Molecular and clinical characteristics of tricarboxylic acid cycle-associated tumors

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a rare, dominantly inherited tumor predisposition syndrome characterized by benign cutaneous and uterine (ULM) leiomyomas, and sometimes renal cell cancer (RCC). A few cases of uterine leiomyosarcoma (ULMS) have also been reported. Mutations in a nuclear gene encoding fumarate hydratase (FH), an enzyme of the mitochondrial tricarboxylic acid cycle (TCA cycle), underlie HLRCC. As a recessive condition, germline mutations in FH predispose to a neurological defect, FH deficiency (FHD). Hereditary paragangliomatosis (HPGL) is a dominant disorder associated with paragangliomas and pheochromocytomas. Inherited mutations in three genes encoding subunits of succinate dehydrogenase (SDH), also a TCA cycle enzyme, predispose to HPGL. Both FH and SDH seem to act as tumor suppressors. One of the consequences of the TCA cycle defect is abnormal activation of HIF1 pathway ( pseudohypoxia ) in the HLRCC and HPGL tumors. HIF1 drives transcription of genes encoding e.g. angiogenetic factors which can facilitate tumor growth. Recently hypoxia/HIF1 has been suggested to be one of the causes of genetic instability as well. One of the aims of this study was to broaden the clinical definers of HLRCC. To determine the cancer risk and to identify possible novel tumor types associated with FH mutations eight Finnish HLRCC/FHD families were extensively evaluated. The extension of the pedigrees and the Finnish Cancer Registry based tumor search yielded genealogical and cancer data of altogether 868 individuals. The standardized incidence ratio-based comparison of HLRCC/FHD family members with general Finnish population revealed 6.5-fold risk for RCC. Moreover, risk for ULMS was highly increased. However, according to the recent and more stringent diagnosis criteria of ULMS many of the HLRCC uterine tumors previously considered malignant are at present diagnosed as atypical or proliferative ULMs (with a low risk of recurrence). Thus, the formation of ULMS (as presently defined) in HLRCC appears to be uncommon. Though increased incidence was not observed, interestingly the genetic analyses suggested possible association of breast and bladder cancer with loss of FH. Moreover, cancer cases were exceptionally detected in an FHD family. Another clinical finding was the conventional (clear cell) type RCC of a young Spanish HLRCC patient. Conventional RCC is distinct from the types previously observed in this syndrome but according to these results, FH mutation may underlie some of young conventional cancer cases. Secondly, the molecular pathway from defective TCA cycle to tumor formation was intended to clarify. Since HLRCC and HPGL tumors display abnormally activated HIF1, the hypothesis on the link between HIF1/hypoxia and genetic instability was of interest to study in HLRCC and HPGL tumor material. HIF1α (a subunit of HIF1) stabilization was confirmed in the majority of the specimens. However, no repression of MSH2, a protein of DNA mismatch repair system, or microsatellite instability (MSI), an indicator of genetic instability, was observed. Accordingly, increased instability seems not to play a role in the tumorigenesis of pseudohypoxic TCA cycle-deficient tumors. Additionally, to study the putative alternative functions of FH, a recently identified alternative FH transcript (FHv) was characterized. FHv was found to contain instead of exon 1, an alternative exon 1b. Differential subcellular distribution, lack of FH enzyme activity, low mRNA expression compared to FH, and induction by cellular stress suggest FHv to have a role distinct from FH, for example in apoptosis or survival. However, the physiological significance of FHv requires further elucidation.

Egg production in furnished cages

In the European Union, conventional cages for laying hens will be faded out at the beginning of 2012. The rationale behind this is a public concern over animal welfare in egg production. As alternatives to conventional cages, the European Union Council Directive 1999/74/EC allows non-cage systems and enriched (furnished) cages. Layer performance, behavior, and welfare in differently sized furnished cages have been investigated quite widely during recent decades, but nutrition of hens in this production system has received less attention. This thesis aims to compare production and feed intake of laying hens in furnished and conventional cages and to study the effects of different dietary treatments in these production systems, thus contributing to the general knowledge of furnished cages as an egg production system. A furnished cage model for 8 hens was compared with a 3-hen conventional cage. Three consecutive experiments each studied one aspect of layer diet: The first experiment investigated the effects of dietary protein/energy ratio, the second dietary energy levels, and the third the effects of extra limestone supplementation. In addition, a fourth experiment evaluated the effects of perches on feed consumption and behavior of hens in furnished cages. The dietary treatments in experiments 1 3 generally had similar effects in the two cage types. Thus, there was no evidence supporting a change in nutrient requirements for laying hens when conventional cages are replaced with small-group furnished cages. Moreover, the results from nutritional experiments conducted in conventional cages can be applied to small-group furnished cage systems. These results support the view that production performance comparable with conventional cages can be achieved in furnished cages. All of the advantages of cages for bird welfare are sustained in the small-group furnished cages used here. In addition, frequent use of perches and nests implies a wider behavioral repertoire in furnished cages than in conventional cages. The increase observed in bone ash content may improve bird welfare in furnished cages. The presence of perches diminished feed consumption during the prelaying period and enhanced the feed conversion ratio during the early laying period in furnished cages. However, as the presence or absence of perches in furnished cages had no significant effect on feed consumption after the prelaying period, the lower feed consumption observed in furnished cages than in conventional cages could be attributed to other factors, such as the presence of wood shavings or a nest box. The wider feed trough space per hen in conventional than in furnished cages may partly explain the higher feed consumption observed in conventional cages.

Atomic Layer Deposition for optical applications: metal fluoride thin films and novel devices

Thin films of various metal fluorides are suited for optical coatings from infrared (IR) to ultraviolet (UV) range due to their excellent light transmission. In this work, novel metal fluoride processes have been developed for atomic layer deposition (ALD), which is a gas phase thin film deposition method based on alternate saturative surface reactions. Surface controlled self-limiting film growth results in conformal and uniform films. Other strengths of ALD are precise film thickness control, repeatability and dense and pinhole free films. All these make the ALD technique an ideal choice also for depositing metal fluoride thin films. Metal fluoride ALD processes have been largely missing, which is mostly due to a lack of a good fluorine precursor. In this thesis, TiF4 precursor was used for the first time as the fluorine source in ALD for depositing CaF2, MgF2, LaF3 and YF3 thin films. TaF5 was studied as an alternative novel fluorine precursor only for MgF2 thin films. Metal-thd (thd = 2,2,6,6-tetramethyl-3,5-heptanedionato) compounds were applied as the metal precursors. The films were grown at 175 450 °C and they were characterized by various methods. The metal fluoride films grown at higher temperatures had generally lower impurity contents with higher UV light transmittances, but increased roughness caused more scattering losses. The highest transmittances and low refractive indices below 1.4 (at 580 nm) were obtained with MgF2 samples. MgF2 grown from TaF5 precursor showed even better UV light transmittance than MgF2 grown from TiF4. Thus, TaF5 can be considered as a high quality fluorine precursor for depositing metal fluoride thin films. Finally, MgF2 films were applied in fabrication of high reflecting mirrors together with Ta2O5 films for visible region and with LaF3 films for UV region. Another part of the thesis consists of applying already existing ALD processes for novel optical devices. In addition to the high reflecting mirrors, a thin ALD Al2O3 film on top of a silver coating was proven to protect the silver mirror coating from tarnishing. Iridium grid filter prototype for rejecting IR light and Ir-coated micro channel plates for focusing x-rays were successfully fabricated. Finally, Ir-coated Fresnel zone plates were shown to provide the best spatial resolution up to date in scanning x-ray microscopy.

Conformal Field Theory Methods for Variants of Schramm-Loewner Evolutions

This thesis consists of an introduction, four research articles and an appendix. The thesis studies relations between two different approaches to continuum limit of models of two dimensional statistical mechanics at criticality. The approach of conformal field theory (CFT) could be thought of as the algebraic classification of some basic objects in these models. It has been succesfully used by physicists since 1980's. The other approach, Schramm-Loewner evolutions (SLEs), is a recently introduced set of mathematical methods to study random curves or interfaces occurring in the continuum limit of the models. The first and second included articles argue on basis of statistical mechanics what would be a plausible relation between SLEs and conformal field theory. The first article studies multiple SLEs, several random curves simultaneously in a domain. The proposed definition is compatible with a natural commutation requirement suggested by Dubédat. The curves of multiple SLE may form different topological configurations, ``pure geometries''. We conjecture a relation between the topological configurations and CFT concepts of conformal blocks and operator product expansions. Example applications of multiple SLEs include crossing probabilities for percolation and Ising model. The second article studies SLE variants that represent models with boundary conditions implemented by primary fields. The most well known of these, SLE(kappa, rho), is shown to be simple in terms of the Coulomb gas formalism of CFT. In the third article the space of local martingales for variants of SLE is shown to carry a representation of Virasoro algebra. Finding this structure is guided by the relation of SLEs and CFTs in general, but the result is established in a straightforward fashion. This article, too, emphasizes multiple SLEs and proposes a possible way of treating pure geometries in terms of Coulomb gas. The fourth article states results of applications of the Virasoro structure to the open questions of SLE reversibility and duality. Proofs of the stated results are provided in the appendix. The objective is an indirect computation of certain polynomial expected values. Provided that these expected values exist, in generic cases they are shown to possess the desired properties, thus giving support for both reversibility and duality.

Methods for Answer Extraction in Textual Question Answering

In this thesis we present and evaluate two pattern matching based methods for answer extraction in textual question answering systems. A textual question answering system is a system that seeks answers to natural language questions from unstructured text. Textual question answering systems are an important research problem because as the amount of natural language text in digital format grows all the time, the need for novel methods for pinpointing important knowledge from the vast textual databases becomes more and more urgent. We concentrate on developing methods for the automatic creation of answer extraction patterns. A new type of extraction pattern is developed also. The pattern matching based approach chosen is interesting because of its language and application independence. The answer extraction methods are developed in the framework of our own question answering system. Publicly available datasets in English are used as training and evaluation data for the methods. The techniques developed are based on the well known methods of sequence alignment and hierarchical clustering. The similarity metric used is based on edit distance. The main conclusions of the research are that answer extraction patterns consisting of the most important words of the question and of the following information extracted from the answer context: plain words, part-of-speech tags, punctuation marks and capitalization patterns, can be used in the answer extraction module of a question answering system. This type of patterns and the two new methods for generating answer extraction patterns provide average results when compared to those produced by other systems using the same dataset. However, most answer extraction methods in the question answering systems tested with the same dataset are both hand crafted and based on a system-specific and fine-grained question classification. The the new methods developed in this thesis require no manual creation of answer extraction patterns. As a source of knowledge, they require a dataset of sample questions and answers, as well as a set of text documents that contain answers to most of the questions. The question classification used in the training data is a standard one and provided already in the publicly available data.

Mies, talli ja moottoripyörä : Etnografinen tutkimus Misfit MC:stä ja bikerkulttuurin eetoksesta

Tutkimuksen punaisena lankana kulkee kysymys siitä, millainen on bikerkulttuurin eetos? Miten se on syntynyt, miten sitä ylläpidetään ja miten Misfit MC:n jäsenet sitä tulkitsevat ja toteuttavat omassa elämässään? Tarkastelen eetosta kahdenlaisen aineiston valossa. i) Kenttätyöllä (vuosina 1995-1998 ja 2000-2001) kerätyn aineiston valossa tarkastelen yhtä pääkaupunkiseudulla toimivaa HD-moottoripyöräkerhoa, vuonna 1989 toimintansa aloittanutta Misfit MC:tä. Jäsenet kutsuvat kerhoaan useimmiten talliksi, joskus pajaksi, kerhoksi tai klubiksi. Puhuessaan tallista, miehet voivat viitata kerhorakennukseen ("tuut sä tallille?") mutta myös ryhmään ("meidän talli") ja sen olemassaoloon ajallisesti ja paikallisesti. Aloittaessani kenttätyön vuonna 1995 Misfit MC:n kuului kymmenen 25-30-vuotiasta miestä. ii) Kenttätyöllä kerätyn aineiston lisäksi käytän materiaalia, joka koostuu Harley-Davidson-moottoripyörän ympärille rakentuneen bikerkulttuurin historiasta ja kulttuurituotteista, kuten kertomuksista, elokuvista, musiikista, kuvataiteesta ja moottoripyörälehdistä. Aineiston avulla valotan bikerkulttuurin eetoksen syntyä, alkuvaiheita, leviämistä ja keskeisiä elementtejä. Lähdeaineiston monimuotoisuus ja runsaus palautuu kenttätyöhöni jolloin vakuutuin siitä, että tutkimusmatka bikerkulttuurin historiaan, perinteisiin ja median välittämiin (mieli)kuviin on välttämätöntä, sillä menneisyys ja Harrikkaan ajan kuluessa varastoituneet merkitykset vaikuttavat ja ovat vahvasti läsnä Misfit MC:n toiminnassa ja talliin kuuluvien miesten elämäntyylissä. Tutkimus etenee seuraavanlaisesti. Luku I on Johdanto. Luvussa II Etnografia käsittelen etnografisen tiedon luonnetta niin tutkimusasenteena kuin kenttätyön valossa. Pohdin kenttätyötä ja sen suhdetta etnografian kirjoittamiseen eli miten kenttätyöllä kerätty aineisto muuntuu etnografiseksi monografiaksi. Käsittelen myös kenttätyöni reunaehtoja, kuten tyttöystävyyden ja sukupuolen merkitystä, ja tarkastelen tutussa kulttuurissa tehdyn kenttätyön ominaispiirteitä. Reunaehtojen kuvailu toimii myös johdatuksena bikerkulttuuriin sellaisena kuin se ilmenee Misfit MC:n tallielämässä ja käytänteissä. Lopuksi pohdin "tiheän kuvauksen" mahdollisuuksia ja vaateita aineistoni puitteissa. Luvussa III Bikerkulttuurin eetosta kartoittamassa, kuvailen Harley-Davidson-moottoripyörän ympärille rakentuneen elämäntavan syntyä, levittäytymistä ja keskeisiä elementtejä. Tarkastelen media- ja populaarikulttuurisia tekstejä (elokuvien kertomat tarinat, musiikkikappaleiden sanoitukset ja HD- ja bikerlehtien artikkelit) ja kuvia (elokuvien audiovisuaaliset aspektit, kuvataide ja HD- ja bikerlehtien kuvitus), jotka ovat vaikuttaneet bikerkulttuurin eetokseen. Luvun keskeisiä - aineistosta nousevia ja miessukupuoleen vahvasti sidoksissa olevia - käsitteitä ovat biker, outlaw ja chopper, jotka ovat bikerkulttuurissa säilyneet alkuperäisessä muodossa maantieteellisestä tai kielialueesta riippumatta. Luvussa IV Misfit MC ja bikerkulttuurin eetos temaattinen painopiste siirtyy Suomeen ja Misfit MC:hen. Aluksi käyn läpi suomalaisen bikerkulttuurin muotoutumista ja ominaispiirteitä. Alkukappaleiden jälkeen keskityn Misfit MC:n jäsenten elämäntyylin sävyihin ja heidän käsityksiinsä bikerkulttuurin eetoksesta. Analyysin kiintopisteitä ovat Misfit MC:n jäsenten näkemys bikeriydestä ja tallitoiminnasta, miesten elämäntyylin moraaliset ja esteettiset sävyt, tallirakennus miesyhteisöllisyyttä ja bikerkulttuurin eetosta luovana ja ylläpitävänä sosiaalisena tilana ja Misfit MC miesten yhteisönä. Luvussa V Eetoksen ytimessä: mies ja Harley-Davidson keskityn bikerkulttuurin ytimeen: miehen ja Harley-Davidson-moottoripyörän väliseen suhteeseen. Luvun alussa esittelen ruotsalaisen yhteiskuntatieteilijä Lars Lagergrenin moottoripyörään soveltamaa työkalu - leikkikalu - toteemi - välittäjä -typologiaa ja tarkastelen moottoripyörän olemusta sukupuolittavana ja sukupuolittuvana artefaktina. Johdanto-osion jälkeen siirryn kuvailemaan Misfit MC:n jäsenten suhdetta Harley-Davidson-moottoripyörään. Lähestyn miesten ja moottoripyörien suhdetta kahden toiminnan - moottoripyörän kunnostamisen ja rakentamisen sekä moottoripyörällä ajamisen - kautta. Avainsanat: aineellinen kulttuuri, arvot, biker, bikerkulttuuri, chopper, eetos, elämäntapa, etnografia, Harley-Davidson-moottoripyörä, Harley-Davidson-moottoripyöräkerho, kenttätyö, maskuliinisuus, mieskulttuuri, mieskuva, moottoripyöräily, osakulttuurit, outlaw, populaarikulttuuri, sukupuoliroolit, yhteisöt

Causes and consequences of variation in nestling immune function

Defence against pathogens is a vital need of all living organisms that has led to the evolution of complex immune mechanisms. However, although immunocompetence the ability to resist pathogens and control infection has in recent decades become a focus for research in evolutionary ecology, the variation in immune function observed in natural populations is relatively little understood. This thesis examines sources of this variation (environmental, genetic and maternal effects) during the nestling stage and its fitness consequences in wild populations of passerines: the blue tit (Cyanistes caeruleus) and the collared flycatcher (Ficedula albicollis). A developing organism may face a dilemma as to whether to allocate limited resources to growth or to immune defences. The optimal level of investment in immunity is shaped inherently by specific requirements of the environment. If the probability of contracting infection is low, maintaining high growth rates even at the expense of immune function may be advantageous for nestlings, as body mass is usually a good predictor of post-fledging survival. In experiments with blue tits and haematophagous hen fleas (Ceratophyllus gallinae) using two methods, methionine supplementation (to manipulate nestlings resource allocation to cellular immune function) and food supplementation (to increase resource availability), I confirmed that there is a trade-off between growth and immunity and that the abundance of ectoparasites is an environmental factor affecting allocation of resources to immune function. A cross-fostering experiment also revealed that environmental heterogeneity in terms of abundance of ectoparasites may contribute to maintaining additive genetic variation in immunity and other traits. Animal model analysis of extensive data collected from the population of collared flycatchers on Gotland (Sweden) allowed examination of the narrow-sense heritability of PHA-response the most commonly used index of cellular immunocompetence in avian studies. PHA-response is not heritable in this population, but is subject to a non-heritable origin (presumably maternal) effect. However, experimental manipulation of yolk androgen levels indicates that the mechanism of the maternal effect in PHA-response is not in ovo deposition of androgens. The relationship between PHA-response and recruitment was studied for over 1300 collared flycatcher nestlings. Multivariate selection analysis shows that it is body mass, not PHA-response, that is under direct selection. PHA-response appears to be related to recruitment because of its positive relationship with body mass. These results imply that either PHA-response fails to capture the immune mechanisms that are relevant for defence against pathogens encountered by fledglings or that the selection pressure from parasites is not as strong as commonly assumed.

Additional binding sites in lysozyme. X-ray analysis of lysozyme complexes with bromophenol red and bromophenol blue

The binding sites in hen egg-white lysozyme for neutral bromophenol red (BPR) and ionized bromophenol blue (BPB) have been characterized at 2 Å resolution. In either case, the dye-bound enzyme is active against the polysaccharide, but not against the cell wall. Both binding sites are outside, but close to, the hexasaccharide binding cleft in the enzyme. The binding site of BPR made up of Arg5, Lys33, Phe34, Asn37, Phe38, Ala122, Trp123 and possibly Arg125, is dose to subsite F while that of BPB made up of Tyr20, Arg21, Asn93, Lys96, Lys97 and Ser100, is close to subsites A and B. The binding sites of the neutral dye and the ionized dye are thus spatially far apart. The peptide component of the bacterial cell wall probably interacts with these cells during enzyme action. Such interactions are perhaps necessary for appropriately positioning the enzyme molecule on the bacterial cell wall.

Adsorption and Unfolding of Lysozyme at a Polarized Aqueous-Organic Liquid Interface

The adsorption of proteins at the interface between two immiscible electrolyte solutions has been found to be key to their bioelectroactivity at such interfaces. Combined with interfacial complexation of organic phase anions by cationic proteins, this adsorption process may be exploited to achieve nanomolar protein detection. In this study, replica exchange molecular dynamics simulations have been performed to elucidate for the first time the molecular mechanism of adsorption and subsequent unfolding of hen egg white lysozyme at low pH at a polarized 1,2-dichloroethane/water interface. The unfolding of lysozyme was observed to occur as soon as it reaches the organic−aqueous interface,which resulted in a number of distinct orientations at the interface. In all cases, lysozyme interacted with the organic phase through regions rich in nonpolar amino acids, such that the side chains are directed toward the organic phase, whereas charged and polar residues were oriented toward the aqueous phase. By contrast, as expected, lysozyme in neat water at low pH does not exhibit significant structural changes. These findings demonstrate the key influence of the organic phase upon adsorption of lysozyme under the influence of an electric field, which results in the unfolding of its structure.

Prospects of riboflavin carrier protein (RCP) as an antifertility vaccine in male and female mammals

Riboflavin carrier protein (RCP) is obligatorily involved in yolk deposition of the vitamin, riboflavin, in the developing oocyte of the hen. The production of this protein is inducible by oestrogen. It is evolutionarily conserved in terms of its physicochemical, immunological and functional characteristics. It is the prime mediator of vitamin supply to the developing fetus in mammals, including primates. Passive immunoneutralization of the protein terminates pregnancy in rats. Active immunization of rats and bonnet monkeys with avian RCP prevents pregnancy without causing any adverse physiological effects of the mother in terms of her vitamin status, reproductive cycles or reproductive-endocrine profile. Denatured, linearized RCP is more effective in eliciting neutralizing antibodies capable of interfering with embryonic viability either before or during peri-implantation stages. Two defined stretches of sequential epitopes, one located at the N-terminus and the other at the C-terminus of the protein have been identified. Active immunization with either of these epitopes conjugated with diptheria toxoid curtails pregnancy in rats and monkeys. Immunohistochemical localization of RCP on ovulated oocytes and early embryos shows that the antibodies cause degeneration only of early embryos. RCP is produced intra-testicularly and becomes localized on acrosomal surface of mammalian spermatozoa. Active immunization of male rats and monkeys with denatured RCP markedly reduces fertility by impairing the fertilizing potential of spermatozoa. These findings suggest that RCP, or its defined fragments, could be a novel, first generation vaccine for regulating fertility in both the sexes.

Functional characterization of MutL homologue mismatch repair proteins and their variants

Mismatch repair (MMR) mechanisms repair DNA damage occurring during replication and recombination. To date, five human MMR genes, MSH2, MHS6, MSH3, MLH1 and PMS2 are known to be involved in the MMR function. Human MMR proteins form 3 different heterodimers: MutSα (MSH2 and MSH6) and MutSβ (MSH2 and MSH3), which are needed for mismatch recognition and binding, and MutLα (MLH1 and PMS2), which is needed for mediating interactions between MutS homologues and other MMR proteins. The other two MutL homologues, MLH3 and PMS1, have been shown to heterodimerize with MLH1. However, the heterodimers MutLγ (MLH1and MLH3) and MutLβ (MLH1 and PMS1) are able to correct mismatches only with low or no efficiency, respectively. A deficient MMR mechanism is associated with the hereditary colorectal cancer syndrome called hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome. HNPCC is the most common hereditary colorectal cancer syndrome and accounts for 2-5% of all colorectal cancer cases. HNPCC-associated mutations have been found in 5 MMR genes: MLH1, MSH2, MSH6, PMS2 and MLH3. Most of the mutations have been found in MLH1 and MSH2 (~90%) and are associated with typical HNPCC, while mutations in MSH6, PMS2 and MLH3 are mainly linked to putative HNPCC families lacking the characteristics of the syndrome. More data of MLH3 mutations are needed to assess the significance of its mutations in HNPCC. In this study, were functionally characterized 51 nontruncating mutations in the MLH1, MLH3 and MSH2 genes to address their pathogenic significance and mechanism of pathogenicity. Of the 36 MLH1 mutations, 22 were deficient in more than one assay, 2 variants were impaired only in one assay, and 12 variants behaved like the wild type protein, whereas all seven MLH3 mutants functioned like the wild type protein in the assays. To further clarify the role and relevance of MLH3 in MMR, we analyzed the subcellular localization of the native MutL homologue proteins. Our immunofluorescence analyses indicated that when all the three MutL homologues are natively expressed in human cells, endogenous MLH1 and PMS2 localize in the nucleus, whereas MLH3 stays in the cytoplasm. The coexpression of MLH3 with MLH1 results in its partial nuclear localization. Only one MSH2 mutation was pathogenic in the in vitro MMR assay. Our study on MLH1 mutations could clearly distinguish nontruncating alterations with severe functional defects from those not or only slightly impaired in protein function. However, our study on MLH3 mutations suggest that MLH3 mutations per se are not sufficient to trigger MMR deficiency and the continuous nuclear localization of MLH1 and PMS2 suggest that MutLα has a major activity in MMR in vivo. Together with our functional assays, this confirms that MutLγ is a less efficient MMR complex than MutLα.

Regulation of Osteoblast Differentiation and Gene Expression by Phosphodiesterases and Bioactive Peptides

Bone is a mineralized tissue that enables multiple mechanical and metabolic functions to be carried out in the skeleton. Bone contains distinct cell types: osteoblasts (bone-forming cells), osteocytes (mature osteoblast that embedded in mineralized bone matrix) and the osteoclasts (bone-resorbing cells). Remodelling of bone begins early in foetal life, and once the skeleton is fully formed in young adults, almost all of the metabolic activity is in this form. Bone is constantly destroyed or resorbed by osteoclasts and then replaced by osteoblasts. Many bone diseases, i.e. osteoporosis, also known as bone loss, typically reflect an imbalance in skeletal turnover. The cyclic adenosine monophosphate (cAMP) and the cyclic guanosine monophosphate (cGMP) are second messengers involved in a variety of cellular responses to such extracellular agents as hormones and neurotransmitters. In the hormonal regulation of bone metabolism, i.e. via parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrp) and prostaglandin E2 signal via cAMP. cAMP and cGMP are formed by adenylate and guanylate cyclases and are degraded by phosphodiesterases (PDEs). PDEs determine the amplitudes of cyclic nucleotide-mediated hormonal responses and modulate the duration of the signal. The activities of the PDEs are regulated by multiple inputs from other signalling systems and are crucial points of cross-talk between the pathways. Food-derived bioactive peptides are reported to express a variety of functions in vivo. The angiotensin-converting enzymes (ACEs) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. The bioactive peptides offer also a nutriceutical and a nutrigenomic aspect to bone cell biology. The aim of this study was to investigate the influence of PDEs and bioactive peptides on the activation and the differentiation of human osteoblast cells. The profile of PDEs in human osteoblast-like cells and the effect of glucocorticoids on the function of cAMP PDEs, were investigated at the mRNA and enzyme levels. The effects of PDEs on bone formation and osteoblast gene expression were determined with chemical inhibitors and siRNAs (short interfering RNAs). The influence of bioactive peptides on osteoblast gene expression and proliferation was studied at the mRNA and cellular levels. This work provides information on how PDEs are involved in the function and the differentiation of osteoblasts. The findings illustrate that gene-specific silencing with an RNA interference (RNAi) method is useful in inhibiting, the gene expression of specific PDEs and further, PDE7 inhibition upregulates several osteogenic genes and increases bALP activity and mineralization in human mesenchymal stem cells-derived osteoblasts. PDEs appear to be involved in a mechanism by which glucocorticoids affect cAMP signaling. This may provide a potential route in the formation of glucocorticoid-induced bone loss, involving the down-regulation of cAMP-PDE. PDEs may play an important role in the regulation of osteoblastic differentiation. Isoleucine-proline-proline (IPP), a bioactive peptide, possesses the potential to increase osteoblast proliferation, differentiation and signalling.

Comparison and integration of MEG and fMRI

MEG directly measures the neuronal events and has greater temporal resolution than fMRI, which has limited temporal resolution mainly due to the larger timescale of the hemodynamic response. On the other hand fMRI has advantages in spatial resolution, while the localization results with MEG can be ambiguous due to the non-uniqueness of the electromagnetic inverse problem. Thus, these methods could provide complementary information and could be used to create both spatially and temporally accurate models of brain function. We investigated the degree of overlap, revealed by the two imaging methods, in areas involved in sensory or motor processing in healthy subjects and neurosurgical patients. Furthermore, we used the spatial information from fMRI to construct a spatiotemporal model of the MEG data in order to investigate the sensorimotor system and to create a spatiotemporal model of its function. We compared the localization results from the MEG and fMRI with invasive electrophysiological cortical mapping. We used a recently introduced method, contextual clustering, for hypothesis testing of fMRI data and assessed the the effect of neighbourhood information use on the reproducibility of fMRI results. Using MEG, we identified the ipsilateral primary sensorimotor cortex (SMI) as a novel source area contributing to the somatosensory evoked fields (SEF) to median nerve stimulation. Using combined MEG and fMRI measurements we found that two separate areas in the lateral fissure may be the generators for the SEF responses from the secondary somatosensory cortex region. The two imaging methods indicated activation in corresponding locations. By using complementary information from MEG and fMRI we established a spatiotemporal model of somatosensory cortical processing. This spatiotemporal model of cerebral activity was in good agreement with results from several studies using invasive electrophysiological measurements and with anatomical studies in monkey and man concerning the connections between somatosensory areas. In neurosurgical patients, the MEG dipole model turned out to be more reliable than fMRI in the identification of the central sulcus. This was due to prominent activation in non-primary areas in fMRI, which in some cases led to erroneous or ambiguous localization of the central sulcus.

Genetic studies on Finnish lupus erythematosus patients with cutaneous manifestations

Lupus erythematosus (LE) is a chronic, heterogeneous autoimmune disorder with abnormal immune responses, including production of autoantibodies and immune complexes. Clinical presentations of the disease range from mild cutaneous manifestations to a more generalised systemic involvement of internal organs. Cutaneous (CLE) forms are further subclassified into discoid LE (DLE), subacute cutaneous LE (SCLE) and acute cutaneous lupus erythematosus (ACLE), and may later progress to systemic disease (SLE). Both genetic and environmental factors contribute to the disease, although the precise aetiology is still elusive. Furthermore, complex gene-gene or gene-environment interactions may result in different subphenotypes of lupus. The genetic background of CLE is poorly known and only a few genes are confirmed, while the number of robust genetic associations in SLE exceeds 30. The aim of this thesis was to characterise the recruited patients clinically, and identify genetic variants conferring susceptibility to cutaneous variants of LE. Given that cutaneous and systemic disease may share underlying genetic factors, putative CLE candidate genes for genotyping were selected among those showing strong evidence of association in SLE. The correlation between relevant clinical manifestations and risk genotypes was investigated in order to find specific subphenotype associations. In addition, epistatic interactions in SLE were studied. Finally, the role of tissue degrading matrix metalloproteinases (MMP) in LE tissue injury was explored. These studies were conducted in Finnish case-control and family cohort, and Swedish case-control cohort. The clinical picture of the patients in terms of cutaneous, haematological and immunological findings resembled that described in the contemporary literature. However, the proportion of daily smokers was very high supporting the role of smoking in disease aetiology. The results confirmed that, even though clinically distinct entities, CLE and SLE share predisposing genetic factors. For the first time it was shown that known SLE susceptibility genes IRF5 and TYK2 also increase the risk of CLE. A tendency toward gene-gene interaction between these genes was found in SLE. As a remarkable novel finding, it was observed that ITGAM polymorphisms associated even more strongly to DLE than SLE, and the risk estimates were substantially higher than those reported for SLE. Several other recently identified SLE susceptibility genes showed signs of good or modest association especially in DLE. Subphenotype analyses indicated possible associations to clinical features, but marginally significant results reflected lack of sufficient power for these studies. Thorough immunohistochemical analyses of several MMPs demonstrated a role in epidermal changes and dermal tissue remodelling in diseased skin, and suggested that targeted action using selective MMP inhibitors may reduce lupus-induced damage in inflamed tissues. In conclusion, the results provide an insight into the genetics of CLE and demonstrate that genetic predisposition is at least in part shared between cutaneous and systemic variants of LE. This doctoral study has contributed IRF5, TYK2, ITGAM and several other novel genes to the so far short list of genes implicated in CLE susceptibility. Detailed examination of the function of these genes in CLE pathogenesis warrants further studies. Furthermore, the results support the need of subphenotype analysis with sample sizes large enough to reveal possible specific disease associations in order to better understand the heterogeneous nature and clinical specificities of the disease. Comprehensive analysis of clinical data suggests that smoking is an environmental triggering factor.

Noninvasive techniques in assessing coronary artery disease

Conventional invasive coronary angiography is the clinical gold standard for detecting of coronary artery stenoses. Noninvasive multidetector computed tomography (MDCT) in combination with retrospective ECG gating has recently been shown to permit visualization of the coronary artery lumen and detection of coronary artery stenoses. Single photon emission tomography (SPECT) perfusion imaging has been considered the reference method for evaluation of nonviable myocardium, but magnetic resonance imaging (MRI) can accurately depict structure, function, effusion, and myocardial viability, with an overall capacity unmatched by any other single imaging modality. Magnetocardiography (MCG) provides noninvasively information about myocardial excitation propagation and repolarization without the use of electrodes. This evolving technique may be considered the magnetic equivalent to electrocardiography. The aim of the present series of studies was to evaluate changes in the myocardium assessed with SPECT and MRI caused by coronary artery disease, examine the capability of multidetector computed tomography coronary angiography (MDCT-CA) to detect significant stenoses in the coronary arteries, and MCG to assess remote myocardial infarctions. Our study showed that in severe, progressing coronary artery disease laser treatment does not improve global left ventricular function or myocardial perfusion, but it does preserve systolic wall thickening in fixed defects (scar). It also prevents changes from ischemic myocardial regions to scar. The MCG repolarization variables are informative in remote myocardial infarction, and may perform as well as the conventional QRS criteria in detection of healed myocardial infarction. These STT abnormalities are more pronounced in patients with Q-wave infarction than in patients with non-Q-wave infarctions. MDCT-CA had a sensitivity of 82%, a specificity of 94%, a positive predictive value of 79%, and a negative predictive value of 95% for stenoses over 50% in the main coronary arteries as compared with conventional coronary angiography in patients with known coronary artery disease. Left ventricular wall dysfunction, perfusion defects, and infarctions were detected in 50-78% of sectors assigned to calcifications or stenoses, but also in sectors supplied by normally perfused coronary arteries. Our study showed a low sensitivity (sensitivity 63%) in detecting obstructive coronary artery disease assessed by MDCT in patients with severe aortic stenosis. Massive calcifications complicated correct assessment of the lumen of coronary arteries.