The function of Bmps and Runx2 in normal tooth development and in the pathogenesis of cleidocranial dysplasia

The development of many embryonic organs is regulated by reciprocal and sequential epithelial-mesenchymal interactions. These interactions are mediated by conserved signaling pathways that are reiteratively used. Cleidocranial dysplasia (CCD) is a congenital syndrome where both bone and tooth development is affected. The syndrome is characterized by short stature, abnormal clavicles, general bone dysplasia, and supernumerary teeth. CCD is caused by mutations in RUNX2, a transcription factor that is a key regulator of osteoblast differentiation and bone formation. The first aim of this study was to analyse the expression of a family of key signal molecules, Bone morphogenetic protein (Bmp) at different stages of tooth development. Bmps have a variety of functions and they were originally discovered as signals inducing ectopic bone formation. We performed a comparative in situ hybridisation analysis of the mRNA expression of Bmp2-7 from initiation of tooth development to differentiation of dental hard tissues. The expression patterns indicated that the Bmps signal between the epithelial and mesenchymal tissues during initiation and morphogenesis of tooth development, as well as during the differentiation of odontoblasts and ameloblasts. Furthermore, they are also part of the signalling networks whereby the enamel knot regulates the patterning of tooth cusps. The second aim was to study the role of Runx2 during tooth development and thereby to gain better understanding of the pathogenesis of the tooth phenotype in CCD. We analysed the tooth phenotype of Runx2 knockout mice and examined the patterns and regulation of Runx2 gene expression.. The teeth of wild-type and Runx2 mutant mice were compared by several methods including in situ hybridisation, tissue culture, bead implantation experiments, and epithelial-mesenchymal recombination studies. Phenotypic analysis of Runx2 -/- mutant tooth development showed that teeth failed to advance beyond the bud stage. Runx2 expression was restricted to dental mesenchyme between the bud and early bell stages of tooth development and it was regulated by epithelial signals, in particular Fgfs. We searched for downstream targets of Runx2 by comparative in situ hybridisation analysis. The expression of Fgf3 was downregulated in the mesenchyme of Runx2 -/- teeth. Shh expression was absent from the enamel knot in the lower molars of Runx2 -/- and reduced in the upper molars. In conclusion, these studies showed that Runx2 regulates key epithelial-mesenchymal interactions that control advancing tooth morphogenesis and histodifferentiation of the epithelial enamel organ. In addition, in the upper molars of Runx2 mutants extra buddings occured at the palatal side of the tooth bud. We suggest that Runx2 acts as an inhibitor of successional tooth formation by preventing advancing development of the buds. Accordingly, we propose that RUNX2 haploinsuffiency in humans causes incomplete inhibition of successional tooth formation and as a result supernumerary teeth.

On linear order and computation : The expressiveness of interactive computations on linear orders and computations indexed by ordinals

We solve the Dynamic Ehrenfeucht-Fra\"iss\'e Game on linear orders for both players, yielding a normal form for quantifier-rank equivalence classes of linear orders in first-order logic, infinitary logic, and generalized-infinitary logics with linearly ordered clocks. We show that Scott Sentences can be manipulated quickly, classified into local information, and consistency can be decided effectively in the length of the Scott Sentence. We describe a finite set of linked automata moving continuously on a linear order. Running them on ordinals, we compute the ordinal truth predicate and compute truth in the constructible universe of set-theory. Among the corollaries are a study of semi-models as efficient database of both model-theoretic and formulaic information, and a new proof of the atomicity of the Boolean algebra of sentences consistent with the theory of linear order -- i.e., that the finitely axiomatized theories of linear order are dense.

On Random Planar Curves and Their Scaling Limits

Planar curves arise naturally as interfaces between two regions of the plane. An important part of statistical physics is the study of lattice models. This thesis is about the interfaces of 2D lattice models. The scaling limit is an infinite system limit which is taken by letting the lattice mesh decrease to zero. At criticality, the scaling limit of an interface is one of the SLE curves (Schramm-Loewner evolution), introduced by Oded Schramm. This family of random curves is parametrized by a real variable, which determines the universality class of the model. The first and the second paper of this thesis study properties of SLEs. They contain two different methods to study the whole SLE curve, which is, in fact, the most interesting object from the statistical physics point of view. These methods are applied to study two symmetries of SLE: reversibility and duality. The first paper uses an algebraic method and a representation of the Virasoro algebra to find common martingales to different processes, and that way, to confirm the symmetries for polynomial expected values of natural SLE data. In the second paper, a recursion is obtained for the same kind of expected values. The recursion is based on stationarity of the law of the whole SLE curve under a SLE induced flow. The third paper deals with one of the most central questions of the field and provides a framework of estimates for describing 2D scaling limits by SLE curves. In particular, it is shown that a weak estimate on the probability of an annulus crossing implies that a random curve arising from a statistical physics model will have scaling limits and those will be well-described by Loewner evolutions with random driving forces.

Neuromagnetic studies on cortical somatosensory functions in infants and children : Normal development and effect of early brain lesions

Until recently, objective investigation of the functional development of the human brain in vivo was challenged by the lack of noninvasive research methods. Consequently, fairly little is known about cortical processing of sensory information even in healthy infants and children. Furthermore, mechanisms by which early brain insults affect brain development and function are poorly understood. In this thesis, we used magnetoencephalography (MEG) to investigate development of cortical somatosensory functions in healthy infants, very premature infants at risk for neurological disorders, and adolescents with hemiplegic cerebral palsy (CP). In newborns, stimulation of the hand activated both the contralateral primary (SIc) and secondary somatosensory cortices (SIIc). The activation patterns differed from those of adults, however. Some of the earliest SIc responses, constantly present in adults, were completely lacking in newborns and the effect of sleep stage on SIIc responses differed. These discrepancies between newborns and adults reflect the still developmental stage of the newborns’ somatosensory system. Its further maturation was demonstrated by a systematic transformation of the SIc response pattern with age. The main early adult­like components were present by age two. In very preterm infants, at term age, the SIc and SIIc were activated at similar latencies as in healthy fullterm newborns, but the SIc activity was weaker in the preterm group. The SIIc response was absent in four out of the six infants with brain lesions of the underlying hemisphere. Determining the prognostic value of this finding remains a subject for future studies, however. In the CP adolescents with pure subcortical lesions, contrasting their unilateral symptoms, the SIc responses of both hemispheres differed from those of controls: For example the distance between SIc representation areas for digits II and V was shorter bilaterally. In four of the five CP patients with cortico­subcortical brain lesions, no normal early SIc responses were evoked by stimulation of the palsied hand. The varying differences in neuronal functions, underlying the common clinical symptoms, call for investigation of more precisely designed rehabilitation strategies resting on knowledge about individual functional alterations in the sensorimotor networks.

Cardiac function before and after treatment for various types of loading conditions and in myocardial restriction : A prospective study on pediatric patients with two- and three-dimensional echocardiography and measurement of serum natriuretic peptides

Background: The incidence of all forms of congenital heart defects is 0.75%. For patients with congenital heart defects, life-expectancy has improved with new treatment modalities. Structural heart defects may require surgical or catheter treatment which may be corrective or palliative. Even those with corrective therapy need regular follow-up due to residual lesions, late sequelae, and possible complications after interventions. Aims: The aim of this thesis was to evaluate cardiac function before and after treatment for volume overload of the right ventricle (RV) caused by atrial septal defect (ASD), volume overload of the left ventricle (LV) caused by patent ductus arteriosus (PDA), and pressure overload of the LV caused by coarctation of the aorta (CoA), and to evaluate cardiac function in patients with Mulibrey nanism. Methods: In Study I, of the 24 children with ASD, 7 underwent surgical correction and 17 percutaneous occlusion of ASD. Study II had 33 patients with PDA undergoing percutaneous occlusion. In Study III, 28 patients with CoA underwent either surgical correction or percutaneous balloon dilatation of CoA. Study IV comprised 26 children with Mulibrey nanism. A total of 76 healthy voluntary children were examined as a control group. In each study, controls were matched to patients. All patients and controls underwent clinical cardiovascular examinations, two-dimensional (2D) and three-dimensional (3D) echocardiographic examinations, and blood sampling for measurement of natriuretic peptides prior to the intervention and twice or three times thereafter. Control children were examined once by 2D and 3D echocardiography. M-mode echocardiography was performed from the parasternal long axis view directed by 2D echocardiography. The left atrium-to-aorta (LA/Ao) ratio was calculated as an index of LA size. The end-diastolic and end-systolic dimensions of LV as well as the end-diastolic thicknesses of the interventricular septum and LV posterior wall were measured. LV volumes, and the fractional shortening (FS) and ejection fraction (EF) as indices of contractility were then calculated, and the z scores of LV dimensions determined. Diastolic function of LV was estimated from the mitral inflow signal obtained by Doppler echocardiography. In three-dimensional echocardiography, time-volume curves were used to determine end-diastolic and end-systolic volumes, stroke volume, and EF. Diastolic and systolic function of LV was estimated from the calculated first derivatives of these curves. Results: (I): In all children with ASD, during the one-year follow-up, the z score of the RV end-diastolic diameter decreased and that of LV increased. However, dilatation of RV did not resolve entirely during the follow-up in either treatment group. In addition, the size of LV increased more slowly in the surgical subgroup but reached control levels in both groups. Concentrations of natriuretic peptides in patients treated percutaneously increased during the first month after ASD closure and normalized thereafter, but in patients treated surgically, they remained higher than in controls. (II): In the PDA group, at baseline, the end-diastolic diameter of LV measured over 2SD in 5 of 33 patients. The median N-terminal pro-brain natriuretic peptide (proBNP) concentration before closure measured 72 ng/l in the control group and 141 ng/l in the PDA group (P = 0.001) and 6 months after closure measured 78.5 ng/l (P = NS). Patients differed from control subjects in indices of LV diastolic and systolic function at baseline, but by the end of follow-up, all these differences had disappeared. Even in the subgroup of patients with normal-sized LV at baseline, the LV end-diastolic volume decreased significantly during follow-up. (III): Before repair, the size and wall thickness of LV were higher in patients with CoA than in controls. Systolic blood pressure measured a median 123 mm Hg in patients before repair (P < 0.001) and 103 mm Hg one year thereafter, and 101 mm Hg in controls. The diameter of the coarctation segment measured a median 3.0 mm at baseline, and 7.9 at the 12-month (P = 0.006) follow-up. Thicknesses of the interventricular septum and posterior wall of the LV decreased after repair but increased to the initial level one year thereafter. The velocity time integrals of mitral inflow increased, but no changes were evident in LV dimensions or contractility. During follow-up, serum levels of natriuretic peptides decreased correlating with diastolic and systolic indices of LV function in 2D and 3D echocardiography. (IV): In 2D echocardiography, the interventricular septum and LV posterior wall were thicker, and velocity time integrals of mitral inflow shorter in patients with Mulibrey nanism than in controls. In 3D echocardiography, LV end-diastolic volume measured a median 51.9 (range 33.3 to 73.4) ml/m² in patients and 59.7 (range 37.6 to 87.6) ml/m² in controls (P = 0.040), and serum levels of ANPN and proBNP a median 0.54 (range 0.04 to 4.7) nmol/l and 289 (range 18 to 9170) ng/l, in patients and 0.28 (range 0.09 to 0.72) nmol/l (P < 0.001) and 54 (range 26 to 139) ng/l (P < 0.001) in controls. They correlated with several indices of diastolic LV function. Conclusions (I): During the one-year follow-up after the ASD closure, RV size decreased but did not normalize in all patients. The size of the LV normalized after ASD closure but the increase in LV size was slower in patients treated surgically than in those treated with the percutaneous technique. Serum levels of ANPN and proBNP were elevated prior to ASD closure but decreased thereafter to control levels in patients treated with the percutaneous technique but not in those treated surgically. (II): Changes in LV volume and function caused by PDA disappeared by 6 months after percutaneous closure. Even the children with normal-sized LV benefited from the procedure. (III): After repair of CoA, the RV size and the velocity time integrals of mitral inflow increased, and serum levels of natriuretic peptides decreased. Patients need close follow-up, despite cessation of LV pressure overload, since LV hypertrophy persisted even in normotensive patients with normal growth of the coarctation segment. (IV): In children with Mulibrey nanism, the LV wall was hypertrophied, with myocardial restriction and impairment of LV function. Significant correlations appeared between indices of LV function, size of the left atrium, and levels of natriuretic peptides, indicating that measurement of serum levels of natriuretic peptides can be used in the clinical follow-up of this patient group despite its dependence on loading conditions.

Acquisition of focus by normal hearing and Cochlear implanted children

Two experiments investigated the perception of compound vs. phrasal stress and narrow focus in normally hearing children and children with Cochlear Implants (CI). Additionally, we investigated whether musical experience would predict children’s performance in these tasks. The results showed no difference between CI and normal-hearing (NH) children in either experiment. However, whereas we found no clear effect of age in the children’s stress detection, there was a clear age related trajectory in the ability to recognise (narrow) focus. Moreover, this trend was similar to what has been found previously for English children. Importantly, prior music experience was significantly linked to CI children’s perception of focus.

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.