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.

Roles of forced nicotine exposure and Comt gene disruption in the development of addiction-related behavioural and neurochemical changes in mice

Activation of midbrain dopamine systems is thought to be critically involved in the addictive properties of abused substances. Drugs of abuse increase dopamine release in the nucleus accumbens and dorsal striatum, which are the target areas of mesolimbic and nigrostriatal dopamine pathways, respectively. Dopamine release in the nucleus accumbens is thought to mediate the attribution of incentive salience to rewards, and dorsal striatal dopamine release is involved in habit formation. In addition, changes in the function of prefrontal cortex (PFC), the target area of mesocortical dopamine pathway, may skew information processing and memory formation such that the addict pays an abnormal amount of attention to drug-related cues. In this study, we wanted to explore how long-term forced oral nicotine exposure or the lack of catechol-O-methyltransferase (COMT), one of the dopamine metabolizing enzymes, would affect the functioning of these pathways. We also wanted to find out how the forced nicotine exposure or the lack of COMT would affect the consumption of nicotine, alcohol, or cocaine. First, we studied the effect of forced chronic nicotine exposure on the sensitivity of dopamine D2-like autoreceptors in microdialysis and locomotor activity experiments. We found that the sensitivity of these receptors was unchanged after forced oral nicotine exposure, although an increase in the sensitivity was observed in mice treated with intermittent nicotine injections twice daily for 10 days. Thus, the effect of nicotine treatment on dopamine autoreceptor sensitivity depends on the route, frequency, and time course of drug administration. Second, we investigated whether the forced oral nicotine exposure would affect the reinforcing properties of nicotine injections. The chronic nicotine exposure did not significantly affect the development of conditioned place preference to nicotine. In the intravenous self-administration paradigm, however, the nicotine-exposed animals self-administered nicotine at a lower unit dose than the control animals, indicating that their sensitivity to the reinforcing effects of nicotine was enhanced. Next, we wanted to study whether the Comt gene knock-out animals would be a suitable model to study alcohol and cocaine consumption or addiction. Although previous work had shown male Comt knock-out mice to be less sensitive to the locomotor-activating effects of cocaine, the present study found that the lack of COMT did not affect the consumption of cocaine solutions or the development of cocaine-induced place preference. However, the present work did find that male Comt knock-out mice, but not female knock-out mice, consumed ethanol more avidly than their wild-type littermates. This finding suggests that COMT may be one of the factors, albeit not a primary one, contributing to the risk of alcoholism. Last, we explored the effect of COMT deficiency on dorsal striatal, accumbal, and prefrontal cortical dopamine metabolism under no-net-flux conditions and under levodopa load in freely-moving mice. The lack of COMT did not affect the extracellular dopamine concentrations under baseline conditions in any of the brain areas studied. In the prefrontal cortex, the dopamine levels remained high for a prolonged time after levodopa treatment in male, but not female, Comt knock-out mice. COMT deficiency induced accumulation of 3,4-dihydroxyphenylacetic acid, which increased further under levodopa load. Homovanillic acid was not detectable in Comt knock-out animals either under baseline conditions or after levodopa treatment. Taken together, the present results show that although forced chronic oral nicotine exposure affects the reinforcing properties of self-administered nicotine, it is not an addiction model itself. COMT seems to play a minor role in dopamine metabolism and in the development of addiction under baseline conditions, indicating that dopamine function in the brain is well-protected from perturbation. However, the role of COMT becomes more important when the dopaminergic system is challenged, such as by pharmacological manipulation.

Language and Letters of the Bluestocking Network : Sociolinguistic Issues in Eighteenth Century Epistolary English

This study deals with language change and variation in the correspondence of the eighteenth-century Bluestocking circle, a social network which provided learned men and women with an informal environment for the pursuit of scholarly entertainment. Elizabeth Montagu (1718 1800), a notable social hostess and a Shakespearean scholar, was one of their key figures. The study presents the reconstruction of Elizabeth Montagu s social networks from her youth to her later years with a special focus on the Bluestocking circle, and linguistic research on private correspondence between Montagu and her Bluestocking friends and family members between the years 1738 1778. The epistolary language use is investigated using the methods and frameworks of corpus linguistics, historical sociolinguistics, and social network analysis. The approach is diachronic and concerns real-time language change. The research is based on a selection of manuscript letters which I have edited and compiled into an electronic corpus (Bluestocking Corpus). I have also devised a network strength scale in order to quantify the strength of network ties and to compare the results of the linguistic research with the network analysis. The studies range from the reconstruction and analysis of Elizabeth Montagu s most prominent social networks to the analysis of changing morphosyntactic features and spelling variation in Montagu s and her network members correspondence. The linguistic studies look at the use of the progressive construction, preposition stranding and pied piping, and spelling variation in terms of preterite and past participle endings in the regular paradigm (-ed, - d, -d, - t, -t) and full / contracted spellings of auxiliary verbs. The results are analysed in terms of social network membership, sociolinguistic variables of the correspondents, and, when relevant, aspects of eighteenth-century linguistic prescriptivism. The studies showed a slight diachronic increase in the use of the progressive, a significant decrease of the stigmatised preposition stranding and increase of pied piping, and relatively informal but socially controlled epistolary spelling. Certain significant changes in Elizabeth Montagu s language use over the years could be attributed to her increasingly prominent social standing and the changes in her social networks, and the strength of ties correlated strongly with the use of the progressive in the Bluestocking Corpus. Gender, social rank, and register in terms of kinship/friendship had a significant influence in language use, and an effect of prescriptivism could also be detected. Elizabeth Montagu s network ties resulted in language variation in terms of network membership, her own position in a given network, and the social factors that controlled eighteenth-century interaction. When all the network ties are strong, linguistic variation seems to be essentially linked to the social variables of the informants.

Adenoviral Gene Therapy for Advanced Head and Neck Cancer

Advanced stage head and neck cancers (HNC) with distant metastasis, as well as prostate cancers (PC), are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment is the use of oncolytic adenoviruses, especially in combination therapy with conventional cancer therapies. The safety of the approach has been tested in many clinical trials. However, antitumor efficacy needs to be improved in order to establish oncolytic viruses as a viable treatment alternative. To be able to test in vivo the effects on anti-tumor efficiency of a multimodal combination therapy of oncolytic adenoviruses with the standard therapeutic combination of radiotherapy, chemotherapy and Cetuximab monoclonal antibody (mAb), a xenograft HNC tumor model was developed. This model mimics the typical clinical situation as it is initially sensitive to cetuximab, but resistance develops eventually. Surprisingly, but in agreement with recent findings for chemotherapy and radiotherapy, a higher proportion of cells positive for HNC cancer stem cell markers were found in the tumors refractory to cetuximab. In vitro as well as in vivo results found in this study support the multimodal combination therapy of oncolytic adenoviruses with chemotherapy, radiotherapy and monoclonal antibody therapy to achieve increased anti-tumor efficiency and even complete tumor eradication with lower treatment doses required. In this study, it was found that capsid modified oncolytic viruses have increased gene transfer to cancer cells as well as an increased antitumor effect. In order to elucidate the mechanism of how oncolytic viruses promote radiosensitization of tumor cells in vivo, replicative deficient viruses expressing several promising radiosensitizing viral proteins were tested. The results of this study indicated that oncolytic adenoviruses promote radiosensitization by delaying the repair of DNA double strand breaks in tumor cells. Based on the promising data of the first study, two tumor double-targeted oncolytic adenoviruses armed with the fusion suicide gene FCU1 or with a fully human mAb specific for human Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) were produced. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Anti-CTLA4 mAb promotes direct killing of tumor cells via apoptosis and most importantly immune system activation against the tumors. These armed oncolytic viruses present increased anti-tumor efficacy both in vitro and in vivo. Furthermore, by taking advantage of the unique tumor targeted gene transfer of oncolytic adenoviruses, functional high tumor titers but low systemic concentrations of the armed proteins were generated. In addition, supernatants of tumor cells infected with Ad5/3-24aCTLA4, which contain anti-CTLA4 mAb, were able to effectively immunomodulate peripheral blood mononuclear cells (PBMC) of cancer patients with advanced tumors. -- In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of advanced and metastatic HNC and PC.

Cellular and network mechanisms generating spontaneous population events in the immature rat hippocampus

Distinct endogenous network events, generated independently of sensory input, are a general feature of various structures of the immature central nervous system. In the immature hippocampus, these type of events are seen as "giant depolarizing potentials" (GDPs) in intracellular recordings in vitro. GABA, the major inhibitory neurotransmitter of the adult brain, has a depolarizing action in immature neurons, and GDPs have been proposed to be driven by GABAergic transmission. Moreover, GDPs have been thought to reflect an early pattern that disappears during development in parallel with the maturation of hyperpolarizing GABAergic inhibition. However, the adult hippocampus in vivo also generates endogenous network events known as sharp (positive) waves (SPWs), which reflect synchronous discharges of CA3 pyramidal neurons and are thought to be involved in cognitive functions. In this thesis, mechanisms of GDP generation were studied with intra- and extracellular recordings in the neonatal rat hippocampus in vitro and in vivo. Immature CA3 pyramidal neurons were found to generate intrinsic bursts of spikes and to act as cellular pacemakers for GDP activity whereas depolarizing GABAergic signalling was found to have a temporally non-patterned facilitatory role in the generation of the network events. Furthermore, the data indicate that the intrinsic bursts of neonatal CA3 pyramidal neurons and, consequently, GDPs are driven by a persistent Na+ current and terminated by a slow Ca2+-dependent K+ current. Gramicidin-perforated patch recordings showed that the depolarizing driving force for GABAA receptor-mediated actions is provided by Cl- uptake via the Na-K-C1 cotransporter, NKCC1, in the immature CA3 pyramids. A specific blocker of NKCC1, bumetanide, inhibited SPWs and GDPs in the neonatal rat hippocampus in vivo and in vitro, respectively. Finally, pharmacological blockade of the GABA transporter-1 prolonged the decay of the large GDP-associated GABA transients but not of single postsynaptic GABAA receptor-mediated currents. As a whole the data in this thesis indicate that the mechanism of GDP generation, based on the interconnected network of bursting CA3 pyramidal neurons, is similar to that involved in adult SPW activity. Hence, GDPs do not reflect a network pattern that disappears during development but they are the in vitro counterpart of neonatal SPWs.

Androgen receptor in transcriptional regulation and carcinogenesis

Androgens control a variety of developmental processes that create the male phenotype and are important for maintaining male fertility and normal functions of tissues and organs that are not directly involved in procreation. Androgen receptor (AR) that mediates the biological actions of androgens is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. Although AR was cloned over 15 years ago, the mechanisms by which it regulates gene expression are not well understood. A growing body of in vitro experimental evidence suggests that a complex network of proteins is involved in the androgen-dependent transcriptional regulation. However, the process of AR-dependent transcriptional regulation under physiological conditions is largely elusive. In the present study, a series of experiments were performed, including quantitative chromatin immunoprecipitation (ChIP) assays, to investigate AR-mediated transcription process using living prostate cancer cells. Our results show that the loading of AR and recruitment of coactivators and RNA polymerase II (Pol II) to both the promoter and enhancer of AR target genes are a transient and cyclic event that in addition to hyperacetylation, also involves dynamic changes in methylation, phosphorylation of core histone H3 in androgen-treated LNCaP cells. The dynamics of testosterone (T)-induced loading of AR onto the proximal promoters of the genes clearly differed from that loaded onto the distal enhancers. Significantly, more holo-AR was loaded onto the enhancers than the promoters, but the principal Pol II transcription complex was assembled on the promoters. By contrast, the pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the PSA promoter, but was unable to load onto the PSA enhancer and was incapable of recruiting Pol II, coactivators and following changes of covalent histone modifications. The partial antagonist cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading onto both the PSA promoter and enhancer at a comparable efficiency with androgen in LNCaP cells expressing mutant AR. However, CPA- and RU486-bound AR not only recruited Pol II and coactivator p300 and GRIP1 onto the promoter and enhancer, but also recruited the corepressor NCoR onto the promoter as efficiently as CDX. In addition, we demonstrate that both proteasome and protein kinases are implicated in AR-mediated transcription. Even though proteasome inhibitor MG132 and protein kinase inhibitor DRB (5, 6-Dichlorobenzimidazole riboside) can block ligand-dependent accumulation of PSA mRNA with same efficiency, their use results in different molecular profiles in terms of the formation of AR-mediated transcriptional complex. Collectively, these results indicate that transcriptional activation by AR is a complicated process, which includes transient loading of holo-AR and recruitment of Pol II and coregulators accompanied by a cascade of distinct covalent histone modifications; This process involves both the promoter and enhancer elements, as well as other general components of the cell machineries e.g. proteasome and protein kinase; The pure antiandrogen CDX and the partial antagonist CPA and RU486 exhibit clearly different profiles in terms of their ability to induce the formation of AR-dependent transcriptional complexes and the histone modifications associated with the target genes in human prostate cancer cells. Finally, by using quantitative RT-PCR to compare the expression of sixteen AR co-regulators in prostate cancer cell lines, xenografts, and clinical prostate cancer specimens we suggest that AR co-regulators protein inhibitor of activated STAT1 (PIAS1) and steroid receptor coactivator 1(SRC1) could be involved in the progression of prostate cancer.

Candidate gene studies on cardiovascular traits

Cardiovascular diseases (CVD) are a major cause of death and disability in Western countries and a growing health problem in the developing world. The genetic component of both coronary heart disease (CHD) and ischemic stroke events has been established in twin studies, and the traits predisposing to CVD, such as hypertension, dyslipidemias, obesity, diabetes, and smoking behavior, are all partly hereditary. Better understanding of the pathophysiology of CVD-related traits could help to target disease prevention and clinical treatment to individuals at an especially high disease risk and provide novel pharmaceutical interventions. This thesis aimed to clarify the genetic background of CVD at a population level using large Nordic population cohorts and a candidate gene approach. The first study concentrated on the allelic diversity of the thrombomodulin (THBD) gene in two Finnish cohorts, FINRISK-92 and FINRISK-97. The results from this study implied that THBD variants do not substantially contribute to CVD risk. In the second study, three other candidate genes were added to the analyses. The study investigated the epistatic effects of coagulation factor V (F5), intercellular adhesion molecule -1 (ICAM1), protein C (PROC), and THBD in the same FINRISK cohorts. The results were encouraging; we were able to identify several single SNPs and SNP combinations associating with CVD and mortality. Interestingly, THBD variants appeared in the associating SNP combinations despite the negative results from Study I, suggesting that THBD contributes to CVD through gene-gene interactions. In the third study, upstream transcription factor -1 (USF1) was analyzed in a cohort of Swedish men. USF1 was associated with metabolic syndrome, characterized by accumulation of different CVD risk factors. A putative protective and a putative risk variant were identified. A direct association with CVD was not observed. The longitudinal nature of the study also clarified the effect of USF1 variants on CVD risk factors followed in four examinations throughout adulthood. The three studies provided valuable information on the study of complex traits, highlighting the use of large study samples, the importance of replication, and the full coverage of the major allelic variants of the target genes to assure reliable findings. Although the genetic basis of coronary heart disease and ischemic stroke remains unknown, single genetic findings may facilitate the recognition of high-risk subgroups.

Identification of the Meckel syndrome gene (MKS1) exposes a novel ciliopathy

Meckel syndrome (MKS, MIM 249000) is an autosomal recessive developmental disorder causing death in utero or shortly after birth. The hallmarks of the disease are cystic kidney dysplasia and fibrotic changes of the liver, occipital encephalocele with or without hydrocephalus and polydactyly. Other anomalies frequently seen in the patients are incomplete development of the male genitalia, club feet and cleft lip or palate. The clinical picture has been well characterized in the literature while the molecular pathology underlying the disease has remained unclear until now. In this study we identified the first MKS gene by utilizing the disease haplotypes in Finnish MKS families linked to the MKS1 locus on chromosome 17q23 (MKS1) locus. Subsequently, the genetic heterogeneity of MKS was established in the Finnish families. Mutations in at least four different genes can cause MKS. These genes have been mapped to the chromosomes 17q23 (MKS1), 11q13 (MKS2), 8q22 (MKS3) and 9q33 (MKS4). Two of these genes have been identified so far: The MKS1 gene (this work) and the MKS3 gene. The identified MKS1 gene was initially a novel human gene which is conserved among species. We found three different MKS mutations, one of them being the Finnish founder mutation. The information available from MKS1 orthologs in other species convinced us that the MKS1 gene is required for normal ciliogenesis. Defects of the cilial system in other human diseases and model organisms actually cause phenotypic features similar to those seen in MKS patients. The MKS3 (TMEM67) gene encodes a transmembrane protein and the gene maps to the syntenic Wpk locus in the rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus. The available information from these two genes suggest that MKS1 would encode a structural component of the centriole required for normal ciliary functions, and MKS3 would be a transmembrane component most likely required for normal ciliary sensory signaling. The MKS4 locus was localized to chromosme 9q32-33 in this study by using an inbred Finnish family with two affected and two healthy children. This fourth locus contains TRIM32 gene, which is associated to another well characterized human ciliopathy, Bardet Biedl syndrome (BBS). Future studies should identify the MKS4 gene on chromosome 9q and confirm if there are more than two genes causing MKS Finnish families. The research on critical signaling pathways in organogenesis have shown that both Wnt and Hedgehog pathways are dependent on functional cilia. The MKS gene products will serve as excellent model molecules for more detailed studies of the functional role of cilia in organogenesis in more detail.

Examination of Genetic Components Affecting Human Obesity-Related Quantitative Traits

Obesity increases the risk for several conditions, including type 2 diabetes mellitus, cardiovascular disease, hypertension, osteoarthirits and certain types of cancer. Twin- and family studies have shown that there is a major genetic component in the determination of body mass. In recent years several technological and scientific advance have been made in obesity research. For instance, novel replicated loci have been revealed by a number of genome wide association studies. This thesis aimed to investigate the association of genetic factors and obesity-related quantitative traits. The first study investigated the role of the lactase gene in anthropometric traits. We genetically defined lactose persistence by genotyping 31 720 individuals of European descent. We found that lactase persistence was significantly correlated with weight and body mass index but not with height. In the second study we performed the largest whole genome linkage scan for body mass index to date. The sample consisted of 4401 twin families and 10 535 individuals from six European countries. We found supporting evidence for two loci (3q29 and 7q36). We observed that the heritability estimate increased substantially when additional family members were removed from the analyses, which suggests reduced environmental variance in the twin sample. In the third study we assessed metabonomic, transcriptomic and genomic variation in a Finnish population cohort of 518 individuals. We formed gene expression networks to portray pathways and showed that a set of highly correlated genes of an inflammatory pathway associated with 80 serum metabolites (of 134 quantified measures). Strong association was found, for example, with several lipoprotein subclasses. We inferred causality by using genetic variation as anchors. The expression of the network genes was found to be dependent on the circulatory metabolite concentrations.

Function of psoriasis susceptibility gene CCHCR1

Psoriasis is a chronic skin disease characterized by abnormal keratinocyte proliferation and differentiation, neoangiogenesis and inflammation. Its etiology is multifactorial, as both the environmental and genetic factors have an important role in the pathogenesis of psoriasis. The exact disease mechanism behind psoriasis still remains unknown. The most important genetic susceptibility region for psoriasis has been located to PSORS1 locus in chromosome 6. The area includes multiply good candidate genes but the strong linkage disequilibrium between them has made genetic studies difficult. One of the candidate genes in PSORS1 is CCHCR1, which has a psoriasis-associated gene form CCHCR1*WWCC. The aim of the study was to elucidate the function of CCHCR1 and its potential role in the pathogenesis of psoriasis. In this study, transgenic mice expressing either the healthy or psoriasis-associated gene form of CCHCR1 were engineered and characterized. Mice were phenotypically normal but their gene expression profiles revealed many similarities to that observed in human psoriatic skin. In addition, the psoriasis-associated gene form had specific impacts on the expression of many genes relevant to the pathogenesis of psoriasis. We also challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoylphorbol-13-acetate (TPA). The experiments revealed that CCHCR1 impacts on keratinocyte proliferation by limiting it. In addition, we demonstrated that CCHCR1 has a role in steroidogenesis and showed that both CCHCR1 forms promote synthesis of steroids. Also many agents relevant either for steroidogenesis or cell proliferation were shown to regulate the expression level of CCHCR1. The present study showed that CCHCR1 has functional properties relevant in the context of psoriasis. Firstly, CCHCR1 affects proliferation of keratinocytes as it may function as a negative regulator of keratinocyte proliferation. Secondly, CCHCR1 also has a role in steroidogenesis, a function relevant both in the pathogenesis of psoriasis and regulation of cell proliferation. This study suggests that aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.