Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a group of inevitably fatal neurodegenerative diseases that occur in mammalian species. Ovine susceptibility to scrapie, the prototypical TSE, is predominantly modulated by nonsynonymous polymorphisms within exon 3 of the ovine prion protein gene (PRNP). Investigation of PRNP exon 3 for two hair-sheep breeds revealed a novel predicted amino acid substitution (P116) associated with the ovine ARQ allele (P116A136R154Q171). Additionally, two novel ovine PRNP genotypes (PARQ/ARR; PARQ/ARQ) also were detected, and most of the hair sheep sampled possessed PRNP exon 3 genotypes associated with some degree of resistance to scrapie and/or experimental BSE (bovine spongiform encephalopathy). Unlike sheep, expression of bovine spongiform encephalopathy (BSE) in cattle and other bovids has not been associated with nucleotide variation within bovine PRNP exon 3. However, BSE susceptibility has been tentatively associated with specific insertion-deletion (indel) polymorphisms within the putative bovine PRNP promoter, and to a lesser extent intron 1, for a few German cattle breeds. Evaluation of the patterns of nucleotide variation associated with bovine PRNP exon 3 provided evidence that strong purifying selection has intensely constrained bovine exon 3 over the long-term evolutionary history of the subfamily Bovinae, as well as evidence for significant purifying selection in regions of bovine PRNP exon 3 that are considered to be of functional, structural, and pathogenic importance in other mammalian species. Evaluation of the frequencies of known indel polymorphisms within the putative bovine PRNP promoter for a panel of U. S. cattle sires revealed no significant differences in the distribution of promoter alleles and/or genotypes between U. S. cattle sires and BSEaffected German cattle. Notably, a nonsynonymous PRNP exon 3 polymorphism (T50C) identified in American bison (Bison bison) was tentatively associated with Brucella spp. seropositivity. Specifically, a significant overabundance (P = 0.021) of Yellowstone National Park bison possessing the CC genotype were Brucella spp. seropositive. Furthermore, the T-allele and TT genotype were observed at significantly higher frequencies in three bison populations that were either founded from Brucella spp. seronegative stock or previously subjected to test-and-slaughter management to eradicate brucellosis.

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Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2016-07-28T18:50:36Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016PricilaGirardi.pdf: 1029142 bytes, checksum: cabbf24ec1d0aa04a64b1668a8ac20cb (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2016-08-30T11:53:59Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016PricilaGirardi.pdf: 1029142 bytes, checksum: cabbf24ec1d0aa04a64b1668a8ac20cb (MD5) / Made available in DSpace on 2016-08-30T11:53:59Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016PricilaGirardi.pdf: 1029142 bytes, checksum: cabbf24ec1d0aa04a64b1668a8ac20cb (MD5) Previous issue date: 2016-07 / Introdução: O transtorno de déficit de atenção e hiperatividade (TDAH) é uma desordem comportamental comum em crianças e que pode persistir na idade adulta. Ele caracteriza-se por sintomas persistentes de desatenção, hiperatividade e impulsividade. Além disso, uma proporção significativa dos pacientes com TDAH também apresenta outras comorbidades envolvidas, especialmente os transtorno por uso de álcool e nicotina. Diversos estudos moleculares têm sido realizados na busca pelos genes envolvidos com o TDAH e alguns estudos têm apontando que fatores genéticos de susceptibilidade ao TDAH são também importantes na dependência de álcool e no uso de nicotina. Objetivo: O objetivo deste estudo é investigar a influência da interação dos polimorfismos rs1799990, no gene PRNP, e rs965560, no gene NCAM1, no TDAH e nas dependências de álcool e nicotina. Metodologia: As amostras foram compostas por pacientes com TDAH, 431 crianças e 535 adultos, 130 homens dependentes de álcool, 639 indivíduos adultos da população geral e 77 crianças controles. Os polimorfismos foram genotipadas através do sistema de discriminação alélica TaqMan. As análises estatísticas envolveram o teste do qui- quadrado, ANOVA e, para o teste da interação gene-gene sobre a susceptibilidade aos transtornos, foram utilizados modelos lineares gerais. Resultados: Não foram detectadas interações significativas entre os polimorfismos investigados sobre a susceptibilidade ao TDAH e às dependências de álcool e nicotina. Foi observada uma associação entre o alelo A do polimorfismo rs1799990 com o TDAH na infância (p=0,04). Em adultos com TDAH, indivíduos heterozigotos AG apresentaram uma maior frequência de transtorno opositor desafiante (TOD) (p=0,017), assim como escores médios dos sintomas de TOD significativamente maiores (p=0,012), em comparação aos indivíduos homozigotos GG. Não foram detectadas associações significativas para o polimorfismo rs965560 com nenhuma das variáveis investigadas. Conclusão: Nossos resultados indicam que o polimorfismo rs1799990 pode estar associado com o TDAH na infância e com a presença de TOD em adultos com TDAH. Esses resultados, no entanto, são preliminares e não foram testados para múltiplos testes. / Introduction: Attention deficit hyperactivity disorder (ADHD) is a common behavioral disorder in children that can persist into adulthood. It characterized by persistent symptoms of inattention, hyperactivity and impulsivity. Moreover, a significant proportion of patients with ADHD present other comorbidities, especially alcohol dependence and nicotine use. Several molecular studies have been employed in the search for the genes involved in ADHD and some studies have pointed that susceptibility genetic factors of ADHD are also important in alcohol dependence and nicotine use. Objective: The objective of this study is to investigate the influence of the interaction between the polymorphisms rs1799990, in the PRNP gene, and rs965560, in the NCAM1 gene, on the susceptibility to ADHD and alcohol and nicotine dependence. Methods: Samples were composed by patients with ADHD, 431 children and 535 adults, 130 alcohol dependent men, 639 adults from the general population and 77 children controls. The polymorphisms were genotyped by the TaqMan allelic discrimination system. The statistical analysis involved the chi- square test, ANOVA and, for the gene-gene interaction tests, general linear models were used. Results: There were no significant interactions between the polymorphisms investigated on the susceptibility to ADHD and to alcohol and nicotine dependences. An association between the A allele of rs1799990 polymorphism and ADHD in childhood (p=0.04) was observed. In adults with ADHD, individuals heterozygous AG had a higher frequency of oppositional defiant disorder (ODD) (p=0.017) and higher mean scores of symptoms of ODD (p=0.012), when compared to homozygous GG. No significant associations were found for the rs965560 polymorphism with the investigated variables. Conclusion: Our results indicate that the polymorphism rs1799990 may be associated with ADHD in childhood and with the presence of ODD in adults with ADHD. These results, however, are preliminary and were not tested for multiple testing.

Cellular prion protein (PrPC ) is a membrane bound glycoprotein. The protein is expressed in all vertebrates, mainly in the nervous system, but it is present also in the cells of gastrointestinal tract, bone marrow, germ cells and heart. PrPC is necessary for pathogenesis of prion diseases, which are deadly and without the possibility of therapy. The pathogenic isoform of prion protein is formed by changing of secondary structure of PrPC and it's the main constituent of infectious prion particles. Pathological form of prion protein accumulates in brain of infected patients and this process is associated with neurodegradation. Physiological function of PrPC is poorly understood. Knock-out of the PrPC gene (PRNP) is not connected with any noticeable phenotype. Potential functions of PrPC are dispersed, protein may have antiapoptotic effect, it can be involved in ions metabolism or in protection against oxidative stress. Latest results show, that PrPC can play important role in cell differentiation. During the differentiation PrPC can influence the development of cells and their typing. It could affect cell cycle and have an influence on formation of nervous system. Aim of the present study was to elucidate, whether the down-regulation of PrPC or infection with prions has an impact on differentiation of...

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Prionerkrankungen sind eine Gruppe seltener, infektiöser neurodegenerativer Erkrankungen, die durch die Aggregation des fehlgefalteten pathologischen Prionproteins ausgelöst werden. Anhand der Ätiologie lassen sich drei Subtypen unterscheiden: die sporadische Creutzfeldt-Jakob Krankheit (CJK), infektiöse Prionerkrankungen, wie zum Beispiel die iatrogene CJK und die neue Variante der CJK sowie genetische Prionerkrankungen. Alle Untergruppen führen zu progredienter Behinderung und schließlich unausweichlich zum Tode des Erkrankten. Genetische Prionerkrankungen werden durch eine Mutation des Prionprotein-Gens (PRNP) ausgelöst. Die zwei häufigsten Mutationen, die E200K- und V210I-Mutation, zeigen einen Phänotyp, der dem klassischen klinischen Bild der sporadischen CJK sehr ähnelt. Patienten entwickeln häufig eine rasch progrediente Demenz, eine Ataxie, visuelle Symptome und Myoklonien. Die definitive Diagnose einer Prionerkrankung kann nur durch die neuropathologische Untersuchung von Hirngewebe nach einer Hirnbiopsie oder post-mortem gestellt werden. Anhand definierter klinischer Diagnosekriterien kann jedoch auch ohne Hirnbiopsie mit relativer Sicherheit eine wahrscheinliche Prionerkrankung diagnostiziert werden. In den Diagnosekriterien finden neben klinischen Symptomen auch Zusatzuntersuchungen wie das EEG, die Liquoruntersuchung und seit kürzerer Zeit auch die kraniale MRT, Berücksichtigung. Ziel dieser Arbeit war es, zu untersuchen, ob die für die sporadische CJK definierten MRT-Kriterien auch auf Patienten mit der E200K- und V210I-Mutation anwendbar sind und ob sich Unterschiede im Verteilungsmuster der Läsionen zeigen, die eine eventuelle Unterscheidung von der sporadischen CJK erlauben. Im vorliegenden Patientenkollektiv von 29 Patienten mit genetischer CJK konnten im Vergleich zur Kontrollgruppe mit sporadischer CJKD keine Unterschiede der Sensitivität der MRT-Veränderungen oder dem Verteilungsmuster nachgewiesen werden. Eine genetische Untersuchung auf das Vorliegen einer möglichen Mutation im PRNP bleibt daher zur Abgrenzung der genetischen CJK von der sporadischen CJK unabdingbar.

The host encoded cellular prion protein (PrPC) is an N-linked glycoprotein tethered to the cell membrane by a glycophosphatidylinositol (GPI) anchor. Under certain conditions, PrPC can undergo conversion into a conformationally-altered isoform (PrPSc) widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Thus, tissues expressing PrPC are potential sites for conversion of PrPSc during TSE pathogenesis. Although much is known about the role of PrPSc in prion diseases, the normal function of PrPC is poorly understood. Lines of mice and cattle in which PrPC has been ablated by gene knockout show no major phenotypical alterations other than resistance to TSE infection. However, recent reports using Prnp-null mouse models have suggested the participation of PrPC in neural stem/progenitor cell proliferation and differentiation. The first objective in our study was to map the expression of PrPC in twenty six somatic and reproductive tissues in ruminants. Our second objective was to characterize the ontogeny of PrPC expression during bovine embryonic and early fetal development. Finally, we used a mouse embryonic stem cell (mESC) model to study the potential role of PrPC during neurogenesis. In adult tissues, intense expression of PrPC was detected in the central nervous system (CNS), thymus and testes, whereas the liver, striated muscle and female reproductive tissues showed the lowest expression. We observed that PrPC was associated with tissues undergoing cellular differentiation including spermatogenesis, lymphocyte activation and hair follicle regeneration. Analyses in bovine embryos and fetuses indicated peaks in expression of PrPC at days 4 and 18 post-fertilization, stages associated with the maternal-zygote transition and the maternal recognition of pregnancy and initiation of placental attachment, respectively. Later in development, PrPC was expressed in the CNS where it was localized in mature neurons of the neuroepithelium and emerging neural trunks. Based on these observations, we hypothesized that PrPC was involved in neurogenesis. We tested this hypothesis in a murine embryonic stem cell model (mESC). mESC were induced to form embryoid bodies (EBs) by placing them in suspension culture under differentiating conditions and allowed to differentiate in vitro for 20 days. We detected increasing levels of PrPC starting on day 12 (8.21- fold higher vs. day 0; P < 0.05) and continuing until day 20 (20.77-fold higher vs. day 0; P < 0.05). PrPC expression was negatively correlated with pluripotency marker Oct-4 (r= -0.85) confirming that mESC had indeed differentiated. To provide a more robust system for assessing the role of PrPC in neural differentiation, mESC were cultured with or without retinoic acid (RA) to encourage differentiation into neural lineages. Induction of EBs with retinoic acid (RA) resulted in an earlier up-regulation of PrPC and nestin (day 12 vs. day 16; P < 0.05). In addition, immunofluorescence studies indicated co-expression of PrPC and nestin in the same cells. The results of these experiments suggested a temporal link between PrPC expression and expression of nestin, a marker of neural progenitor cells. We next tested whether PrPC was involved in RA-enhanced neural differentiation from mESC using a PrPC knockdown model. Plasmid vectors designed to express either a PrP-targeted shRNA or scrambled, control shRNA were transfected into mESC. Stable transfectants were selected under G418 and cloned. PrP-targeted and control shRNA clones, as well as wild-type mESC, were differentiated in presence of RA and sampled as above. PrPC expression was knocked down in PrP-targeted shRNA cultures between days 12 and 20 (62.2 % average reduction vs. scrambled shRNA controls). Nestin expression was reduced at days 16 and 20 in PrPC knockdown cells (61.3% and 70.7%, respectively vs. scrambled shRNA controls). These results provide evidence that PrPC plays a role in the neural differentiation at a point up-stream from the stages at which nestin is expressed. In conclusion, the widely distributed expression of PrPC in ruminant tissues suggests an important biological role for this protein. In the present work we have provided evidence for the participation of PrPC in the differentiation of mESC along the neurogenic pathway. / Ph. D.

En el gen de la proteïna priònica, o PRNP, hem observat que el particular patró de variació que hem trobat basant-nos en dades de seqüenciació en humans es deu a selecció positiva, i que el mètode utilitzat per detectar selecció és crític. Utilitzant dades basades en SNPs es pot introduir un biaix al aplicar tests de neutralitat basats en diversitat de seqüències, i això pot portar a conclusions errònies. A més, hem vist que els polimorfismes en els codons 129 i 219 presenten gran diferències de freqüència en diferents poblacions humanes i també hem vist que aquestes posicions estan fixades en ximpanzés. La variació trobada en controls ha estat comparada amb el patró de variació existent en pacients per la mateixa regió. La reseqüenciació del gen PRNP en un gran nombre de mostres humanes i de ximpanzés ens ha permès obtenir un gran nombre d´informació d´aquest gen. / In the prion gene or PRNP, we have observed that the particular pattern of variation that we have found in this gene based on sequencing data in humans is due to positive selection, and that the method and the approach used to detect this selection critical. Ascertainment bias can be introduced by using SNP data and applying neutrality tests based on sequence diversity, therefore leading to anomalous conclusions being drawn. Moreover, we have seen that polymorphisms in codon 129 and 219 have big differences in frequency in different human populations and we have also seen that these positions are fixed in chimpanzees. The normal variation that we found in controls have been then compared with patients for the same region. The resequencing of PRNP in a very large sample of humans and chimpanzees has provided a great deal of information on this gene.