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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1151

Bedeutung des cytosolischen Teils des großen Hüllproteins für die Umhüllung des Hepatitis B Virus Nukleokapsids / Relevance of the cytosolic range of the large surface protein for the envelopment of hepatitis b virus nucleocapsid

Schläger, Michaela 24 April 2002 (has links)
No description available.
1152

Improvement of the jpHMM approach to recombination detection in viral genomes and its application to HIV and HBV / Verbesserung des jpHMM-Ansatzes zur Rekombinationsvorhersage in viralen Genomen und dessen Anwendung auf HIV und HBV

Schultz, Anne-Kathrin 27 April 2011 (has links)
No description available.
1153

El virus de l'hepatitis C i la ribonucleasa Phumana: aspectes biològics i terapèutics

Nadal i Matamala, Anna 26 January 2004 (has links)
El virus de l'hepatitis C (VHC) provoca una hepatitis crònica que afecta a més de 170 milions de persones d'arreu del món. És un virus petit que es classifica dins de la família Flaviviridae i és un virus d'RNA de cadena positiva amb un genoma d'aproximadament 9.600 nucleòtids. A l'extrem 5' del genoma viral s'hi troba una regió no codificant (5'NCR) que comprèn els primers 341 nucleòtids i la seva funció està relaciona amb la traducció. Immediatament després hi ha una pauta de lectura oberta ORF que acaba en un únic codó d'aturada i codifica una poliproteïna de 3.010 aminoàcids. A continuació l'extrem 3' no codificant (3'NCR), que malgrat es desconeixen les seves funcions exactes, s'ha demostrat que és essencial per a la replicació vírica. La única poliproteïna generada és processada co- i postraduccionalment mitjançant proteases de l'hoste i víriques, donant lloc a les proteïnes estructurals (Core, E1 i E2-p7) i no estructurals (NS2-NS5B). Igual que la majoria de virus RNA, el VHC es caracteritza per tenir una taxa de mutació elevada. De fet, el genoma del virus no es pot definir com una única seqüència sinó per una població de variants molt relacionades entre sí. A aquesta manera d'organitzar la informació genètica se l'anomena quasiespècie viral i una de les seves implicacions principals és la facilitat amb què sorgeixen resistents al tractament. Els tractaments disponibles són llargs, cars, provoquen efectes secundaris considerables i només es resolen completament el 40% dels casos. Per aquesta raó es busquen altres solucions terapèutiques per combatre el virus entre les quals s'hi inclouen diferents estratègies. Una de les més innovadores i prometedores és la utilització de ribozims dirigits directament contra el genoma del virus. Aquest treball es centra en l'estudi de les noves estratègies terapèutiques basades en ribozims, concretament la ribonucleasa P. La ribonucleasa P és un ribozim que està present en tots els organismes ja que és l'enzim responsable de la maduració dels precursors d'RNA de transferència. El més interessant a nivell terapèutic és que s'ha demostrat que es pot dirigir la seva activitat cap a qualsevol RNA utilitzant una seqüència guia d'RNA que quan hibrida amb l'RNA diana, l'híbrid imita l'estructura secundària del substrat natural. En el cas del VHC, s'han estudiat ribozims dependents de seqüència (ribozims derivats d'RNAs satèl·lits i de viroides de plantes), sempre dirigits contra la regió més conservada del virus per evitar una disminució de l'eficiència del ribozim deguda a la variació de la diana. La ribonucleasa P és una endonucleasa d'activitat molt específica i es diferencia dels altres ribozims naturals en el sistema de reconeixement del substrat, reconeix elements estructurals i no de seqüència. L'objectiu final del treball és tallar in vitro l'RNA del VHC aprofitant la propietat que presenta aquest ribozim de reconèixer elements estructurals i no de seqüència ja que per a un mateix nombre de seqüències, el nombre d'estructures viables que pot adoptar l'RNA genòmic és molt més petit i per tant la variabilitat de la diana disminueix. S'han estudiat dos models d'RNasa P, la RNasa P humana guiada per seqüència guia externa (EGS) i l'RNA M1 de l'RNasa P d'E.coli unit a la seqüència guia per l'extrem 3' (ribozim M1GS). Abans però de dirigir el ribozim, s'han estudiat l'estructura i la variabilitat d'una regió del genoma del virus ja que s'ha descrit que són factors que poden limitar l'eficiència de qualsevol ribozim. Derivat d'aquests estudis s'aporten dades sobre accessibilitat i variabilitat d'una regió interna del genoma del virus de l'hepatitis C, la zona d'unió de la regió E2/NS2 (regió 2658-2869). L'estudi d'accessibilitat revela que la regió 2658-2869 del genoma del virus conté dominis oberts i tancats i que la transició entre uns i altres no és brusca si es compara amb altres regions d'estructura coneguda (regió 5' no codificant). Els resultats dels assajos in vitro amb els dos models de RNasa P mostren que s'ha aconseguit dirigir tant la ribonucleasa P humana com el ribozim M1GS cap a una zona, predeterminada segons l'estudi d'accessibilitat, com a poc estructurada i tallar l'RNA del virus. De l'anàlisi de mutacions, però, es dedueix que la regió estudiada és variable. Tot i dirigir el ribozim cap a la zona més accessible, la variació de la diana podria afectar la interacció amb la seqüència guia i per tant disminuir l'eficiència de tall. Si es proposés una estratègia terapèutica consistiria en un atac simultani de vàries dianes.D'altra banda i derivat d'un resultat inesperat on s'ha observat en els experiments control que l'extracte de RNasa P humana tallava l'RNA viral en absència de seqüències guia externes, s'ha caracteritzat una nova interacció entre l'RNA del VHC i la RNasa P humana. Per a la identificació de l'enzim responsable dels talls s'han aplicat diferents tècniques que es poden dividir en mètodes directes (RNA fingerprinting) i indirectes (immunoprecipitació i inhibicions competitives). Els resultats demostren que la ribonucleasa P humana, i no un altre enzim contaminant de l'extracte purificat, és la responsable dels dos talls específics observats i que es localitzen, un a l'entrada interna al ribosoma (IRES) i molt a prop del codó AUG d'inici de la traducció i l'altre entre la regió codificant estructural i no estructural. La ribonucleasa P és un dels enzims del metabolisme del tRNA que s'utilitza per identificar estructures similars al tRNA en substrats diferents del substrat natural. Així doncs, el fet que la ribonucleasa P reconegui i talli el genoma del VHC en dues posicions determinades suggereix que, a les zones de tall, el virus conté estructures semblants al substrat natural, és a dir estructures tipus tRNA. A més, tot i que el VHC és molt variable, els resultats indiquen que aquestes estructures poden ser importants per el virus, ja que es mantenen en totes les variants naturals analitzades. Creiem que la seva presència podria permetre al genoma interaccionar amb factors cel·lulars que intervenen en la biologia del tRNA,particularment en el cas de l'estructura tipus tRNA que es localitza a l'element IRES. Independentment però de la seva funció, es converteixen en unes noves dianes terapèutiques per a la RNasa P. S'ha de replantejar però l'estratègia inicial ja que la similitud amb el tRNA les fa susceptibles a l'atac de la ribonucleasa P, directament, en absència de seqüències guia externes. / Hepatitis C virus is a human pathogen causing chronic liver disease in 170 million people worldwide. The virus is classified within the family Flaviviridae. The RNA genome is single-stranded and functions as the sole mRNA species for translation. It comprises a 5'-untranslated region, which functions as an internal ribosome entry site, and a long open reading frame, which encodes a polyprotein precursor of about 3010 amino acids, that is cleaved into structural (core, envelope 1, envelope 2 and p7) and non-structural (NS-2, NS-3, NS-4 and NS-5) proteins; followed by a 3' non-coding region. Analyzing significant numbers of cDNA clones of hepatitis C virus (HCV) from single isolates provides unquestionable proof that the viral genome cannot be defined by a single sequence, but rather by a population of variant sequences closely related to one another. In the infected patient, a master (the most frequently represented sequence) and a spectrum of mutant sequences may be isolated at any given time during chronic infection. This manner of organizing genetic information, which characterizes most RNA viruses, is referred to as quasispecies. HCV resistance to treatment (either alone or in combination with ribavirin) is one of the most important clinical implications predicted by the quasispecies model suggesting the necessity to seek new therapies. HCV therapeutic strategies based on ribozyme cleavage are leading candidates. The ribozyme activity of Ribonuclease P (RNase P) is among proposed antiviral agents. RNase P is a ubiquitous cellular endonuclease and one of the most abundant and efficient enzymes in the cell. This enzyme is a ribonucleoprotein complex that catalyzes a hydrolysis reaction to remove the leader sequence of precursor tRNA to generate the mature tRNA. Substrate recognition by the RNase P ribozyme does not rely on sequence requirements but on structural features of the RNA substrate. Custom-designed ribo-oligonucleotides, which hybridize with the target, called external guide sequences (EGSs), may provide the RNA structure which RNase P recognizes and cleaves in the hybridized complex. Recognition of structures instead of sequences may represent a great advantage in the fight against variable viruses because single or even double mutations in the target may be tolerated for RNase P recognition. One of the major aims of this work is to cleave HCV RNA using the RNase P ribozyme guided by EGS. To expand investigation of targeting in the HCV genome we assessed accessibility and low potential of variation of the target RNA since it is described that are crucial requirements for ribozyme therapy against viral infections. In the hepatitis C virus, the sequence of the 5' non coding region is conserved but the highly folded RNA structure severely limits the number of accessible sites. We have considered an internal genomic region whose sequence variation has been widely investigated. We have first mapped the accessibility of the genomic RNA to complementary DNAs within an internal genomic region. We performed a kinetic and thermodynamic study. Accordingly, we have designed and assayed four RNase P ribozymes targeted to the selected sites. Considerations on RNA structural accessibility and sequence variation indicate that several target sites should be defined for simultaneous attack. While performing targeting experiments on HCV RNA transcripts with RNase P we have found that, surprisingly, purified RNase P (peak activity) from HeLa cells cleaved HCV genomic RNA efficiently at two sites in the absence of EGSs. We report the techniques used to prove that the cleavage is specific to human RNase P (indirect methods: immunoprecipitation and competitive inhibition), and to show where cleavage occurs (direct method: RNA fingerprinting). We have confirmed that human RNase P is responsible for HCV RNA processing and that the two cleavages sites are in the IRES HCV domain, close to AUG initiator triplet, and in the E2/NS2 junction fragment (between structural and non structural coding region). To define cleavage by RNase P as a general property of HCV, viral sequences obtained from different patients were compared for RNase P cleavage accessibility. Cleavage was consistently observed in all sequences tested although with different efficiencies. Since RNase P recognizes and cleaves tRNA-like structures, we believe that such recognition by RNase P is an indication for the presence of two possible tRNA-like structures in the HCV genome. Comparison of such results at the two HCV RNase P cleavage sites should help us to understand in greater detail HCV substrate structure, tRNA mimicry, rules underlying recognition by human RNase P, and, in the particular case of the IRES motif, possible participation in translation. Whatever the role of such tRNA-like structures, such a strong tendency to maintain them might be important in the development of therapeutic strategies against the virus because they can represent highly susceptible targets for RNase P.
1154

Estudo da Hepatite B oculta em doadores de sangue de Vitória, Espírito Santo.

Tovar, Thais Tristão 29 February 2012 (has links)
Made available in DSpace on 2016-12-23T13:49:05Z (GMT). No. of bitstreams: 1 Hepatite_B_Oculta_2012.pdf: 1487094 bytes, checksum: 098e451e5a2e2af112aa8508df956a77 (MD5) Previous issue date: 2012-02-29 / Occult hepatitis B (OHB) is defined as the presence of low levels of HBV DNA in the liver or serum of individuals testing hepatitis B surface antigen (HBsAg) negative. In most cases of OHB, sera are positive for hepatitis B core antibody. The literature contains quite a few studies on the prevalence of OHB in Brazil, as well as in the worldwide population. Such reports, often controversial, demonstrate that the OHB prevalence varies among healthy individuals or patients with diseases unrelated to the liver and patients with chronic liver disease. Despite efforts, it is necessary a better understanding of: the reasons for the persistence of low levels of HBV-DNA in the absence of detectable HBsAg, the potential risk of OHB transmission and its role in the progression and aggravation of some liver diseases. Therefore, it is interesting to know the prevalence of OHB indifferent population samples which allows de monitoring of carriers of the occult infection, followed prospectively in order to try to surprise the possible effects of the presence of low levels of HBV-DNA in these individuals. In this study we investigated the presence of Occult Hepatitis B in peripheral blood obtained from 520 healthy donors of Vitoria, Espirito Santo, with the aim of guiding policies to include or not the sensitive HBV-DNA nucleic acid amplification technique (NAT) screening in blood donations with a detection limit of 54UI/mL. In order to enable the molecular detection we had also developed a method that screens plasma samples in pools which is capable of detecting the presence of HBV in the ratio of 1:40.Through the technique of nested-PCR we found that 0,2% (1/520) had occult HBV in serum samples. Despite the low prevalence of OHB detected in the study, a considerable number of patients with occult HBV infection may not have been detected if the blood units were only tested for serological markers of HBV infection. So it is important to know the prevalence of OHB in one or more additional population groups, in order to follow up carriers of occult infection prospectively to determine possible effects of the presence of HBV-DNA in low concentrations in these individuals / A Hepatite B Oculta (HBO) é definida pela presença do DNA do VHB com carga viral geralmente baixa (<200UI/mL), no fígado ou soro de pessoas com antígeno de superfície (HBsAg) indetectável. Na maioria dos casos de HBO há positividade para o anticorpo contra o antígeno core da Hepatite B (anti-HBc). Na literatura constam poucos estudos sobre a prevalência da HBO no Brasil, bem como na população mundial. Existem relatos, muitas vezes controversos, que reportam frequências variáveis da HBO em indivíduos sem doença ou portadores de doença não relacionada com o fígado e em hepatopatas crônicos. Apesar dos esforços, faz-se necessário ainda uma melhor compreensão das razões para a persistência da baixa viremia do VHB na ausência de HBsAg detectável, do potencial risco de transmissão da HBO e do seu papel na progressão e agravamento de algumas hepatopatias. No estudo investigou-se a presença de HBO em 520 doadores de sangue de Vitória, Espírito Santo, com o objetivo de nortear políticas para incluir ou não a triagem molecular NAT (Nucleic Acid Amplification Technique ) de bolsas de sangue. Para viabilizar a detecção molecular desenvolveu-se também uma metodologia de extração em pool de amostrascapaz de detectar a presença do VHB na proporção de 1:40 com limite de detecção de 54UI/mL. Através da técnica de nested-PCR detectou-se a presença do VHB oculto em 0,2% (1/520) das amostras. Apesar da baixa prevalência de HBO detectada no estudo, um número considerável de portadores da infecção oculta podem não estar sendo detectados, se apenas a pesquisa de marcadores sorológicos da presença do VHB esta sendo realizada nas bolsas de sangue. Sendo assim é importante conhecer a prevalência da HBO em diferentes amostras da população para que se possa ter em mãos portadores da infecção oculta acompanhados prospectivamente a fim de tentar surpreender possíveis efeitos da presença do DNA do VHB em baixas concentrações nesses indivíduos
1155

Avaliação da via do hedgehog nas hepatites crônicas B e C : da fibrose zero até a cirrose associada ou não ao carcinoma hepatocelular

Pereira, Thiago de Almeida 17 March 2010 (has links)
Made available in DSpace on 2016-12-23T13:56:07Z (GMT). No. of bitstreams: 1 Dissertacao de Thiago de Almeida Pereira.pdf: 7083594 bytes, checksum: b087de1012807ea82b96d6e7ada7b3cc (MD5) Previous issue date: 2010-03-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Hedgehog (Hh) pathway activation promotes many processes that occur during fibrogenic liver repair. Whether the Hh pathway modulates the outcomes of virally-mediated liver injury has never been examined. Gene-profiling studies of human hepatocellular carcinomas (HCC) demonstrate Hh pathway activation in HCCs related to chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). Because most HCC develop in cirrhotic livers, we hypothesized that Hh pathway activation occurs during fibrogenic repair of liver damage due to chronic viral hepatitis, and that Hh-responsive cells mediate disease progression and hepatocarcinogenesis in chronic viral hepatitis. Methods Immunohistochemistry and qRT-PCR analysis were used to analyze Hh pathway activation and identify Hhresponsive cell types in archival liver biopsies from 45 patients with chronic HBV or HCV with different stages of fibrosis (F0-4), some of which also presented HCC. Hh signaling was manipulated with cyclopamine in primary human hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC). Angiogenesis assay was used to investigate if the pathway plays a role in tube formation. SECs were incubated with recombinant Shh, conditioned media from HSC, cyclopamine, short hairpin RNA against smoothened or their respective controls and the length of vascular tubes were measured by morphometry. Hedgehog production was investigated in Huh7 cells infected with JFH-1 and in a rat hepatoma cell line that express the HBV X gene by qRT-PCR. Results We found increased hepatic expression of Hh ligands (Shh and Ihh) in all patients with chronic viral hepatitis (p<0.005), and demonstrated that infection with JFH-1 or ectopic expression of the HBV X gene stimulated cultured hepatocytes to produce Shh (p<0.005). The major cell populations that expanded during cirrhosis and HCC (i.e., liver myofibroblasts, activated endothelial cells, and progenitors expressing markers of tumor stem/initiating cells Krt7, CD133, Epcam and survivin) were Hh-responsive (Patched and Gli-2 positive) and higher levels of Hh pathway activity associated with cirrhosis and HCC (p<0.005). Inhibiting pathway activity in Hh-responsive target cells (HSC and SEC) reduced fibrogenesis (p<0.005 for &#945;SMA and p<0.05 for col1&#945;1 expression) and angiogenesis (p<0.05). Conclusions HBV/HCV infection increases hepatocyte production of Hh ligands in hepatocytes and expands types of Hh-responsive cells, including myofibroblasts and xiv sinusoidal endothelial cells that associate with fibrosis progression to cirrhosis and hepatocellular carcinoma. Moreover, hedgehog-responsive progenitors that are undergoing epithelial-to-mesenchymal transition accumulate during the progression of viral hepatitis and may play a role in the development or progression of hepatocellular carcinoma. In vitro inhibition of Hh-signaling in primary human hepatic stellate cells and sinusoidal endothelial cells suggest the possibility of using Hh inhibitors as potential tools to prevent cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis B and C / Ativação da via do Hedgehog (Hh) promove vários processos que ocorrem durante o reparo fibrogênico hepático. O papel da via do Hh na lesão causada pela hepatite crônica B e C ainda não foi investigado. Estudos de expressão global em carcinomas hepatocelulares (CHC) demostraram a ativação da via do Hh em pacientes com CHC relacionados à infecção crônica com o vírus da hepatite B (VHB) ou vírus da hepatite C (VHC). Como a maioria dos CHCs desenvolve em fígados cirróticos, levantamos a hipótese de que a ativação da via do Hh ocorre durante o reparo fibrogênico relacionado à hepatite viral crônica B e C e que células Hh-reatoras poderiam orquestrar a progressão da doença e a hepatocarcinogênese na hepatite viral crônica. Métodos Immunohistoquímica e análise por PCR quantitativo em tempo real (qRTPCR) foram utilizados para investigar a ativação da via do Hh e identificar os tipos celulares que respondem aos ligantes dessa via em biópsias arquivadas de 45 pacientes com hepatite crônica B ou C, de diferentes graus de fibrose (F0-4), sendo que 7 também apresentavam CHC. A via do Hh foi manipulada com Ciclopamina em células estreladas hepáticas (HSC) e em células endoteliais sinusoidais hepáticas (SEC) primárias humanas. O ensaio da angiogênese foi usado para investigar o papel da via do Hh na formação de tubos. SECs foram incubadas com Shh recombinante, meio condicionado de HSC, ciclopamina, short hairpin RNA (shRNA) contra o Smoothened ou os seus controles respectivos e o comprimento dos tubos vasculares foram quantificados por morfometria. A produção de ligantes Hh foi investigada por qRT-PCR em células Huh7 infectadas com o virus JFH-1 e em uma linhagem de hepatoma de rato que expressa o gene da proteína X do VHB. Resultados Observamos um aumento dos níveis de expressão de ligantes Hh (Shh e Ihh) em todos os pacientes com hepatite crônica viral (p<0,005) e demonstramos que a infecção pelo vírus da hepatite C JFH-1 ou a expressão ectópica do gene X do VHB estimulam culturas de hepatoma a produzirem Shh (p<0,005). Os principais tipos celulares que proliferam durante a cirrose e CHC (miofibroblastos, células endoteliais ativadas e progenitores que expressam marcadores de células tronco/iniciadoras de tumor CD133, Krt7, EpCAM e survivina) são reatores aos ligantes Hh e os níveis mais altos de expressão de componentes da via do Hh (Shh, Ihh, Gli-2 e Ptch) estão associados xii com cirrose e CHC (p<0,005). Inibição da via do Hh em células reatoras (HSC e SEC) reduziu fibrogênese (p<0,005 para expressão de &#945;SMA e p<0,05 para expressão de col1&#945;1) e angiogênese (p<0,05). Conclusões A Infecção pelo VHB/VHC aumenta a produção de ligantes hedgehog em hepatócitos e expande o número de células estreladas hepáticas e células endoteliais sinusoidais primárias humanas reatoras a esses ligantes, em relação direta com a progressão da fibrose para a cirrose e o carcinoma hepatocelular. Também induz o acúmulo progressivo de células progenitoras reatoras a via do Hh e que estão em processo de transição epitélio mesenquimal, o que pode estar relacionado com o desenvolvimento ou a progressão do carcinoma hepatocelular. A inibição in vitro da ativação da via do Hedgehog em células estreladas e em células sinusoidais endoteliais hepáticas primárias humanas sugere a possibilidade de investigar inibidores dessa via como potenciais ferramentas para reduzir a progressão da fibrose hepática para cirrose e carcinoma hepatocelular nas hepatites crônicas produzidas pelos vírus B e C
1156

Desenvolvimento de plataformas nanotecnológicas para a construção de biossensores: diagnóstico molecular de doenças infecciosas e inflamatórias / Development of nanotech platforms for the construction of biosensors: molecular diagnosis of infectious diseases and inflammatory

Oliveira, Danielle Alves de 28 July 2017 (has links)
CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Na presente tese foram desenvolvidas três plataformas para a construção de biossensores visando o diagnóstico molecular da hepatite C, hepatite B e artrite reumatoide, por técnicas eletroquímicas, ópticas e microscópicas, usando amostras reais. Os genossensores para hepatites C e B foram desenvolvidos sobre a superfície de um eletrodo de ouro modificado com nanomateriais, sendo esses o óxido de grafeno e o óxido de grafeno reduzido, respectivamente. Em todos os biossensores propostos a interação da sonda com o alvo foi efetivamente verificada pelas diferentes técnicas. No caso do genossensor para hepatite C, o óxido de grafeno foi modificado quimicamente com etilenodiamina e apresentou limites de detecção e quantificação de 1:483 (v/v) e 1:145 (v/v), respectivamente, usando amostras de soro de pacientes positivos. A interação da sonda específica do HCV: gRNA causou uma redução na amplitude de resposta de corrente de cerca de 2,9 vezes quando comparada ao controle negativo, usando a VPD. O genossensor para a hepatite B a sonda foi imobilizada sobre eletrodo de ouro contendo óxido de grafeno reduzido, ouro descoberto e nanoparticulas de ouro. A análise usando VPD indica que a adição de DNA genômico de HBV provocou um aumento de cerca de 1,4 vezes na amplitude de corrente de pico quando comparado ao controle negativo. Em adição, análises de SPR mostraram que as amostras positivas de HBV resultaram em uma alteração de cerca de 15 vezes em comparação com as amostras negativas. No biossensor desenvolvido para o diagnóstico da artrite reumatoide foi utilizado um eletrodo de grafite modificado com um filme poli(3-hidroxibenzóico), no qual foi imobilizado um peptídeo mimético que reconhece o anticorpo anti-CAIII. O sensor mimético desenvolvido permitiu a distinção entre amostras positivas e negativas para a artrite reumatóide, uma vez que apresentou uma diminuição expressiva no sinal de corrente de cerca de 2,2 vezes, quando comparado ao soro negativo. Assim, foi possível desenvolver plataformas analíticas, seletivas e específicas fornecendo novas abordagens para o diagnóstico clínico e aplicações point-of-care para o monitoramento de doenças inflamatórias e infecciosas. / In the present thesis, three biosensing platforms aiming the molecular diagnosis of hepatitis C, hepatitis B and rheumatoid arthritis were developed by electrochemical, optical and microscopic techniques using real samples. The genosensors for the diagnosis of hepatitis C and B were developed on a gold electrode modified with nanomaterials, being these graphene oxide and reduced graphene oxide, respectively. In all proposed biosensors the interaction of the probe with the target was effectively verified by the different techniques. In the case of the genossensor for hepatitis C, graphene oxide was chemically modified with ethylenediamine and showed limits of detection and quantification of 1:483 (v/v) and 1:145 (v/v), respectively, using serum samples from positive patients. The interaction of the HCV probe and the gRNA caused a reduction in current response amplitude of about 2.9 fold as compared to the negative control, using the DPV. The genossensor for hepatitis B probe was immobilized on a gold electrode containing reduced graphene oxide, gold disks and gold nanoparticles. Analysis using DPV indicates that the addition of HBV gDNA caused an increase of about 1.4 times in peak current amplitude, when compared to the negative control. In addition, SPR analyzes showed that positive samples of HBV resulted in a change of about 15- fold compared to negative samples. In the biosensor developed for the diagnosis of rheumatoid arthritis, a graphite electrode modified with a poly (3-hydroxybenzoic) film was used, in which a mimetic peptide that recognizes the anti-CAIII antibody was immobilized. The developed mimetic sensor allowed the distinction between positive and negative samples for rheumatoid arthritis, since it presented an decrease in the current signal of about 2.2 times, when compared to the negative serum. Thus, it was possible to develop analytical, selective and specific platforms, providing new approaches for clinical diagnosis and point-of-care applications, for the monitoring of inflammatory and infectious diseases. / Tese (Doutorado)
1157

Custo do teste Anti-HBs pós-vacinação primária em relação ao manejo para hepatite B pós-exposição à material biológico entre trabalhadores da área da saúde / The cost of primary post-vaccination Anti-HBs testing in relation to hepatitis B post-exposure practices among health care workers

Souza, Camila Lucas de 11 May 2018 (has links)
Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2018-07-09T12:16:28Z No. of bitstreams: 2 Dissertação - Camila Lucas de Souza - 2018.pdf: 2326676 bytes, checksum: 8ce05f9e4402bf801ab775da922be861 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-07-10T10:59:46Z (GMT) No. of bitstreams: 2 Dissertação - Camila Lucas de Souza - 2018.pdf: 2326676 bytes, checksum: 8ce05f9e4402bf801ab775da922be861 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-07-10T10:59:46Z (GMT). No. of bitstreams: 2 Dissertação - Camila Lucas de Souza - 2018.pdf: 2326676 bytes, checksum: 8ce05f9e4402bf801ab775da922be861 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-05-11 / INTRODUCTION: Hepatitis B virus (HBV) infection is a worldwide public health problem and the cost of treatment for hepatitis B is high. Work related accidents with biological material among health care workers present a risk of transmission of this virus. The primary preventive measure against HBV is vaccination, followed by an anti-HBs test one to two months after the final dose of the vaccine, however this test is not part of the pre-exposure routine in Brazilian public health. OBJECTIVES: To compare the direct medical cost, from the public health perspective, of the evaluation of pre-exposure serological status vs the post-exposure management of hepatitis B virus-related occupational exposure among exposed health care workers. The specific objectives were to describe sociodemographic and job characteristics, to classify the profile of accidents, to identify the vaccination history and serological status for hepatitis B, and to quantify the direct cost of post-exposure management and preventive measures for hepatitis B. METHODOLOGY: A cross-sectional, descriptive study with a partial economic evaluation of the cost analysis type, focusing on direct medical costs among health care workers exposed to biological material in the municipality of Goiania, accidents registered in the SINAN-NET database in the period 2006-2016. RESULTS: This study included 7,265 recorded accidents with biological material, being predominantly female health care workers (80.5%), nursing staff (55.2%) with secondary education (43.0%), with accidents involving blood (74.4%), those being percutaneous (72.4%). The mean age was 34 years, with the age group 21-30 years being the most predominant. Among those exposed, 85.1% received all three doses of the vaccine, and of those, 44.6% performed the anti-HBs test at the time of the accident. The prevalence of HBsAg among known source patients was 1.8% (95% CI 1.0- 3.2). The costs analyzed in this study were the anti-HBs test, HBsAg test, IGHAHB and a worker health office visit. Considering this history, the direct cost of occupational post- exposure management for hepatitis B among victims was evaluated in four different scenarios, and the direct cost ranged from R$ 3,615.52 (Int$ 6.573,67) to R$ 835,751.52 (Int$1.519.548,22). The post-exposure scenarios for HBV that presented the greatest direct medical cost impact for public health were the scenarios in which the porkers had anti-HBs <10 IU/ml and were exposed to an HBsAg-positive, unknown source patient. The direct cost of performing the anti-HBs test after primary vaccination among 7.265 workers was R$ 207,415.75 (Int$ 377,119.54). CONCLUSION: The direct per capita cost of post-exposure management was higher than the direct cost of performing the pre-exposure anti-HBs test, money that could be invested in policies that protect the health of the worker. / INTRODUÇÃO: A infecção pelo Vírus da Hepatite B (HBV) é um problema mundial de saúde pública, e o custo do tratamento para hepatite B é elevado. Os acidentes de trabalho com material biológico entre Trabalhadores da Área da Saúde (TAS) apresentam risco de transmissão desse vírus. A principal prevenção contra o HBV é a vacinação seguida da realização do teste anti-HBs após um a dois meses da última dose da vacina, entretanto o exame não é uma rotina pós-vacinação primária no Sistema Único de Saúde (SUS), no Brasil. OBJETIVOS: Comparar o custo direto médico, sob a perspectiva do SUS, da avaliação do status sorológico após as três doses da vacina contra hepatite B ou após vacinação primária para hepatite B com o manejo pós-exposição ocupacional, relacionado ao vírus da hepatite B entre trabalhadores da área da saúde, vítimas de acidente com material biológico. Os objetivos específicos: descrever características sociodemográficas e laborais, identificar o perfil dos acidentes, identificar o histórico vacinal e status sorológico para hepatite B e quantificar o custo direto do manejo pós-exposição e das medidas de prevenção para hepatite B. METODOLOGIA: estudo transversal, descritivo e de avaliação econômica parcial do tipo análise de custo, com enfoque no custo direto médico entre TAS, vítimas de acidentes com material biológico no município de Goiânia, acidentes registrados no banco de dados do SINAN-NET, no período de 2006 a 2016. RESULTADOS: foram registrados 7.265 acidentes com material biológico, sendo, predominantemente, entre TAS do sexo feminino (80,5%), da equipe de enfermagem (55,2%), com ensino médio (43,0%), acidentes que envolveram sangue (74,4%) e acidentes percutâneos (72,4%). A média de idade entre os TAS foi de 34 anos, sendo a faixa etária de 21 a 30 anos a mais predominante. Entre os TAS expostos, 85,1% possuíam as três doses da vacina e desses, 44,6% realizaram o teste anti-HBs no momento do acidente. A prevalência de HBsAg entre pacientes-fonte conhecidos foi de 1,8% (IC 95% 1,0 – 3,2). Os custos analisados, neste estudo, foram o teste anti-HBs, teste HBsAg, IGHAHB e consulta médica em saúde do trabalhador. Considerando esse histórico, o custo direto do manejo pós- exposição ocupacional para hepatite B entre vítimas foi avaliado em quatro diferentes cenários, nominados A, B, C e D, e o custo direto variou de R$ 3.615,52 (Int$ 6.573,67) a R$ 835.751,52 (Int$ 1.519.548,22). Os cenários de pós-exposição ao HBV que apresentaram maior impacto de custo direto médico para o SUS foram os cenários em que os TAS possuíam anti-HBs < 10 UI/ml e foram expostos a paciente-fonte HBsAg positivo e a paciente-fonte desconhecido. O custo direto da realização do teste anti-HBs após vacinação primária entre 7.265 TAS foi de R$ 207.415,75 (Int$ 377.119,54). CONCLUSÃO: O custo direto per capita do manejo pós-exposição foi mais elevado do que o custo direto da realização do teste anti-HBs pós-vacinação primária entre TAS, vítimas de acidente com material biológico, dinheiro que poderia ser investido em políticas para saúde do trabalhador.
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Identificação de polimorfismos e mutações primárias de resistência aos inibidores de protease (NS3/NS4A) no vírus da hepatite C em pacientes com hepatite C crônica monoinfectados e coinfectados pelo vírus da imunodeficiência humana / Characterization of NS3/NS4A polymorphisms and hepatitis C protease inhibitors resistance-associated mutations in hepatitis C virus monoinfected and human immunodeficiency virus coinfected patients

Gaspar Lisbôa Neto 12 April 2017 (has links)
INTRODUÇÃO: A hepatite C crônica é uma das principais causas de hepatopatia em todo mundo. A coinfecção pelo vírus C (VHC) e o HIV não é incomum, pois ambos compartilham vias similares de transmissão. Recentemente, a terapêutica da hepatite C crônica foi radicalmente modificada com o advento das drogas antivirais de ação direta (DAAs), elevando as taxas de RVS mesmo na população coinfectada. O VHC é caracterizado pela sua alta taxa replicativa e por grande diversidade populacional. Substituições de ocorrência natural na protease viral associadas a resistência podem comprometer a terapêutica em alguns regimes baseados no uso de inibidores de protease (IPs). OBJETIVOS: Estimar a prevalência de polimorfismos e mutações de ocorrência natural associadas a resistência aos IPs em pacientes monoinfectados e coinfectados pelo VHC e HIV e identificar fatores clínicos e virológicos associados a presença de tais substituições. MATERIAIS E MÉTODOS: Dados epidemiológicos e clínicos foram obtidos de 247 pacientes (135 monoinfectados e 112 coinfectados pelo VHC e HIV). VHC RNA foi extraído do plasma dos indivíduos participantes e um fragmento de 765 pares de base da região NS3 foi amplificado e sequenciado por metodologia populacional (técnica de Sanger). O estadiamento da fibrose hepática foi realizado pelo escore não invasivo FIB- 4. RESULTADOS: 54 indivíduos (21,9%) apresentaram pelo menos uma substituição na região NS3/NS4A do VHC. Somente 14 pacientes (5,7%) apresentaram pelo menos uma mutação de resistência aos IPs (T54S, V55A ou Q80R). A Q80K não foi identificada em nenhuma das amostras. Não houve diferença entre monoinfectados e coinfectados quanto à ocorrência de polimorfismos ou mutações associadas a resistência. As variáveis independentemente associadas com substituições na região da protease foram infecção pelo VHC genótipo 1b, bilirrubinas totais > 1,5 vezes o LSN e níveis de albumina < 3,5 g/dL. Fibrose hepática avançada (FIB-4 > 3.25) não esteve associada a presença de substituições. A análise de diversidade nucleotídica na protease viral revelou maior heterogeneidade do VHC genótipo 1b em relação ao 1a. Contudo, a análise de pressão seletiva não demonstrou maior variabilidade de quasiespécies no grupo de hepatopatia avançada, achado este compatível com uma sequência genômica relativamente conservada. CONCLUSÕES: As substituições na região NS3/NS4 do VHC consistiram majoritariamente por polimorfismos naturais sem impacto clínico num eventual tratamento que envolva o uso de IPs. A prevalência de substituições associadas a resistência foi baixa e compatível com os valores informados pela maioria dos estudos nacionais e internacionais. A coinfecção pelo HIV não parece elevar a frequência de substituições na protease do VHC. A região NS3 do genótipo 1b foi altamente variável em relação ao genótipo 1a, reforçando o conceito de possíveis diferenças geográficas em relação ao perfil genético deste vírus / INTRODUCTION: Chronic hepatitis C is a major cause of liver disease worldwide. Hepatitis C vírus (HCV) and HIV coinfection is not uncommon due to similar transmission routes. Recently developed direct-acting antivirals drugs (DAAs) have increased the rate of SVR even in coinfected patients. HCV has a high replication rate and a lack of proofreading activity, leading to a greatly diverse viral population. Baseline spontaneously occurring resistance substitutions in the protease region may impair the rate of success in some protease inhibitors (PI) based regimens. OBJECTIVE: to determine the prevalence of naturally occurring polymorphisms and resistance associated variants to HCV PIs in mono and coinfected HCV HIV patients and to evaluate potential associations between amino acid substitutions in protease domain and clinical / virological features of those patients. METHODS: Clinical and epidemiological data were retrieved from medical records of 247 subjects in Brazil (135 HCV monoinfected and 112 HIV HCV coinfected patients). HCV-RNA was extracted from plasma and a fragment of 765 base pairs from the NS3 region was amplified and sequenced with Sanger-based technology. Fibrosis staging was assessed by non invasive score (FIB-4). RESULTS: Overall, 54 patients (21.9%) had at least one amino acid substitution in the NS3 region; only 14 patients (5.7%) harboured at least one resistance mutation (T54S, V55A, Q80R). Q80K mutation was not found in any sample. There was no difference between monoinfected and coinfected patients regarding the frequency of natural polymorphisms and resistance mutations. Variables independently associated with amino acid substitution were HCV subtype 1b, total bilirubin level > 1.5 ULN and albumin level < 3.5 g/dL. Advanced liver fibrosis (FIB-4 > 3.25) was not related to NS3 polymorphisms nor resistance associated variants. Examination of HCV protease nucleotide diversity revealed greater heterogeneity in subtype 1b than subtype 1a. Analysis of selective pressure did not reveal a greater quasispecies variability in advanced liver fibrosis group, being such finding consistent with a relatively conserved gene in this setting. CONCLUSION: Baseline HCV NS3 amino acid substitutions depicted herein were considered mostly natural polymorphisms with no clinical impact in a PI based therapy. The prevalence of resistance-associated substitutions was low and compatible with values reported by most national and international studies. HIV coinfection was not associated with a greater frequency of such substitutions in the studied sample. The NS3 region of genotype 1b was highly variable in relation to genotype 1a, highlighting geographic differences concerning HCV genetic profile
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Interactions entre le métabolisme hépatique des sels biliaires et des lipoprotéines et les infections par les virus des hépatites B et C / Interactions between hepatic metabolism of bile acids and lipoproteins and Hepatitis B and C infections

Ramière, Christophe 23 February 2012 (has links)
Les virus des hépatites B et C (VHB et VHC) entretiennent des liens étroits avec le métabolisme lipidique des hépatocytes. Ainsi, la réplication du VHB est dépendante de certains récepteurs nucléaires hépatiques, tels que HNF4α et PPARα, impliqués dans ce métabolisme. L’assemblage des particules virales du VHC dépend lui de la voie de synthèse des lipoprotéines de très faible densité (VLDL) et le virus circule dans le sang sous forme de lipo-viro-particules associé notamment à l’apolipoprotéine B, un composant essentiel des VLDL. Dans ce travail, nous avons d’abord étudié le rôle de FXRα, le récepteur nucléaire des sels biliaires, sur la réplication du VHB. Nous avons montré, in vitro, que les sels biliaires, via FXRα, activaient le promoteur de Core du VHB qui contrôle le niveau de réplication virale. Puis dans l’étude des liens entre les lipoprotéines et le VHC, nous avons montré que l’apoB présente sur certaines particules virales jouaient un rôle important dans l’infectiosité du virus in vitro, et que la protéine Cideb, présente en surface des gouttelettes lipidiques et impliquée dans l’assemblage des VLDL, était impliquée dans l’association du VHC avec l’apoB et influençait l’infectiosité des virions sécrétés. De plus nous avons mis en évidence l’existence de particules sub-virales chez les patients infectés, de nature lipoprotéique mais ne portant que les protéines d’enveloppes du VHC. Tous ces résultats renforcent l’idée d’une adaptation du VHB et du VHC au métabolisme lipidique hépatique. Les bénéfices éventuels qu’en retirent ces deux virus, ainsi que l’existence de possibles thérapeutiques anti-virales ciblant le métabolisme lipidique, restent à explorer / Hepatitis B and C viruses (HBV and HCV) infections are tightly linked with hepatic lipid metabolism. HBV replication depends on specific nuclear receptors, such as HNF4α and PPARα, both implicated in this metabolism. HCV assembly depends on the synthesis of Very-Low-Density Lipoproteins (VLDL), and the virus circulates in the blood as lipo-viral-particles associated in particular with apoB, an essential component of VLDL. In this study, we first studied the influence of FXRα, the nuclear receptor for bile acids, on HBV replication. We showed that, in vitro, bile acids, via FXRα, were able to activate the HBV Core promoter which controls the level of viral replication. Then, in the study of the interactions between HCV and lipoproteins, we demonstrated that apoB, which is associated with a proportion of viral particles, played an important role in HCV infectivity in vitro, and that Cideb, a protein involved in VLDL assembly, was implicated in the association between HCV and apoB and influenced the infectivity of secreted viral particles. Finally, we showed that, besides HCV infectious particles, sub-particles bearing only viral envelope glycoproteins circulated in the blood of infected patients. Interactions of HBV with the metabolism of bile acids, and of HCV with the metabolism of lipoproteins, are two examples of adaptation of a parasite to its host. The potential benefits from these interactions are still to be determined, as well as the possibility to develop anti-viral strategies targeting lipid metabolism
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Virus de l'hépatite C, Nétrine-1 et réponse aux protéines mal repliées en contexte hépatique / Hepatitis C virus, Netrin-1 and the unfolded protein response in a hepatic context

Lahlali, Thomas 16 December 2014 (has links)
Les connaissances actuelles en pathologie hépatique suggèrent que HCV n'est pas directement oncogénique mais expose les patients au risque de cancer du foie dans un contexte inflammatoire associé à une réponse UPR (Unfolded Protein Response) et une régénération hépatique. La nétrine-1, le ligand canonique de la famille des DRs (Récepteurs à dépendance), est une protéine anti-apoptotique impliquée dans le développement, l'inflammation et la tumorigenèse. Les DRs induisent l'apoptose en absence de leurs ligands. A ce jour, il n'existe aucune donnée reliant le concept de DR et les virus oncogènes. Au cours de ma thèse, j'ai contribué à démontrer que la fonctionnalité des DRs était altérée au cours de l'infection par HCV in vitro et in vivo. Nous avons montré que la surexpression de la nétrine-1 augmente l'infectivité des virions et promeut leur entrée via l'activation et la diminution du recyclage de l'EGFR. De son coté, HCV augmente l'expression de la nétrine-1 suite à l'activation de l'épissage de son ARN pré-messager. Nous avons aussi montré que l'expression du récepteur à la nétrine-1, UNC5A, était diminuée au cours de l'infection suite à des diminutions transcriptionnelle et traductionnelle. Dans ce cadre, la nétrine-1 joue le rôle de facteur proviral en inhibant une potentielle voie de signalisation antivirale induite par le récepteur UNC5A non lié. Nous avons ensuite voulu savoir quelles conséquences cette surexpression de nétrine-1 pourrait avoir en physiopathologie hépatique en contexte non infectieux. Un stress du RE (Réticulum Endoplasmique) est observé au cours de l'infection par HCV. Le stress du RE entraîne l'activation de la réponse UPR qui induit l'apoptose médiée par la DAPK1 en cas de stress prolongé. Le fait que le récepteur UNC5B active aussi l'apoptose via l'activation de la DAPK1 nous a conduit à étudier l'implication de la nétrine-1 dans la survie cellulaire au cours de la réponse UPR en contexte hépatique. Nous avons démontré à la fois in vitro et in vivo que l'expression de la nétrine-1 pourrait protéger les cellules contre l'apoptose induite par la réponse UPR suite à sa liaison aux récepteurs UNC5A et C qui entraîne l'inhibition de la DAPK1. De nombreuses études ont également reporté des rôles de la nétrine-1 dans l'inflammation et la néoangiogenèse. Nous avons montré que la nétrine-1 inhibe la migration transendothéliale hépatique des PBMCs (Peripheral Blood Mononucleated Cells) et accélère la tubulogenèse des cellules endothéliales intrasinusoïdales hépatiques. Dans leur ensemble, mes travaux de thèse suggèrent que la nétrine-1 via ses récepteurs UNC5s joue des rôles délétères en pathophysiologie hépatique favorables à la persistance virale et à la résistance à la mort cellulaire / Current knowledge in hepatic pathology suggests that HCV is not directly oncogenic but puts patients at risk for liver cancer in a context associated with a chronic inflammation, UPR (Unfolded Protein Response) and liver regeneration. Netrin-1, the canonical ligand of the DR (Dependence Receptor) family, is an antiapoptotic secreted factor implicated in development, cancer and cancer-associated inflammatory diseases. DRs induce cell death when unbound. No data linking the DR system to oncogenic viruses are available to date. During the first part of my PhD, I contributed to demonstrate that HCV infection alters DR functionality both in vitro and in vivo. We found that Netrin-1 conditions HCV virion infectivity and promotes virion entry by increasing the activation and decreasing the recycling of the EGFR. In turn, HCV increases Netrin-1 expression through enhanced Netrin-1 pre-mRNA splicing. The Netrin-1 UNC5A receptor expression was decreased upon HCV infection through diminished transcription and translation. In this setting, Netrin-1 acts as a proviral factor by inhibiting a putative antiviral signaling pathway conveyed by the unbound UNC5A receptor. In this context, we wanted to determine what consequences such Netrin-1 up-regulation could induce in non-infectious hepatic pathophysiology. Chronic ER (endoplasmic reticulum) stress is observed during HCV infection. ER stress leads to UPR activation which triggers apoptosis via DAPK1 activation upon prolonged stress. The fact that the UNC5B receptor induces apoptosis through DAPK1 activation led us to investigate Netrin-1 implication in cell survival upon UPR in the liver. During the second part of my PhD, I have demonstrated both in vitro and in vivo in mice that Netrin-1 translation during UPR could protect cells against UPR-related cell death after binding to UNC5A and C, in a DAPK1-mediated fashion. Several studies have also identified Netrin-1 roles in inflammation and neo-angiogenesis. We found that Netrin-1 inhibits hepatic transendothelial migration of PBMCs (Peripheral Blood Mononucleated Cells) and accelerates tubulogenesis of liver sinusoidal endothelial cells. Netrin-1’s role in a hepatic inflammation and neoangiogenesis, both events being tightly associated with viral hepatitis, remains to be thoroughly elucidated. Altogether, our results suggest that Netrin-1 plays UNC5-dependent deleterious roles in hepatic pathophysiology, leading to viral persistence as well as resistance to cell death

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