Controlling transduction and replication of oncolytic adenoviruses

Despite progress in conventional cancer treatment regimes, metastatic disease essentially remains incurable and new treatment alternatives are needed. Virotherapy is a relatively novel approach in cancer treatment. It harnesses the natural ability of oncolytic viruses to kill the cells they proliferate in and to spread to neighboring cells, thereby amplifying the therapeutic effect of the initial input dose. The use of replicating, oncolytic viruses for cancer treatment necessitates introduction of various genetic modifications to the viral genome, thereby restraining replication exclusively to tumor cells and eventually obtaining selective eradication of the tumor without side effects to healthy tissue. Furthermore, various modifications can be applied to the viral capsid in hope of gaining effective transduction of target tissue. In other words, the entry of viruses into tumor tissue can be augmented by allowing the virus to utilize non-native receptors for entry. Genetic capsid modifications may also help to avoid some major hurdles in systemic delivery that ultimately lead to the rapid clearance of the virus from the blood and virus induced toxicity. In addition to genetic modifications that alter the phenotype of the virus, some pharmacologic agents may be utilized to enhance the virus entry to target site. Liver kupffer cells (KC) are responsible for the majority of viral clearance after systemic viral delivery and they play a major role in adenovirus induced acute toxicity. The therapeutic window could possibly be widened by transiently depleting KCs, allowing smaller viral input doses and diminishing KC related toxicity. The transductional efficacy of various capsid modified viruses was analyzed in vitro and in vivo in murine orthotopic breast cancer model. The effect of capsid modifications on the oncolytic efficacy, i.e. the ability of the viruses to kill cancer cells, was evaluated in vitro and in vivo in murine cancer models. We concluded that capsid modifications result in transductional enhancement, and that enhanced transduction translates into more potent oncolysis in vitro and in vivo. When KC depleting agents were used in vivo prior to viral injections, enhanced tumor transduction was seen, but this effect was not translated into enhanced antitumor activity. Transcriptional regulation of replicative oncolytic viruses is a prerequisite for virotherapy. Tumor or tissue specific promoters can be used to control the transcription of adenoviral early genes to gain cancer specific viral replication. Specific deletions in viral regions essential for virus replication in normal cells can further increase the safety by allowing viral genome replication in cancer cells featuring specific mutations. Genetically modified viruses were shown to be able to kill putative cancer stem cells that are thought to be responsible for post treatment relapses and metastasis. Further, pharmacologic intervention reduced viral replication and thereby might offer an additional safety switch in case viral replication related side effects are encountered.

Genetic alterations in microsatellite-unstable colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in Finland. Of all CRC tumors, 15% display microsatellite-instability (MSI) caused by defective cellular mismatch repair. Cells displaying MSI accumulate a high number of mutations genome-wide, especially in short repeat areas, microsatellites. When targeting genes essential for cell growth or death, MSI can promote tumorigenesis. In non-coding areas, microsatellite mutations are generally considered as passenger events. Since the discovery of MSI and its linkage to cancer, more that 200 genes have been investigated for a role in MSI tumorigenesis. Although various criteria have been suggested for MSI target gene identification, the challenge has been to distinguish driver mutations from passenger mutations. This study aimed to clarify these key issues in the research field of MSI cancer. Prior to this, background mutation rate in MSI cancer has not been studied in a large-scale. We investigated the background mutation rate in MSI CRC by analyzing the spectrum of microsatellite mutations in non-coding areas. First, semenogelin I was studied for a possible role in MSI carcinogenesis. The intronic T9 repeat of semenogelin I was frequently mutated but no evidence for selection during tumorigenesis was obtained. Second, a sequencing approach was utilized to evaluate the general background mutation rate in MSI CRC. Both intronic and intergenic repeats harbored extremely high mutation rates of ≤ 87% and intergenic repeats were more unstable than the intronic repeats. As mutation rates of presumably neutral microsatellites can be high in MSI CRC in the absence of apparent selection pressure, high mutation frequency alone is not sufficient evidence for identification of driver MSI target genes. Next, an unbiased approach was designed to identify the mutatome of MSI CRC. By combining expression array data and a database search we identified novel genes possibly related to MSI CRC carcinogenesis. One of the genes was studied further. In the functional analysis this gene was observed to cause an abnormal cancer-prone cellular phenotype, possibly through altered responses to DNA damage. In our recent study, smooth muscle myosin heavy chain 11 (MYH11) was identified as a novel MSI CRC gene. Additionally, MYH11 has a well established role in acute myeloid leukemia (AML) through an oncogenic fusion protein CBFB-MYH11. We investigated further the role of MYH11 in AML by sequencing. Three novel missense variants of MYH11 were identified. None of the variants were present in the population-based control material. One of the identified variants, V71A, lies in the N-terminal SH3-like domain of MYH11 of unknown function. The other two variants, K1059E and R1792Q are located in the coil-coiled myosin rod essential for the regulation and filament formation of MYH11. The variant K1059E lies in the close proximity of the K1044N that has been functionally assessed in our earlier work of CRC and has been reported to cause total loss of MYH11 protein regulation. As the functional significance of the three novel variants examined in this work remains unknown, future studies should clarify the further role of MYH11 in AML leukaemogenesis and in other malignancies.

Molecular profiling of malignant pleural mesothelioma and pulmonary fibrosis

Malignant mesothelioma (MM) is a rare, usually incurable, disease mainly caused by former exposure to asbestos. Even though MM has a strong etiological link, genetic factors may play a role, since not all cases can be linked to former asbestos exposure. This thesis focuses on lung diseases, mainly malignant mesothelioma (MM), and idiopathic pulmonary fibrosis (IPF), which resembles asbestosis. The specific asbestos-related pathways associated with malignant as well as non-malignant lung diseases, still need to be clarified. Since most patients diagnosed with MM or asbestosis/fibrosis have a dismal prognosis and few therapeutic options are available, early diagnosis and better understanding of the disease pathogenesis are of the utmost importance. The first objective of this thesis was to identify asbestos specific differentially expressed genes. This was approached by using high-resolution gene expression arrays, and three different human lung cell lines, as well as with three different bioinformatics approaches. Since the first study aimed to elucidate potential early changes, the second study was used to screen DNA copy number changes in MM tumour samples. This was performed using genome wide microarrays for identification of DNA copy number changes characterstic for MM. Study III focused on the role of gremlin in the regulation of bone morphogenetic protein (BMPs) in IPF. Further studies were conducted in asbestos-exposed cell cultures as well as in an asbestos-induced mouse model. Furthermore, GATA-6 was studied in MM and metastatic pleural adenocarcinoma. The GATA transcription factors are important during embryonic development, but their role in cancer is still unclear. GATA-6 is a co-factor/target of thyroid transcription factor 1 (TTF-1), which is used in differential diagnostics of pleural MM and adenocarcinoma. Bioinformatics probed the genes and biological processes ordered in terms of significance, clusters, and highly enriched chromosomal regions. The study revealed several already identified targets, produced new ideas about genes which are central for asbestos exposure, as well as provided supplementary data for researchers to check their own novel findings or ideas. The analysis revealed DNA copy number changes characteristic for MM tumors. The most common regions of loss were detected in 1p, 3p, 6q, 9p, 13, 14, and 22, and gains at 17q. The histological features in asbestosis and IPF are very similar, wherefore IPF can be studied in asbestos models. The BMP antagonist gremlin was up-regulated by asbestos exposure in human epithelial cell lines, which was also observed in Study I. The transforming growth factor (TGF) -β and BMP expression and signaling activities were measured from murine and human fibrotic lungs. BMP-7 signaling was down-regulated in response to up-regulation of gremlin, and restoration of BMP-7 signaling prevented progression of fibrosis in mice. Therefore, the study suggests that the restoration of BMP-7 signaling in fibrotic lung could potentially aid in the treatment of IPF patients. Study IV revealed that GATA-6 was strongly expressed in the majority of the MM cases, and correlated statistically significant with longer survival in subgroups of MM.

Regulation of strawberry growth and development

Strawberries (Fragaria sp.) are adapted to diverse environmental conditions from the tropics to about 70ºN, so different responses to environmental conditions can be found. Most genotypes of garden strawberry (F. x ananassa Duch.) and woodland strawberry (F. vesca L.) are short-day (SD) plants that are induced to flowering by photoperiods under a critical limit, but also various photoperiod x temperature interactions can be found. In addition, continuously flowering everbearing (EB) genotypes are found. In addition to flowering, axillary bud differentiation in strawberry is regulated by photoperiod. In SD conditions, axillary buds differentiate to rosette-like structures called "branch crowns", whereas in long-day conditions (LD) they form runners, branches with 2 long internodes followed by a daughter plant (leaf rosette). The number of crown branches determines the yield of the plant, since inflorescences are formed from the apical meristems of the crown. Although axillary bud differentiation is an important developmental process in strawberries, its environmental and hormonal regulation has not been characterized in detail. Moreover, the genetic mechanisms underlying axillary bud differentiation and regulation of flowering time in these species are almost completely unresolved. These topics have been studied in this thesis in order to enhance strawberry research, cultivation and breeding. The results showed that 8-12 SD cycles suppressed runner initiation from the axillary buds of the garden strawberry cv. Korona with the concomitant induction of crown branching, and 3 weeks of SD was sufficient for the induction of flowering in the main crown. Furthermore, a second SD treatment given a few weeks after the first SD period can be used to induce flowering in the primary branch crowns and to induce the formation of secondary branches. Thus, artificial SD treatments effectively stimulate crown branching, providing one means for the increase of cropping (yield) potential in strawberry. It was also shown by growth regulation applications, quantitave hormone analysis and gene expression analysis that gibberellin (GA) is one of the key signals involved in the photoperiod control of shoot differentiation. The results indicate that photoperiod controls GA activity specifically in axillary buds, thereby determining bud fate. It was further shown that chemical control of GA biosynthesis by prohexadione-calcium can be utilized to prevent excessive runner formation and induce crown branching in strawberry fields. Moreover, ProCa increased berry yield up to 50%, showing that it is an easier and more applicable alternative to artificial SD treatments for controlling strawberry crown development and yield. Finally, flowering gene pathways in Fragaria were explored by searching for homologs of 118 Arabidopsis thaliana flowering-time genes. In total, 66 gene homologs were identified, and they distributed to all known flowering pathways, suggesting the presence of these pathways also in strawberry. Expression analysis of selected genes revealed that the mRNA of putative floral identity gene APETALA1 accumulated in the shoot apex of the EB genotype after the induction of flowering, whereas it was absent in vegetative SD genotype, indicating the usefulness of this gene product as the marker of floral initiation. The present data enables the further exploration of strawberry flowering pathways with genetic transformation, gene mapping and transcriptomics methods.

Computational Methods for Locating and Analyzing Conserved Gene Regulatory DNA Elements

This thesis presents methods for locating and analyzing cis-regulatory DNA elements involved with the regulation of gene expression in multicellular organisms. The regulation of gene expression is carried out by the combined effort of several transcription factor proteins collectively binding the DNA on the cis-regulatory elements. Only sparse knowledge of the 'genetic code' of these elements exists today. An automatic tool for discovery of putative cis-regulatory elements could help their experimental analysis, which would result in a more detailed view of the cis-regulatory element structure and function. We have developed a computational model for the evolutionary conservation of cis-regulatory elements. The elements are modeled as evolutionarily conserved clusters of sequence-specific transcription factor binding sites. We give an efficient dynamic programming algorithm that locates the putative cis-regulatory elements and scores them according to the conservation model. A notable proportion of the high-scoring DNA sequences show transcriptional enhancer activity in transgenic mouse embryos. The conservation model includes four parameters whose optimal values are estimated with simulated annealing. With good parameter values the model discriminates well between the DNA sequences with evolutionarily conserved cis-regulatory elements and the DNA sequences that have evolved neutrally. In further inquiry, the set of highest scoring putative cis-regulatory elements were found to be sensitive to small variations in the parameter values. The statistical significance of the putative cis-regulatory elements is estimated with the Two Component Extreme Value Distribution. The p-values grade the conservation of the cis-regulatory elements above the neutral expectation. The parameter values for the distribution are estimated by simulating the neutral DNA evolution. The conservation of the transcription factor binding sites can be used in the upstream analysis of regulatory interactions. This approach may provide mechanistic insight to the transcription level data from, e.g., microarray experiments. Here we give a method to predict shared transcriptional regulators for a set of co-expressed genes. The EEL (Enhancer Element Locator) software implements the method for locating putative cis-regulatory elements. The software facilitates both interactive use and distributed batch processing. We have used it to analyze the non-coding regions around all human genes with respect to the orthologous regions in various other species including mouse. The data from these genome-wide analyzes is stored in a relational database which is used in the publicly available web services for upstream analysis and visualization of the putative cis-regulatory elements in the human genome.

Lymphatic vessels in health and disease: Role of the VEGF-C/VEGFR-3 pathway and the transcription factor FOXC2

The circulatory system consists of two vessel types, which act in concert but significantly differ from each other in several structural and functional aspects as well as in mechanisms governing their development. The blood vasculature transports oxygen, nutrients and cells to tissues whereas the lymphatic vessels collect extravasated fluid, macromolecules and cells of the immune system and return them back to the blood circulation. Understanding the molecular mechanisms behind the developmental and functional regulation of the lymphatic system long lagged behind that of the blood vasculature. Identification of several markers specific for the lymphatic endothelium, and the discovery of key factors controlling the development and function of the lymphatic vessels have greatly facilitated research in lymphatic biology over the past few years. Recognition of the crucial importance of lymphatic vessels in certain pathological conditions, most importantly in tumor metastasis, lymphedema and inflammation, has increased interest in this vessel type, for so long overshadowed by its blood vascular cousin. VEGF-C (Vascular Endothelial Growth Factor C) and its receptor VEGFR-3 are essential for the development and maintenance of embryonic lymphatic vasculature. Furthermore, VEGF-C has been shown to be upregulated in many tumors and its expression found to positively correlate with lymphatic metastasis. Mutations in the transcription factor FOXC2 result in lymphedema-distichiasis (LD), which suggests a role for FOXC2 in the regulation of lymphatic development or function. This study was undertaken to obtain more information about the role of the VEGF-C/VEGFR-3 pathway and FOXC2 in regulating lymphatic development, growth, function and survival in physiological as well as in pathological conditions. We found that the silk-like carboxyterminal propeptide is not necessary for the lymphangiogenic activity of VEGF-C, but enhances it, and that the aminoterminal propeptide mediates binding of VEGF-C to the neuropilin-2 coreceptor, which we suggest to be involved in VEGF-C signalling via VEGFR-3. Furthermore, we found that overexpression of VEGF-C increases tumor lymphangiogenesis and intralymphatic tumor growth, both of which could be inhibited by a soluble form of VEGFR-3. These results suggest that blocking VEGFR-3 signalling could be used for prevention of lymphatic tumor metastasis. This might prove to be a safe treatment method for human cancer patients, since inhibition of VEGFR-3 activity had no effect on the normal lymphatic vasculature in adult mice, though it did lead to regression of lymphatic vessels in the postnatal period. Interestingly, in contrast to VEGF-C, which induces lymphangiogenesis already during embryonic development, we found that the related VEGF-D promotes lymphatic vessel growth only after birth. These results suggest, that the lymphatic vasculature undergoes postnatal maturation, which renders it independent of ligand induced VEGFR-3 signalling for survival but responsive to VEGF-D for growth. Finally, we show that FOXC2 is necessary for the later stages of lymphatic development by regulating the morphogenesis of lymphatic valves, as well as interactions of the lymphatic endothelium with vascular mural cells, in which it cooperates with VEGFR-3. Furthermore, our study indicates that the absence of lymphatic valves, abnormal association of lymphatic capillaries with mural cells and an increased amount of basement membrane underlie the pathogenesis of LD. These findings have given new insight into the mechanisms of normal lymphatic development, as well as into the pathogenesis of diseases involving the lymphatic vasculature. They also reveal new therapeutic targets for the prevention and treatment of tumor metastasis and lymphatic vascular failure in certain forms of lymphedema. Several interesting questions were posed that still need to be addressed. Most importantly, the mechanism of VEGF-C promoted tumor metastasis and the molecular nature of the postnatal lymphatic vessel maturation remain to be elucidated.

Cytokinin signalling in the regulation of cambial development

Secondary growth of plants is of pivotal importance in terrestrial ecosystems, providing a significant carbon sink in the form of wood. As plant biomass accumulation results largely from the cambial growth, it is surprising that quite little is known about the hormonal or genetic control of this important process in any plant species. The central aim of my thesis studies was to explore the function of cytokinin in the regulation of cambial development. Since their discovery as regulators of plant cell divisions, cytokinins have been assumed to participate in the control of cambial development. Evidence for this action was deduced from hormone treatment experiments, where exogenously applied cytokinin was shown to enhance cambial cell divisions in diverse plant organs and species. In my thesis work, the conservation of cytokinin signalling and homeostasis genes between a herbaceous plant, Arabidopsis, and a hardwood tree species, Populus trichocarpa. Presumably reflecting the ancient origin of cytokinin signalling system, the Populus genome contains orthologs for all Arabidopsis cytokinin signalling and homeostasis genes. Thus, genes belonging to five main families of isopentenyl transferases (IPTs), cytokinin oxidases (CKXs), two-component receptors, histidine containing phosphotransmitters (HPts) and response regulators (RRs) were identified from the Populus genome. Three subfamilies associated with cytokinin signal transduction, the CKI1-like family of two-component receptors, the AHP4-like HPts, and the ARR22-like atypical RRs, were significantly larger in Populus genome than in Arabidopsis. Potential contribution to the extensive secondary development of Populus by the members of these considerably expanded gene families will be discussed. Representatives of all cytokinin signal transduction elements were expressed in the Populus cambial zone, and most of the expressed genes appeared to be slightly more abundant on the phloem side of the meristem. The abundance of cytokinin related genes in the cambium emphasizes the important role of this hormone in the regulation of the extensive secondary growth characteristic of tree species. The function of the pseudo HPts in primary vascular development was studied in Arabidopsis root vasculature. It was demonstrated that the pseudo HPt AHP6 has a role in locally inhibiting cytokinin signalling in the protoxylem position in the Arabidopsis root, thus enabling differentiation of the protoxylem cell file. The possible role of pseudo HPts in cambial development will be discussed. The expression peak of cytokinin signalling genes in the tree cambial zone strongly indicates that cytokinin has a role in the regulation of this meristem function. To address whether cytokinin signalling is required for cambial activity, transgenic Populus trees with modified cytokinin signalling were produced. These trees were expressing a cytokinin catabolic gene from Arabidopsis, CYTOKININ OXIDASE 2, (AtCKX2) under the promoter of a Betula CYTOKININ RECEPTOR 1 (BpCRE1). The pBpCRE1::CKX2 transgenic Populus trees showed a reduced concentration of a biologically active cytokinin, correlating with their impaired cytokinin response. Furthermore, the radial growth of these trees was compromised, as illustrated by a smaller stem diameter than in wild-type trees of the same height. Moreover, the level of cambial cytokinin signalling was down-regulated in these thin-stemmed trees. The reduced signalling correlated with a decreased number of meristematic cambial cells, implicating cytokinin activity as a direct regulator of cambial cell division activity. Together, the results of my study indicate that cytokinins are major hormonal regulators required for cambial development.

Neuronal K-Cl Cotransporter : Transcriptional Mechanisms of KCC2 Gene Regulation

K-Cl cotransporter 2 (KCC2) maintains a low intracellular Cl concentration required for fast hyperpolarizing responses of neurons to classical inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Decreased Cl extrusion observed in genetically modified KCC2-deficient mice leads to depolarizing GABA responses, impaired brain inhibition, and as a consequence to epileptic seizures. Identification of mechanisms regulating activity of the SLC12A5 gene, which encodes the KCC2 cotransporter, in normal and pathological conditions is, thus, of extreme importance. Multiple reports have previously elucidated in details a spatio-temporal pattern of KCC2 expression. Among the characteristic features are an exclusive neuronal specificity, a dramatic upregulation during embryonic and early postnatal development, and a significant downregulation by neuronal trauma. Numerous studies confirmed these expressional features, however transcriptional mechanisms predetermining the SLC12A5 gene behaviour are still unknown. The aim of the presented thesis is to recognize such transcriptional mechanisms and, on their basis, to create a transcriptional model that would explain the established SLC12A5 gene behaviour. Up to recently, only one KCC2 transcript has been thought to exist. A particular novelty of the presented work is the identification of two SLC12A5 gene promoters (SLC12A5-1a and SLC12A5-1b) that produce at least two KCC2 isoforms (KCC2a and KCC2b) differing by their N-terminal parts. Even though a functional 86Rb+ assay reveals no significant difference between transport activities of the isoforms, consensus sites for several protein kinases, found in KCC2a but not in KCC2b, imply a distinct kinetic regulation. As a logical continuation, the current work presents a detailed analysis of the KCC2a and KCC2b expression patterns. This analysis shows an exclusively neuron-specific pattern and similar expression levels for both isoforms during embryonic and neonatal development in rodents. During subsequent postnatal development, the KCC2b expression dramatically increases, while KCC2a expression, depending on central nervous system (CNS) area, either remains at the same level or moderately decreases. In an attempt to explain both the neuronal specificity and the distinct expressional kinetics of the KCC2a and KCC2b isoforms during postnatal development, the corresponding SLC12A5-1a and SLC12A5-1b promoters have been subjected to a comprehensive bioinformatical analysis. Binding sites of several transcription factors (TFs), conserved in the mammalian SLC12A5 gene orthologs, have been identified that might shed light on the observed behaviour of the SLC12A5 gene. Possible roles of these TFs in the regulating of the SLC12A5 gene expression have been elucidated in subsequent experiments and are discussed in the current thesis.

GATA Transcription Factors in Tissue Homeostasis and Pathology of the Gastrointestinal Tract and Liver

Mammalian gastrointestinal tract and liver are self-renewing organs that are able to sustain themselves due to stem cells present in their tissues. In constant, inflammation-related epithelial damage, vigorous activation of stem cells may lead to their uncontrolled proliferation, and further, to cancer. GATA-4, GATA-5, and GATA-6 regulate cell proliferation and differentiation in many mammalian organs. Lack of GATA-4 or GATA-6 leads to defective endodermal development and cell differentiation. GATA-4 and GATA-5 are considered the ones with tumor suppressive functions, whereas GATA-6 is more related to tumor promotion. In the digestive system their roles in inflammation and tumor-related molecular pathways remain unclear. In this study, we examined the GATA-related molecular pathways involved in normal tissue organization and renewal and in inflammation-related epithelial repair in the gastrointestinal tract and liver. The overall purpose of this study was to elucidate the relation of GATA factors to gastrointestinal and hepatic disease pathology and to evaluate their possible clinical significance in tumor biology. The results indicated distinct expression patterns for GATA-4, GATA-5, and GATA-6 in the human and murine gastrointestinal tract and liver, and their involvement in the regulation of intestine-specific genes. GATA-5 was confined to the intestines of suckling mice, suggesting an association with postnatal enzymatic changes. GATA-4 was upregulated in bowel inflammation concomitantly with TGF-β signaling. In gastrointestinal tumors, GATA-4 was restricted to benign neoplasias of the stomach, while GATA-6 was detected especially at the invasive edges of malignant tumors throughout the gut. In the liver, GATA-4 was upregulated in pediatric tumors along with erythropoietin (Epo), which was detected also in the sera of tumor patients. Furthermore, GATA-4 was enhanced in areas of vigorous hepatic regeneration in patients with tyrosinemia type I. These results suggest a central role for GATA-4 in pediatric tumor biology of the liver. To conclude, GATA-4, GATA-5, and GATA-6 are associated with normal gastrointestinal and hepatic development and regeneration. The appearance of GATA-4 along with TGF-β-signaling in the inflammatory bowel suggests a protective role in the response to inflammation-related epithelial destruction. However, in extremely malignant pediatric liver tumors, GATA-4 function is unlikely to be tumor-suppressing, probably due to the nature of the very primitive multipotent tumor cells. GATA-4, along with its possible downstream factor Epo, could be utilized as novel hepatic tumor markers to supplement the present diagnostics. They could also serve a function in future biological therapies for aggressive pediatric tumors.

Hematopoietic Stem Cell Development and Transcriptional Regulation

The continuous production of blood cells, a process termed hematopoiesis, is sustained throughout the lifetime of an individual by a relatively small population of cells known as hematopoietic stem cells (HSCs). HSCs are unique cells characterized by their ability to self-renew and give rise to all types of mature blood cells. Given their high proliferative potential, HSCs need to be tightly regulated on the cellular and molecular levels or could otherwise turn malignant. On the other hand, the tight regulatory control of HSC function also translates into difficulties in culturing and expanding HSCs in vitro. In fact, it is currently not possible to maintain or expand HSCs ex vivo without rapid loss of self-renewal. Increased knowledge of the unique features of important HSC niches and of key transcriptional regulatory programs that govern HSC behavior is thus needed. Additional insight in the mechanisms of stem cell formation could enable us to recapitulate the processes of HSC formation and self-renewal/expansion ex vivo with the ultimate goal of creating an unlimited supply of HSCs from e.g. human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPS) to be used in therapy. We thus asked: How are hematopoietic stem cells formed and in what cellular niches does this happen (Papers I, II)? What are the molecular mechanisms that govern hematopoietic stem cell development and differentiation (Papers III, IV)? Importantly, we could show that placenta is a major fetal hematopoietic niche that harbors a large number of HSCs during midgestation (Paper I)(Gekas et al., 2005). In order to address whether the HSCs found in placenta were formed there we utilized the Runx1-LacZ knock-in and Ncx1 knockout mouse models (Paper II). Importantly, we could show that HSCs emerge de novo in the placental vasculature in the absence of circulation (Rhodes et al., 2008). Furthermore, we could identify defined microenvironmental niches within the placenta with distinct roles in hematopoiesis: the large vessels of the chorioallantoic mesenchyme serve as sites of HSC generation whereas the placental labyrinth is a niche supporting HSC expansion (Rhodes et al., 2008). Overall, these studies illustrate the importance of distinct milieus in the emergence and subsequent maturation of HSCs. To ensure proper function of HSCs several regulatory mechanisms are in place. The microenvironment in which HSCs reside provides soluble factors and cell-cell interactions. In the cell-nucleus, these cell-extrinsic cues are interpreted in the context of cell-intrinsic developmental programs which are governed by transcription factors. An essential transcription factor for initiation of hematopoiesis is Scl/Tal1 (stem cell leukemia gene/T-cell acute leukemia gene 1). Loss of Scl results in early embryonic death and total lack of all blood cells, yet deactivation of Scl in the adult does not affect HSC function (Mikkola et al., 2003b. In order to define the temporal window of Scl requirement during fetal hematopoietic development, we deactivated Scl in all hematopoietic lineages shortly after hematopoietic specification in the embryo . Interestingly, maturation, expansion and function of fetal HSCs was unaffected, and, as in the adult, red blood cell and platelet differentiation was impaired (Paper III)(Schlaeger et al., 2005). These findings highlight that, once specified, the hematopoietic fate is stable even in the absence of Scl and is maintained through mechanisms that are distinct from those required for the initial fate choice. As the critical downstream targets of Scl remain unknown, we sought to identify and characterize target genes of Scl (Paper IV). We could identify transcription factor Mef2C (myocyte enhancer factor 2 C) as a novel direct target gene of Scl specifically in the megakaryocyte lineage which largely explains the megakaryocyte defect observed in Scl deficient mice. In addition, we observed an Scl-independent requirement of Mef2C in the B-cell compartment, as loss of Mef2C leads to accelerated B-cell aging (Gekas et al. Submitted). Taken together, these studies identify key extracellular microenvironments and intracellular transcriptional regulators that dictate different stages of HSC development, from emergence to lineage choice to aging.

Mitochondrial helicase Twinkle in mtDNA copy number regulation and a late-onset disease model

Mitochondrial diseases are caused by disturbances of the energy metabolism. The disorders range from severe childhood neurological diseases to muscle diseases of adults. Recently, mitochondrial dysfunction has also been found in Parkinson s disease, diabetes, certain types of cancer and premature aging. Mitochondria are the power plants of the cell but they also participate in the regulation of cell growth, signaling and cell death. Mitochondria have their own genetic material, mtDNA, which contains the genetic instructions for cellular respiration. Single cell may host thousands of mitochondria and several mtDNA molecules may reside inside single mitochondrion. All proteins needed for mtDNA maintenance are, however, encoded by the nuclear genome, and therefore, mutations of the corresponding genes can also cause mitochondrial disease. We have here studied the function of mitochondrial helicase Twinkle. Our research group has previously identified nuclear Twinkle gene mutations underlying an inherited adult-onset disorder, progressive external ophthalmoplegia (PEO). Characteristic for the PEO disease is the accumulation of multiple mtDNA deletions in tissues such as the muscle and brain. In this study, we have shown that Twinkle helicase is essential for mtDNA maintenance and that it is capable of regulating mtDNA copy number. Our results support the role of Twinkle as the mtDNA replication helicase. No cure is available for mitochondrial disease. Good disease models are needed for studies of the cause of disease and its progression and for treatment trials. Such disease model, which replicates the key features of the PEO disease, has been generated in this study. The model allows for careful inspection of how Twinkle mutations lead to mtDNA deletions and further causes the PEO disease. This model will be utilized in a range of studies addressing the delay of the disease onset and progression and in subsequent treatment trials. In conclusion, in this thesis fundamental knowledge of the function of the mitochondrial helicase Twinkle was gained. In addition, the first model for adult-onset mitochondrial disease was generated.

Transcription factor GATA4 and apoptotic TRAIL pathway in granulosa cell function and tumors

Normal growth and development require the precise control of gene expression. Transcription factors are proteins that regulate gene expression by binding specific sequences of DNA. Abnormalities in transcription are implicated in a variety of human diseases, including cancer, endocrine disorders and birth defects. Transcription factor GATA4 has emerged as an important regulator of normal development and function in a variety of endoderm- and mesoderm- derived tissues, including gut, heart and several endocrine organs, such as gonads. Mice harboring a null mutation of Gata4 gene die during embryogenesis due to failure in heart formation, complicating the study of functional role of GATA4 in other organs. However, the expression pattern of GATA4 suggests it may play a role in the regulation of ovarian granulosa cell development, function and apoptosis. This premise is supported by in vitro studies showing that GATA4 regulates several steroidogenic enzymes as well as auto-, para- and endocrine signaling molecules important for granulosa cell function. This study assessed the in vivo role of GATA4 for granulosa cell function by utilizing two genetically modified mouse strains. The findings in the GATA4 deficient mice included delayed puberty, impaired fertility and signs of diminished estrogen production. At the molecular level, the GATA4 deficiency leads to attenuated expression of central steroidogenic genes, Steroidogenic acute regulatory protein (StAR), Side-chain cleavage (SCC), and aromatase as a response to stimulations with exogenous gonadotropins. Taken together, these suggest GATA4 is necessary for the normal ovarian function and female fertility. Programmed cell death, apoptosis, is a crucial part of normal ovarian development and function. In addition, disturbances in apoptosis have been implicated to pathogenesis of human granulosa cell tumors (GCTs). Apoptosis is controlled by extrinsic and intrinsic pathways. The intrinsic pathway is regulated by members of Bcl-2 family, and its founding member, the anti-apoptotic Bcl-2, is known to be important for granulosa cell survival. This study showed that the expression levels of GATA4 and Bcl-2 correlate in the human GCTs and that GATA4 regulates Bcl-2 expression, presumably by directly binding to its promoter. In addition, disturbing GATA4 function was sufficient to induce apoptosis in cultured GCT- derived cell line. Taken together, these results suggest GATA4 functions as an anti-apoptotic factor in GCTs. The extrinsic apoptotic pathway is controlled by the members of tumor necrosis factor (TNF) superfamily. An interesting ligand of this family is TNF-related apoptosis-inducing ligand (TRAIL), possessing a unique ability to selectively induce apoptosis in malignant cells. This study characterized the previously unknown expression of TRAIL and its receptors in both developing and adult human ovary, as well as in malignant granulosa cell tumors. TRAIL pathway was shown to be active in GCTs suggesting it may be a useful tool in treating these malignancies. However, more studies are required to assess the function of TRAIL pathway in normal ovaries. In addition to its ability to induce apoptosis in GCTs, this study revealed that GATA4 protects these malignancies from TRAIL-induced apoptosis. GATA4 presumably exerts this effect by regulating the expression of anti-apoptotic Bcl-2. This is of particular interest as high expression of GATA4 is known to correlate to aggressive GCT behavior. Thus, GATA4 seems to protect GCTs from endogenous TRAIL by upregulating anti-apoptotic factors such as Bcl-2.

Sterol-binding proteins in late endosomes : Regulation of endosome motility and lipid metabolism

Despite its bad reputation in the mass media, cholesterol is an indispensable constituent of cellular membranes and vertebrate life. It is, however, also potentially lethal as it may accumulate in the arterial intima causing atherosclerosis or elsewhere in the body due to inherited conditions. Studying cholesterol in cells, and research on how the cell biology of cholesterol affects on system level is essential for a better understanding of the disease states associated with cholesterol and for the development of new therapies for these conditions. On its way to the cell, exogenous cholesterol traverses through endosomes, transport vesicles involved in internalizing material to cells, and needs to be transported out of this compartment. This endosomal pool of cholesterol is important for understanding both the common disorders of metabolism and the more rare hereditary disorders of cholesterol metabolism. The study of cholesterol in cells has been hampered by the lack of bright fluorescent sterol analogs that would resemble cholesterol enough to be used in cellular studies. In the first study of my thesis, we present a new sterol analog, Boron-Dipyrromethene (BODIPY)-cholesterol for visualizing sterols in living cells and organism. This fluorescent cholesterol derivative is shown to behave similarly to cholesterol both by atomic scale computer simulations and biochemical experiments. We characterize its localization inside different types of living cells and show that it can be used to study sterol trafficking in living organisms. Two sterol binding proteins associated with the endosomal membrane; the Niemann-Pick type C disease protein 1 (NPC1) and the Oxysterol Binding Protein Related Protein 1 (ORP1) are the subjects of the rest of this study. Sensing cholesterol on endosomes, transporting lipids away from this compartment and the effects these lipids play on cellular metabolism are considered. In the second study we characterize how the NPC1 protein affects lipid metabolism. We show that this cholesterol binding protein affects synthesis of triglycerides and that genetic polymorphisms or a genetic defect in the NPC1 gene affect triglyceride on the whole body level. These effects take place via regulation of carbon fluxes to different lipid classes in cells. In the third part we characterize the effects of another endosomal sterol binding protein, ORP1L on the function and motility of endosomes. Specifically we elucidate how a mutation in the ability of ORP1L to bind sterols affects its behavior in cells, and how a change in ORP1L levels in cells affects the localization, degradative capacity and motility of endosomes. In addition we show that ORP1L manipulations affect cholesterol balance also in macrophages, a cell type important for the development of atherosclerosis.

DNA damage recognition in the normal epithelium of human prostate and seminal vesicles

Prostate cancer is one of the most prevalent cancer types in men. The development of prostate tumors is known to require androgen exposure, and several pathways governing cell growth are deregulated in prostate tumorigenesis. Recent genetic studies have revealed that complex gene fusions and copy - number alterations are frequent in prostate cancer, a unique feature among solid tumors. These chromosomal aberrations are though to arise as a consequence of faulty repair of DNA double strand breaks (DSB). Most repair mechanisms have been studied in detail in cancer cell lines, but how DNA damage is detected and repaired in normal differentiated human cells has not been widely addressed. The events leading to the gene fusions in prostate cancer are under rigorous studies, as they not only shed light on the basic pathobiologic mechanisms but may also produce molecular targets for prostate cancer treatment and prevention. Prostate and seminal vesicles are part of the male reproductive system. They share similar structure and function but differ dramatically in their cancer incidence. Approximately fifty primary seminal vesicle carcinomas have been reported worldwide. Surprisingly, only little is known on why seminal vesicles are resistant to neoplastic changes. As both tissues are androgen dependent, it is a mystery that androgen signaling would only lead to tumors in prostate tissue. In this work, we set up novel ex vivo human tissue culture models of prostate and seminal vesicles, and used them to study how DNA damage is recognized in normal epithelium. One of the major DNA - damage inducible pathways, mediated by the ATM kinase, was robustly activated in all main cell types of both tissues. Interestingly, we discovered that secretory epithelial cells had less histone variant H2A.X and after DNA damage lower levels of H2AX were phosphorylated on serine 139 (γH2AX) than in basal or stromal cells. γH2AX has been considered essential for efficient DSB repair, but as there were no significant differences in the γH2AX levels between the two tissues, it seems more likely that the role of γH2AX is less important in postmitotic cells. We also gained insight into the regulation of p53, an important transcription factor that protects genomic integrity via multiple mechanisms, in human tissues. DSBs did not lead to a pronounced activation of p53, but treatments causing transcriptional stress, on the other hand, were able to launch a notable p53 response in both tissue types. In general, ex vivo culturing of human tissues provided unique means to study differentiated cells in their relevant tissue context, and is suited for testing novel therapeutic drugs before clinical trials. In order to study how prostate and seminal vesicle epithelial cells are able to activate DNA damage induced cell cycle checkpoints, we used primary cultures of prostate and seminal vesicle epithelial cells. To our knowledge, we are the first to report isolation of human primary seminal vesicle cells. Surprisingly, human prostate epithelial cells did not activate cell cycle checkpoints after DSBs in part due to low levels of Wee1A, a kinase regulating CDK activity, while primary seminal vesicle epithelial cells possessed proficient cell cycle checkpoints and expressed high levels of Wee1A. Similarly, seminal vesicle cells showed a distinct activation of the p53 - pathway after DSBs that did not occur in prostate epithelial cells. This indicates that p53 protein function is under different control mechanisms in the two cell types, which together with proficient cell cycle checkpoints may be crucial in protecting seminal vesicles from endogenous and exogenous DNA damaging factors and, as a consequence, from carcinogenesis. These data indicate that two very similar organs of male reproductive system do not respond to DNA damage similarly. The differentiated, non - replicating cells of both tissues were able to recognize DSBs, but under proliferation human prostate epithelial cells had deficient activation of the DNA damage response. This suggests that prostate epithelium is most vulnerable to accumulating genomic aberrations under conditions where it needs to proliferate, for example after inflammatory cellular damage.

Dissection of the genetic background of childhood onset progressive myoclonic epilepsies

The progressive myoclonic epilepsies (PMEs) are a clinically and etiologically heterogeneous group of symptomatic epilepsies characterized by myoclonus, tonic-clonic seizures, psychomotor regression and ataxia. Different disorders have been classified as PMEs. Of these, the group of neuronal ceroid lipofuscinoses (NCLs) comprise an entity that has onset in childhood, being the most common cause of neurodegeneration in children. The primary aim of this thesis was to dissect the molecular genetic background of patients with childhood onset PME by studying candidate genes and attempting to identify novel PME-associated genes. Another specific aim was to study the primary protein properties of the most recently identified member of the NCL-causing proteins, MFSD8. To dissect the genetic background of a cohort of Turkish patients with childhood onset PME, a screen of the NCL-associated genes PPT1, TPP1, CLN3, CLN5, CLN6, MFSD8, CLN8 and CTSD was performed. Altogether 49 novel mutations were identified, which together with 56 mutations found by collaborators raised the total number of known NCL mutations to 364. Fourteen of the novel mutations affect the recently identified MFSD8 gene, which had originally been identified in a subset of mainly Turkish patients as the underlying cause of CLN7 disease. To investigate the distribution of MFSD8 defects, a total of 211 patients of different ethnic origins were evaluated for mutations in the gene. Altogether 45 patients from nine different countries were provided with a CLN7 molecular diagnosis, denoting the wide geographical occurrence of MFSD8 defects. The mutations are private with only one having been established by a founder-effect in the Roma population from the former Czechoslovakia. All mutations identified except one are associated with the typical clinical picture of variant late-infantile NCL. To address the trafficking properties of MFSD8, lysosomal targeting of the protein was confirmed in both neuronal and non-neuronal cells. The major determinant for this lysosomal sorting was identified to be an N-terminal dileucine based signal (9-EQEPLL-14), recognized by heterotetrameric AP-1 adaptor proteins, suggesting that MFSD8 takes the direct trafficking pathway en route to the lysosomes. Expression studies revealed the neurons as the primary cell-type and the hippocampus and cerebellar granular cell layer as the predominant regions in which MFSD8 is expressed. To identify novel genes associated with childhood onset PME, a single nucleotide polymorphism (SNP) genomewide scan was performed in three small families and 18 sporadic patients followed by homozygosity mapping to determine the candidate loci. One of the families and a sporadic patient were positive for mutations in PLA2G6, a gene that had previously been shown to cause infantile neuroaxonal dystrophy. Application of next-generation sequencing of candidate regions in the remaining two families led to identification of a homozygous missense mutation in USP19 for the first and TXNDC6 for the second family. Analysis of the 18 sporadic cases mapped the best candidate interval in a 1.5 Mb region on chromosome 7q21. Screening of the positional candidate KCTD7 revealed six mutations in seven unrelated families. All patients with mutations in KCTD7 were reported to have early onset PME, rapid disease progression leading to dementia and no pathologic hallmarks. The identification of KCTD7 mutations in nine patients and the clinical delineation of their phenotype establish KCTD7 as a gene for early onset PME. The findings presented in this thesis denote MFSD8 and KCTD7 as genes commonly associated with childhood onset symptomatic epilepsy. The disease-associated role of TXNDC6 awaits verification through identification of additional mutations in patients with similar phenotypes. Completion of the genetic spectrum underlying childhood onset PMEs and understanding of the gene products functions will comprise important steps towards understanding the underlying pathogenetic mechanisms, and will possibly shed light on the general processes of neurodegeneration and nervous system regulation, facilitating the diagnosis, classification and possibly treatment of the affected cases.