A doença hepática crônica causada pelo vírus da hepatite C (HCV) tornou-se, nos últimos anos, uma das principais comorbidades dos pacientes portadores do vírus da imunodeficiência humana (HIV) nos países desenvolvidos. Os pacientes coinfectados com HIV/HCV apresentam uma progressão mais rápida para a cirrose e as suas complicações que os pacientes monoinfectados com HCV. Embora os mecanismos responsáveis por esta evolução não estejam totalmente esclarecidos, a expressão da molécula de HLA-G, um HLA de classe Ib não clássico, que tem propriedades bem reconhecidas na regulação negativa da resposta imune, pode estar relacionada à progressão da doença hepática. Os objetivos deste trabalho foram analisar o perfil de expressão de HLA-G em tecido hepático de pacientes coinfectados HIV/HCV e identificar possíveis variáveis do hospedeiro, do HCV e do HIV que possam estar relacionadas com a expressão de HLA-G na biópsia hepática. Para isso, 57 amostras de biópsia hepática de pacientes coinfectados com HIV/HCV, nas quais a imuno-histoquímica para HLA-G foi realizada, foram analisadas retrospectivamente quanto à expressão desta molécula no tecido hepático. Avaliaram-se também outras características histopatológicas da biópsia como grau de fibrose, atividade inflamatória, deposição de ferro e gordura. Determinou-se o polimorfismo de inserção ou deleção de 14 pares de bases da região 3` não traduzida do exon 8 do gene do HLA-G, que está relacionada com a produção de RNA-mensageiro, em 43 destes pacientes, além do polimorfismo de IL-28B, relacionado com a resposta ao tratamento do HCV, em 44 deles. Características bioquímicas e virológicas, tanto do HIV quanto do HCV também foram avaliadas. O genótipo 1 do HCV foi o mais prevalente (87,75%), especialmente o subgenótipo 1a (60%). A expressão do HLA-G foi observada em 38 (66,7%) amostras de fígado, e foi mais frequente em estágios moderados e severos de fibrose do que em estágios mais leves (94,1% x 55%, P < 0,01). Não houve relação entre a expressão do HLA-G e os outros parâmetros estudados. Embora a progressão para a cirrose no contexto da coinfecção por HIV/ HCV seja um processo complexo, modulado por muitos factores, a associação da intensidade de fibrose com a expressão do HLA-G pode indicar que a expressão desta proteína desempenha um importante papel nos mecanismos que contribuem para a progressão da doença, por meio da regulação negativa da resposta imune contra o HCV na coinfecção pelo HIV. / Chronic liver disease induced by hepatitis C virus (HCV) infection has recently become one of the most common comorbidities in patients who are infected with the human immunodeficiency virus (HIV) in developed countries. HIV/HCV coinfected patients show faster progression to cirrhosis and its complications than the HCV monoinfected patients. Even though the responsible mechanisms for this evolution have not been entirely clarified yet, the expression of the HLA-G molecule, a HLA from the non-classic Ib class, with well-known properties of negatively regulating the immune response, may be related to the liver disease progression. The aims of the present work were to analyze the HLA-G expression profile in the liver micro ambience of HIV/HCV coinfected patients and to identify possible host factors, HIV or HCV, that may be related to the HLA-G expression on the liver biopsy. For this purpose, 57 liver biopsies of HIV/HCV coinfect patients, in which immunohistochemistry for HLA-G had been performed, were retrospectively analyzed according the HLA-G expression on the hepatic tissue. Other histopathological features in the liver biopsies, such as fibrosis degree, inflammatory activity, iron deposition and fat were also evaluated. The polymorphism of insertion or deletion in 14-base pairs of the 3`non-translated region of exon 8 of the HLA-G gene, which is related to the production of HLA-G messenger RNA, was evaluated in 43 of the patients. Also, the polymorphism of IL-28B, related to the response to HCV treatment, was evaluated in 44 of them. Biochemical and virological features of HIV and HCV were also evaluated. The HCV genotype 1 was the most prevalent (87.75%), especially the subgenotype 1a (60%). The expression of HLA-G was observed in 38 (66.7%) samples of the liver biopsies, and it was most frequent in moderate and severe stages of fibrosis than in the mild stages (94.1% x 55%, P < 0.01). There was no established relationship between HLA-G and other parameters studied. Although the progression to cirrhosis in the context of HIV/HCV coinfection is a complex process modulated by many factors, the association of HLA-G expression with the intensity of the liver fibrosis may indicate the protein expression play an important role in the mechanisms that contribute to the progression of the disease, through the negative regulation of the immune response against HCV setting of a coinfection with HIV.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-07-21T13:54:37Z No. of bitstreams: 1 aecioflaviomeirellesdesouza.pdf: 588675 bytes, checksum: 84d528796e59a48f1e76239e179980a4 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-22T15:30:38Z (GMT) No. of bitstreams: 1 aecioflaviomeirellesdesouza.pdf: 588675 bytes, checksum: 84d528796e59a48f1e76239e179980a4 (MD5) / Made available in DSpace on 2016-07-22T15:30:38Z (GMT). No. of bitstreams: 1 aecioflaviomeirellesdesouza.pdf: 588675 bytes, checksum: 84d528796e59a48f1e76239e179980a4 (MD5) Previous issue date: 2011-10-10 / Objetivos: verificar a prevalência de resistência insulínica (RI) em portadores de hepatite crônica C não diabéticos e analisar o impacto da mesma sobre os parâmetros laboratoriais e histológicos. Sujeitos e métodos: foram incluídos no estudo 82 pacientes e amostras de sangue foram coletadas para determinação de glicose, perfil lipídico, ALT, AST, ferritina, HOMA-IR, carga viral e genótipo do VHC. HOMA-IR superior a 2,5 foi considerado resistência insulínica. Resultados: RI foi observada em 27% dos pacientes e foi associada a idade, circunferência abdominal e índice de massa corpórea. Quando comparado a pacientes sem RI, aqueles com HOMA-IR superior a 2,5 apresentaram graus mais acentuados de fibrose hepática e atividade necroinflamatória, maiores níveis de aminotransferases e esteatose hepática mais freqüente. Conclusões: É comum a presença de RI em portadores de hepatite crônica C e esta tem como resultado a acentuação da fibrose hepática induzida pelo vírus da hepatite C. DESCRITORES: Hepatite crônica C, fibrose hepática, resistência insulínica, HOMA-IR / Objectives: To determine the prevalence of insulin resistance (IR) in patients nondiabetic with chronic hepatitis C and analyze the impact of insulin resistance on laboratory and histology parameters. Subjects and methods: The study included 82 patients and blood samples were collected for glucose, lipid profile, CRP, ferritin, ALT, AST, HOMA-IR, viral load and HCV genotype. HOMA-IR greater than 2.5 were considered insulin resistance. Results: Insulin resistance was observed in 27% of patients and was associated with age, waist circumference and body mass index. When compared with patients without RI those with insulin resistance had more pronounced stage of hepatic fibrosis and necroinflammatory activity, higher levels of aminotransferases and liver steatosis more frequently. Conclusions: It is often the presence of insulin resistance in patients with chronic hepatitis C and this has resulted in the exacerbation of liver fibrosis induced by HCV.

La fibrose hépatique est un problème de santé publique. Cette pathologie est caractérisée par une accumulation excessive de matrice extracellulaire, composée principalement de collagène, augmentant la rigidité du foie. Environ 90% des hépatocarcinomes se développent sur un foie fibrotique / cirrhotique, laissant présager une relation entre la rigidité tissulaire et le développement tumoral. Pour étudier le rôle des forces exercées par la matrice extracellulaire sur le phénotype des cellules hépatiques, nous avons développé un modèle de culture 3D de cellules hépatiques dans des gels de collagène de rigidités variables. Dans ces conditions, les cellules hépatiques présentent une forte prolifération et un maintien de la différenciation dans les matrices les plus rigides. En parallèle, les cellules hépatiques transformées peuvent modifier la matrice de collagène pour former des signatures de collagène TACS (Tumor Associated Collagen Signatures). Une analyse des voies de signalisation impliquées dans la formation des TACS 3 nous a permis de déterminer 2 voies indispensables pour ces mécanismes: MEK/ERK et MLCK. Le bon maintien des fonctions différenciées et de biotransformation des cellules hépatiques font des cultures 3D en gel de collagène un excellent modèle pour des applications en biotechnologie. Nous avons également développé une technique de quantification standardisée et automatisée du collagène, dans un modèle murin de fibrose hépatique, par utilisation de la microscopie SHG, qui permet de détecter de faibles variations de quantité de collagène. Cette technique permet également de caractériser qualitativement, après analyse d'images, le collagène et de renforcer la discrimination entre les différents stades fibrotiques. La caractérisation des cross-links de collagène, par cette approche, est actuellement en cours d'étude et permettrait d'appréhender les capacités de réversion. / Liver fibrosis is a real public health problem. This pathology is characterized by an excessive accumulation of extracellular matrix, mainly composed of collagen, increasing liver rigidity. Approximately 90% of hepatocellular carcinomas develop from a fibrotic/cirrhotic liver, suggesting a relationship between tissue rigidity and tumor development. To investigate the role of stiffness on the hepatic phenotype, we have developed a 3D culture model of collagen gels of varying stiffness. Our results show a better survival, an increase of proliferation and differentiation of liver cells in rigid matrices. In addition, the cells are able to modify the collagen matrix and to form collagen signatures TACS (Tumor Associated Collagen Signatures). An analysis of the signaling pathways involved in the formation of TACS 3 allowed us to determine that 2 pathways are important for these mechanisms: MEK/ERK and MLCK. The high level of differentiated functions and biotransformation of the hepatic cells make 3D collagen cultures an excellent model for applications in biotechnology. Using the SHG microscopy, we have also developed a standardized and automated quantification of collagen to detect small amount of collagen in a mouse liver fibrosis model. This technique allows us to characterize qualitatively the collagen and to strengthen the discrimination between fibrotic scores. The characterization of the collagen cross-links by this approach is under study and would allow to study the reversion capacity.

Natural Killer (NK) cells, members of the innate lymphoid cells (ILCs), are known to play an important role in the defense against foreign cells and abnormal host cells that have arisen due to viral infection or cancer inducing mutations. The typical immune response of NK cells involves the release of cytotoxic granules containing perforin and granzyme, and the secretion of immune-regulatory cytokines such as interferon gamma (IFN-γ). Unlike the adaptive lymphocytes such as T cells and B cells, NK cells do not require prior sensitization, enabling them to initiate an immune response much faster. This unique feature of NK cells is made possible by the utilization of an array of germline encoded receptors; but on the other hand, it limits NK cells ability to respond against rapidly evolving pathogens. NK cells overcome this shortcoming with an antibody-assisted process called antibody dependent cellular cytotoxicity (ADCC). A novel subset of human NK cells, which displays potent and broad antiviral responsiveness in concert with virus-specific antibodies, was recently discovered in cytomegalovirus positive (CMV+) individuals. This NK cell subset, called g-NK cell, was characterized by a deficiency in the expression of FcεRIγ, an adaptor protein that associates with CD16 which enables ADCC. Surprisingly, despite this deficiency, g-NK cells displayed an enhanced ADCC as compared to their conventional counterparts. Furthermore, having a long-lasting memory-like NK-cell phenotype suggests a role for g-NK cells in chronic infections. This study investigates the importance of g-NK-cells in clinical settings, first by investigating whether the presence of g-NK cells is associated with the magnitude of liver disease during chronic hepatitis C virus (HCV) infection. Analysis of g-NK cell proportions and function in the peripheral blood mononuclear cells (PBMCs) of healthy controls and chronic HCV subjects showed that chronic HCV subjects had slightly lower proportions of g-NK cells, while having similarly enhanced ADCC responses compared to conventional NK cells. Notably, among CMV+ chronic HCV patients, lower levels of liver enzymes and fibrosis were found in those possessing g-NK cells. g-NK cells were predominant among the CD56neg NK cell population often found in chronic HCV patients, suggesting their involvement in the immune response against HCV. Rituximab is a chimeric anti-CD20 antibody used to treat B cell lymphoma patients; and studies have suggested that its efficacy is associated with the ADCC potency and CD16 affinity. Since g-NK cells are characterized by their superior ADCC compared to their conventional counterpart, I decided to investigate whether the presence of g-NK cells can improve the effectiveness of rituximab against malignant B cells in the context of lymphoma and leukemia. The analysis of g-NK cells’ ADCC response against rituximab-coated lymphoma cell lines and B cells from a CLL patient indicated a superior ADCC by g-NK cells compared to their conventional NK cell counterparts. Taken together, for the first time, my findings indicate that the presence of g-NK cells in CMV+ individuals is associated with milder liver disease in chronic HCV infection. In addition, an enhanced ADCC response by g-NK cells upon encountering rituximab coated target cells suggests the beneficial roles of g-NK cells, and opens an avenue for novel therapeutic approaches where g-NK cells can be utilized to treat persistent diseases such as chronic viral infection and cancer.

O colágeno XVIII é um componente das membranas basais com diversos domínios funcionais, como a endostatina e o domínio frizzled, que têm importante papel em processos celulares como proliferação e diferenciação. COL18A1 possui dois promotores alternativos: o promotor 1, que regula a síntese da variante NC11-303, e o promotor 2 responsável pelas variantes NC11- 728 e NC11-493, expressas por hepatócitos. Existe uma variação interindividual da endostatina circulante e da expressão do colágeno XVIII no fígado. A expressão do colágeno XVIII/endostatina foi correlacionada com a progressão tanto do hepatocarcinoma (HCC), quanto da fibrose/cirrose hepática. Elucidar a regulação da expressão de COL18A1 pode auxiliar na compreensão dessa variação interindividual e da progressão dessas doenças. Neste trabalho demos início a caracterização do promotor 2 do COL18A1. Identificamos na seqüência predita como promotora cinco regiões conservadas entre humanos e camundongos. A análise in silico e funcional dessas regiões revelou que os fatores de transcrição, Sp1, Sp3, YY1, Oct-1, C/EBP&#945; e C/EBP&#946;, interagem com as mesmas. Demonstramos que C/EBP&#946;&#61472;aumenta a taxa de transcrição do promotor 2 em hepatócitos, e que existe uma correlação positiva da expressão de NC11-493 com C/EBP&#945;&#61472;e C/EBP&#946;&#61472;em tecido hepático cirrótico e tumoral. As expressões de C/EBP&#945;&#61472;em tecido hepático cirrótico e tumoral estão diretamente correlacionadas, enquanto que os níveis de NC11-493 nos tumores estão inversamente correlacionados com o tamanho dos mesmos. Mostramos a existência de diversos SNPs no promotor 2. O SNP-700T/G, funcional in vitro, afeta a interação de Sp3 e YY1 com essa região regulatória. A deleção da região do SNP indicou que ela possui elementos importantes para a transcrição em hepatócitos, apesar deste SNP não estar relacionado com o nível de expressão do colágeno XVIII em fígado fibrótico ou com susceptibilidade a HCC. O SNP- 700T/G está em desequilíbrio de ligação com o SNPc.1135C/T, no domínio frizzled do colágeno XVIII. Não foi possível elucidar a funcionalidade do SNPs c.1135C/T in vitro, mas os haplótipos formados por esses dois SNPs têm diferentes frequências entre descendentes de europeus e de africanos. Nosso trabalho traz importantes contribuições e abre novas perspectivas para a compreensão da regulação do colágeno XVIII em fígado humano, tanto em situações fisiológicas, quanto em processos fibrogênicos e tumorigênicos¶ / Collagen XVIII is a basal membrane component with several funcional domains, such as endostatin and frizzled domains, which have important roles in cellular processes such as proliferation and differentiation. COL18A1 has two promoter regions: promoter 1, that regulates the synthesis of NC11-303 isoform, and promoter 2, localized in intron 2, responsible for NC11-728 and NC11-493 isoforms expressed by hepatocytes. There is a large interindividual variation in circulating endostatin and in collagen XVIII liver expression. Collagen XVIII/endostatin levels were correlated with hepatocellular carcinoma (HCC) progression, as well as liver fibrosis/cirrhosis, conditions that precede HCC. Elucidating the mechanisms that regulate COL18A1 expression in hepatocytes may help understanding its variation among individuals and liver disease stages, as well as contribute to new treatment strategies. In this work we began to characterize COL18A1 promoter region 2. We identified in the predicted promoter sequence five conserved regions between human and mouse. The in silico and functional analysis of these regions revealed that transcription factors Sp1, Sp3, YY1, Oct-1, C/EBP&#945; and C/EBP&#946; interact with them. We have demonstrated that C/EBP&#946; increases promoter 2 transcription rate in hepatocytes, and that there is a positive correlation of NC11-493 expression with that of C/EBP&#945; and C/EBP&#946; in cirrhotic and tumor liver samples. Non-tumor and tumor C/EBP&#945; expressions positively correlate between themselves, while NC11-493 tumor expression inversely correlates with tumor size. We also showed that there are several SNPs in COL18A1 promoter 2 region. SNP-700T/G, functional in vitro, affects Sp3 and YY1 interaction with the promoter 2 region and deletion of the SNP region indicated that this sequence has important hepatocyte regulatory elements. Our results suggest that this SNP does not significantly affects COL18A1 expression in fibrotic/cirrhotic liver and is not associated with HCC susceptibility. SNP-700T/G is in linkage disequilibrium with SNPc.1135C/T, at collagen XVIII frizzled domain. We could not elucidate SNPc.1135C/T functionality in vitro, but the haplotypes formed by these two SNPs have different frequencies in European and African descendants. In conclusion, our work brings important contributions and opens new perspectives for the comprehension of collagen XVIII regulation in human liver in physiological situations, as well as in fibrotic/cirrhotic and tumorigenic process.

Este trabalho teve como objetivo desenvolver um protocolo para a diferenciação in vitro de células-tronco mesenquimais (CTM) em hepatócitos e a padronização de um modelo animal de fibrose hepática induzida por dimetilnitrosamina (DMN) para ensaios pré-clínicos de transplante de CTM. CTM isoladas de fontes variadas apresentaram morfologia fibroblastóide e aderência ao plástico e o padrão de marcadores de superfície celular esperado na análise por citometria de fluxo. A capacidade de diferenciação osteogênica e adipogênica dessas células foi comprovada pelas colorações de vermelho de alizarina, oil red e azul de toluidina, respectivamente, confirmando, que as células isoladas para este estudo se comportaram como CTM conforme proposto pela Sociedade Internacional de Pesquisa em Células-tronco. A diferenciação hepática foi avaliada quanto à morfologia e capacidade das células diferenciadas de estocar glicogênio confirmada por PAS (ácido periódico-Schiff), de sintetizar albumina confirmada por imunofluorescência, além da capacidade de expressar genes hepato-específicos verificada por ensaios de PCR em tempo real. Com base na literatura para diferenciação hepática, diferentes protocolos de um, dois e três passos foram testados. CTM humanas mostraram capacidade de produzir e estocar glicogênio e de sintetizar albumina, apenas quando diferenciadas com protocolos de três etapas, porém sem uma expressão aumentada dos genes hepato-específicos albumina, &#945;-fetoproteína e c-Met. Uma etapa de diferenciação endodérmica, previamente aplicada à diferenciação hepática, aumentou a capacidade de produzir e estocar glicogênio das CTM diferenciadas. Para a padronização do modelo de fibrose hepática induzida por DMN, foram realizados experimentos de dose-resposta e foi verificado o efeito da hepatectomia em modelos mistos DMN/hepatectomia. A injúria hepática e o efeito do transplante de CTM foram avaliados por análise macroscópica dos fígados, histologia das biópsias de fígados corados com HE e tricromo de Masson e parâmetros bioquímicos séricos. Alterações macroscópicas, histológicas e nos níveis séricos de fosfatase alcalina indicam a indução da fibrose hepática nos ratos Wistar tratados com DMN na dose de 10 &#181;g/g de peso animal por três dias consecutivos durante quatro semanas, mas não observamos nenhum efeito induzido pela hepatectomia. Porém, este modelo com DMN se mostra semelhante a estágios iniciais de uma fibrose hepática. O transplante de 1 x 107 CTM de veia de cordão umbilical humano (VCUH) no modelo de injúria hepática induzida por DMN não resultou em melhora da fibrose, diminuição dos níveis séricos de fosfatase alcalina e nem em ganho de peso dos animais quando comparados aos animais tratados com PBSA após a injúria hepática (grupo placebo). Em conjunto, esses resultados sugerem que CTM humanas se diferenciam após tratamentos mais complexos, onde os indutores hepatogênicos são sequencialmente adicionados ao meio de modo a mimetizar a sinalização durante o desenvolvimento embrionário. O transplante de CTM de VCUH parece não ter efeito positivo em um modelo pré-clínico de injúria hepática similar a estágios iniciais de fibrose. Financiado por CNPq (573578/2008-7) e FAPESP (2007/54260-2). / This study aimed to develop an in vitro differentiation protocol of mesenchymal (MSC) stem cells to hepatocytes and to standardize an animal model for hepatic fibrosis induced by dimethylnitrosamine (DMN) for preclinical transplant assays of MSC. MSC isolated from various sources presented fibroblastoid morphology, plastic adherence, and the expected pattern of cell surface markers by flow cytometry analysis. The capacity of osteogenic, adipogenic and chondrogenic differentiation of these cells was confirmed by alizarin red, oil red and toluidine blue staining, respectively, confirming that the cells isolated for this study behave as MSC, as proposed by the International Society for Stem Cell Research. Hepatogenic differentiation was evaluated by analysis of cell morphology, capacity to store glycogen confirmed by PAS (periodic acid-Schiff), albumin synthesis confirmed by immunofluorescence, as well as hepatic-specific gene expression verified by real time PCR assays. Based on the published literature on hepatic differentiation, several protocols of one, two, and three steps were tested. Human MSC differentiated solely when treated in a three step-protocol, showing the ability to produce and store glycogen and synthesize albumin; however the expression of hepatic-specific genes such as albumin, &#945;-fetoprotein and c-Met was not increased. An endoderm differentiation stage, added to the hepatic differentiation protocol, increased the capacity to produce and store glycogen of differentiated MSC. In order to standardize the model of liver fibrosis induced by DMN, dose-response experiments were performed and the effect of hepatectomy in mixed models DMN/hepatectomy was observed. Severity of liver injury and the effect of cell transplantation were evaluated by macroscopic analysis of the livers, histology of liver biopsies stained with HE and Masson\'s trichrome, and evaluation of serum biochemical parameters. The macroscopic and histological observations, and altered alkaline phosphatase serum levels indicated the success in inducing liver fibrosis in DMN-treated rats at a dose of 10 &#181;g/g of animal weight for three consecutive days, during four weeks, without any additional effect upon hepatectomy. Transplanting 1 x 107 umbilical cord MSC in the model of liver injury induced by DMN did not result in improvement of the fibrosis, decrease of alkaline phosphatase serum levels, or in weight gain of the treated animals compared to animals treated with PBSA after liver injury (placebo group). Together, these results suggest that human MSC are capable of differentiating to hepatocyte-like cells after more complex protocols, where hepatogenic inducers are sequentially added to the medium in order to mimic signaling that occurs during fetal development. Transplantation of undifferentiated umbilical cord MSC did not have any positive effect in a preclinical liver injury model characterized by an early stage of fibrosis. Supported by CNPq (573578/2008-7) and FAPESP (2007/54260-2).

No description available.

Die Elastografie ist ein diagnostisches bildgebendes Verfahren zur Bestimmung der Geschwindigkeit oder Amplitude der ins Gewebe eingebrachten Scherwellen. Die Scherwellengeschwindigkeit c in der Leber ist ein Marker zur Fibrosegraduierung. Pathologische Elastizitätsveränderungen, wie sie bei der Herzinsuffizienz auftreten können mittels der Scherwellenamplitude in der Herzwand erfasst werden. Bisherige Verfahren sind durch eine geringe Eindringtiefe/Zeitauflösung limitiert. Dies erschwert die Untersuchung der Leber bei Patienten mit Adipositas oder Aszites und am Herzen. Daher wird in dieser Arbeit die zeitharmonische Vibrationen zur Anregung von Scherwellen im tiefen Gewebe mit der zeiteffizienten Ultraschallelastografie kombiniert. In der Leber wurde c als Maß für die Elastizität und die Dispersion von c als Maß für die Viskosität in 11 Probanden und 24 Patienten mit Leberzirrhose gemessen. Im Herzen wurde die Amplitude als Maß für die relative Elastizitätsänderung des Herzmuskels in Echtzeit bei 11 Probanden und einer Patientin mit Herzinsuffizienz bestimmt. In der Leber ergab sich für Probanden c=1,77(15)ms und für Patienten mit Zirrhose c=3,10(55)ms. Diese 100%ige Separation bestätigt die Stabilität und das diagnostische Potential des neuen Verfahrens. Für die Probanden ergab sich eine Abnahme der Scherwellenamplitude in der Herzwand 121(34)ms vor der Kontraktion des Herzens und ein Anstieg 56(29)ms vor der Relaxation entsprechend den isovolumetrischen Kontaktions- und Relaxationszeiten. Die zeitharmonische Ultraschallelastografie erlaubt erstmalig die Bestimmung der Scherwellengeschwindigkeit der gesamten Leber auch bei Adipositas und Aszites. Dies erweitert den Anwendungsbereich der Ultraschallelastografie in der klinischen Diagnostik wesentlich. Zudem wurde die Anspannungsänderung des Herzmuskels in vivo und in Echtzeit bestimmt. Diese neue Modalität bietet erstmalig die Möglichkeit pathologische Herzveränderungen mittels Elastografie zu diagnostizieren. / Elastography is a diagnostic imaging technology for the quantification of speed or amplitude of external induced shear waves. The shear wave speed c is used as diagnostic marker for the staging of liver fibrosis. In contrast, the shear wave amplitude in the myocardial wall is sensitive to pathologic elasticity alterations induced by cardiac insufficiency. Current methods have a less penetration depth or low spatial resolution. This limits the investigation of liver in patients with ascites or adiposity and of the heart. Therefore the time harmonic vibration, inducing shear waves in deep tissue is combined with the time efficient ultrasound elastography in this work. In the liver, c and the dispersion of c as marker for elasticity and for viscosity respectively was measured in 11 volunteers and 24 patient with cirrhosis. Relative elasticity changes of the myocardial muscle were assessed by measuring shear wave amplitude in real-time in 11 volunteers. As an outlook, one patient with diastolic dysfunction was investigated. The shear wave speed in liver is c=1.77(15)ms for the volunteers and c=3.10(55)ms for the patients with cirrhosis. The 100%age separation between both groups confirms the stability and the diagnostic potential of these new method. In volunteers the shear wave amplitude in the myocardial wall decreases by 121(34)ms prior to heart contraction and increases by 56(29)ms prior to relaxation. This times correlate to the cardiac isovolumetric contraction and relaxation times. for the first times, the new time harmonic ultrasound elastography allows the quantification of shear wave speed in the whole liver even in patients with ascites or adiposity. This extends the field of application for liver-elastography in clinical diagnostics substantially. Furthermore the tension changes in the myocardial wall can be measured in vivo and in real-time. For the first time, this new modality opens the possibility to diagnose pathological alterations by elastography.

Magnetresonanzelastographie (MRE) bietet die Möglichkeit, über die Aufnahme mechanischer Scherwellen im Körper auf die mechanischen Eigenschaften lebender Gewebe zu schließen. Dabei werden in der klassischen MRE Wellen mittels Einkopplung externer Vibrationen einer einzelnen Frequenz angeregt. Wegen der starken Absorption der Vibrationsenergie in biologischen Geweben und der damit verbundenen Dispersion der Phasengeschwindigkeit sowie der Dämpfung der Wellen können mit dieser Methode nur frequenzabhängige Größen, jedoch keine Materialkonstanten bestimmt werden. Die in dieser Arbeit entwickelte Methode erlaubt die synchrone Einkopplung und Aufnahme multipler Gewebeschwingungen, wodurch viskoelastische Gewebekenngrößen in einer zeitlich-zyklisierten MRE-Untersuchung erfasst werden können. Diese Technik wird in Phantomstudien, an Gewebeproben sowie am Menschen evaluiert. Mittels verschiedener rheologischer Modelle werden erstmalig die viskoelastischen Eigenschaften der Leber und des Gehirns in ihrem intakten, lebenden Umfeld bestimmt. Dabei zeigt sich die Überlegenheit eines zweiparametrischen Modells, mit dessen Hilfe die gewonnene, spektrale Information des komplexen Moduls des Gewebes in eine einzige diagnostisch-relevante Kenngröße zusammengefasst werden kann. / Magnetic resonance elastography (MRE) is capable of measuring the mechanical properties of living tissue by using externally introduced vibrations and phase contrast magnetic resonance imaging techniques. Until now, monofrequency shear wave excitation techniques have been used in conventional MRE. However, since biological tissue is highly dispersive due to its strong damping characteristics, the study of tissue rheology requires knowledge of wave propagation at multiple frequencies. The multifrequency-MRE method, which was engineered in this thesis, applies a superposition of multiple harmonics as the shear wave excitation signal. All vibrations are acquired simultaneously, which enables the determination of viscoelastic tissue parameters in one time-resolved MRE experiment. This technique is evaluated in studies on gel phantoms and excised tissue samples, as well as in human in-vivo studies. The viscoelastic properties of human brain and liver are determined in their in-vivo environment using several rheological models. A two-parameter fractional model demonstrates excellent stability and allows for combining the spectral information of the complex modulus acquired by multifrequency-MRE, which then results in a single viscoelastic parameter that is diagnostically relevant.

Le carcinome hépatocellulaire (CHC) est un cancer au pronostic sombre, car il est souvent diagnostiqué trop tardivement pour entreprendre un traitement curatif. Il se développe dans 80-90% des cas sur fond de cirrhose. On connait mal comment la fibrose, étape préliminaire à la cirrhose, et son principal constituant, le collagène de type 1 (COL1), peuvent jouer un rôle dans le processus du CHC. Nous avons tout d’abord étudié le développement de la fibrose dans un modèle utilisant la souris nue. Nous avons déterminé qu’après 16 semaines d’administration de thioacétamide dans l’eau de boisson, il est possible d’obtenir une fibrose suffisante pour induire une hépatoprotection en présence de différents hépatotoxiques (AST dans le sérum de souris fibrotiques vs non-fibrotiques : Anti-Fas JO2 (4665 ± 2596 vs. 13953 ± 2260 U/L; P<0.05), acétaminophène (292 ± 66 vs. 4087 ± 2205 U/L; P<0.01) et CCL4 (888 ± 268 vs. 15673 ± 2782 U/L; P<0.001)). Ces résultats confirment que la présence de COL1 et de fibrose favorise la survie des hépatocytes normaux tel qu’observé précédemment au laboratoire. Par la suite, nous avons sélectionné in vivo, par injection intrasplénique de la lignée de CHC Hepa1-6, une lignée à forte tumorigénicité nommée dt-Hepa1-6 (28±12 lésions vs. 0±0 lésions à 21 jours). Cette lignée était composée d’une sous-population cellulaire arborant la protéine de surface EpCAM (34.0±0.1%). Par tri cellulaire, nous avons démontré que ces cellules étaient partiellement responsables de la tumorigénicité accrue (EpCAM + (86.7±2.3%) :1093±74 lésions vs. EpCAM- (15.3±1.0%) :473±100 lésions; P<0.01). Nous avons alors démontré que la présence de fibrose favorise le développement de la lignée dt-Hepa1-6 in vivo (604±242 vs 22±9 lésions; P<0.05). De plus, la présence de fibrose réduit l’efficacité du traitement au cisplatin in vivo (44.5±4.9 vs. 78.7±6.9%; P<0.01) confirmant les résultats obtenus in vitro (Apoptose : COL1 13.75±0.44% vs. plastique 31.45±1.37%; P<0.001). En conclusion, la présence de fibrose et de son principal constituant, le COL1, favorise la survie et la progression du CHC. / Hepatocellular carcinoma (HCC) is a dreadful pathology, often diagnosed too late to be cured. In 80-90% of cases, it arises in the context of liver cirrhosis. Little is known on the implication of liver fibrosis, one of the key elements of cirrhosis, and its major constituent, type I collagen (COL1), on the development of HCC. We first studied the development of fibrosis in a nude mouse model. We determined that, after 16 weeks of thioacetamide administration in drinking water, we obtained a sufficient degree of fibrosis to reach a hepatoprotective state when animals were exposed to different hepatotoxic agents (Serum AST of fibrotic vs non-fibrotic mice : Anti-Fas JO2 (4665 ± 2596 vs. 13953 ± 2260 U/L; P<0.05), acetaminophen (292 ± 66 vs. 4087 ± 2205 U/L; P<0.01) et CCL4 (888 ± 268 vs. 15673 ± 2782 U/L; P<0.001)). This confirmed that COL1 and the presence of fibrosis protects normal hepatocytes as observed previously in our laboratory. Next, we selected in vivo, by intrasplenic injection of the murine HCC cell line Hepa1-6, a highly tumorigenic cell line that we named dt-Hepa1-6 (28±12 lesions vs. 0±0 lesions at 21 days). This cell line was constituted of cell subsets expressing EpCAM protein at their surface (34.0±0.1%). Through cell sorting, we demonstrated that these cells were partially responsible for the enhanced tumorigenicity observed (EpCAM + (86.7±2.3%) :1093±74 lesions vs. EpCAM- (15.3±1.0%) :473±100 lesions; P<0.01). We then showed that the presence of liver fibrosis increases the development of dt-Hepa1-6 cell line in vivo (604±242 vs 22±9 lesions; P<0.05). Moreover, fibrosis reduced the anti-neoplastic efficacy of cisplatinum in vivo (44.5±4.9 vs. 78.7±6.9%; P<0.01) confirming in vitro results (Apototic index : COL1 13.75±0.44% vs. plastic 31.45±1.37%; P<0.001). In conclusion, fibrosis and its major constituent, COL1, favor the survival and progression of HCC.