106-128) amyloidogenesis. Our results showed that the cholesterol inhibits the peptide aggregation and delays the formation of fibrils in a concentration-dependent manner. Cholesterol-SO4 dramatically facilitates the aggregation at high concentrations but has the potential to slow down the fibrillization at low concentrations, whereas cationic DC-cholesterol vesicles can effectively inhibit peptide fibril formation at high concentrations. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

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Despite intensive research, the cellular and molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined, in part due to the continuing lack of cultured human cells that are susceptible to infection with human prions. Such culture models would present distinct advantages including speed and expense compared with animal models, and would provide systems in which to investigate the interaction between PrPC and PrPSc, the basis of cellular susceptibility, the nature of the species barrier and the mechanism of prion propagation in situ. This study sought to examine whether non-neuronal cells might provide opportunities to establish human cell lines replicating human prions. A human follicular dendritic cell-like cell line (termed HK) was obtained, further characterised and then tested for its ability to support human prion replication. The mechanisms of internalisation, intracellular trafficking and the eventual fate of exogenous PrPSc taken up by these cells were also examined. This thesis similarly examined the cellular response of human embryonic stem cells (hESC) to acute exposure to human and animal prions. PrPC was found to be abundantly expressed by HK cells and HK cell extracts were found to support conversion to PrPSc in a cell-free conversion assay. However, HK cells exposed to infectious brain homogenates failed to accumulate PrPSc or become infected in vitro. Exposed HK and hESC did display a readily detectable, time dependent uptake of PrPSc from medium spiked with prion-infected brain homogenates that was independent of the species, disease phenotype and PRNP codon 129 genotype of the human source and the recipient cells. The exposed cells showed intensely labelled intracellular accumulations of PrPSc with coarse granular morphology, largely in the juxtanuclear region of cytoplasm. However, when the brain-spiked medium was withdrawn and cells were given control medium, the intensity and extent of PrPSc immunostaining rapidly diminished. Co-localisation studies implicated caveolae-mediated endocytic uptake of exogenous PrPSc, apparently preceding uptake via clathrin coated pits in HK cells. Evidence suggesting that the endosomal recycling compartment and lysosomes are involved in intracellular trafficking and degradation of exogenous PrPSc was also found. Understanding the cell biology of these processes may help to explain why the majority of cultured cells are refractory to prion infection in vitro. Internalization of misfolded PrP and its subsequent degradation in the lysosomal compartment might function as a self-protective cellular mechanism, serving to eliminate non-native, presumably dysfunctional and potentially dangerous PrP conformers, whether generated endogenously or acquired through exposure to exogenous prion infectivity.

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are lethal and infectious neurodegenerative diseases of humans and animals. The misfolding of the normal, or cellular isoform of the prion protein (PrPC) into the abnormal disease-associated isoform of PrP (PrPSc) could change the properties of PrP, consequently, PrPSc has lethal infectivity to transmit diseases. The proteinaceous infectious particle consisting mainly of PrPSc is called prion. Transmissibility of prions is strongly influenced by multiple factors including PrP polymorphisms, species barriers (PrP sequence specificity) and prion strains (conformational specificity) by unknown mechanisms. Even though the ability of prions to cross a species barrier has been recognized, the precise mechanisms of interspecies prion transmission remain unclear. This dissertation research was conducted in order to learn more about the molecular mechanisms of conversion, propagation and transmission of PrPSc; about determinants of genetic susceptibility to infection in prion diseases; and about understanding those mechanisms, which might govern the zoonotic potential of prion diseases. First, we investigated the transmissibility risk of multiple strains of Chronic Wasting Disease, which is a cervid TSE, with humanized transgenic mice and showed that the transmission barriers between cervid and the humanized mice are high. Next, the structural factors underlying the species barrier of prion diseases were studied using cell culture systems by systematically introducing amino acid substitutions in the regions of PrP, where the most divergences of different PrP species are recognized. Thirdly, we investigated the effects of the genetic susceptibility to prions as well as conversion kinetics and properties of PrPSc using Tg mice expressing ovine PrP polymorphism (OvPrP) at codon 136 either alanine (A) or valine (V). The templating characteristics of OvPrPSc-V136 were dominant over OvPrPSc-A136 under co-expressions of OvPrPC-A136 and OvPrPC-V136. Finally, the function of PrP was studied in relation to the pathogenesis of Alzheimer’s disease. These studies demonstrated that the conformational compatibility between PrPC and PrPSc contributed to the conversion kinetics and species barrier. We concluded that the conformational compatibility of PrPC to PrPSc is controlled not only by the PrP sequence specificity but also by the tertiary structure of PrPC.