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.

Analysis of proteins with the `hot dog' fold: Prediction of function and identification of catalytic residues of hypothetical proteins

Background: The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. Results: We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. Conclusion: The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.

Novel conditional mouse models for targeted kidney research : Emphasis on the analysis of the biological function of nephrin

Nephrin is a transmembrane protein belonging to the immunoglobulin superfamily and is expressed primarily in the podocytes, which are highly differentiated epithelial cells needed for primary urine formation in the kidney. Mutations leading to nephrin loss abrogate podocyte morphology, and result in massive protein loss into urine and consequent early death in humans carrying specific mutations in this gene. The disease phenotype is closely replicated in respective mouse models. The purpose of this thesis was to generate novel inducible mouse-lines, which allow targeted gene deletion in a time and tissue-specific manner. A proof of principle model for succesful gene therapy for this disease was generated, which allowed podocyte specific transgene replacement to rescue gene deficient mice from perinatal lethality. Furthermore, the phenotypic consequences of nephrin restoration in the kidney and nephrin deficiency in the testis, brain and pancreas in rescued mice were investigated. A novel podocyte-specific construct was achieved by using standard cloning techniques to provide an inducible tool for in vitro and in vivo gene targeting. Using modified constructs and microinjection procedures two novel transgenic mouse-lines were generated. First, a mouse-line with doxycycline inducible expression of Cre recombinase that allows podocyte-specific gene deletion was generated. Second, a mouse-line with doxycycline inducible expression of rat nephrin, which allows podocyte-specific nephrin over-expression was made. Furthermore, it was possible to rescue nephrin deficient mice from perinatal lethality by cross-breeding them with a mouse-line with inducible rat nephrin expression that restored the missing endogenous nephrin only in the kidney after doxycycline treatment. The rescued mice were smaller, infertile, showed genital malformations and developed distinct histological abnormalities in the kidney with an altered molecular composition of the podocytes. Histological changes were also found in the testis, cerebellum and pancreas. The expression of another molecule with limited tissue expression, densin, was localized to the plasma membranes of Sertoli cells in the testis by immunofluorescence staining. Densin may be an essential adherens junction protein between Sertoli cells and developing germ cells and these junctions share similar protein assembly with kidney podocytes. This single, binary conditional construct serves as a cost- and time-efficient tool to increase the understanding of podocyte-specific key proteins in health and disease. The results verified a tightly controlled inducible podocyte-specific transgene expression in vitro and in vivo as expected. These novel mouse-lines with doxycycline inducible Cre recombinase and with rat nephrin expression will be useful for conditional gene targeting of essential podocyte proteins and to study in detail their functions in the adult mice. This is important for future diagnostic and pharmacologic development platforms.

Single kernel NIR assessment for QTL identification

Development and evaluation of a single kernel NIR assessment method for improving baley malting quality QTL identification.

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.

Dynamic structure factor across the liquid-solid interface: appearance of a delta-function elastic peak

We present a theoretical calculation of the dynamic structure factor, S(k, ω), at the liquid-solid interface for large values of the wavevector k. An analytic expression is derived which shows the evolution of the elastic peak as the solid surface is approached from the liquid side.

Age-related changes in hepatic function: An update on implications for drug therapy

The accumulation of deficits with increasing age results in a decline in the functional capacity of multiple organs and systems. These changes can have a significant influence on the pharmacokinetics and pharmacodynamics of prescribed drugs. Although alterations in body composition and worsening renal clearance are important considerations, for most drugs the liver has the greatest effect on metabolism. Age-related change in hepatic function thereby causes much of the variability in older people’s responses to medication. In this review, we propose that a decline in the ability of the liver to inactivate toxins may contribute to a proinflammatory state in which frailty can develop. Since inflammation also downregulates drug metabolism, medication prescribed to frail older people in accordance with disease-specific guidelines may undergo reduced systemic clearance, leading to adverse drug reactions, further functional decline and increasing polypharmacy, exacerbating rather than ameliorating frailty status. We also describe how increasing chronological age and frailty status impact liver size, blood flow and protein binding and enzymes of drug metabolism. This is used to contextualise our discussion of appropriate prescribing practices. For example, while the general axiom of ‘start low, go slow’ should underpin the initiation of medication (titrating to a defined therapeutic goal), it is important to consider whether drug clearance is flow or capacity-limited. By summarising the effect of age-related changes in hepatic function on medications commonly used in older people, we aim to provide a guide that will have high clinical utility for practising geriatricians.

Brownian particles in stationary and moving traps: The mean and variance of the heat distribution function

A recent theoretical model developed by Imparato et al. Phys of the experimentally measured heat and work effects produced by the thermal fluctuations of single micron-sized polystyrene beads in stationary and moving optical traps has proved to be quite successful in rationalizing the observed experimental data. The model, based on the overdamped Brownian dynamics of a particle in a harmonic potential that moves at a constant speed under a time-dependent force, is used to obtain an approximate expression for the distribution of the heat dissipated by the particle at long times. In this paper, we generalize the above model to consider particle dynamics in the presence of colored noise, without passing to the overdamped limit, as a way of modeling experimental situations in which the fluctuations of the medium exhibit long-lived temporal correlations, of the kind characteristic of polymeric solutions, for instance, or of similar viscoelastic fluids. Although we have not been able to find an expression for the heat distribution itself, we do obtain exact expressions for its mean and variance, both for the static and for the moving trap cases. These moments are valid for arbitrary times and they also hold in the inertial regime, but they reduce exactly to the results of Imparato et al. in appropriate limits. DOI: 10.1103/PhysRevE.80.011118 PACS.

The Myopathic Protein Myotilin in Developing Mouse and in Muscle Function

Skeletal muscle cells are highly specialised in order to accomplish their function. During development, the fusion of hundreds of immature myoblasts creates large syncytial myofibres with a highly ordered cytoplasm filled with packed myofibrils. The assembly and organisation of contractile myofibrils must be tightly controlled. Indeed, the number of proteins involved in sarcomere building is impressive, and the role of many of them has only recently begun to be elucidated. Myotilin was originally identified as a high affinity a-actinin binding protein in yeast twohybrid screen. It was then found to interact also with filamin C, actin, ZASP and FATZ-1. Human myotilin is mainly expressed in striated muscle and induces efficient actin bundling in vitro and in cells. Moreover, mutations in myotilin cause different forms of muscle disease, now collectively known as myotilinopathies. In this thesis, consisting of three publications, the work on the mouse orthologue is presented. First, the cloning and molecular characterisation of the mouse myotilin gene showed that human and mouse myotilin share high sequence homology and a similar expression pattern and gene regulation. Functional analysis of the mouse promoter revealed the myogenic factor-binding elements that are required for myotilin gene transcription. Secondly, expression of myotilin was studied during mouse embryogenesis. Surprisingly, myotilin was expressed in a wide array of tissues at some stages of development; its expression pattern became more restricted at perinatal stages and in adult life. Immunostaining of human embryos confirmed broader myotilin expression compared to the sarcomeric marker titin. Finally, in the third article, targeted deletion of myotilin gene in mice revealed that it is not essential for muscle development and function. These data altogether indicate that the mouse can be used as a model for human myotilinopathy and that loss of myotilin does not alter significantly muscle structure and function. Therefore, disease-associated mutant myotilin may act as a dominant myopathic factor.

A Branch and Bound Algorithm for a Transportation Type Problem with Piecewise Linear Convex Objective Function

A branch and bound type algorithm is presented in this paper to the problem of finding a transportation schedule which minimises the total transportation cost, where the transportation cost over each route is assumed to be a piecewice linear continuous convex function with increasing slopes. The algorithm is an extension of the work done by Balachandran and Perry, in which the transportation cost over each route is assumed to beapiecewise linear discontinuous function with decreasing slopes. A numerical example is solved illustrating the algorithm.

Time evolution of graphene growth on SiC as a function of annealing temperature

We followed by X-ray Photoelectron Spectroscopy (XPS) the time evolution of graphene layers obtained by annealing 3C SiC(111)/Si(111) crystals at different temperatures. The intensity of the carbon signal provides a quantification of the graphene thickness as a function of the annealing time, which follows a power law with exponent 0.5. We show that a kinetic model, based on a bottom-up growth mechanism, provides a full explanation to the evolution of the graphene thickness as a function of time, allowing to calculate the effective activation energy of the process and the energy barriers, in excellent agreement with previous theoretical results. Our study provides a complete and exhaustive picture of Si diffusion into the SiC matrix, establishing the conditions for a perfect control of the graphene growth by Si sublimation.

Northern challanges for profitable production of quality naked oat

Naked oat (Avena sativa f.sp. nuda L.) is the highest quality cereal in northern growing conditions. However the cultivation area of naked oat is remarkably small. Major challenges for naked oat production are to observe its nakedness. The caryopsis of naked oat is sensitive to mechanical damage at harvest, especially at high grain moisture content. The greater the grain moisture content of naked oat at harvest, the more loses of germination capacity was caused by threshing. For producing high quality naked oat seed, it is recommended that harvesting be done at as low grain moisture content as possible. However, if this is not possible, better germination can be ensure with gentle harvest by reducing the cylinder speed. In spite of conventional oat s excellent fat and amino acid composition in animal feed use, as far as nutritional value is concerned, the total energy yield of oat is weaker than other cereals because of the hulls. Also with naked oat the dehulling is not complete, while hull content on different cultivars mostly varied between one to six percent. In addition to genotype, environmental conditions markedly control the expression of nakedness. Thresher settings had only limited effects on hull content. The function of hulls is to protect the groat, but this was confirmed only for Finnish, small grain, cultivar Lisbeth. The oat kernel is generally covered with fine silky hairs termed trichomes. The trichomes of naked oat are partly lost during threshing and handling of grains. Trichomes can cause itchiness in those handling the grains and also accumulate and form fine dust and can block-up machinery. The cultivars differed considerably in pubescence. Some thresher settings, including increased cylinder speed, slightly increased grain polishing such that grains had some areas completely free of trichomes. Adjusting thresher settings was generally not an efficient means of solving the problems associated with naked oat trichomes. The main differences in cultivation costs between naked and conventional oat lie in the amount of seeds required and the drying costs. The main differences affecting the economic result lie in market prices, yield level and feed value. The results indicate that naked oat is financially more profitable than conventional oat, when the crop is sold at a specific price at all yield levels and when the crop is used as feed at highest yield level. At lower yield levels, conventional oat is, in spite of its lower feed value, the more profitable option for feed use. Dehulled oat did not achieve the same economic result as naked oat, as the cost of dehulling, including the hull waste, was considerable. According to this study naked oat can be cultivated successfully under northern conditions, when taking into consideration the soft, naked grain through cultivation chain.