Molecular biology and functions of epilysin (MMP-28)

Epilysin (MMP-28) is the most recently identified member of the matrix metalloproteinase (MMP) family of extracellular proteases. Together these enzymes are capable of degrading almost all components of the extracellular matrix (ECM) and are thus involved in important biological processes such as development, wound healing and immune functions, but also in pathological processes such as tumor invasion, metastasis and arthritis. MMPs do not act solely by degrading the ECM. They also regulate cell behavior by releasing growth factors and biologically active peptides from the ECM, by modulating cell surface receptors and adhesion molecules and by regulating the activity of many important mediators in inflammatory pathways. The aim of this study was to define the unique role of epilysin within the MMP-family, to elucidate how and when it is expressed and how its catalytic activity is regulated. To gain information on its essential functions and substrates, the specific aim was to characterize how epilysin affects the phenotype of epithelial cells, where it is biologically expressed. During the course of the study we found that the epilysin promoter contains a well conserved GT-box that is essential for the basic expression of this gene. Transcription factors Sp1 and Sp3 bind this sequence and could hence regulate both the basic and cell type and differentiation stage specific expression of epilysin. We cloned mouse epilysin cDNA and found that epilysin is well conserved between human and mouse genomes and that epilysin is glycosylated and activated by furin. Similarly to in human tissues, epilysin is normally expressed in a number of mouse tissues. The expression pattern differs from most other MMPs, which are expressed only in response to injury or inflammation and in pathological processes like cancer. These findings implicate that epilysin could be involved in tissue homeostasis, perhaps fine-tuning the phenotype of epithelial cells according to signals from the ECM. In view of these results, it was unexpected to find that epilysin can induce a stable epithelial to mesenchymal transition (EMT) when overexpressed in epithelial lung carcinoma cells. Transforming growth factor b (TGF-b) was recognized as a crucial mediator of this process, which was characterized by the loss of E-cadherin mediated cell-cell adhesion, elevated expression of gelatinase B and MT1-MMP and increased cell migration and invasion into collagen I gels. We also observed that epilysin is bound to the surface of epithelial cells and that this interaction is lost upon cell transformation and is susceptible to degradation by membrane type-1-MMP (MT1-MMP). The wide expression of epilysin under physiological conditions implicates that its effects on epithelial cell phenotype in vivo are not as dramatic as seen in our in vitro cell system. Nevertheless, current results indicate a possible interaction between epilysin and TGF-b also under physiological circumstances, where epilysin activity may not induce EMT but, instead, trigger less permanent changes in TGF-b signaling and cell motility. Epilysin may thus play an important role in TGF-b regulated events such as wound healing and inflammation, processes where involvement of epilysin has been indicated.

Aspiration-based utility functions in a planning model for timber flow management.

The study presents a theory of utility models based on aspiration levels, as well as the application of this theory to the planning of timber flow economics. The first part of the study comprises a derivation of the utility-theoretic basis for the application of aspiration levels. Two basic models are dealt with: the additive and the multiplicative. Applied here solely for partial utility functions, aspiration and reservation levels are interpreted as defining piecewisely linear functions. The standpoint of the choices of the decision-maker is emphasized by the use of indifference curves. The second part of the study introduces a model for the management of timber flows. The model is based on the assumption that the decision-maker is willing to specify a shape of income flow which is different from that of the capital-theoretic optimum. The utility model comprises four aspiration-based compound utility functions. The theory and the flow model are tested numerically by computations covering three forest holdings. The results show that the additive model is sensitive even to slight changes in relative importances and aspiration levels. This applies particularly to nearly linear production possibility boundaries of monetary variables. The multiplicative model, on the other hand, is stable because it generates strictly convex indifference curves. Due to a higher marginal rate of substitution, the multiplicative model implies a stronger dependence on forest management than the additive function. For income trajectory optimization, a method utilizing an income trajectory index is more efficient than one based on the use of aspiration levels per management period. Smooth trajectories can be attained by squaring the deviations of the feasible trajectories from the desired one.

Koneellisen annosjakelupalvelun aloittaminen - lääkehoidon tarkistus ja sen vaikutus potilaan lääkehoitoon

Farmaseuttisilla palveluilla tarkoitetaan apteekkien palveluita, joissa hyödynnetään apteekin farmaseuttisen henkilökunnan tietoja ja taitoja. Farmaseuttiset palvelut voidaan jakaa farmaseuttisiin perus- ja erityispalveluihin. Farmaseuttiset peruspalvelut kattavat apteekkien lakisääteiset tehtävät, kun taas farmaseuttisilla erityispalveluilla pyritään ottamaan aktiivisempi rooli asiakkaan terveyden edistämisessä. Koneellinen annosjakelupalvelu on farmaseuttinen erityispalvelu. Koneellisessa annosjakelupalvelussa lääkkeet jaetaan kerta-annospusseihin annostusajankohdan mukaan. Kun uusi asiakas aloittaa koneellinen annosjakelupalvelun, tarkistetaan asiakkaan lääkitys yhteensopimattomien ja turhien lääkkeiden osalta. Palvelun aloitushetkellä huomioidaan myös lääkevalmisteiden sopivuus koneelliseen annosjakeluun sekä tarkistetaan valmisteiden annosteluajankohdat. Koneellisessa annosjakelupalvelussa asiakkaan lääkehoidosta muodostetaan lääkityskortti, josta kokonaislääkehoito on helppo tarkistaa. Erikoistyön tavoitteena oli selvittää millainen lääkehoidon arviointi tai tarkistus koneellisen annosjakelupalvelun aloittamisen yhteydessä tehdään ja miten palvelun aloittavien asiakkaiden kokonaislääkehoitotieto saadaan selvitettyä. Lisäksi selvitettiin millaisia muutoksia lääkehoitoihin tehdään palvelun aloittamisen yhteydessä, mitkä ovat muutosten syyt sekä millainen on asiakkaan kokonaislääkehoito. Kyselylomake lähetettiin kaikkiin apteekkeihin, jotka tilasivat koneellista annosjakelua sopimusvalmistuksena Espoonlahden apteekilta syyskuussa 2010. Tutkimus suoritettiin semistrukturoidulla kirjallisella kyselyllä, joka sisälsi sekä avoimia kysymyksiä että monivalintakysymyksiä. Kyselyyn saatiin 147 vastausta ja vastausprosentiksi muodostui 45. Vastauksia kyselyyn saatiin koko Mannersuomen alueelta ja kaikkien kokoluokkien apteekeilta. Koneellisen annosjakelupalvelun aloittavat henkilöt ovat pääasiassa iäkkäitä, jotka ovat kotihoidon piirissä, asuvat hoitokodissa tai palveluasumisen yksikössä. Asiakkaiden lääkitystietojen keräämisessä hyödynnetään lääkityskorttia, mutta lääkityskortin tietoja päivitetään myös muista lähteistä. Asiakkaiden lääkityksille tehdään useimmiten lääkehoidon tarkistus moniammatillisena yhteistyönä. Lääkehoidolle tehdyt muutokset johtuvat pääasiassa lääkevaihdosta, annosjakelukoneen lääkevalikoimasta tai puolittamisen välttämisestä. Lääkehoidoissa on vain vähän yhteisvaikutuksia, jotka johtavat lääkevalmisteen käytön lopettamiseen. Lääkehoidon tarkistuksella ei ollut suurta vaikutusta asiakkaiden käyttämien lääkevalmisteiden määrään. Palvelun aloittamisen jälkeen asiakkaalla on käytössään keskimäärin 11 lääkevalmistetta, joista seitsemää jaellaan koneellisesti. Lääkeaineryhmistä eniten käytettyjä ovat hermostoon vaikuttavat sekä sydän- ja verisuonisairauksien lääkkeet, joita kumpaakin on käytössä keskimäärin kolme jokaisella uudella koneellisen annosjakelupalvelun asiakkaalla sekä palvelun aloittamista ennen että sen jälkeen.

Monetary policy rules and estimated reaction functions for the European Central Bank

This thesis studies the interest-rate policy of the ECB by estimating monetary policy rules using real-time data and central bank forecasts. The aim of the estimations is to try to characterize a decade of common monetary policy and to look at how different models perform at this task.The estimated rules include: contemporary Taylor rules, forward-looking Taylor rules, nonlinearrules and forecast-based rules. The nonlinear models allow for the possibility of zone-like preferences and an asymmetric response to key variables. The models therefore encompass the most popular sub-group of simple models used for policy analysis as well as the more unusual non-linear approach. In addition to the empirical work, this thesis also contains a more general discussion of monetary policy rules mostly from a New Keynesian perspective. This discussion includes an overview of some notable related studies, optimal policy, policy gradualism and several other related subjects. The regression estimations are performed with either least squares or the generalized method of moments depending on the requirements of the estimations. The estimations use data from both the Euro Area Real-Time Database and the central bank forecasts published in ECB Monthly Bulletins. These data sources represent some of the best data that is available for this kind of analysis. The main results of this thesis are that forward-looking behavior appears highly prevalent, but that standard forward-looking Taylor rules offer only ambivalent results with regard to inflation. Nonlinear models are shown to work, but on the other hand do not have a strong rationale over a simpler linear formulation. However, the forecasts appear to be highly useful in characterizing policy and may offer the most accurate depiction of a predominantly forward-looking central bank. In particular the inflation response appears much stronger while the output response becomes highly forward-looking as well.

Die Eigenschaften der Benennungen und ihr Einfluss auf deren Verwendung am Beispiel der Euro-Währung : Eine quantitative und kontrastive Analyse der Terminologie im Finnischen und im Deutschen

This dissertation deals with the terminology of the Euro currency. Its aims are to determine the characteristics of the designations in a certain LSP and to discover whether the recommendations and rules that have been given to the formation of designations and 'ideal' designations have any influence on the usage of the designations. The characteristics analysed include length of the designation, part of speech, form, formation method, constancy, monosemy, suitability to a concept system and degree of specialty. The study analyses the actual usage of the designations in texts and the implementation of the designations. The study is an adaptation of a terminometric survey and uses concept analysis and quantitative analysis as its basic methods. The frequency of each characteristic is measured in terms of statistics. It is assumed that the 'ideality' of a designation influences its usage, for example that if a designation is short, it is used more than its longer rivals (synonyms). The results are analysed in a corpus consisting of a compilation of different texts concerning the Euro. The corpus is divided according to three features: year (1998-2003), genre (judicial texts, annual reports and brochures) and language (Finnish and German). Each analysis is performed according to each of these features and compared with the others. The results indicate that some of the characteristics of the designations indeed seem to have an influence on the usage of the designations. For example, monosemy and suitability to the concept system often lead to the implementation of the designation having the ideal or certain value in these characteristics in the analysed Finnish material. In German material, an 'ideal' value in the characteristics leads to the implementation of the designations more often than in Finnish. The contrastive study indicates that, for example, suitability to a concept system leads to implementation of a designation in judicial texts more often than in other genres. The recommendations given to an 'ideal' designation are thus often acceptable, but they cannot be generalized for all languages in the same extent.

Vyra enie obob čёnno-ličnogo značenija v russkom jazyke

Expressing generalized-personal meaning in Russian Based on data from Russian, this doctoral dissertation examines generalized-personal meaning that is, generic expressions referring to all human beings, people in general, each or any person (e.g. S vozrastom načinae cenit prostye ve či With age you start to appreciate simple things ). The study shares its basic theoretical orientation with functional approaches going from meaning to form . The objective of the thesis is to determine and describe the various linguistic means which can be used by the speaker to express generalized-personal meaning. The main material of the study consists of 2,000 examples collected from modern Russian literature, newspapers, and magazines. The linguistic means of expressing generalized-personal meaning are divided into three main classes. Morphological and lexico-grammatical means (22% of the material) include the use of personal pronouns and personal verbal endings. In Russian, all personal forms except the 3rd person singular can be used in a generalized-personal meaning. Lexical means (14% of the material) involve, above all, pronouns like vse all , ka dyj everyone , nikto no one , as well as the nouns čelovek man and ljudi people . In emotional speech, generalized-personal meaning can also be conveyed lexically by using utterances like da e idiot znaet even an idiot knows . In rhetorical questions the pronoun kto who can appear in this meaning (cf. Kto ne ljubit moro enoe?! Who doesn t like ice cream?! ). The third main class, syntactic means (64% of the material), consists of constructions in which the generic person is not expressed at the surface level. This class mainly includes two-component structures in which the infinitive relates to a modal predicative adverb (e.g. mo no can, be allowed to , nado must ), modal verb (e.g. stoit be worth(while) , sleduet must, be obliged to ), or predicative adverb ending in -о (e.g. trudno it is hard to , neprilično is not appropriate ). Other syntactic means are: one-component infinitive structures, so-called embedded structures, structures with a processual noun, passive constructions, and gerund constructions. The different forms of expression available in Russian are not interchangeable in all contexts. Even if a given context tolerates the substitution of one construction for another, the two expressions are never entirely synonymous. In addition to determining the range of forms which can express generalized-personal meaning, the study aims to compare these forms and to specify the conditions and possible restrictions (contextual, semantic, syntactic, stylistic, etc.) associated with the use of each construction. In Russian linguistics, the generalized-personal meaning has not been extensively studied from a functional perspective. The advantage of a meaning-based functional approach is that it gives a comprehensive picture of the diversity and distribution of the phenomenon.

Mauwake reference grammar

This dissertation provides a synchronic grammatical description of Mauwake, a Papuan (Trans-New Guinea) language of about 2000 speakers on the North Coast of the Madang Province in Papua New Guinea. The theoretical background is that of Basic Linguistic Theory (BLT), used extensively in analysing and writing descriptive grammars. The chapters from morphology to clause level are described from form to function; in the later chapters the function is taken more often as the starting point. Any theory-specific terminology is kept to the minimum and formalisms have been avoided in accordance with BLT principles. Mauwake has a classic 5-vowel system and 14 consonant phonemes. With its simple phonology it is a typical representative of the Madang North Coast languages. For a Papuan language there are relatively few morphophonological alternations. Nouns are either alienably or inalienably possessed. There is no obligatory number marking in nouns or noun phrases. Pronouns have several different forms: five for case and three for other functions. The dative pronouns are treated as [+human] locatives, and they have also grammaticalised as possessives. The verbal morphology is agglutinative and mainly suffixal. Unusual features include two distributive suffixes, and the interaction of the derivational benefactive and the inflectional beneficiary suffixes. The applicative suffix has either transitivising or causative but not benefactive function. The switch-reference system distinguishes between simultaneous and sequential action, as well as same or different subject in relation to the following clause. There are several verbs denoting coming and going, and they may combine with one of three prefixes to indicate bringing and taking. Mauwake is a nominative-accusative type language, and the basic constituent order in a clause is SOV. Subject and object are the only syntactic arguments. There is no indirect object, but a clause can have two or even three objects. A nominalised clause with a finite verb functions as a relative clause or a complement clause; one with a nominalised verb has several different functions. Functional domains described include modality, negation, deixis, quantification, possession and comparison. As there are four negators, Mauwake has more variation in negative expressions than is usual in Papuan languages. Clause chaining is the preferred strategy for joining clauses into sentences, but coordination and subordination of finite clauses are also common. The form of a complement clause depends on whether it is of the fact, action or potential type. Tail-head linkage is used as a cohesive device between sentences. The discourse-level features described are topic and focus.

Questions to Artificial Nature: A Philosophical Study of Interdisciplinary Models and their Functions in Scientific Practice

This thesis presents an interdisciplinary analysis of how models and simulations function in the production of scientific knowledge. The work is informed by three scholarly traditions: studies on models and simulations in philosophy of science, so-called micro-sociological laboratory studies within science and technology studies, and cultural-historical activity theory. Methodologically, I adopt a naturalist epistemology and combine philosophical analysis with a qualitative, empirical case study of infectious-disease modelling. This study has a dual perspective throughout the analysis: it specifies the modelling practices and examines the models as objects of research. The research questions addressed in this study are: 1) How are models constructed and what functions do they have in the production of scientific knowledge? 2) What is interdisciplinarity in model construction? 3) How do models become a general research tool and why is this process problematic? The core argument is that the mediating models as investigative instruments (cf. Morgan and Morrison 1999) take questions as a starting point, and hence their construction is intentionally guided. This argument applies the interrogative model of inquiry (e.g., Sintonen 2005; Hintikka 1981), which conceives of all knowledge acquisition as process of seeking answers to questions. The first question addresses simulation models as Artificial Nature, which is manipulated in order to answer questions that initiated the model building. This account develops further the "epistemology of simulation" (cf. Winsberg 2003) by showing the interrelatedness of researchers and their objects in the process of modelling. The second question clarifies why interdisciplinary research collaboration is demanding and difficult to maintain. The nature of the impediments to disciplinary interaction are examined by introducing the idea of object-oriented interdisciplinarity, which provides an analytical framework to study the changes in the degree of interdisciplinarity, the tools and research practices developed to support the collaboration, and the mode of collaboration in relation to the historically mutable object of research. As my interest is in the models as interdisciplinary objects, the third research problem seeks to answer my question of how we might characterise these objects, what is typical for them, and what kind of changes happen in the process of modelling. Here I examine the tension between specified, question-oriented models and more general models, and suggest that the specified models form a group of their own. I call these Tailor-made models, in opposition to the process of building a simulation platform that aims at generalisability and utility for health-policy. This tension also underlines the challenge of applying research results (or methods and tools) to discuss and solve problems in decision-making processes.

Double-Edged Imitation : Theories and Practices of Pastiche in Literature

This study concentrates on the contested concept of pastiche in literary studies. It offers the first detailed examination of the history of the concept from its origins in the seventeenth century to the present, showing how pastiche emerged as a critical concept in interaction with the emerging conception of authorial originality and the copyright laws protecting it. One of the key results of this investigation is the contextualisation of the postmodern debate on pastiche. Even though postmodern critics often emphasise the radical novelty of pastiche, they in fact resuscitate older positions and arguments without necessarily reflecting on their historical conditions. This historical background is then used to analyse the distinction between the primarily French conception of pastiche as the imitation of style and the postmodern notion of it as the compilation of different elements. The latter s vagueness and inclusiveness detracts from its value as a critical concept. The study thus concentrates on the notion of stylistic pastiche, challenging the widespread prejudice that it is merely an indication of lack of talent. Because it is multiply based on repetition, pastiche is in fact a highly ambiguous or double-edged practice that calls into question the distinction between repetition and original, thereby undermining the received notion of individual unique authorship as a fundamental aesthetic value. Pastiche does not, however, constitute a radical upheaval of the basic assumptions on which the present institution of literature relies, since, in order to mark its difference, pastiche always refers to a source outside itself against which its difference is measured. Finally, the theoretical analysis of pastiche is applied to literary works. The pastiches written by Marcel Proust demonstrate how it can become an integral part of a writer s poetics: imitation of style is shown to provide Proust with a way of exploring the role of style as a connecting point between inner vision and reality. The pastiches of the Sherlock Holmes stories by Michael Dibdin, Nicholas Meyer and the duo Adrian Conan Doyle and John Dickson Carr illustrate the functions of pastiche within a genre detective fiction that is itself fundamentally repetitive. A.S. Byatt s Possession and D.M. Thomas s Charlotte use Victorian pastiches to investigate the conditions of literary creation in the age of postmodern suspicion of creativity and individuality. The study thus argues that the concept of pastiche has valuable insights to offer to literary criticism and theory, and that literary pastiches, though often dismissed in reviews and criticism, are a particularly interesting object of study precisely because of their characteristic ambiguity.

Cholesterol Disturbances in Inherited Neurodegenerative Diseases : Studies on Npc1, Ppt1 and Ctsd deficient mice

The central nervous system (CNS) is the most cholesterol-rich organ in the body. Cholesterol is essential to CNS functions such as synaptogenesis and formation of myelin. Significant differences exist in cholesterol metabolism between the CNS and the peripheral organs. However, the regulation of cholesterol metabolism in the CNS is poorly understood compared to our knowledge of the regulation of cholesterol homeostasis in organs reached by cholesterol-carrying lipoprotein particles in the circulation. Defects in CNS cholesterol homeostasis have been linked to a variety of neurodegenerative diseases, including common diseases with complex pathogenetic mechanisms such as Alzheimer s disease. In spite of intense effort, the mechanisms which link disturbed cholesterol homeostasis to these diseases remain elusive. We used three inherited recessive neurodegenerative disorders as models in the studies included in this thesis: Niemann-Pick type C (NPC), infantile neuronal ceroid lipofuscinosis and cathepsin D deficiency. Of these three, NPC has previously been linked to disturbed intracellular cholesterol metabolism. Elucidating the mechanisms with which disturbances of cholesterol homeostasis link to neurodegeneration in recessive inherited disorders with known genetic lesions should shed light on how cholesterol is handled in the healthy CNS and help to understand how these and more complex diseases develop. In the first study we analyzed the synthesis of sterols and the assembly and secretion of lipoprotein particles in Npc1 deficient primary astrocytes. We found that both wild type and Npc1 deficient astrocytes retain significant amounts of desmosterol and other cholesterol precursor sterols as membrane constituents. No difference was observed in the synthesis of sterols and the secretion of newly synthesized sterols between Npc1 wild type, heterozygote or knockout astrocytes. We found that the incorporation of newly synthesized sterols into secreted lipoprotein particles was not inhibited by Npc1 mutation, and the lipoprotein particles were similar to those excreted by wild type astrocytes in shape and size. The bulk of cholesterol was found to be secreted independently of secreted NPC2. These observations demonstrate the ability of Npc1 deficient astrocytes to handle de novo sterols, and highlight the unique sterol composition in the developing brain. Infantile neuronal ceroid lipofuscinosis is caused by the deficiency of a functional Ppt1 enzyme in the cells. In the second study, global gene expression studies of approximately 14000 mouse genes showed significant changes in the expression of 135 genes in Ppt1 deficient neurons compared to wild type. Several genes encoding for enzymes of the mevalonate pathway of cholesterol biosynthesis showed increased expression. As predicted by the expression data, sterol biosynthesis was found to be upregulated in the knockout neurons. These data link Ppt1 deficiency to disturbed cholesterol metabolism in CNS neurons. In the third study we investigated the effect of cathepsin D deficiency on the structure of myelin and lipid homeostasis in the brain. Our proteomics data, immunohistochemistry and western blotting data showed altered levels of the myelin protein components myelin basic protein, proteolipid protein and 2 , 3 -cyclic nucleotide 3 phosphodiesterase in the brains of cathepsin D deficient mice. Electron microscopy revealed altered myelin structure in cathepsin D deficient brains. Additionally, plasmalogen-derived alkenyl chains and 20- and 24-carbon saturated and monounsaturated fatty acids typical for glycosphingolipids were found to be significantly reduced, but polyunsaturated species were significantly increased in the knockout brains, pointing to a decrease in white matter. The levels of ApoE and ABCA1 proteins linked to cholesterol efflux in the CNS were found to be altered in the brains of cathepsin D deficient mice, along with an accumulation of cholesteryl esters and a decrease in triglycerols. Together these data demonstrate altered myelin architecture in cathepsin D deficient mice and link cathepsin D deficiency to aberrant cholesterol metabolism and trafficking. Basic research into rare monogenic diseases sheds light on the underlying biological processes which are perturbed in these conditions and contributes to our understanding of the physiological function of healthy cells. Eventually, understanding gained from the study of disease models may contribute towards establishing treatment for these disorders and further our understanding of the pathogenesis of other, more complex and common diseases.

Androgen receptor-dependent and independent functions of ARIP4

Androgen receptor (AR) is necessary for normal male phenotype development and essential for spermatogenesis. AR is a classical steroid receptor mediating actions of male sex steroids testosterone and 5-alpha-dihydrotestosterone. Numerous coregulators interact with the receptor and regulate AR activity on target genes. This study deals with the characterization of androgen receptor-interacting protein 4 (ARIP4). ARIP4 binds DNA, interacts with AR in vitro and in cultured yeast and mammalian cells, and modulates AR-dependent transactivation. ARIP4 is an active DNA-dependent ATPase, and this enzymatic activity is essential for the ability of ARIP4 to modulate AR function. On the basis of sequence homology in its ATPase domain, ARIP4 belongs to the SNF2 family of proteins involved in chromatin remodeling, DNA repair, and homologous recombination. Similar to its closest homologs ATRX and Rad54, ARIP4 does not seem to be a classical chromatin remodeling protein in that it does not appear to form large protein complexes in vivo or remodel mononucleosomes in vitro. However, ARIP4 is able to generate superhelical torsion on linear DNA fragments. ARIP4 is covalently modified by SUMO-1, and mutation of six potential SUMO attachment sites abolishes the ability of ARIP4 to bind DNA, hydrolyze ATP, and activate AR function. ARIP4 expression starts in early embryonic development. In mouse embryo ARIP4 is present mainly in the neural tube and limb buds. In adult mouse tissues ARIP4 expression is virtually ubiquitous. In mouse testis ARIP4 is expressed in the nuclei of Sertoli cells in a stage-dependent manner. ARIP4 is also present in the nuclei of Leydig cells, spermatogonia, pachytene and diplotene spermatocytes. Testicular expression pattern of ARIP4 does not differ significantly in wild-type, FSHRKO, and LuRKO mice. In the testis of hpg mice, ARIP4 is found mainly in interstitial cells and has very low, if any, expression in Sertoli and germ cells. Heterozygous Arip4+/ mice are fertile and appear normal; however, they are haploinsufficient with regard to androgen action in Sertoli cells. In contrast, Arip4 / embryos are not viable. They have significantly reduced body size at E9.5 and die by E11.5. Compared to wild-type littermates, Arip4 / embryos possess a higher percentage of apoptotic cells at E9.5 and E10.5. Fibroblasts derived from Arip4 / embryos cease growing after 2-3 passages and exhibit a significantly increased apoptosis and decreased proliferation rate than cells from wild-type embryos. Our findings demonstrate that ARIP4 plays an essential role in mouse embryonic development. In addition, testicular expression and AR coregulatory activity of ARIP4 suggest a role of ARIP4-AR interaction in the somatic cells of the testis.

Orphan nuclear receptor subfamily NR4A their interplay with other nuclear receptors and functions in osteoblasts

Nurr1, NGFI-B and Nor1 (NR4A2, NR4A1 and NR4A3, respectively) belong to the NR4A subfamily of nuclear receptors. The NR4A receptors are orphan nuclear receptors which means that activating or repressing ligands for these receptors have not been found. NR4A expression is rapidly induced in response to various stimuli including growth factors and the parathyroid hormone (PTH). The studies concerning the NR4A receptors in the central nervous system have demonstrated that they have a major role in the development and function of the dopaminergic neurons of the midbrain and in regulating hypothalamus-pituitary-adrenal-axis. However, the peripheral functions of the NR4A family are largely unknown. Cultured mouse primary osteoblasts, a preosteoblastic cell line and several osteoblastic cell lines were used to investigate the role of NR4A receptors in osteoblasts. NR4A receptors were shown to directly bind to and activate the promoter of the osteopontin gene (OPN) in osteoblastic cells, thus regulating its expression. OPN is a major bone matrix protein expressed throughout the differentiation of preosteoblastic cells into osteoblasts. The activation of the OPN promoter was shown to be dependent on the activation function-1 located in the N-terminal part of Nurr1 and to occur in both monomeric and RXR heterodimeric forms of NR4A receptors. Furthermore, PTH was shown to upregulate OPN expression through the NR4A family. It was also demonstrated that the fibroblast growth factor-8b (FGF-8b) induces the expression of NR4A receptors in osteoblasts as immediate early genes. This induction involved phosphatidylinositol-3 kinase, protein kinase C, and mitogen activated protein kinase, which are all major pathways of FGF signalling. Nurr1 and NGFI-B were shown to induce the proliferation of preosteoblastic cells and to reduce their apoptosis. FGF-8b was shown to stimulate the proliferation of osteoblastic cells through the NR4A receptors. These results suggest that NR4A receptors have a role both in the differentiation of osteoblasts and in the proliferation and apoptosis of preosteoblast. The NR4A receptors were found to bind to the same response element on OPN as the members of the NR3B family of orphan receptors do. Mutual repression was observed between the NR4A receptors and the NR3B receptors. This repression was shown to be dependent on the DNA-binding domains of both receptor families, but to result neither from the competition of DNA binding nor from the competition for coactivators. As the repression was dependent on the relative expression levels of the NR4As and NR3Bs, it seems likely that the ratio of the receptors mediates their activity on their response elements. Rapid induction of the NR4As in response to various stimuli and differential expression of the NR3Bs can effectively control the gene activation by the NR4A receptors. NR4A receptors can bind DNA as monomers, and Nurr1 and NGFI-B can form permissive heterodimers with the retinoid X receptor (RXR). Permissive heterodimers can be activated with RXR agonists, unlike non-permissive heterodimers, which are formed by RXR and retinoic acid receptor or thyroid hormone receptor (RAR and TR, respectively). Non-permissive heterodimers can only be activated by the agonists of the heterodimerizing partner. The mechanisms behind differential response to RXR agonists have remained unresolved. As there are no activating or repressing ligands for the NR4A receptors, it would be important to find out, how they are regulated. Permissiviness of Nurr1/RXR heterodimers was linked to the N-terminal part of Nurr1 ligand-binding domain. This region has previously been shown to mediate the interaction between NRs and corepressors. Non-permissive RAR and TR, permissive Nurr1 and NGFI-B, and RXR were overexpressed with corepressors silencing mediator for retinoic acid and thyroid hormone receptors (SMRT), and with nuclear receptor corepressor in several cell lines. Nurr1 and NGFI-B were found to be repressed by SMRT. The interaction of RXR heterodimers with corepressors was weak in permissive heterodimers and much stronger in non-permissive heterodimers. Non-permissive heterodimers also released corepressors only in response to the agonist of the heterodimeric partner of RXR. In the permissive Nurr1/RXR heterodimer, however, SMRT was released following the treatment with RXR agonists. Corepressor release in response to ligands was found to differentiate permissive heterodimers from non-permissive ones. Corepressors were thus connected to the regulation of NR4A functions. In summary, the studies presented here linked the NR4A family of orphan nuclear receptors to the regulation of osteoblasts. Nurr1 and NGFI-B were found to control the proliferation and apoptosis of preosteoblasts. The studies also demonstrated that cross-talk with the NR3B receptors controls the activity of these orphan receptors. The results clarified the mechanism of permissiviness of RXR-heterodimers. New information was obtained on the regulation and functions of NR4A receptors, for which the ligands are unknown.