Fine-tuning of the signalling network controlling morphogenesis and stem cell development in teeth

Tooth development is regulated by sequential and reciprocal interactions between epithelium and mesenchyme. The molecular mechanisms underlying this regulation are conserved and most of the participating molecules belong to several signalling families. Research focusing on mouse teeth has uncovered many aspects of tooth development, including molecular and evolutionary specifi cs, and in addition offered a valuable system to analyse the regulation of epithelial stem cells. In mice the spatial and temporal regulation of cell differentiation and the mechanisms of patterning during development can be analysed both in vivo and in vitro. Follistatin (Fst), a negative regulator of TGFβ superfamily signalling, is an important inhibitor during embryonic development. We showed the necessity of modulation of TGFβ signalling by Fst in three different regulatory steps during tooth development. First we showed that tinkering with the level of TGFβ signalling by Fst may cause variation in the molar cusp patterning and crown morphogenesis. Second, our results indicated that in the continuously growing mouse incisors asymmetric expression of Fst is responsible for the labial-lingual patterning of ameloblast differentiation and enamel formation. Two TGFβ superfamily signals, BMP and Activin, are required for proper ameloblast differentiation and Fst modulates their effects. Third, we identifi ed a complex signalling network regulating the maintenance and proliferation of epithelial stem cells in the incisor, and showed that Fst is an essential modulator of this regulation. FGF3 in cooperation with FGF10 stimulates proliferation of epithelial stem cells and transit amplifying cells in the labial cervical loop. BMP4 represses Fgf3 expression whereas Activin inhibits the repressive effect of BMP4 on the labial side. Thus, Fst inhibits Activin rather than BMP4 in the cervical loop area and limits the proliferation of lingual epithelium, thereby causing the asymmetric maintenance and proliferation of epithelial stem cells. In addition, we detected Lgr5, a Wnt target gene and an epithelial stem cell marker in the intestine, in the putative epithelial stem cells of the incisor, suggesting that Lgr5 is a marker of incisor stem cells but is not regulated by Wnt/β-catenin signalling in the incisor. Thus the epithelial stem cells in the incisor may not be directly regulated by Wnt/β-catenin signalling. In conclusion, we showed in the mouse incisors that modulating the balance between inductive and inhibitory signals constitutes a key mechanism regulating the epithelial stem cells and ameloblast differentiation. Furthermore, we found additional support for the location of the putative epithelial stem cells and for the stemness of these cells. In the mouse molar we showed the necessity of fi ne-tuning the signalling in the regulation of the crown morphogenesis, and that altering the levels of an inhibitor can cause variation in the crown patterning.

Crystallization as a Tool for Controlling and Designing Properties of Pharmaceutical Solids

The ability to deliver the drug to the patient in a safe, efficacious and cost-effective manner depends largely on the physicochemical properties of the active pharmaceutical ingredient (API) in the solid state. In this context, crystallization is of critical importance in pharmaceutical industry, as it defines physical and powder properties of crystalline APIs. An improved knowledge of the various aspects of crystallization process is therefore needed. The overall goal of this thesis was to gain better understanding of the relationships between crystallization, solid-state form and properties of pharmaceutical solids with a focus on a crystal engineering approach to design technological properties of APIs. Specifically, solid-state properties of the crystalline forms of the model APIs, erythromycin A and baclofen, and the influence of solvent on their crystallization behavior were investigated. In addition, the physical phenomena associated with wet granulation and hot-melting processing of the model APIs were examined at the molecular level. Finally, the effect of crystal habit modification of a model API on its tabletting properties was evaluated. The thesis enabled the understanding of the relationship between the crystalline forms of the model APIs, which is of practical importance for solid-state control during processing and storage. Moreover, a new crystalline form, baclofen monohydrate, was discovered and characterized. Upon polymorph screening, erythromycin A demonstrated high solvate-forming propensity thus emphasizing the need for careful control of the solvent effects during formulation. The solvent compositions that yield the desirable crystalline form of erythromycin A were defined. Furthermore, new examples on solvent-mediated phase transformations taking place during wet granulation of baclofen and hot-melt processing of erythromycin A dihydrate with PEG 6000 are reported. Since solvent-mediated phase transformations involve the crystallization of a stable phase and hence affect the dissolution kinetics and possibly absorption of the API these transformations must be well documented. Finally, a controlled-crystallization method utilizing HPMC as a crystal habit modifier was developed for erythromycin A dihydrate. The crystals with modified habit were shown to posses improved compaction properties as compared with those of unmodified crystals. This result supports the idea of morphological crystal engineering as a tool for designing technological properties of APIs and is of utmost practical interest.

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.

The efficacy and potential risks of controlling sprouting in Finnish birches (Betula spp.) with the fungal decomposer Chondrostereum purpureum

Sprouting of fast-growing broad-leaved trees causes problems in young coniferous stands, under power transmission lines and along roads and railways. Public opinion and the Finnish Forest Certification System oppose the use of chemical herbicides to control sprouting, which means that most areas with problems rely on mechanical cutting. However, cutting is a poor control method for many broad-leaved species because the removal of leaders can stimulate the sprouting of side branches and cut stumps quickly re-sprout. In order to be effective, cutting must be carried out frequently but each cut increases the costs, making this control method increasingly difficult and expensive once begun. As such, alternative methods for sprout control that are both effective and environmentally sound represent a continuing challenge to managers and research biologists. Using biological control agents to prevent sprouting has been given serious consideration recently. Dutch and Canadian researchers have demonstrated the potential of the white-rot fungus Chondrostereum purpureum (Pers. ex Fr.) Pouzar as a control agent of stump sprouting in many hardwoods. These findings have focused the attention of the Finnish forestry community on the utilization of C. purpureum for biocontrol purposes. Primarily, this study sought determines the efficacy of native C. purpureum as an inhibitor of birch stump sprouting in Finland and to clarify its mode of action. Additionally, genotypic variation in Finnish C. purpureum was examined and the environmental risks posed by a biocontrol program using this fungus were assessed. Experimental results of the study demonstrated that C. purpureum clearly affects the sprouting of birch: both the frequency of living stumps and the number of living sprouts per stump were effectively reduced by the treatment. However, the treatment had no effect on the maximum height of new sprouts. There were clear differences among fungal isolates in preventing sprouting and those that possessed high oxidative activities as measured in the laboratory inhibited sprouting most efficiently in the field. The most effective treatment time during the growing season was in early and mid summer (May July). Genetic diversity in Nordic and Baltic populations of C. purpureum was found to be high at the regional scale but locally homogeneous. This natural distribution of diversity means that using local genotypes in biocontrol programs would effectively prevent the introduction of novel genes or genotypes. While a biocontrol program using local strains of C. purpureum would be environmentally neutral, pruned birches that are close to the treatment site would have a high susceptibility to infect by the fungus during the early spring.

Kannustaminen ja moraali : Kannustamisen idea suomalaisessa yhteiskuntapolitiikassa 1990-luvulta alkaen

Even though the concept of incentive has become very popular in Finnish welfare politics since the economic crisis of the 1990s, the content of this concept is not clear. Fundamentally, it is a matter of controlling the behaviour of individuals to accord with the authorities' objectives and interests in gaining cooperative benefits. As early as in Plato's Republic, citizens were encouraged to use their abilities and skills in a way most beneficial to the society. Similarly, in today's welfare society citizens are urged to produce common goods and distribute welfare to enable a better life for all through cooperation. The fundamental question is to what extent society can shape individuals' preferences with incentives, and encourage them without external coercion to choose actions beneficial for both the society and the individuals themselves. The objective of the incentive institution is to gain cooperative benefits, but there are different views on how it should be implemented. For example, the incentive system in the Finnish welfare society includes several economic and social conceptions which adjust the distribution of welfare. From an economic perspective, the objective of the incentive system is economic efficiency, while from a social perspective it is the securing of social rights and citizens' equality. The market mechanism, for example, can at best lead to economically efficient activity, but it might sacrifice fairness and equality. In this research, the idea of activation policy expands to cover normative and social incentives, in addition to the economic factors affecting human choice and social actions. Desirable co-living and meaningful cooperation have some prerequisites. We need the expanded idea of activation to study them, and to maintain them in society. The themes discussed in all the ten chapters aim at evaluating the preconditions of a just society. This study provides tools to examine the changes in the welfare state, also from the viewpoint of normative ethics. This offers a morally and conceptually wider perspective than a normative viewpoint of economics alone. In terms of the values of our welfare society, it makes a difference how the relationship between the legalities of economics and citizens' well-being is understood. The research asks whether economic benefits to the society should be allowed to supersede the principles of human dignity Key words:incentives, activation policy, morality, social philosophy, social justice, policy paradigm

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.

The Human UDP-Glucuronosyltransferases: Studies on Substrate Binding and Catalytic Mechanism

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Mechanism of RNA Translocation by a Viral Packaging Motor

Molecular motors are proteins that convert chemical energy into mechanical work. The viral packaging ATPase P4 is a hexameric molecular motor that translocates RNA into preformed viral capsids. P4 belongs to the ubiquitous class of hexameric helicases. Although its structure is known, the mechanism of RNA translocation remains elusive. Here we present a detailed kinetic study of nucleotide binding, hydrolysis, and product release by P4. We propose a stochastic-sequential cooperative model to describe the coordination of ATP hydrolysis within the hexamer. In this model the apparent cooperativity is a result of hydrolysis stimulation by ATP and RNA binding to neighboring subunits rather than cooperative nucleotide binding. Simultaneous interaction of neighboring subunits with RNA makes the otherwise random hydrolysis sequential and processive. Further, we use hydrogen/deuterium exchange detected by high resolution mass spectrometry to visualize P4 conformational dynamics during the catalytic cycle. Concerted changes of exchange kinetics reveal a cooperative unit that dynamically links ATP binding sites and the central RNA binding channel. The cooperative unit is compatible with the structure-based model in which translocation is effected by conformational changes of a limited protein region. Deuterium labeling also discloses the transition state associated with RNA loading which proceeds via opening of the hexameric ring. Hydrogen/deuterium exchange is further used to delineate the interactions of the P4 hexamer with the viral procapsid. P4 associates with the procapsid via its C-terminal face. The interactions stabilize subunit interfaces within the hexamer. The conformation of the virus-bound hexamer is more stable than the hexamer in solution, which is prone to spontaneous ring openings. We propose that the stabilization within the viral capsid increases the packaging processivity and confers selectivity during RNA loading. Finally, we use single molecule techniques to characterize P4 translocation along RNA. While the P4 hexamer encloses RNA topologically within the central channel, it diffuses randomly along the RNA. In the presence of ATP, unidirectional net movement is discernible in addition to the stochastic motion. The diffusion is hindered by activation energy barriers that depend on the nucleotide binding state. The results suggest that P4 employs an electrostatic clutch instead of cycling through stable, discrete, RNA binding states during translocation. Conformational changes coupled to ATP hydrolysis modify the electrostatic potential inside the central channel, which in turn biases RNA motion in one direction. Implications of the P4 model for other hexameric molecular motors are discussed.