Introduction and methods: Hepatocellular carcinoma (HCC) remains a difficult disease to study even after a decade of genomic analysis. Metabolic and cell-cycle perturbations are known, large changes in tumors that add little to our understanding of the development of tumors, but generate “noise” that obscures potentially important smaller scale expression changes in “driver genes”. Recently, some researchers have suggested that HCC shares pathways involving the master regulators of embryonic development. Here, we investigated the involvement and specificity of developmental genes in HCV-cirrhosis and HCV-HCC. We obtained microarray studies from 30 patients with HCV-cirrhosis and 49 patients with HCV-HCC and compared to 12 normal livers. Differential gene expression is specific to liver development genes: 86 of 202 (43%) genes specific to liver development had differential expression between normal and cirrhotic or HCC samples. Of 60 genes with paralogous function, which are specific to development of other organs and have known associations with other cancer types, none were expressed in either adult normal liver or tumor tissue. Developmental genes are widely differentially expressed in both cirrhosis and early HCC, but not late HCC: 69 liver development genes were differentially expressed in cirrhosis, and 58 of these (84%) were also dysregulated in early HCC. 19/58 (33%) had larger-magnitude changes in cirrhosis and 5 (9%) had larger-magnitude changes in early HCC. 16 (9%) genes were uniquely altered in early tumors, while only 2 genes were uniquely changed in late-stage (T3 and T4) HCC. Together, these results suggest that the involvement of the master regulators of liver development are active in the pre-cancerous cirrhotic liver and in cirrhotic livers with emerging tumors but play a limited role in the transition from early to late stage HCC. Common patterns of coordinated developmental gene expression include: (1) Dysregulation of BMP2 signaling in cirrhosis followed by overexpression of BMP inhibitors in HCC. BMP inhibitor GPC3 was overexpressed in nearly all tumors, while GREM1 was associated specifically with recurrence-free survival after ablation and transplant. (2) Cirrhosis tissues acquire a progenitor-like signature including high expression of Vimentin, EPCAM, and KRT19, and these markers remain over-expressed to a lesser extent in HCC. (3) Hepatocyte proliferation inhibitors (HPI) E-cadherin (CDH1), BMP2, and MST1 were highly expressed in cirrhosis and remained over-expressed in 16 HCC patients who were transplanted with excellent recurrence-free survival (94% survival after 2 years; mean recurrence-free survival = 5.6 yrs), while loss in early HCC was associated with early recurrence and (2 year). Loss of HPI overexpression was also correlated with overexpression of c-MET and loss of STAT3, LAMA2, FGFR2, CITED2, KIT, SMAD7, GATA6, ERBB2, and NOTCH2.

Statisticians are used to model interactions between variables on the basis of observed data. In a lot of emerging fields, like bioinformatics, they are confronted with datasets having thousands of variables, a lot of noise, non-linear dependencies and, only, tens of samples. The detection of functional relationships, when such uncertainty is contained in data, constitutes a major challenge. Our work focuses on variable selection and network inference from datasets having many variables and few samples (high variable-to-sample ratio), such as microarray data. Variable selection is the topic of machine learning whose objective is to select, among a set of input variables, those that lead to the best predictive model. The application of variable selection methods to gene expression data allows, for example, to improve cancer diagnosis and prognosis by identifying a new molecular signature of the disease. Network inference consists in representing the dependencies between the variables of a dataset by a graph. Hence, when applied to microarray data, network inference can reverse-engineer the transcriptional regulatory network of cell in view of discovering new drug targets to cure diseases. In this work, two original tools are proposed MASSIVE (Matrix of Average Sub-Subset Information for Variable Elimination) a new method of feature selection and MRNET (Minimum Redundancy NETwork), a new algorithm of network inference. Both tools rely on the computation of mutual information, an information-theoretic measure of dependency. More precisely, MASSIVE and MRNET use approximations of the mutual information between a subset of variables and a target variable based on combinations of mutual informations between sub-subsets of variables and the target. The used approximations allow to estimate a series of low variate densities instead of one large multivariate density. Low variate densities are well-suited for dealing with high variable-to-sample ratio datasets, since they are rather cheap in terms of computational cost and they do not require a large amount of samples in order to be estimated accurately. Numerous experimental results show the competitiveness of these new approaches. Finally, our thesis has led to a freely available source code of MASSIVE and an open-source R and Bioconductor package of network inference.

The emergence of drug-resistance of tumor cells is a major complication for succesful chemotherapy. In this study, the molecular mechanisms of resistance to prednisone, vincristine and melphalan in multiple myeloma cell lines, RPMI-8226 and U-266 were investigated. Drug resistance was induced by application of the drugs by stepwise dose increments and confirmed by XTT cytotoxicity assay. Gene expression analysis demostrated that MDR1 gene is one of the most important factor causing the multidrug resistance phenotype in prednisone, vincristine and melphalan resistant multiple myeloma cell lines. According to microarray analysis alterations in laminin, integrin and collagen genes were detected. Additionally, upregulation of some oncogenes and growth factors (Rho family of GTPases, YES1, ACT2, TGFBR, EPS15, PDGF) was shown to have a role in MDR in multiple myeloma. Significant downregulation of suppressors of cytokine signalling gene expressions and upregulation of different types of interleukine and interferon gene expressions (IL3 and interferon-gamma receptor) which are related to JAK-STAT signalling pathay was shown. Alterations in expression levels of genes related to ceramide metabolism were shown especially for melphalan resistance in multiple myeloma. The data from vincristine/prednisone and vincristine/melphalan drug combination studies were shown that the usage of vincristine on prednisone and melphalan resistant multiple myeloma cell lines increase the efficacy of the chemotherapy. On the other hand the cross-resistance development of prednisone and melphalan resistant sublines to irradiation was detected. These results may help to understand the molecular mechanisms of prednisone, vincristine and melphalan resistance in multiple myeloma model cell lines RPMI-8226 and U-266.

Adult schistosomes depend for growth and development on hormonal signals from the mammalian host, which may include the insulin signalling pathway. In this project, I firstly used microarray analysis to demonstrate that human insulin can be utilised by adult S. japonicum in culture, resulting in the modulation of distinct metabolic effects as reflected in transcriptional levels of parasite genes. The addition of insulin resulted in the differential expression of 1,101 genes with many related to functions corresponding to the biological and metabolic effects of insulin reported for mammalian cells. Those identified genes in male or female S. japonicum worms that were up or down regulated after exposure to insulin were predominantly involved in growth and development, with significant sex-specific responses evident. Insulin appeared to play a similar role in male parasites as those seen in classical mammalian systems including an increase in protein synthesis though gene transcription and the stimulation of mRNA translation and control protein degradation via the ubiquitin proteasome pathway. Microarray analysis indicated that insulin not only leads to increased gene expression of the PI3-K pathway, which enhances parasite growth, but may also play a role in the sexual differentiation and fecundity of female worms by activating the MAPK pathway. As the insulin target proteins, two types of insulin receptors from Schistosoma japonicum were isolated, S. japonicum insulin receptors 1 (SjIR-1) and 2 (SjIR-2), with features similar to insulin receptors from other taxa. The sequences share 70% and 74% identity to S. mansoni insulin receptor 1 and 2 (SmIR-1 and SmIR-2), respectively. SjIR-1 and SjIR-2 are highly conserved in their tyrosine kinase domain to other IRs from Homo, Mus musculus and Drosophila melanogaster. SjIR-2 is located in the parenchyma in males and in the vitelline glands of female worms, which occupy most of male or female tissue and play an important role in growth or fecundity. In contrast, SjIR-1 was located in the tegument and intestinal epithelium of adult worms, representing much smaller cellular regions compared with the voluminous vitelline tissue or parenchyma. This observation was further confirmed by real time PCR showing that SjIR-2 was more abundantly expressed in S. japonicum adult worm than SjIR-1. Phylogenetic analysis showed that SjIR-2 and SmIR-2 are closer to EmIR than to SjIR-1 and SmIR-1, indicating that SjIR-1 and SmIR-1 might perform specific functions in schistosomes, while SjIR-2, SmIR-2 and EmIR might share similar roles in parasite growth and development in the three parasitic flatworms. Structure modelling recovered the conserved structure between the SjIRs and Homo sapiens IR (HIR) implying a common predicted binding mechanism in the ligand domain and the same downstream signal transduction processing in the tyrosine kinase domain as in HIR. Two-hybrid analysis was used to confirm that the ligand domains of SjIR-1 and SjIR-2 contain the insulin binding site. Incubation of adult worms in vitro, both with a specific insulin receptor inhibitor and anti-SjIRs antibodies, resulted in a significant decrease in worm glucose levels, suggesting again the same function for SjIRs in regulating glucose uptake as described for mammalian cells. Adult worms of S. japonicum possess insulin receptors that can specifically bind to insulin, indicating that the parasite can utilize host insulin for development and growth by sharing the same pathway as mammalian cells in regulating glucose uptake. In vaccination/challenge trials, there was no significant reduction in adult worm burdens with either of the SjLD vaccines. However, there were significant reductions in mean lengths of adult worms ranging from 22-25% in the SjLD1 vaccinated group to 37-42% in the SjLD2 vaccinated groups, significant reductions in faecal eggs in both the SjLD1 (66%) and SjLD2 (68%) vaccinated groups, and a reduction in liver egg numbers in the SjLD1(33%) vaccinated group. These results show that although the SjLDs vaccines were unable to reduce adult worm numbers by clearing them from the vaccinated mice, nevertheless, they significantly depressed the growth of male and female adult worms and affected female egg production. The protective efficacy obtained in terms of the substantial decrease in faecal eggs exceeded that of many of the recently available schistosome antigens and prototype vaccine formulations, which, at best, elicit 40–50% protection in animals using the standard readouts of reduced worm burden or egg production and viability. Overall, disruption of this insulin pathway leading to parasite starvation through the prevention of glucose uptake thereby affecting parasite growth, development and female fecundity, provides a new intervention target and transmission blocking approach to combat schistosomiasis and may be applicable for the control of other debilitating parasitic infections as well.

Diss. (sammanfattning) Göteborg : Univ., 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 4 uppsatser.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Partial embargo. Vita. Bibliography: References located at the end of each study.

Thesis--University of Oklahoma. / Bibliography: leaves 82-83.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 134 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 163-183).