Urease de Helicobacter pylori e taupatias : abordagens in vitro e in vivo
Autor(a) principal: | |
---|---|
Data de Publicação: | 2020 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da PUC_RS |
Texto Completo: | http://tede2.pucrs.br/tede2/handle/tede/9307 |
Resumo: | Alzheimer's disease (AD) is considered the most common neurodegenerative disorder in people over the age of 60, characterized by dementia combined with memory deficit. Its characteristic histopathological changes in the patients’ brain are the formation of senile plaques - deposits of beta-amyloid peptide (Aβ) - and neurofibrillary tangles constituted by the deposition of hyperphosphorylated tau protein. The function of the tau protein, normally associated with microtubules in neuronal axons, is regulated by phosphorylation and dephosphorylation mechanisms, with more than 80 reported phosphorylation sites. The literature reported that the Helicobacter pylori culture’s filtrate, obtained without causing lysis of the bacteria, induces hyperphosphorylation of the tau protein at different sites, in vivo and in vitro. As there was no significant cerebral inflammatory response, a hypothesis was raised that exotoxins produced by the bacteria, capable of penetrating the blood-brain barrier (BBB), would directly induce tau phosphorylation. The gram-negative bacterium Helicobacter pylori is a gastric pathogen responsible for chronic gastritis, peptic and duodenal ulcers, and gastric cancer, infecting approximately 60% of the world population. This bacterium produces large amounts of urease (HPU), an enzyme considered as an important virulence factor of the bacterium. Our group has previously demonstrated the ability of HPU to induce the activation of human neutrophils, protecting them against apoptosis, and contributing to the promotion of tissue damage. The present work sought to investigate possible changes promoted by HPU in phosphorylation sites of tau protein in vivo and possible pro-inflammatory mechanisms in vitro on cells of the nervous system. In in vivo tests, male 30-days Wistar rats received an intraperitoneal dose of 5 g of this protein daily, for 7 days, then euthanized on the eighth day, and had their brains stored at -80 ºC for further analysis. In the control group, only the vehicle (sterile saline) was administered. The treatment regimen was designed to mimic a chronic H. pylori infection in young individuals. Western blotting assays were performed, using anti-Tau5, anti-pTau396, anti-pTau199 and anti-pTau205 antibodies, specific for the identification of tissue total tau protein and phosphorylated tau at Ser396, Ser199 and Thr205 sites, respectively. The levels of GSK-3β and NFκβ-p65 and the presence of HPU in brain tissue were also evaluated by Western blotting. The results indicated that HPU significantly increased tau phosphorylation at Thr205, Ser396 and Ser199 sites, however there was no detection of HPU in brain tissue, suggesting that HPU may interfere in tau phosphorylation indirectly. After in vivo treatment, HPU did not alter or decrease the GSK-3β total protein content in brain tissue. However, it increased the levels of NFκβp65 in brain tissue, indicating neuroinflammation. Such findings reinforced an association between H. pylori infection and Alzheimer's disease tauopathy, as indicated in epidemiological studies, and suggest a contribution of HPU to the pathogenesis of this neurodegenerative disease. |
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Carlini, Celia Regina Ribeiro da Silvahttp://lattes.cnpq.br/5587901760491970Uberti, Augusto Frantzhttp://lattes.cnpq.br/6403074358587624http://lattes.cnpq.br/7187898528471580Silva, Natalia Callai da2020-10-30T17:02:54Z2020-03-30http://tede2.pucrs.br/tede2/handle/tede/9307Alzheimer's disease (AD) is considered the most common neurodegenerative disorder in people over the age of 60, characterized by dementia combined with memory deficit. Its characteristic histopathological changes in the patients’ brain are the formation of senile plaques - deposits of beta-amyloid peptide (Aβ) - and neurofibrillary tangles constituted by the deposition of hyperphosphorylated tau protein. The function of the tau protein, normally associated with microtubules in neuronal axons, is regulated by phosphorylation and dephosphorylation mechanisms, with more than 80 reported phosphorylation sites. The literature reported that the Helicobacter pylori culture’s filtrate, obtained without causing lysis of the bacteria, induces hyperphosphorylation of the tau protein at different sites, in vivo and in vitro. As there was no significant cerebral inflammatory response, a hypothesis was raised that exotoxins produced by the bacteria, capable of penetrating the blood-brain barrier (BBB), would directly induce tau phosphorylation. The gram-negative bacterium Helicobacter pylori is a gastric pathogen responsible for chronic gastritis, peptic and duodenal ulcers, and gastric cancer, infecting approximately 60% of the world population. This bacterium produces large amounts of urease (HPU), an enzyme considered as an important virulence factor of the bacterium. Our group has previously demonstrated the ability of HPU to induce the activation of human neutrophils, protecting them against apoptosis, and contributing to the promotion of tissue damage. The present work sought to investigate possible changes promoted by HPU in phosphorylation sites of tau protein in vivo and possible pro-inflammatory mechanisms in vitro on cells of the nervous system. In in vivo tests, male 30-days Wistar rats received an intraperitoneal dose of 5 g of this protein daily, for 7 days, then euthanized on the eighth day, and had their brains stored at -80 ºC for further analysis. In the control group, only the vehicle (sterile saline) was administered. The treatment regimen was designed to mimic a chronic H. pylori infection in young individuals. Western blotting assays were performed, using anti-Tau5, anti-pTau396, anti-pTau199 and anti-pTau205 antibodies, specific for the identification of tissue total tau protein and phosphorylated tau at Ser396, Ser199 and Thr205 sites, respectively. The levels of GSK-3β and NFκβ-p65 and the presence of HPU in brain tissue were also evaluated by Western blotting. The results indicated that HPU significantly increased tau phosphorylation at Thr205, Ser396 and Ser199 sites, however there was no detection of HPU in brain tissue, suggesting that HPU may interfere in tau phosphorylation indirectly. After in vivo treatment, HPU did not alter or decrease the GSK-3β total protein content in brain tissue. However, it increased the levels of NFκβp65 in brain tissue, indicating neuroinflammation. Such findings reinforced an association between H. pylori infection and Alzheimer's disease tauopathy, as indicated in epidemiological studies, and suggest a contribution of HPU to the pathogenesis of this neurodegenerative disease.A Doença de Alzheimer (DA) é considerada a desordem neurodegenerativa mais comum em pessoas com idade acima de 60 anos, caracterizada por demência aliada a déficit de memória. Tem como alterações histopatológicas características a formação de placas senis - depósitos do peptídeo beta-amiloide (Aβ) - e emaranhados neurofibrilares constituídos pela deposição da proteína tau hiperfosforilada. A função da proteína Tau, encontrada associada a microtúbulos nos axônios neuronais, é regulada por mecanismos de fosforilação e desfosforilação, com mais de 80 sítios de fosforilação relatados. A literatura reportou que o filtrado da cultura de Helicobacter pylori, obtido sem provocar a lise da bactéria, induz a hiperfosforilação da proteína tau em diferentes sítios, in vivo e in vitro. Como não houve resposta inflamatória cerebral significante, levantou-se a hipótese de que exotoxinas produzidas pela bactéria, capazes de penetrar a barreira hematoencefálica (BHE), induziriam diretamente a fosforilação da Tau. A bactéria gram-negativa Helicobacter pylori é um patógeno gástrico responsável pela gastrite crônica, úlcera péptica e duodenal, e câncer gástrico, infectando aproximadamente 60% da população mundial. Essa bactéria produz grandes quantidades de urease (HPU), enzima considerada um importante fator de virulência da bactéria. Nosso grupo já demostrou a capacidade da HPU de induzir a ativação de neutrófilos humanos, protegendo-os contra apoptose e contribuindo para promoção de dano tecidual. O presente trabalho buscou investigar possíveis alterações promovidas por HPU em sítios de fosforilação da proteína tau in vivo e os possíveis mecanismos pró-inflamatórios in vitro sobre células do sistema nervoso. Em ensaios realizados in vivo, ratos machos Wistar de 30 dias receberam diariamente uma dose intraperitoneal de 5 g dessa proteína durante 7 dias, sendo eutanasiados no oitavo dia, e seus cérebros armazenado a -80 ºC para posterior análise. No grupo controle foi administrado somente o veículo (solução salina estéril). O esquema de tratamento foi concebido para mimetizar uma infecção crônica por H. pylori em indivíduos jovens. Ensaios de Western blotting foram realizados, utilizando anticorpos anti-Tau5, anti-pTau396, anti-pTau199 e antipTau205, específicos para identificação da proteína tau total do tecido e tau fosforilada nos sítios Ser396, Ser199 e Thr205, respectivamente. Foram avaliados também os níveis de GSK-3β e NFκβ-p65, e a presença de HPU no tecido cerebral por Western blotting. Os resultados indicam que a HPU aumentou significativamente a fosforilação da tau nos sítios Thr205, Ser396 e Ser199, entretanto não houve detecção da HPU no tecido cerebral, sugerindo que a HPU pode atuar sobre a fosforilação da tau indiretamente. O tratamento in vivo com a HPU não alterou os níveis proteicos da GSK3β total no tecido cerebral. Entretanto, aumentou os níveis de NFκβ-p65 no tecido cerebral, evidenciando neuroinflamação. Tais achados reforçam uma associação entre infecção por H. pylori e a taupatia da Doença de Alzheimer, como indicada em estudos epidemiológicos, e sugerem uma possível contribuição da HPU na patogênese dessa doença neurodegenerativa.Submitted by PPG Medicina e Ciências da Saúde (medicina-pg@pucrs.br) on 2020-07-24T13:48:40Z No. of bitstreams: 1 Dissertação Mestrado final _ Natalia Callai da Silva.pdf: 5217279 bytes, checksum: b5e8671f3f195c70705f5269044e03e6 (MD5)Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2020-10-30T16:57:12Z (GMT) No. of bitstreams: 1 Dissertação Mestrado final _ Natalia Callai da Silva.pdf: 5217279 bytes, checksum: b5e8671f3f195c70705f5269044e03e6 (MD5)Made available in DSpace on 2020-10-30T17:02:54Z (GMT). No. of bitstreams: 1 Dissertação Mestrado final _ Natalia Callai da Silva.pdf: 5217279 bytes, checksum: b5e8671f3f195c70705f5269044e03e6 (MD5) Previous issue date: 2020-03-30Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESConselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPqapplication/pdfhttp://tede2.pucrs.br:80/tede2/retrieve/179244/DIS_NATALIA_CALLAI_DA_SILVA_CONFIDENCIAL.pdf.jpgporPontifícia Universidade Católica do Rio Grande do SulPrograma de Pós-Graduação em Medicina e Ciências da SaúdePUCRSBrasilEscola de MedicinaHelicobacter PyloriUreaseAlzheimer’s DiseaseAlzheimerNeuinflamaçãoTaupatiasCIENCIAS DA SAUDE::MEDICINAUrease de Helicobacter pylori e taupatias : abordagens in vitro e in vivoinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisTrabalho será publicado como artigo ou livro60 meses30/10/2025-721401722658532398500500500600600-224747486637135387-96936945230878662735904625501369753661802873727776104890info:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da PUC_RSinstname:Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)instacron:PUC_RSTHUMBNAILDIS_NATALIA_CALLAI_DA_SILVA_CONFIDENCIAL.pdf.jpgDIS_NATALIA_CALLAI_DA_SILVA_CONFIDENCIAL.pdf.jpgimage/jpeg4121http://tede2.pucrs.br/tede2/bitstream/tede/9307/4/DIS_NATALIA_CALLAI_DA_SILVA_CONFIDENCIAL.pdf.jpga26222dedd25713c5b0852164b0a4066MD54TEXTDIS_NATALIA_CALLAI_DA_SILVA_CONFIDENCIAL.pdf.txtDIS_NATALIA_CALLAI_DA_SILVA_CONFIDENCIAL.pdf.txttext/plain1846http://tede2.pucrs.br/tede2/bitstream/tede/9307/3/DIS_NATALIA_CALLAI_DA_SILVA_CONFIDENCIAL.pdf.txt44c5c61042304c382f2f34e7eb3af485MD53ORIGINALDIS_NATALIA_CALLAI_DA_SILVA_CONFIDENCIAL.pdfDIS_NATALIA_CALLAI_DA_SILVA_CONFIDENCIAL.pdfapplication/pdf616230http://tede2.pucrs.br/tede2/bitstream/tede/9307/2/DIS_NATALIA_CALLAI_DA_SILVA_CONFIDENCIAL.pdfc2a400be6711649435ad79795c7e208aMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-8590http://tede2.pucrs.br/tede2/bitstream/tede/9307/1/license.txt220e11f2d3ba5354f917c7035aadef24MD51tede/93072020-10-30 21:00:11.061oai:tede2.pucrs.br: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Biblioteca Digital de Teses e Dissertaçõeshttp://tede2.pucrs.br/tede2/PRIhttps://tede2.pucrs.br/oai/requestbiblioteca.central@pucrs.br||opendoar:2020-10-30T23:00:11Biblioteca Digital de Teses e Dissertações da PUC_RS - Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)false |
dc.title.por.fl_str_mv |
Urease de Helicobacter pylori e taupatias : abordagens in vitro e in vivo |
title |
Urease de Helicobacter pylori e taupatias : abordagens in vitro e in vivo |
spellingShingle |
Urease de Helicobacter pylori e taupatias : abordagens in vitro e in vivo Silva, Natalia Callai da Helicobacter Pylori Urease Alzheimer’s Disease Alzheimer Neuinflamação Taupatias CIENCIAS DA SAUDE::MEDICINA |
title_short |
Urease de Helicobacter pylori e taupatias : abordagens in vitro e in vivo |
title_full |
Urease de Helicobacter pylori e taupatias : abordagens in vitro e in vivo |
title_fullStr |
Urease de Helicobacter pylori e taupatias : abordagens in vitro e in vivo |
title_full_unstemmed |
Urease de Helicobacter pylori e taupatias : abordagens in vitro e in vivo |
title_sort |
Urease de Helicobacter pylori e taupatias : abordagens in vitro e in vivo |
author |
Silva, Natalia Callai da |
author_facet |
Silva, Natalia Callai da |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Carlini, Celia Regina Ribeiro da Silva |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/5587901760491970 |
dc.contributor.advisor-co1.fl_str_mv |
Uberti, Augusto Frantz |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/6403074358587624 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/7187898528471580 |
dc.contributor.author.fl_str_mv |
Silva, Natalia Callai da |
contributor_str_mv |
Carlini, Celia Regina Ribeiro da Silva Uberti, Augusto Frantz |
dc.subject.eng.fl_str_mv |
Helicobacter Pylori Urease Alzheimer’s Disease |
topic |
Helicobacter Pylori Urease Alzheimer’s Disease Alzheimer Neuinflamação Taupatias CIENCIAS DA SAUDE::MEDICINA |
dc.subject.por.fl_str_mv |
Alzheimer Neuinflamação Taupatias |
dc.subject.cnpq.fl_str_mv |
CIENCIAS DA SAUDE::MEDICINA |
description |
Alzheimer's disease (AD) is considered the most common neurodegenerative disorder in people over the age of 60, characterized by dementia combined with memory deficit. Its characteristic histopathological changes in the patients’ brain are the formation of senile plaques - deposits of beta-amyloid peptide (Aβ) - and neurofibrillary tangles constituted by the deposition of hyperphosphorylated tau protein. The function of the tau protein, normally associated with microtubules in neuronal axons, is regulated by phosphorylation and dephosphorylation mechanisms, with more than 80 reported phosphorylation sites. The literature reported that the Helicobacter pylori culture’s filtrate, obtained without causing lysis of the bacteria, induces hyperphosphorylation of the tau protein at different sites, in vivo and in vitro. As there was no significant cerebral inflammatory response, a hypothesis was raised that exotoxins produced by the bacteria, capable of penetrating the blood-brain barrier (BBB), would directly induce tau phosphorylation. The gram-negative bacterium Helicobacter pylori is a gastric pathogen responsible for chronic gastritis, peptic and duodenal ulcers, and gastric cancer, infecting approximately 60% of the world population. This bacterium produces large amounts of urease (HPU), an enzyme considered as an important virulence factor of the bacterium. Our group has previously demonstrated the ability of HPU to induce the activation of human neutrophils, protecting them against apoptosis, and contributing to the promotion of tissue damage. The present work sought to investigate possible changes promoted by HPU in phosphorylation sites of tau protein in vivo and possible pro-inflammatory mechanisms in vitro on cells of the nervous system. In in vivo tests, male 30-days Wistar rats received an intraperitoneal dose of 5 g of this protein daily, for 7 days, then euthanized on the eighth day, and had their brains stored at -80 ºC for further analysis. In the control group, only the vehicle (sterile saline) was administered. The treatment regimen was designed to mimic a chronic H. pylori infection in young individuals. Western blotting assays were performed, using anti-Tau5, anti-pTau396, anti-pTau199 and anti-pTau205 antibodies, specific for the identification of tissue total tau protein and phosphorylated tau at Ser396, Ser199 and Thr205 sites, respectively. The levels of GSK-3β and NFκβ-p65 and the presence of HPU in brain tissue were also evaluated by Western blotting. The results indicated that HPU significantly increased tau phosphorylation at Thr205, Ser396 and Ser199 sites, however there was no detection of HPU in brain tissue, suggesting that HPU may interfere in tau phosphorylation indirectly. After in vivo treatment, HPU did not alter or decrease the GSK-3β total protein content in brain tissue. However, it increased the levels of NFκβp65 in brain tissue, indicating neuroinflammation. Such findings reinforced an association between H. pylori infection and Alzheimer's disease tauopathy, as indicated in epidemiological studies, and suggest a contribution of HPU to the pathogenesis of this neurodegenerative disease. |
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2020 |
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2020-10-30T17:02:54Z |
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