Eine Monotherapie mit Tenofovir disoproxil fumarate (TDF) stellt eine hoch effiziente Therapie-option für multipel vorbehandelte Patienten mit chronischer Hepatitis-B-Virusinfektion (HBV) dar. Eine Resistenz gegen TDF wurde bislang nicht beschrieben, jedoch wird ein möglicher negativer Einfluss von Adefovir dipivoxil (ADV)-Resistenzvarianten auf die TDF-Ansprechrate diskutiert. Diese retrospektive Kohortenstudie untersucht die Dynamik von Nukleos(t)id-Analoga (NA)-Resistenzvarianten im HBV-Polymerasegen mit Fokus auf ADV-Resistenzvarianten bei 18 chronisch HBV-infizierten Patienten mit Therapieversagen auf eine vorangegangene Lamivudin (LAM)- und ADV-Therapie, sowie nur partiellem Therapieansprechen auf eine TDF-Monotherapie. Zur Detektion von NA-Resistenzvarianten wird eine HBV-Genomsequenzierung mit Tiefenpyrosequenzierung (Genome Sequencer FLX, Roche Diagnostics, Germany) (UDPS), direkte Sequenzierung (TRUGENETM HBV Genotyping Kit, OpenGeneTM DNA Sequencing Sys-tem, Siemens Healthcare Diagnostic, USA) (TG) und Line Probe Assay (INNO-LiPa DRv2 und v3, Innogenetics, Belgium) (INNO-LiPA) durchgeführt. Unter TDF kommt es zu einer quantitati-ven Shift zugunsten der ADV-Resistenzvarianten mit konstant bleibendem Anteil und deutlich höher persistierender Virämie zu Monat 12 im Vergleich zu Patienten ohne ADV-Resistenzvarianten. Vor allem werden die Varianten rtA181V und rtN236T selektiert, jedoch nicht die Variante rtA181T. Die absolute Anzahl der LAM-Resistenzvarianten hingegen halbiert sich. Varianten mit einem initial per UDPS detektierten Anteil von >20% der patientenspezifi-schen HBV-Population werden meist selektiert und nehmen im Verlauf den Hauptanteil der Quasispezies ein. UDPS stellte ein potentes Medium der Detektion, Identifikation und Quantifi-zierung von HBV-Varianten dar und ist INNO-LiPa und TG überlegen. Es ergibt sich kein Hin-weis auf TDF-Resistenzvarianten, jedoch zeigt das Vorliegen von ADV-Resistenzvarianten ei-nen tendentiell negativen Einfluss auf die virale Kinetik. Weitere größere Langzeitstudien sind zur Bestätigung dieser Beobachtung notwendig. / Tenofovir disoproxil fumarate (TDF) is a highly efficient treatment option for nucleos(t)ide analogue (NA) pre-treated patients with chronic hepatitis B virus (HBV) infection. Little is known about the reasons for persistent virus replication in some rare cases. As of today, no TDF resistance variants have been identified, but a possible linkage to Adefovir dipivoxil (ADV) resistance associated variants negatively influencing HBV-DNA suppression by TDF has been suspected, based on the similarity of the chemical structure. In this retrospective cohort study the dynamics of NA resistance variants in the HBV polymerase gene with focus on ADV resistance variants were assessed. For this, we have chosen a cohort including patients with multiple failures to treatment with different NAs. Thus, data of 18 patients with previous treatment failure to LAM and ADV was analysed, showing a persistent viremia (HBV-DNA >35 copies/mL) despite switch to TDF monotherapy (median HBV-DNA at month 12 3,5±0,8 (2,1-4,9) log10 copies/mL). Sequencing analysis was performed with ultra-deep pyrosequencing (UDPS) (Genome Sequencer FLX, 454 Life Science, Roche Diagnostic, Branford, CT), direct sequencing (TG) (TRUGENETM HBV Genotyping Kit, OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) and line probe assay (INNO-LiPA) (INNO-LiPa DRv2/v3, Innogenetics, Belgium). Using TDF monotherapy, a quantitative shift in favour to ADV resistance variants was observed in this cohort. The percentage of substitutions conferring resistance to ADV at baseline (BL) and at the time of the last sequencing endpoint (EP) of the HBV genome remained constant (BL 35%, 13/37, EP 36%, 9/25). The variants rtA181V and rtN236T were mostly selected, whereas rtA181T was not selected. The total amount of substitutions conferring resistance to Lamivudin (LAM) showed a strong decline, however remained the majority part of all NA resistance variants (BL 51% (19/37), EP 40% (10/25)). The percentage of ETV resistance variants increased slightly (BL 14% (5/37), EP 24% (6/25)). Known ADV, Lam and ETV resistance variants emerged in variable abundance (1,0-99,6%) of quasispecies during TDF therapy. A homogenization of HBV quasispecies took place. Especially mutations occurring in higher abundance (>20% of viral population) were mostly selected (BL 51% (19/37), EP 80% (20/25)). No new HBV variants with possible association to resistance against TDF were identified, but patients with ADV resistance variants showed the highest HBV-DNA level at month 12 of TDF therapy (median HBV-DNA 3,57±0,72 (2,14-3,96) log10 copies/mL, not significant). A negative influence of ADV resistance variants on viral suppression with TDF monotherapy may be assumed, however more long-term studies are needed to confirm the role of ADV resistance variants in TDF therapy. UDPS is a potent medium for detection, identification and quantification of dominant to low level variants in HBV-DNA. It is superior to direct sequencing and line probe assay in the detection of variants.

Hepatitis B Virus (HBV) Infektionen verursachen entzündliche Erkrankungen der Leber. Insbesondere die frühen Phasen des HBV Lebenszyklus sind noch nicht geklärt, so ist z.B. der Rezeptorkomplex an den HBV bindet unbekannt. Mittlerweile stehen neue Infektionsmodelle zur Verfügung um den HBV Lebenszyklus zu untersuchen. Dies erfordert eine effiziente Zellkultur basierende Methode um große Mengen infektiöser Partikel zu generieren. Ein Ziel der Arbeit war durch Kultivierung auf Mikrocarrier die HBV Produktion der Zelllinie HepG2.2.15 zu steigern. Die Analyse von Protein und HBV Sekretion, Infektiösität und MAP Signalübertragung ergab eine 18x höhere HBV Produktion bei einer reduzierten Sekretion von subviralen Partikeln durch HepG2.2.15 die auf Mikrocarrier kultiviert wurden. Der Anstieg der Virusproduktion korreliert mit einer verstärkten Aktivierung der MAP Kinase ERK-2, die mit HBV Replikation in Verbindung steht. Ein weiterer wenig verstandener Teil des HBV Lebenszyklus ist der Kernimport des HBV Genoms. Spuren der viralen Polymerase finden sich im Zellkern von HBV infizierten Zellen. Ziel der Arbeit war, Motive in der HBV Polymerase zu finden, die in der Lage sind Zelllokalisation zu beeinflussen. Durch Sequenzvergleich wurde eine konservierte zweiseitige Kernlokalisationssequenz im Terminalen Protein der HBV Polymerase identifiziert, die eine Proteinkinase CKII Erkennungsstelle enthält. Inhibition der CKII Aktivität in HBV infizierten primären Hepatozyten sowie die Zerstörung der CKII Erkennungsstelle im Terminalen Protein inhibieren die HBV Replikation. Die Funktionalität der Kernlokalisationssequenz wurde durch Fusion an GFP bestätigt und war Abhängig von CKII Aktivität in der Zelle. Dies wurde in vitro durch Bindung des Adapterproteins Karyopherin-alpha an CKII-phosphoryliertes Terminales Protein bestätigt. Die HBV Polymerase enthält eine konservierte zweiseitige Kernlokalisationssequenz deren Funktionalität durch CKII Phosphorilierung vermittelt wird. / Hepatitis B virus (HBV) infection causes acute and chronic liver inflammation. Especially the early phase of the HBV life cycle is not clearly understood. For example the receptor complex that mediates viral entry is not known. Novel infection models to study the HBV lifecycle are described that demand for a large amount of cell culture generated infectious HBV particles. One aim was to enhance HBV production of the cell line HepG2.2.15 by cultivation on microcarrier substrate. Analysis of protein and viral particle secretion, infectivity, and cellular MAP kinase signaling revealed an up to 18x increased HBV production and a decreased subviral particle secretion by HepG2.2.15 when cultivated on microcarrier. The observed effect was due to an enhanced phospho-activation of MAP kinase ERK-2 that is tightly associated with HBV replication. Another poorly understood part of the HBV lifecycle is the mechanism that delivers the HBV genome into the nucleus. Traces of HBV polymerase can be found in HBV infected cells. The second objective was to identify motifs on the HBV polymerase that determine its subcellular localization. By sequence alignment a conserved bipartite nuclear localization signal was found in the terminal protein of the HBV polymerase encompassing a protein kinase CKII recognition site. Inhibition of CKII kinase in infected primary hepatocytes and destruction of the identified CKII recognition site in the viral polymerase impaired virus production. The functionality of the putative nuclear localization signal was confirmed by fusion to GFP. Moreover, its functionality was depended on CKII activity that was verified by in vitro binding experiments of terminal protein to the import adaptor karyopherin-alpha. This data identified a nuclear localization signal in the HBV polymerase, which functionality is mediated by CKII phosphorylation.

Le virus de l’hépatite B (HBV) est l’agent étiologique de l’hépatite B. Ce virus est responsable d’hépatite chronique B, de cirrhose et de cancer du foie au niveau mondial. L’absence d’activation de la voie Interféron (IFN) suite à l’infection par HBV est encore mal comprise. Récemment, le senseur cellulaire cytosolic GMP-AMP synthase (cGAS) a été décrit comme un senseur efficace de DNA double brin possédant également une activité antivirale envers des virus à ADN et à ARN. Le but de mes travaux de thèse a été de contribuer à la compréhension des relations existants entre le HBV et cGAS, à des stades précoces et tardifs de l’infection HBV en utilisant des expériences de perte- et gain- de function ainsi que du profilage génomique des génes apparentés à cGAS dans un modéle cellulaire permissif au HBV. Mes travaux ont démontré (1) que cGAS exerce une forte activité antivirale envers le HBV incluant une réduction de la forme nucléaire du génome, le cccDNA; (2) alors que le rcDNA génomique nu est reconnu par la voie cGAS/STING et induit une réponse IFN efficace, la nucléocapside virale protège le DNA génomique viral et l’empêche d’être détecté par la réponse immunitaire innée; et (3) que l’infection par HBV diminue l’expression des acteurs de la voie cGAS-STING et des gènes impliqués dans la réponse immunitaire innée in vitro et in vivo. Ce dernier point met en lumière le rôle de cGAS dans un nouveau mécanisme d’échappement du HBV au système immunitaire inné dans les cellules hépatocytaires et dans ce mécanisme. / Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic GMP-AMP synthase (cGAS) was identified as a DNA sensor. In this PhD work, we aimed to investigate the functional role of cGAS in sensing of HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss- and gain-of-function experiments combined with cGAS effector gene expression profiling in an HBV infection-susceptible cell culture model. Collectively, our data show that (1) the cGAS-STING pathway exhibits robust antiviral activity against HBV infection including reduction of viral cccDNA levels; (2) naked HBV genomic rcDNA is sensed in a cGAS-dependent manner whereas packaging of the viral genome during infection abolishes host cell recognition of viral nucleic acids; (3) HBV infection down-regulates the cGAS/STING pathway actors as well as innate immune effector gene expression in vitro and vivo. Overall, this work led to describing new aspects of the complex interaction between HBV and the DNA sensor cGAS in hepatocytes.

L'infection chronique ou occulte par le virus de l'hépatite B (HBV) est l'un des facteurs de risque les plusimportants pour le développement de carcinome hépatocellulaire viro-induit. Malgré la petite taille dugénome et les contraintes imposées par l'organisation de ce génome, le HBV présente une grandevariabilité génétique qui est le résultat des erreurs générées lors de l’étape de reverse transcription maisaussi, lors des processus de sélection et d’adaptation. Durant l'infection, une relation étroite hôte-viruss'établit. La population virale est caractérisée par la présence de quasi-espèces pouvant influencerl'évolution de la maladie hépatique et favoriser les phénomènes d'échappement. Le but de notre travail aété 1) d'étudier la variabilité virale associée aux infections occultes et l'impact de certaines modificationsde l'AgHBs (sY100C) sur la reconnaissance des tests de détection enzymatiques, et l'impact des mutations(sK122R, sD144E) sur la reconnaissance des anticorps anti-HBs d'un patient 2) de développer un nouveloutil d'amplification de génomes entiers de HBV que nous avons appliqué à l'étude préliminaire de quasiespècesd'un mutant Core. Cet outil d'amplification est plus sensible que les techniques existantes, et ilpermet l'étude des propriétés moléculaires et fonctionnelles des isolats virales. En conclusion, nous avonspu montrer que les processus d'échappement sont complexes, et font intervenir des facteurs du virus et del'hôte. Ce travail contribue à l'amélioration de la compréhension de la biologie de l'infection par le HBV,et illustre la complexité des interactions hôte-virus. / Pas de résumé en anglais / A infecção crônica e a infecção oculta pelo vírus da hepatite B (HBV) representam fatores de riscoconhecidos para o desenvolvimento de carcinoma hepatocelular. Apesar do pequeno tamanho do genomae as restrições impostas pela organização do genoma, o HBV tem uma alta variabilidade genética que é oresultado dos erros gerados durante a etapa de transcrição reversa e também durante o processo de seleçãoe adaptação do vírus. Durante a infecção uma relação de proximidade se estabelece entre o vírus e ohospedeiro. A população viral é caracterizada pela presença de quasispecies que poderiam influenciar aevolução da doença hepática, e também promover a seleção de variantes de escape imunológico. Osobjetivos do nosso estudo foram 1) estudar a variabilidade do HBV associada às infecções ocultas e oimpacto de certas modificações do HBsAg (sY100C) no reconhecimento de testes de detecçãoenzimáticos. Estudar também o impacto das mutações (sK122R, sD144E) sobre o reconhecimento deanticorpos anti-HBs de um paciente, 2) desenvolver um novo método para amplificar o genoma completodo HBV que foi aplicado a um estudo para identificação de quasispecies de um mutante da região do coreviral. Este método de amplificação é mais sensível do que as técnicas existentes e permite o estudo depropriedades moleculares e funcionais dos virus. Em conclusão, mostramos que os processos de escapeviral são complexas e envolvem fatores relacionados tanto com o vírus como com o hospedeiro. Estetrabalho contribui de maneira significativa para a compreensão da biologia da infecção pelo HBV eilustra a complexidade das interações vírus-hospedeiro.

I denna rapport undersöks hur mängden organiskt kol, TOC (Totalt organiskt kol), varierar i tid och rum i Mälarens avrinningsområde, samt vad det är som styr TOC-halten i Mälaren. Det är viktigt att förstå dynamiken hos TOC i Mälaren och i dess avrinningsområde eftersom ökat TOC i vattnet påverkar vattenkvaliteten och orsakar problem vid beredning av dricksvatten. TOC kan bland annat reagera med klor/UV-ljus och bilda cancerframkallande ämnen. Det kan också öka antal mikrober i vattnets distributionssystem. Arbetet omfattar analys av samband mellan elementen, transportberäkningar per ytenhet av elementen till Mälaren och en modelleringsansats för ett av avrinningsområdena. Rapporten innehåller även en jämförelse mellan de olika vattenföringsmodellerna samt uppmätt vattenföring för analys av eventuella systematiska skillnader mellan dessa som påverkar beräkningen av TOC och de andra elementens transport till Mälaren. Analysen av sambanden mellan variablerna TOC (mg/l), kaliumpermanganat förbrukning (KMnO4, mg/l), absorbans_F (F=filtrerad), järn (mg/l), mangan (mg/l) och SO4_IC (sulfat mätt med hjälp av jonkromatografi, mg/l), visade att vissa av dessa variabler är korrelerade med varandra. TOC mot KMnO4 och TOC mot absorbans_F hade de bästa anpassningarna med respektive R2- värden 0,65 och 0,59 och p-värden <0,001. Årsnederbörd är positivt korrelerad med TOC per ytenhet för Kolbäcksån med R2-värde 0,63 och p-värde <0,01, vilket innebär att sambandet är signifikant. Ökad årsnederbörd leder till ökad tillförsel av TOC till Mälaren. Det finns däremot inget signifikant samband mellan TOC-transport per ytenhet och årsmedeltemperatur. Arealflödesberäkningar tyder på att den största tillförseln av TOC- transport per ytenhet kommer från den nordöstra delen av Mälaren. Fyrisån står för den största tillförseln av TOC. Hydrologiska, kemiska och meteorologiska data inkluderades i modeller för att kunna skatta TOC-halten i Mälaren. Temperatur-, evapotranspirations- och nederbördsdata användes i en hydrologisk modell, HBV- modellen, för att simulera vattenföringen från avrinningsområdet. Sedan användes en processbaserad modell, INCA- C, som drivs av hydrologisk data och beräknade grundvattenbildning och markfuktighet för att simulera tidsmässiga mönster i TOC. Invariablerna till INCA-modellen, markfuktigheten och HER (grundvattenbildning), simulerades med hjälp av HBV- modellen. Dessa modeller tillämpades i Kolbäcksån (ett av Mälarens största avrinningsområden). Modelleringen av Kolbäcksåns TOC- halt resulterade i en modell som anpassade dynamiken mellan 1996 och 2009, men missar den mellan 2009 och juni 2010, med bäst anpassning mellan 2006 och 2008. R2- och NS värden som erhölls för modellen var 0,086 och -0,059. / In this report, it has been investigated how the amount of organic carbon, TOC, varies in time and space in the basin of Mälaren, and what controls the TOC content in the lake. It is important to understand the dynamics of the TOC in the lake and its catchment because increased TOC in the water affects water quality and causes problems in the preparation of drinking water. Particularly, it can react with chlorine / UV- light and form carcinogenic substances. It can also increase the number of microbes in water distribution systems. In addition the work includes analysis of the relation between water chemistry variables, annual fluxes calculations (g/m2/year) of element flows to the lake and a modeling approach to a watershed. Annual fluxes calculations (g/m2/year) indicate that the largest supply of TOC to the lake comes from the northeast of the lake. Fyrisån accounts for the largest input of TOC to the lake. The high TOC-flux is due to a small proportion of open water in the catchment. Hydrological, chemical and meteorological data have been included in models to estimate the TOC content in the Mälaren. Input data processing, especially precipitation data, has been an important part of the work as it affects the whole model. Temperature, evapotranspiration and precipitation data were used in a hydrological model, HBV model, to simulate the flow from the catchment area. Then a process-based model, INCA-C, operated by the hydrological data and soil moisture, has been used to simulate the temporal patterns in TOC. The input variables to INCA-C- model, soil moisture and HER (Hydrological effective rainfall), have been simulated using the HBV- model. Those models were applied in Kolbäcksån, one of the lake's largest catchments. The modeling of Kolbäcksån resulted in a model that captured the dynamics of a few periods of the whole time series. The modeling of Kolbäcksån TOC-concentration resulted in a model that captured the dynamics between 1996 and 2009, but misses it between 2009 and June 2010. R2 and NS values obtained for the model were 0.086 and -0.059, respectively.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-29T14:55:41Z No. of bitstreams: 2 Dissertação - Marina Pedroso de Oliveira - 2013.pdf: 1546029 bytes, checksum: 047574069d3a0ee543cf485aa4e61edd (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-30T10:52:04Z (GMT) No. of bitstreams: 2 Dissertação - Marina Pedroso de Oliveira - 2013.pdf: 1546029 bytes, checksum: 047574069d3a0ee543cf485aa4e61edd (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-30T10:52:04Z (GMT). No. of bitstreams: 2 Dissertação - Marina Pedroso de Oliveira - 2013.pdf: 1546029 bytes, checksum: 047574069d3a0ee543cf485aa4e61edd (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-02-28 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / It is estimated that about 10% of individuals infected with human immunodeficiency virus (HIV) throughout the world have chronic infection with hepatitis B virus (HBV). The HIV-HBV co-infection increases the risk for developing cirrhosis and hepatocellular carcinoma. Also, co-infected individuals have atypical serological profiles more often than monoinfected patients, many of whom may have occult hepatitis B (presence of HBV-DNA in serum and/or liver in the absence of HBsAg) in addition to mutations in the viral genome that may have implications for the prognosis and therapeutics of hepatitis B. This study aimed to estimate the prevalence of occult HBV infection in HIV-infected treatment-naive patients, in Goiânia, Goiás, to determine the viral load of HBV-DNA positive samples, to identify circulating genotypes/subgenotypes and to investigate the occurrence of mutations in P and S regions of the HBV genome. This is a cross-sectional study conducted in HIV-infected treatment-naive patients attended at a reference hospital for infectious diseases in the Goiania city. After serological screening of the population (n = 505), two groups of patients were selected for this study: (i) 99 anti-HBc positive/HBsAg negative and (ii) 29 HBsAg reactive individuals. HBV-DNA was detected by semi-nested PCR and quantified by real time PCR. The amplicons were genotyped by RFLP and nucleotide sequencing. The identification of mutations in the viral genome was made by deduction of the amino acid sequences from the nucleotides sequences. The search for occult HBV infection performed in 99 anti-HBc positive/HBsAg negative samples (Group 1) showed a prevalence of 16.2% (16/99, 95% CI: 9.8-25.2). Of 29 HBsAg positive patients (Group 2), HBV-DNA was detected in 69% (20/29). As expected, DNA levels in the HBsAg reactive patients were significantly higher than those found in patients with occult infection (mean 2.89 x 108 copies/ml vs 6.59 x 102 copies/ml, p < 0.01). The genotypes (subgenotypes) A (A1 and A2), D (D2 and D3) and F (F2) were identified, being A/A1 the most frequently found. Mutations in the S region of HBV were found in both groups of patients, whereas mutations in the P region were observed only in patients HBsAg positive. These results revealed a high prevalence of occult HBV infection, the predominance of genotype A (A1), and the presence of mutations in the viral genome of HBV-HIV co-infected treatment-naive patients in Goiânia, Goiás. / Estima-se que cerca de 10% dos indivíduos infectados pelo vírus da imunodeficiência humana (HIV) em todo o mundo apresentam infecção crônica pelo vírus da hepatite B (HBV). A coinfecção HIV-HBV aumenta o risco para desenvolvimento de cirrose e hepatocarcinoma. Ainda, indivíduos coinfectados apresentam perfis sorológicos atípicos mais frequentemente do que os monoinfectados, sendo que muitos deles podem apresentar hepatite B oculta (presença do HBV-DNA no soro e/ou fígado na ausência do HBsAg), além de mutações no genoma viral que podem ter implicações no prognóstico da hepatite B e na terapêutica. Este estudo teve como objetivos estimar a prevalência de infecção oculta pelo HBV em indivíduos infectados pelo HIV, virgens de tratamento, em Goiânia-Goiás, determinar a carga viral das amostras HBV-DNA positivas, identificar os genótipos/subgenótipos circulantes e investigar a ocorrência de mutações nas regiões S e P do genoma do HBV. Este é um estudo transversal conduzido em pacientes infectados pelo HIV, virgens de tratamento, atendidos em um hospital de referência para doenças infecciosas na cidade de Goiânia. Após triagem sorológica da população (n=505), dois grupos de pacientes foram selecionados para o presente estudo: (i) 99 anti-HBc positivos/HBsAg negativos e (ii) 29 indivíduos HBsAg reagentes. O HBV-DNA foi detectado por semi-nested PCR e quantificado por PCR em tempo real. Os amplicons foram genotipados por RFLP e sequenciamento de nucleotídeos. A identificação de mutações no genoma viral foi feita pela dedução de aminoácidos a partir de sequências de nucleotídeos. Ao pesquisar a infecção oculta para o HBV nas 99 amostras anti-HBc positivas/HBsAg negativas (Grupo 1), estimou-se uma prevalência de 16,2% (16/99; IC 95%: 9,8-25,2). Dos 29 pacientes HBsAg positivos (Grupo 2), o HBV-DNA foi detectado em 69% (20/29). Como esperado, os níveis de HBV-DNA nos pacientes HBsAg reagentes foram significativamente maiores do que os encontrados nos pacientes com infecção oculta (média: 2,89 x 108 cópias/mL vs 6,59 x 102 cópias/mL; p < 0,01). Os genótipos (subgenótipos) A (A1 e A2), D (D2 e D3) e F (F2) foram identificados, sendo A/A1 os mais frequentes. Mutações na região S do HBV foram encontradas nos dois grupos de pacientes, enquanto que as mutações na região P foram evidenciadas somente nos pacientes HBsAg positivos. Estes resultados revelaram uma prevalência alta de infecção oculta pelo HBV, a predominância do genótipo A (A1), bem como a presença de mutações no genoma viral dos pacientes HBV-HIV coinfectados, virgens de tratamento, em Goiânia-Goiás.

Made available in DSpace on 2015-04-11T13:57:17Z (GMT). No. of bitstreams: 1 Lucinete Okamura Kimura.pdf: 3604811 bytes, checksum: 944fcd9a6fb1e06fc9a8b1ff83801f92 (MD5) Previous issue date: 2011-02-03 / Hepatitis B virus (HBV) infection is one of the most serious public health problems in the world. At least two billion people are infected, with over 350 million showing serological markers of active infection, despite prevention by vaccination. In Brazil, the HBV endemicity is heterogeneous, with the most prevalent disease in the north region. Among the indigenous population, epidemiological serum studies have reported high rates of hepatitis B prevalence in the Brazilian component of the Amazon rainforest. Studies have shown that in some cases the serological markers are not enough to detect viral activity, and in these situations, molecular tests are more sensitive and specific. The proposal of this study was to determine the prevalence of DNA (deoxyribonucleic acid) of hepatitis B virus in indigenous ethnic groups (Kanamary, Matis, Mayoruna, Marubo, Kulina and Korubo) in habitants of the rivers Curuçá and Itaquaí at the Javari Valley, Amazon, Brazil. This was a descriptive, cross-sectional study, such as used in case detection. One hundred and eighty (180) samples were analyzed from the indigenous communities of São Sebastião, Volta Grande, Pedro Lopes, Massapê, Remancinho and Bananeira. The samples were subjected to polymerase chain reaction (PCR) and semi-nested PCR to Hepatitis B virus, S gene. The prevalence for HBV-DNA of S gene was 51.1% (92/180). Among the PCR positive samples for HBV-DNA, 18/49 (36.7%) were from Marubo, 68/125 (54.4%) from Kanamary and 6/6 (100%) from other ethnicities. With regards to socio-demographic data, no significant difference was found (p=0.889) in relation to gender (statistical analysis at 5%). However, when analyzing age it was observed that the natives had lower median age (p<0.001) of 23 years old, suggesting that sexual activity was the main form of HBV transmission. There was no statistical difference found in relation to sources of infection and the presence of HBV DNA, as well as clinical aspects, with the exception of fever (p<0.001). The high prevalence of HBV-DNA of 75% (15/20) in pregnant women (p=0,009) demonstrates association with vertical transmission. The results confirm the high prevalence of HBV DNA in the Javari Valley, making it important to devise strategies for control and a more effective prevention in combating the spread of HBV / A infecção pelo vírus da hepatite B (VHB) é um dos mais sérios problemas de Saúde Pública do mundo. Estima-se que dois bilhões de pessoas estejam infectadas, com mais de 350 milhões apresentando marcadores sorológicos de infecção ativa, apesar da prevenção pela vacinação. No Brasil, a endemicidade do VHB é heterogênea, sendo a doença mais prevalente na região norte do país. Entre a população indígena, estudos soro epidemiológicos relatam altas taxas de prevalência de hepatite B na Amazônia brasileira. Pesquisas têm demonstrado que, em alguns casos, os marcadores sorológicos não são suficientes para detectar uma atividade viral e, nessas situações, os testes moleculares se mostram mais sensíveis e específicos. O presente estudo se propôs determinar a prevalência do DNA (ácido desoxirribonucléico) do vírus da hepatite B em povos de etnias indígenas habitantes dos rios Curuçá e Itaquaí no Vale do Javari, Amazonas, Brasil, pertencentes às etnias Kanamary, Matis, Mayoruna, Marubo, Kulina e Korubo. Tratou-se de um estudo descritivo, transversal, do tipo detecção de caso. Foram analisadas 180 amostras pertencentes às comunidades indígenas de São Sebastião, Volta Grande, Pedro Lopes, Massapê, Remancinho e Bananeira. As amostras foram submetidas à reação em cadeia da polimerase (PCR) e semi-nested para vírus da hepatite B, gene S. A prevalência encontrada para o DNA-VHB gene S foi de 51,1% (92/180). Entre as amostras positivas para DNA-VHB PCR, 18/49 (36,7%) pertenciam à etnia Marubo, 68/125 (54,4%) à Kanamary e 6/6 (100%) a outras etnias. Quanto aos dados sóciodemográficos dos casos positivos para DNA-VHB PCR, pôde-se verificar que não houve diferença significante ao nível de 5% em relação ao gênero (p= 0,889). No entanto, quando se analisou a idade foi observado que os indígenas com PCR positiva para DNA-VHB apresentavam menores mediana de idade (p<0,001) de 23 anos, sugerindo ser a atividade sexual uma das principais formas de transmissão do VHB. Não foi constatada nenhuma diferença estatística em relação às fontes de contágio e à presença do DNA-VHB, como também aos aspectos clínicos, com exceção da febre (p<0,001). A alta prevalência do DNA-VHB de 75% (15/20) em gestantes (p=0,009) demonstra associação com a transmissão vertical. Os resultados comprovam a alta prevalência do DNA-VHB no Vale do Javari, tornando-se importante traçar estratégias de controle e prevenção mais eficazes no combate à disseminação do VHB

Sogeti is an IT consulting company that’s active in large parts of the world. One of their biggest clients in Sundsvall is SCA Skog. In case of fire risk, SCA parti- cipates in consultation meetings with contractors to determine fire prevention measures. SMHI’s fire forecasts and warnings are a few of the bases that deter- mine when a consultation meeting is to begin. The problem is that the contractor today does not receive an automatic indication of fire risk. The purpose of the project was therefore to investigate SMHI’s services and find a solution to the problem and to find a limit for when a consultation meeting should be initia- ted based on the risk within an area. The solution presented is illustrated by a proof-of-concept model. The work has been carried agile in sprinting. Data has been taken from SMHI for a more detailed documentation over warnings and against MSB for the raw data that is behind fire forecasts so that those can be included in the solution. The project has resulted in an application that supports indication of risk based on a list of coordinates as well as automatic indication to the user’s position. An implementation guide was also created to facilitate a possible further development of the solution. The investigation shows that fire forecasts give a more precise indication of danger, but warnings should not be overlooked, as they indicate a risk of other warnings than fire risk. The solution is only intended to give the contractor an additional tool when making a deci- sion. The responsibility remains with the contractor, but the tool will hopefully allow the right decision to be made. / Sogeti är ett IT-konsultföretag med kontor i många länder. En av deras största klienter i Sundsvall är SCA Skog. Vid brandrisk deltar SCA tillsammans deras entreprenörer i samrådsmöten för att bestämma brandförebyggande åtgärder. SMHI:s brandprognoser och varningar är ett par av de underlag som avgör när ett samrådsmöte ska inledas. Problemet är att entreprenören idag inte får en automatisk indikation för brandrisk. Syftet med projektet blev därför att undersöka SMHI:s tjänster och finna en automatisk lösning samt att hitta en gräns för när ett samrådsmöte bör inledas baserat på brandrisken inom ett område. Den lösning som tagits fram illustreras genom en Proof-of-Concept modell. Arbetet har utförts agilt i sprintar. En datainsamling har utförts mot SMHI för en mer detaljerad dokumentation och mot MSB för den rådata som ligger bakom brandprognoser så att detta kan inkluderas i lösningen. Projektet har resulterat i en applikation som stödjer indikation av risk baserat på geografiska positioner samt automatisk indikation av risk vid en användares position. En implementationsguide togs även fram för att underlätta en möjlig vidareutveckling av lösningen. Undersökningen visar att brandprognoser ger en mer precis indikation på fara däremot bör varningar inte helt förbises då dessa kan indikera risk vid andra varningar än just brandrisk. Lösningen är enbart avsedd att ge entreprenören ett extra verktyg när denne ska ta ett beslut. Ansvaret ligger fortfarande hos entreprenören men en implementation möjliggör att beslut kan tas med gott un- derlag.

Es gibt wenige Erkenntnisse über den Zusammenhang zwischen Lamivudin induzierten Resistenzmutationen und Hepatitis B Genotypen. Die vorliegende Studie untersucht das Verhältnis zwischen diesen Mutationen und den Hepatitis B Genotypen A-D. Die Datenbank der US-amerikanischen Kongressbibliothek (Pubmed) wurde nach den Begriffen „HBV OR hepatitis B”, „YMDD”, „genotype”, und „lamivudine” durchsucht. Alle in dieser Suche gefundenen Arbeiten, die bis Juni 2009 veröffentlicht worden waren, wurden in die Studie eingeschlossen. Die Ergebnisse der Literaturanalyse wurden mit den Hepatitis B-Genomdaten zweier Referenzlabore in Tübingen und Melbourne verglichen. Insgesamt konnten 29 Arbeiten aus der Datenbankrecherche in die Literaturanalyse eingeschlossen werden. Diese Studien enthielten Daten zu insgesamt 827 Patienten, deren Hepatitis B Genotyp bekannt war und die eine Lamivudinresistenzmutation aufwiesen. In statistischen Untersuchungen konnte nachgewiesen werden, dass die rtM204V-Mutation die dominierende Mutation bei Infektionen mit Genotyp A ist. Dieses Ergebnis konnte durch die Analyse der Genomdaten der Referenzlabore bestätigt werden. Ferner konnte gezeigt werden, dass bei den Genotypen A, B, und D die rtL180M-Mutation hochsignifikant mit der rtM204V-Mutation verknüpft ist. Die Dissertationsschrift enthält neben dem Artikel „Mutation pattern of lamivudine resistance in relation to hepatitis B genotypes: hepatitis B genotypes differ in their lamivudine resistance associated mutation pattern“ (Damerow, H, Yuen L et al.; J Med Virol. 2010 Nov; 82(11):1850-8) eine Einführung in die Rationale der Studie, eine Zusammenfassung der Ergebnisse sowie ein Fazit.:1. Einleitung 1.1. Bibliografische Beschreibung und Kurzzusammenfassung, Seite 4 1.2. Inhalt und Aufbau der Arbeit, Seite 5 2. Hintergründe und Rationale 2.1. Therapie der chronischen Hepatitis B-Infektion, Seite 6 2.2. Resistenzen unter Therapie mit Nukleos(t)idanaloga, Seite 7 2.3. Hepatitis B-Genotypen, Seite 9 2.4. Die vorliegende Studie 2.4.1. Rationale der Studie, Seite 10 2.4.2. Arbeitsverteilung, Seite 11 3. Studie “Mutation pattern of lamivudine resistance in relation to hepatitis B genotypes: hepatitis B genotypes differ in their lamivudine resistance associated mutation pattern” 3.1. Abstract, Seite 14 3.2. Introduction, Seite 15 3.3. Methods, Seite 16 3.4. Results, Seite 17 3.5. Discussion, Seite 20 3.6. Reference List, Seite 22 3.7. Appendix, Seite 25 4. Zusammenfassung der vorliegenden Studie 4.1. Mutationsverhalten im YMDD-Motiv 4.1.1. Literaturanalyse, Seite 31 4.1.2. SeqHepB-Datenbank, Seite 32 4.1.3. Tübinger Datenbank, Seite 32 4.2. rtL180M-Mutation in Relation zu HBV-Genotyp und rtM204I/V-Mutation 4.2.1. Literaturanalyse, Seite 34 4.2.2. SeqHepB-Datenbank, Seite 34 4.2.3. Tübinger Datenbank, Seite 35 4.3. Analyse der Polymerase-Mutationen rtL80I/V, rtV173L und rtA181V/T in Abhängigkeit vom HBV-Genotyp, Seite 35 4.4. Analyse der Mutationen im Surface-Antigen, die mit Polymerasemutationen assoziiert sind, Seite 36 5. Fazit 5.1. Literaturübersicht, Seite 37 5.2. Mögliche klinische Relevanz, Seite 38 6. Anhang 6.1. Literaturverzeichnis, Seite 41 6.2. Abbildungs- und Tabellenverzeichnis, Seite 45 6.3.Selbstständigkeitserklärung, Seite 47 6.4. Publikationsverzeichnis, Seite 48 6.5. Danksagungen, Seite 49

The aim of this thesis was to investigate the processes behind an unexpected runoff behaviour after a clear-cut in a boreal forest in northern Sweden (Balsjö). The risks of increased flooding, erosion, nutrient leakage and changes in the local ecosystems are some reasons why it is important to fully understand the effect of clear-cuts on the water balance. In northern boreal forests the snow is of great importance as it results in the main hydrological event of the year, the spring flood. In general, open areas accumulate more snow, have a lower evapotranspiration and therefore maintain a higher runoff than a forest. In a recent paired catchment study at Balsjö the expected pattern after a clear-cut was only shown in three out of five years (2007-2011). The expected increase in runoff did not occur in 2010 and 2011. Two hypothesized alternatives were year-to-year variation of ET or changes in soil water storage. In order to investigate this further the rainfall-runoff model HBV was used. First, the model was calibrated for the forest catchment (Ref) and the clear-cut catchment (CC), using observed data from Balsjö. To account for parameter uncertainty the calibration was performed using parameter optimization, resulting in 100 different parameter sets. Model results were evaluated using observed snow data from Balsjö and ET from Flakaliden, a nearby forest. Both the simulated snow and ET were quite consistent with the observed values. Finally the annual and the spring water balance were studied, using the simulated data. The simulated results did not detect the unexpected runoff behavior for the two years as clearly as the observations. The reason for this was that the model was calibrated for all five years, which meant that annual variations were not taken into account. The hypothesis, that higher ET could be the reason for the unexpected runoff behavior, could neither be dismissed nor confirmed by this thesis. This was because there were no observed data for the clear-cut area and limitations within the HBV model, which meant that sublimation and interception processes could not be analyzed separately. The model results indicated that the change in soil water storage was a more likely explanation for the unexpected runoff behavior. The simulation result showed that the meltwater was stored in the soil water storage. However, this theory does not seem likely since a clear-cut is normally wetter than a forest. The results of this thesis are consistent with other studies as they indicate that clear-cut effects should be studied seasonally as well as annually. The special feature of this thesis was the opportunity to study observed ET and investigate its influence on the water balance.