Hepatitis C Virus (HCV) infects 170 million people worldwide. The current treatment regimen, which is combination therapy with pegylated interferon (PegIFN) and Ribavirin (Rib), cures only 50% of the patients infected with the most prevalent HCV genotype. Therefore, there is a pressing need to understand the molecular mechanism of interferon resistance and to develop a prognostic tool to predict who will respond to treatment before initiation of therapy. It has been firmly established that the virus-host interaction plays an important role in determining treatment outcomes. My thesis investigated the host factors that are involved in interferon resistance with an aim to provide insights into the molecular mechanism of IFN resistance. cDNA microarray analysis identified 18 differentially expressed hepatic genes from pretreatment liver tissues of responders (Rs) and non-responders (NRs). Based on the differential expression levels of these 18 genes, a prognostic tool was developed to predict who will respond to therapy, with a positive predicting value (PPV) of 96%. Most of these 18 genes are interferon stimulated genes (ISGs) and they are more highly expressed in NR livers, indicating that preactivation of interferon signaling in the pre-treatment liver tissues contributes to NR. 3 out of the 18 genes are involved in an ubiquitin-like ISG15/USP18 signaling pathway that plays an important role in interferon response. Over-expression of USP18 and ISG15 in the pretreatment liver tissues of NR promotes HCV production and blunts interferon anti-HCV activity. There exists a distinct cell-type specific ISG activation in the pretreatment liver tissues of Rs and NRs. Up-regulation of the two ISGs that I tested (ISG15 and MxA) was found mainly in hepatocytes in NRs while ISG activation was preferentially observed in macrophages in Rs. Taking all these data together, pre-activation of interferon signaling and cell-type specific gene activation in the pretreatment liver tissues of patients infected with HCV are associated with treatment non-response. HCV exploits the host interferon system to favour its persistence by enhanced replication /secretion stimulated by a few ISGs (ISG15, USP18) in response to IFN. The developed prognostic tool can be used to stratify patients for treatment and the novel insights of the molecular mechanism of IFN resistance in HCV patients offer potential drug targets for future development.

Despite improvements in the clinical management of soft tissue sarcomas (STS), 50% of patients will die of metastatic disease that is largely unresponsive to conventional chemotherapeutic agents. The aims of this study were to identify genes and pathways that are dysregulated in progressive and metastatic STS. In addition to this, cell lines from fresh tumours were initiated and established, thus increasing the repository of cell lines available for functional studies. Recent advances in the understanding of the molecular biology of STS have thus far not resulted in the use of molecular markers for clinical prognostication. Identifying novel genes and pathways will lead to molecular diagnostic methods to better stratify prognostic groups and could identify cellular targets for more efficacious treatments. Gene expression profiling of sarcoma cell lines of increasing metastatic potential revealed over-expression of genes involved in the epidermal growth factor (EGF) and transforming growth factor beta (TGFb) pathways. Factors involved in invasion and metastasis such as integrins and MMPs were over-expressed in the cell lines with higher metastatic potential. The developmental Notch pathway and cell cycle regulators were also dysregulated. NDRG1 was significantly over-expressed in the high grade sarcoma cell line, a novel finding in sarcomas. The expression of EGFR, NDRG1 and other genes from the above pathways was validated using quantitative RT-PCR in real time (qRT-PCR). A tissue microarray (TMA) comprising STS of varying tumour grades was constructed for high throughput assessment of target proteins. EGFR, its activated form and its signal transducers were investigated using immunohistochemistry (IHC). Activated EGFR (HR 2.228, p < 0.001) and phosphorylated Akt (HR 2.032, p = 0.003) were found to be independent predictors of overall survival and both correlated with tumour grade. Of the several STS cultures initiated and maintained, two of these cell lines were fully characterised in terms of cytogenetics, telomerase and alternate lengthening of 5 telomeres (ALT) status, KIT and TP53 mutation and the expression of certain biomarkers using both qRT-PCR and IHC. In summary, transcript profiling identified several potential biomarkers of tumour progression and metastasis in STS. Crucially, activated EGFR and pAkt were found in a cohort of STS samples to correlate with clinical outcome, identifying them as potential diagnostic and therapeutic targets in the treatment of STS. Activated EGFR can be used as a diagnostic marker for patient selection, as well as for target effect monitoring. Furthermore, the cell lines established in this project will serve as valuable tools in future preclinical studies.

Despite improvements in the clinical management of soft tissue sarcomas (STS), 50% of patients will die of metastatic disease that is largely unresponsive to conventional chemotherapeutic agents. The aims of this study were to identify genes and pathways that are dysregulated in progressive and metastatic STS. In addition to this, cell lines from fresh tumours were initiated and established, thus increasing the repository of cell lines available for functional studies. Recent advances in the understanding of the molecular biology of STS have thus far not resulted in the use of molecular markers for clinical prognostication. Identifying novel genes and pathways will lead to molecular diagnostic methods to better stratify prognostic groups and could identify cellular targets for more efficacious treatments. Gene expression profiling of sarcoma cell lines of increasing metastatic potential revealed over-expression of genes involved in the epidermal growth factor (EGF) and transforming growth factor beta (TGFb) pathways. Factors involved in invasion and metastasis such as integrins and MMPs were over-expressed in the cell lines with higher metastatic potential. The developmental Notch pathway and cell cycle regulators were also dysregulated. NDRG1 was significantly over-expressed in the high grade sarcoma cell line, a novel finding in sarcomas. The expression of EGFR, NDRG1 and other genes from the above pathways was validated using quantitative RT-PCR in real time (qRT-PCR). A tissue microarray (TMA) comprising STS of varying tumour grades was constructed for high throughput assessment of target proteins. EGFR, its activated form and its signal transducers were investigated using immunohistochemistry (IHC). Activated EGFR (HR 2.228, p < 0.001) and phosphorylated Akt (HR 2.032, p = 0.003) were found to be independent predictors of overall survival and both correlated with tumour grade. Of the several STS cultures initiated and maintained, two of these cell lines were fully characterised in terms of cytogenetics, telomerase and alternate lengthening of 5 telomeres (ALT) status, KIT and TP53 mutation and the expression of certain biomarkers using both qRT-PCR and IHC. In summary, transcript profiling identified several potential biomarkers of tumour progression and metastasis in STS. Crucially, activated EGFR and pAkt were found in a cohort of STS samples to correlate with clinical outcome, identifying them as potential diagnostic and therapeutic targets in the treatment of STS. Activated EGFR can be used as a diagnostic marker for patient selection, as well as for target effect monitoring. Furthermore, the cell lines established in this project will serve as valuable tools in future preclinical studies.

Despite improvements in the clinical management of soft tissue sarcomas (STS), 50% of patients will die of metastatic disease that is largely unresponsive to conventional chemotherapeutic agents. The aims of this study were to identify genes and pathways that are dysregulated in progressive and metastatic STS. In addition to this, cell lines from fresh tumours were initiated and established, thus increasing the repository of cell lines available for functional studies. Recent advances in the understanding of the molecular biology of STS have thus far not resulted in the use of molecular markers for clinical prognostication. Identifying novel genes and pathways will lead to molecular diagnostic methods to better stratify prognostic groups and could identify cellular targets for more efficacious treatments. Gene expression profiling of sarcoma cell lines of increasing metastatic potential revealed over-expression of genes involved in the epidermal growth factor (EGF) and transforming growth factor beta (TGFb) pathways. Factors involved in invasion and metastasis such as integrins and MMPs were over-expressed in the cell lines with higher metastatic potential. The developmental Notch pathway and cell cycle regulators were also dysregulated. NDRG1 was significantly over-expressed in the high grade sarcoma cell line, a novel finding in sarcomas. The expression of EGFR, NDRG1 and other genes from the above pathways was validated using quantitative RT-PCR in real time (qRT-PCR). A tissue microarray (TMA) comprising STS of varying tumour grades was constructed for high throughput assessment of target proteins. EGFR, its activated form and its signal transducers were investigated using immunohistochemistry (IHC). Activated EGFR (HR 2.228, p < 0.001) and phosphorylated Akt (HR 2.032, p = 0.003) were found to be independent predictors of overall survival and both correlated with tumour grade. Of the several STS cultures initiated and maintained, two of these cell lines were fully characterised in terms of cytogenetics, telomerase and alternate lengthening of 5 telomeres (ALT) status, KIT and TP53 mutation and the expression of certain biomarkers using both qRT-PCR and IHC. In summary, transcript profiling identified several potential biomarkers of tumour progression and metastasis in STS. Crucially, activated EGFR and pAkt were found in a cohort of STS samples to correlate with clinical outcome, identifying them as potential diagnostic and therapeutic targets in the treatment of STS. Activated EGFR can be used as a diagnostic marker for patient selection, as well as for target effect monitoring. Furthermore, the cell lines established in this project will serve as valuable tools in future preclinical studies.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. Stockholm : Karolinska institutet, 2007.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.