Crystallization as a Tool for Controlling and Designing Properties of Pharmaceutical Solids

The ability to deliver the drug to the patient in a safe, efficacious and cost-effective manner depends largely on the physicochemical properties of the active pharmaceutical ingredient (API) in the solid state. In this context, crystallization is of critical importance in pharmaceutical industry, as it defines physical and powder properties of crystalline APIs. An improved knowledge of the various aspects of crystallization process is therefore needed. The overall goal of this thesis was to gain better understanding of the relationships between crystallization, solid-state form and properties of pharmaceutical solids with a focus on a crystal engineering approach to design technological properties of APIs. Specifically, solid-state properties of the crystalline forms of the model APIs, erythromycin A and baclofen, and the influence of solvent on their crystallization behavior were investigated. In addition, the physical phenomena associated with wet granulation and hot-melting processing of the model APIs were examined at the molecular level. Finally, the effect of crystal habit modification of a model API on its tabletting properties was evaluated. The thesis enabled the understanding of the relationship between the crystalline forms of the model APIs, which is of practical importance for solid-state control during processing and storage. Moreover, a new crystalline form, baclofen monohydrate, was discovered and characterized. Upon polymorph screening, erythromycin A demonstrated high solvate-forming propensity thus emphasizing the need for careful control of the solvent effects during formulation. The solvent compositions that yield the desirable crystalline form of erythromycin A were defined. Furthermore, new examples on solvent-mediated phase transformations taking place during wet granulation of baclofen and hot-melt processing of erythromycin A dihydrate with PEG 6000 are reported. Since solvent-mediated phase transformations involve the crystallization of a stable phase and hence affect the dissolution kinetics and possibly absorption of the API these transformations must be well documented. Finally, a controlled-crystallization method utilizing HPMC as a crystal habit modifier was developed for erythromycin A dihydrate. The crystals with modified habit were shown to posses improved compaction properties as compared with those of unmodified crystals. This result supports the idea of morphological crystal engineering as a tool for designing technological properties of APIs and is of utmost practical interest.

Ultrasound-assisted surface engineering of pharmaceutical powders

Effective processing of powdered particles can facilitate powder handling and result in better drug product performance, which is of great importance in the pharmaceutical industry where the majority of active pharmaceutical ingredients (APIs) are delivered as solid dosage forms. The purpose of this work was to develop a new ultrasound-assisted method for particle surface modification and thin-coating of pharmaceutical powders. The ultrasound was used to produce an aqueous mist with or without a coating agent. By using the proposed technique, it was possible to decrease the interparticular interactions and improve rheological properties of poorly-flowing water-soluble powders by aqueous smoothing of the rough surfaces of irregular particles. In turn, hydrophilic polymer thin-coating of a hydrophobic substance diminished the triboelectrostatic charge transfer and improved the flowability of highly cohesive powder. To determine the coating efficiency of the technique, the bioactive molecule ��-galactosidase was layered onto the surface of powdered lactose particles. Enzyme-treated materials were analysed by assaying the quantity of the reaction product generated during enzymatic cleavage of the milk sugar. A near-linear increase in the thickness of the drug layer was obtained during progressive treatment. Using the enzyme coating procedure, it was confirmed that the ultrasound-assisted technique is suitable for processing labile protein materials. In addition, this pre-treatment of milk sugar could be used to improve utilization of lactose-containing formulations for populations suffering from severe lactose intolerance. Furthermore, the applicability of the thin-coating technique for improving homogeneity of low-dose solid dosage forms was shown. The carrier particles coated with API gave rise to uniform distribution of the drug within the powder. The mixture remained homogeneous during further tabletting, whereas the reference physical powder mixture was subject to segregation. In conclusion, ultrasound-assisted surface engineering of pharmaceutical powders can be effective technology for improving formulation and performance of solid dosage forms such as dry powder inhalers (DPI) and direct compression products.

Primestoimenno-otnositel'nye konstrukcii v sovremennom russkom jazyke

Relative Constructions with Pronominal Heads in Contemporary Russian Chapter 1 introduces the distinctive syntactic and semantic properties of Russian relative constructions (RCs), which are then divided into two main classes according to the type of the head phrase. The study concentrates on RCs with pronominal heads, which are systematically compared with noun-headed RCs. Chapter 2 clarifies the categorization of pronouns in Russian. The conclusion is that Russian pronouns include only personal, reflexive and wh-pronouns. The remaining words that are traditionally seen as pronouns are actually functional equivalents of determiners. This idea leads to the suggestion that RCs with these determiner-like words as the only constituent of the head phrase are actually headed by zero pronouns. In the other type of RCs with pronominal heads, the head position is occupied by wh-pronouns with clitics expressing different types of indefiniteness and quantification. Comparison of the two types of pronoun-headed RCs shows that the wh-heads and zero-heads share a number of common properties with respect to the grammatical gender, number and person as well as to the semantic distinction between animates and inanimates. The rest of Chapter 2 gives an overview of various uses of wh-pronouns in Russian and an experimental analysis of RCs headed by pronominal adverbs. Chapter 3 discusses fundamental differences between RCs with noun and pronominal heads. One of the main findings is that the choice of the relative pronoun (kto 'who' and chto 'what' versus kotoryj 'which') is motivated by a tendency to reproduce maximally the essential grammatical and semantic properties of the antecedent. Chapter 4 gives a detailed description of the determiner-like words and wh-based heads used in the two types of RCs with pronominal heads. In addition, several issues related to the syntax and semantics of free relatives are discussed. The conclusion is that there is no need to establish a separate category of free relatives in Russian. Chapter 5 discusses the syntax and semantics of correlative and free concessive constructions. They share a number of properties with pronoun-headed RCs and the two are often confused in Russian linguistics. However, a detailed analysis shows that these constructions must be distinguished from RCs. The study combines the methods of functionally-oriented Russian structuralism with some insights from generative syntax.

Speech Rhythm in an Interlanguage Perspective : Finnish Adolescents Speaking English

Speech rhythm is an essential part of speech processing. It is the outcome of the workings of a combination of linguistic and non-linguistic parameters, many of which also have other functions in speech. This study focusses on the acoustic and auditive realization of two linguistic parameters of rhythm: (1) sentence stress, and (2) speech rate and pausing. The aim was to find out how well Finnish comprehensive school pupils realize these two parameters in English and how native speakers of English react to Finnish pupils English rhythm. The material was elicited by means of a story-telling task and questionnaires. Three female and three male pupils representing different levels of oral skills in English were selected as the experimental group. The control group consisted of two female and two male native speakers of English. The stories were analysed acoustically and auditorily with respect to interstress intervals, weak forms, fundamental frequency, pausing, and speech as well as articulation rate. In addition, 52 native speakers of English were asked to rate the intelligibility of the Finnish pupils English with respect to speech rhythm and give their attitudes on what the pupils sounded like. Results showed that Finnish pupils can produce isochronous interstress intervals in English, but that too large a proportion of these intervals contain pauses. A closer analysis of the pauses revealed that Finnish pupils pause too frequently and in inappropriate places when they speak English. Frequent pausing was also found to cause slow speech rates. The findings of the fundamental frequency (F0) measurements indicate that Finnish pupils tend to make a slightly narrower F0 difference between stressed and unstressed syllables than the native speakers of English. Furthermore, Finnish pupils appear to know how to reduce the duration and quality of unstressed sounds, but they fail to do it frequently enough. Native listeners gave lower intelligibility and attitude scores to pupils with more anomalous speech rhythm. Finnish pupils rhythm anomalies seemed to derive from various learning- or learner-related factors rather than from the differences between English and Finnish. This study demonstrates that pausing may be a more important component of English speech rhythm than sentence stress as far as Finnish adolescents are concerned and that interlanguage development is affected by various factors and characterised by jumps or periods of stasis. Other theoretical, methodological and pedagogical implications of the results are also discussed.

��voljucija sistemy glasnyh fonem v nekotoryh russkih govorah Vologodskoj oblasti

The present dissertation analyses 36 local vernaculars of villages surrounding the northern Russian city of Vologda in relation to the system of the vowels in the stressed syllables and those preceding the stressed syllables by using the available dialectological researches. The system in question differs from the corresponding standard Russian system by that the palatalisation of the surrounding consonants affects the vowels much more significantly in the vernaculars, whereas the phonetic difference between the stressed and non-stressed vowels is less obvious in them. The detailed information on the local vernaculars is retrieved from the Dialektologi��eskij Atlas Russkogo Jazyka dialect atlas, the data for which were collected, for the most part, in the 1940 s and 1950 s. The theoretical framework of the research consists of a brief cross-section of western sociolinguistic theory related to language change and that of historical linguistics related to the Slavonic vowel development, which includes some new theories concerning the development of the Russian vowel phonemes. The author has collected dialect data in one of the 36 villages and three villages surrounding it. During the fieldwork, speech of nine elderly persons and ten school children was recorded. The speech data were then transcribed with coded information on the corresponding etymological vowels, the phonetic position, and the factual pronunciation at each appearance of vowels in the phonetic positions named above. The data from both of the dialect strata were then systematised to two corresponding systems that were compared with the information retrievable from the dialect atlas and other dialectological literature on the vowel phoneme system of the traditional local vernacular. As a result, it was found out (as hypothesised) that the vernacular vowel phoneme system has approached that of the standard language but has nonetheless not become similar to it. The phoneme quantity of the traditional vernacular is by one greater than that of the standard language, whereas the vowel phoneme quantity in the speech of the school children coincides with that in the standard language, although the phonetic realisations differ to some extent. The analysis of the speech of the elderly people resulted in that it is quite difficult to define the exact phoneme quantity of this stratum due to the fluctuation and irregularities in the realisation of the old phoneme that has ceased to exist in the newest stratum. It was noticed that the effect of the quality of the surrounding consonants on the phonetic realisation of the vowel phonemes has diminished, and the dependence of the phonetic realisation of a vowel phoneme on its place in a word in relation to the word stress has become more and more obvious, which is the state of affairs in the standard language as well.

Talking About Time in Russian and Finnish

In this study I look at what people want to express when they talk about time in Russian and Finnish, and why they use the means they use. The material consists of expressions of time: 1087 from Russian and 1141 from Finnish. They have been collected from dictionaries, usage guides, corpora, and the Internet. An expression means here an idiomatic set of words in a preset form, a collocation or construction. They are studied as lexical entities, without a context, and analysed and categorized according to various features. The theoretical background for the study includes two completely different approaches. Functional Syntax is used in order to find out what general meanings the speaker wishes to convey when talking about time and how these meanings are expressed in specific languages. Conceptual metaphor theory is used for explaining why the expressions are as they are, i.e. what kind of conceptual metaphors (transfers from one conceptual domain to another) they include. The study has resulted in a grammatically glossed list of time expressions in Russian and Finnish, a list of 56 general meanings involved in these time expressions and an account of the means (constructions) that these languages have for expressing the general meanings defined. It also includes an analysis of conceptual metaphors behind the expressions. The general meanings involved turned out to revolve around expressing duration, point in time, period of time, frequency, sequence, passing of time, suitable time and the right time, life as time, limitedness of time, and some other notions having less obvious semantic relations to the others. Conceptual metaphor analysis of the material has shown that time is conceptualized in Russian and Finnish according to the metaphors Time Is Space (Time Is Container, Time Has Direction, Time Is Cycle, and the Time Line Metaphor), Time Is Resource (and its submapping Time Is Substance), Time Is Actor; and some characteristics are added to these conceptualizations with the help of the secondary metaphors Time Is Nature and Time Is Life. The limits between different conceptual metaphors and the connections these metaphors have with one another are looked at with the help of the theory of conceptual integration (the blending theory) and its schemas. The results of the study show that although Russian and Finnish are typologically different, they are very similar both in the needs of expression their speakers have concerning time, and in the conceptualizations behind expressing time. This study introduces both theoretical and methodological novelties in the nature of material used, in developing empirical methodology for conceptual metaphor studies, in the exactness of defining the limits of different conceptual metaphors, and in seeking unity among the different facets of time. Keywords: time, metaphor, time expression, idiom, conceptual metaphor theory, functional syntax, blending theory

Die deutsche Sprache als Teil der sprachlichen und beruflichen Identit��t : Eine Studie unter Germanistikstudierenden der Universit��t Helsinki

Tutkielmassa tarkastellaan saksan kielen merkityst�� osana kielellist�� ja ammatillista identiteetti��. Tutkimuskohteena ovat Helsingin Yliopiston germaanisen filologian opiskelijat, jotka ovat aloittaneet opintonsa vuosina 2008 ja 2009. Identiteetti�� on tutkittu monilla eri tieteen aloilla, mutta yksimielisyytt�� identiteetin m����ritelm��st�� ei ole, sill�� eri tieteenalat painottavat tarkastelussaan eri n��k��kulmia. Tutkielman teoriaosassa pyrit����nkin luomaan yleiskuva vallalla olevista k��sityksist�� ja k��sitell����n identiteetti�� tarkemmin kielitieteellisest�� n��k��kulmasta. Teoriaosassa kuvataan my��s saksan kielen t��m��nhetkist�� kansainv��list�� asemaa, sill�� kielen asema vaikuttaa vahvasti siihen, kuinka ko. kieleen suhtaudutaan ja halutaanko sit�� opiskella vieraana kielen��. Vaikka identiteetti�� onkin tutkittu paljon, ei vieraan kielen yliopisto-opiskelijoiden suhteesta opiskelemaansa kieleen ole kattavia tutkimuksia. Opiskelijoiden suhtautuminen vieraaseen kieleen heijastuu kuitenkin heid��n oppimiseensa, motivaatioonsa ja laaja-alaisemmin koko opiskeluun, joten t��t�� tutkimalla voidaan saavuttaa t��rke���� tietoa, jota voidaan hy��dynt���� mm. yliopisto-opintojen kehitt��misess��. Tutkielman tavoitteena onkin kuvata t��t�� erityislaatuista suhdetta ja selvitt����, miten opiskelijat kuvaavat suhdettaan saksan kieleen. Kielellisen identiteetin n��k��kulmasta tutkitaan, miten opiskelijat suhtautuvat saksan kieleen, miten he kuvaavat omaa kielitaitoaan ja mitk�� tekij��t ovat vaikuttaneet kielellisen identiteetin kehittymiseen senhetkiseen muotoonsa. Ammatillisen identiteetin kannalta selvitet����n, kuinka suuri merkitys saksan kielell�� on osana opiskelijoiden ammatillista identiteetti�� ja miten opiskelijat haluavat k��ytt���� hyv��kseen saksan kielen taitoaan ty��el��m��ss��. Tutkielman korpus koostuu opiskelijoiden kirjoitelmista, jotka ovat osa TAITO-hanketta, sek�� Saksalaisen laitoksen (laitos on vuodesta 2010 osa Nykykielten laitosta) lukuvuosina 2008 ja 2009 uusille opiskelijoilleen teett��mist�� l��ht��kyselyist��. Analyysi on kaksiosainen, jotta saavutetaan mahdollisimman kattava kuva opiskelijoiden identiteetist��. Ensimm��isess�� osassa kirjoitelmia analysoidaan autobiografisen metodin avulla. Toisessa osassa korpuksia verrataan toisiinsa kvantitatiivisesti ja tutkitaan ovatko opiskelijat kuvanneet suhdettaan saksan kieleen samalla tavalla molemmissa korpuksissa. Tutkimuksesta k��y ilmi, ett�� opiskelijat aloittavat opintonsa hyvin erilaisista l��ht��kohdista, mik�� ilmenee opiskelijoiden eritasoisen kielitaidon ja erilaisten odotusten lis��ksi my��s eroina identiteeteiss��. Saksan kielell�� on t��rke�� merkitys suurimmalle osalle tutkituista opiskelijoista. Heid��n kielellinen identiteettins�� on selv��sti muotoutunut, ja he ovat motivoituneita opiskeluun. Heid��n ammatillinen identiteettins�� on sit�� vastoin ep��tarkempi, mik�� osoittaa tarvetta opiskelijoiden tukemiseen ja ty��el��m��orientaatioon jo opintojen alusta l��htien.

Sananlaskujen k��ytt��j��t ja k��ytt��yhteys : Sananlaskut k��ytt��kontekstissaan 'Perinne el��m��ss��ni' -kilpakirjoituksen aineistossa

Tutkielmassa l��hestyt����n sananlaskuja niiden k��ytt��kontekstin kautta. Tarkastelun kohteena on kirjoitettu kertova aineisto, jossa esiintyy sananlaskuja muun kerronnan yhteydess��. Sananlaskuja l��hestyt����n kuulijan n��k��kulmasta, kuulijan tulkintaan tukeutuen. Aineistona on Kalevalan juhlavuoden 1985 kilpakirjoitus "Perinne el��m��ss��ni". Tarkasteltavina ovat kilpakirjoituksen aihepiirit 1. "Koti ja suku" sek�� 5. "Sukupuoliroolit ja kasvatus avioliittoon". Tutkielman aineiston muodostavat vastaukset, joissa kerronta k��ytt���� sananlaskuja. T��llaisia vastauksia on yhteens�� 170 ker����j��n aineistossa (188 vastaajalta) koko kilpakeruun ko. aihepiirien sis��lt��ess�� 439 ker��yst��. Vastaajien ik�� vaihtelee 13-90 ik��vuoden v��lill��. Muisteluaineisto k��sittelee vuosia 1900-1985. Kilpakeruun vastaukset ovat muistelukerrontaa. T��m��n tutkielman aineistossa kertojat muistelevat lapsuuttaan ja tuovat esille kuulijan n��k��kulman sananlaskujen k��ytt��tilanteista. Sek�� arkistotietoon ett�� muistelukerrontaan liittyy olennaisesti aineiston l��hdekriittinen tarkastelu. Tutkimuksessa sananlasku on yleisnimitys. Vastaajat ovat k��sitelleet yhdenvertaisesti sananlaskuna niin sananlaskut, sananparret, sanonnat kuin raamatunlauseetkin, joten tutkielmassakin ne on k��sitelty sananlaskuina. Tutkimusteht��v��n�� on ollut selvitt���� yht����lt�� kuka sananlaskuja k��ytti ja toisaalta miss�� tarkoituksessa sananlaskuja k��ytettiin. N��k��kulma on kuulijan. Ensimm��iseen kysymykseen vastaus haettiin kertomuksissa olleista maininnoista, kuten esimerkiksi "��iti sanoi ...", "kuulin mummoltani ...", "lapsuudessani neuvottiin ...", "is��ll��ni oli tapana sanoa ...". Toista kysymyst�� l��hestyttiin k��ytt��yhteyden teonsanalla. Oletuksena oli, ett�� kuulija k��ytt��m��ll����n verbill�� kertoi, miss�� mieless�� h��n sananlaskuja koki k��ytetyn. Kertomusten mukaan sananlaskujen k��yt��n koettiin liittyv��n neuvoviin ja ohjaaviin tapahtumiin, kotona tapahtuneeseen kasvatukseen. Ne olivat osa kasvatuksessa k��ytetty�� puhetta. Toinen aineistossa esiin tullut sananlaskujen k��ytt��yhteys oli sananlaskujen k��ytt�� retorisena tehokeinona. V��itteit�� voitiin todentaa, kumota, vahvistaa jne. sananlaskujen avulla. Sananlaskuja kerrotiin kuullun ennen kaikkea kotona, kotiymp��rist��ss��. Niiden alkuper�� voi olla joko personoimaton ryhm�� tai k��ytt��j�� oli nimetty. Kun kyseess�� oli ryhm��n personoimaton puhe tai tieto, oli kyse ennen kaikkea vanhojen tiedosta, viisaista lauseista. Milloin sananlaskut on kuultu nimetylt�� henkil��lt��, oli kyseess�� useimmiten ��iti tai iso��iti. T��m�� selittynee osin sill��, ett�� lasten kasvatuksesta ovat kotiymp��rist��ss�� huolehtineet suvun ja perheen naiset.

Kielipes�� ja revitalisaatio : Karjalaisten ja inarinsaamelaisten kielipes��toiminta

Pro gradu -ty�� k��sittelee karjalan ja inarinsaamen kielipesi��. Kielipes�� on kielellisen v��hemmist��n tai alkuper��iskansan lapsille tarkoitettu p��iv��hoitopaikka, jossa lapsille puhutaan alusta asti kaikissa tilanteissa pelk��st����n ko. v��hemmist��kielt��. Tavoitteena on s��ilytt���� uhanalainen kieli tilanteessa, jossa vanhemmat eiv��t usein pysty siirt��m����n kielt�� seuraavalle sukupolvelle kotona. Kielipes��n idea on per��isin Uuden Seelannin maoreilta. Inarinsaamelaisten kielipes��toiminta alkoi vuonna 1997 ja karjalaisten vuonna 2000. Ty��n karjalan kielipes���� koskeva osuus liittyy Vienan Karjalan Uhtualla vuonna 1999 alkaneeseen kielipes��projektiin, jota olen vet��nyt. Ensimm��isess�� luvussa esittelen ty��n aihetta, tavoitteita, ty��hypoteesej��, metodeja ja aineistoa. Esit��n ty��hypoteesein��ni, ett�� kielipes�� vaikuttaa v��hemmist��yhteis����n kolmella eri tasolla, joita ovat lapset, heid��n perheens�� ja muu yhteis��. Lis��ksi esit��n, ett�� kielipes��ll�� on edellytykset muodostua merkitt��v��ksi kielellisen revitalisaation tekij��ksi. Tavoitteenani on kuvata kielipesi�� monipuolisesti sek�� revitalisaation ett�� lasten kielenoppimisen l��ht��kohdista. Toisessa luvussa esittelen ty��n kannalta olennaisia taustatietoja. Kerron karjalaisten ja inarinsaamelaisten nykytilanteesta ja historiasta painottaen kielitilanteen kehitykseen vaikuttaneita seikkoja. Esittelen tutkimuksen kielisosiologisen viitekehyksen, kielten uhanalaisuuden ja revitalisaation k��sitteet. Kolmannessa luvussa kerron kielipes��menetelm��n juurista ja esittelen karjalaisten ja inarinsaamelaisten kielipes��toiminnan taustaa, historiaa ja tulevaisuutta. P����paino on karjalan kielipes��n osuudessa. Kerron ennen kielipes��toimintaa vallinneesta kielitilanteesta, kyselytutkimuksestani, suomen ja karjalan kielen suhteesta ja hankkeen kulusta. Lopuksi k��sittelen Uhtuan kielipes��hankkeen tulevaisuudenn��kymi��. Inarinsaamelaisia koskevassa luvussa esittelen kielipes��toimintaa l��hteiden perusteella ja kerron kielipes��n vaikutuksista inarinsaamen puhujayhteis����n. Nelj��s luku koskee kielipes��lasten kielt��. Esittelen kielikylvyn ja kaksikielisyyden k��sitteit��. Kuvaan karjalan kielipes��lasten kielenoppimisprosessia ensimm��isen vuoden ajalta ja tarkastelen lasten kielen piirteit��. Kuvaan inarinsaamen kielipes��ss�� nelj��nnen toimintavuoden kev����ll�� vallinnutta kielitilannetta ja tarkastelen samoin lasten kielen piirteit��. Tarkastelen erityisesti kahden kielen yhdistymist�� lasten puheessa. Yhteenvetoluvussa k��sittelen sit��, mist�� kielipesien erilaiset tulokset johtuvat. Viides luku koostuu aineistosta, jota olen ker��nnyt karjalan kielipes��lasten vanhempien haastatteluista. Kuvaan vanhempien kielellisi�� taustoja ja asenteita, perheen kielenk��ytt���� ja kielipes��n vaikutusta siihen. Lopuksi tarkastelen tuloksia kielisosiologisessa kontekstissa. Kuudennessa luvussa tarkastelen ty��hypoteesieni toteutumista. Tulen siihen tulokseen, ett�� kielipes��n positiiviset vaikutukset v��hemmist��kielen tilaan ovat kiistattomat. Moni tutkimuksessa esilletuomani kysymys vaatii jatkotutkimusta mm. tutkijan positioon liittyvien seikkojen takia, ja koska kielipesien vaikutusta on tutkittava pitk��ll�� aikav��lill��.

The effect of interrogative function on intonation in spontaneous and read Finnish

Goals This study aims to map the effect of interrogative function on the intonation of spontaneous and read Finnish. Earlier research shows that the most prominent feature in Finnish question intonation is an appeal to the listener. Question word questions typically start with a high peak which is followed by falling intonation. In yes/no questions, F0 remains on a high level until the word carrying sentence stress and then falls. Final rises are mainly found in intonation clich��s such as "Ai mit��?" ("What?") These earlier results are based on read speech and enacted dialogues. In this study, questions and statements found in spontaneous dialogues were compared. These utterances were also compared with read versions of the same utterances. Fundamental frequency values were compared using a mixed model. Contours were also grouped using auditory and visual inspection. Thus it was possible to compare frequencies of contour types according to utterance type and speech style. The position of questions in the F0 distribution of the whole material was also investigated in this study. Method The material consisted of four spontaneous dialogues and their read versions. The speakers were young adults from the Helsinki metropolitan area, four females and four males. The whole material was first divided into broad dialogue function categories arising from the material and F0 curves were calculated for each category. After this, 277 questions and 244 statements were selected for closer inspection. Values reflecting F0 distribution and contour shape were measured from the F0 contours of these utterances. A mixed model was used to analyse the differences. Utterance type, question type, speech style and speaker gender were used as fixed effects. The frequencies of F0 contour types were compared using a Chi square test. Additional material in this study came from eight young female speakers in central Finland. Results and conclusions In the mixed model analysis, significant differences were found both between questions and statements and between spontaneous and read speech. Generally, utterance type affected the variables reflecting contour type while speech style affected the variables reflecting F0 distribution. The effect of question type was not clearly visible. In read speech the contours resembled earlier results more closely. Speakers had different strategies in differentiating between questions and statements. In the whole material, F0 was slightly higher in questions than in statements. The effect of dialectal background could be seen in the contour types. The results show that interrogative function affects intonation in both spontaneous and read Finnish.

Probiotic activity of Lactobacillus delbrueckii subsp. bulgaricus in the oral cavity

Yogurt consumption has been related to longevity of some populations living on the Balkans. Yogurt starter L. delbrueckii subsp. bulgaricus and Str. thermophilus have been recognized as probiotics with verified beneficial health effects. The oral cavity emerges as a arget for probiotic applications. Probiotics have demonstrated promising results in controlling dental diseases and oral yeast infections. However, L. bulgaricus despite its broad availability in dairy products has not been evaluated for probiotic activity in the mouth. These series of studies investigated in vitro properties of L. bulgaricus to outline its potential as an oral probiotic. Prerequisite probiotic properties in the mouth are resistance to oral defense mechanisms, adherence to saliva-coated surfaces, and inhibition of oral pathogens. L. bulgaricus strains showed a strain-dependent inhibition of oral streptococci and Aggregatibacter actinomycetemcomitans, whereas none of the dairy starter strains could affect growth of Porphyromonas gingivalis and Fusobacterium nucleatum. Adhesion is a factor contributing to colonization of the species at the target site. Radiolabeled L. bulgaricus strains and L. rhamnosus GG were tested for their ability to adhere to saliva-coated surfaces. The effects of lysozyme on adhesion and adhesion of Streptococcus sanguinis after lactobacilli pretreatment were also assessed. Adhesion of L. bulgaricus remained lower in comparison to L. rhamnosus GG. One L. bulgaricus strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment significantly increased Lactobacillus adhesion. Low gelatinolytic activity was observed for all strains and no conversion of proMMP-9 to its active form was induced by L. bulgaricus. Safety assessment ruled out deleterious effects of L. bulgaricus on extracellular matrix structures. Cytokine response of oral epithelial cells was assessed by measuring IL-8 and TNF-�� in cell culture supernatants. The effect of P. gingivalis on cytokine secretion after lactobacilli pretreatment was also assessed. A strain- and time-dependent induction of IL-8 was observed with live bacteria inducing the highest levels of cytokine secretion. Levels of TNF-�� were low and only one of ten L. bulgaricus strains stimulated TNF-�� secretion similar to positive control. The addition of P. gingivalis produced immediate reduction of cytokine levels within the first hours of incubation irrespective of lactobacilli strains co-cultured with epithelial cells. According to these studies strains among the L. delbrueckii subsp. bulgaricus species may have beneficial probiotic properties in the mouth. Their potential in prevention and management of common oral infectious diseases needs to be further studied.

Oral Actinomyces Species in Health and Disease: Identification, Occurence and Importance of Early Colonization

The genus Actinomyces consists of a heterogeneous group of gram-positive, mainly facultatively anaerobic or microaerobic rods showing various degrees of branching. In the oral cavity, streptococci and Actinomyces form a fundamental component of the indigenous microbiota, being among initial colonizers in polymicrobial biofilms. The significance of the genus Actinomyces is based on the capability of species to adhere to surfaces such as on teeth and to co-aggregate with other bacteria. Identification of Actinomyces species has mainly been based on only a few biochemical characteristics, such as pigmentation and catalase production, or on the use of a single commercial kit. The limited identification of oral Actinomyces isolates to species level has hampered knowledge of their role both in health and disease. In recent years, Actinomyces and related organisms have attracted the attention of clinical microbiologists because of a growing awareness of their presence in clinical specimens and their association with disease. This series of studies aimed to amplify the identification methods for Actinomyces species. With the newly developed identification scheme, the age-related occurrence of Actinomyces in healthy mouths of infants and their distribution in failed dental implants was investigated. Adhesion of Actinomyces species to titanium surfaces processed in various ways was studied in vitro. The results of phenotypic identification methods indicated a relatively low applicability of commercially available test kits for reliable identification within the genus Actinomyces. However, in the study of conventional phenotypic methods, it was possible to develop an identification scheme that resulted in accurate differentiation of Actinomyces and closely related species, using various different test methods. Genotypic methods based on 16S rRNA sequence analysis of Actinomyces proved to be a useful method for genus level identification and further clarified the species level identification with phenotypic methods. The results of the study of infants showed that the isolation frequency of salivary Actinomyces species increased according to age: thirty-one percent of the infants at 2 months but 97% at 2 years of age were positive for Actinomyces. A. odontolyticus was the most prominent Actinomyces colonizer during the study period followed in frequency by A. naeslundii and A. viscosus. In the study of explanted dental implants, Actinomyces was the most prevalent bacterial genus, colonizing 94% of the fixtures. Also in the implants A. odontolyticus was revealed as the most common Actinomyces species. It was present in 84% of Actinomyces -positive fixtures followed in frequency by A. naeslundii, A. viscosus and A. israelii. In an in vitro study of titanium surfaces, different Actinomyces species showed variation regarding their adhesion to titanium. Surface roughness as well as albumin coating of titanium had significant effects on adhesion. The use of improved phenotypic and molecular diagnostic methods increased the accuracy of the identification of the Actinomyces to species level. This facilitated an investigation of their occurrence and distribution in oral specimens in both health and disease.

Latent TGF-beta Binding Proteins : Adhesive functions and matrix association of LTBP-2 and potential functions of LTBP-1 and LTBP-3 in mesothelioma

Latent transforming growth factor-beta (TGF-beta) binding proteins (LTBPs) -1, -3 and -4 are ECM components whose major function is to augment the secretion and matrix targeting of TGF-beta, a multipotent cytokine. LTBP-2 does not bind small latent TGF-beta but has suggested functions as a structural protein in ECM microfibrils. In the current work we focused on analyzing possible adhesive functions of LTBP-2 as well as on characterizing the kinetics and regulation of LTBP-2 secretion and ECM deposition. We also explored the role of TGF-beta binding LTBPs in endothelial cells activated to mimic angiogenesis as well as in malignant mesothelioma. We found that, unlike most adherent cells, several melanoma cell lines efficiently adhered to purified recombinant LTBP-2. Further characterization revealed that the adhesion was mediated by alpha3beta1 and alpha6beta1 integrins. Heparin also inhibited the melanoma cell adhesion suggesting a role for heparan sulphate proteoglycans. LTBP-2 was also identified as a haptotactic substrate for melanoma cell migration. We used cultured human embryonic lung fibroblasts to analyze the temporal and spatial association of LTBP-2 into ECM. By We found that LTBP-2 was efficiently assembled to the ECM only in confluent cultures following the deposition of fibronectin (FN) and fibrillin-1. In early, subconfluent cultures it remained primarily in soluble form after secretion. LTBP-2 colocalized transiently with FN and fibrillin-1. Silencing of fibrillin-1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP-2 indicating that the ECM association of LTBP-2 depends on a pre-formed fibrillin-1 network. Considering the established role of TGF-beta as a regulator of angiogenesis we induced morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) and followed the fate of LTBP-1 in the endothelial ECM. This resulted in profound proteolytic processing of LTBP-1 and release of latent TGF-beta complexes from the ECM. The processing was coupled with increased activation of MT-MMPs and specific upregulation of MT1-MMP. The major role of MT1-MMP in the proteolysis of LTBP-1 was confirmed by suppressing the expression with lentivirally induced short-hairpin RNAs as well as by various metalloproteinases inhibitors. TGF-beta can promote tumorigenesis of malignant mesothelioma (MM), which is an aggressive tumor of the pleura with poor prognosis. TGF-beta activity was analyzed in a panel of MM tumors by immunohistochemical staining of phosphorylated Smad-2 (P-Smad2). The tumor cells were strongly positive for P-Smad2 whereas LTBP-1 immunoreactivity was abundant in the stroma, and there was a negative correlation between LTBP-1 and P-Smad2 staining. In addition, the high P-Smad2 immunoreactivity correlated with shorter survival of patients. mRNA analysis revealed that TGF-beta1 was the most highly expressed isoform in both normal human pleura and MM tissue. LTBP-1 and LTBP-3 were both abundantly expressed. LTBP-1 was the predominant isoform in established MM cell lines whereas the expression of LTBP-3 was high in control cells. Suppression of LTBP-3 expression by siRNAs resulted in increased TGF-beta activity in MM cell lines accompanied by decreased proliferation. Our results suggest that decreased expression of LTBP-3 in MM could alter the targeting of TGF-beta to the ECM and lead to its increased activation. The current work emphasizes the coordinated process of the assembly and appropriate targeting of LTBPs with distinct adhesive or cytokine harboring properties into the ECM. The hierarchical assembly may have implications in the modulation of signaling events during morphogenesis and tissue remodeling.

The role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia

Identification of genes predisposing to tumor syndromes has raised general awareness of tumorigenesis. Genetic testing of tumor susceptibility genes aids the recognition of individuals at increased risk of tumors. Identification of novel predisposing genes enables further studies concerning the classification of potential associated tumors and the definition of target patient group. Pituitary adenomas are common, benign neoplasms accounting for approximately 15% of all intracranial tumors. Accurate incidence estimation is challenging since a great portion of these adenomas are small and asymptomatic. Clinically relevant adenomas, that cause symptoms due to the expansion of the cell mass or the over-secretion of normally produced hormones, occur in approximately one of 1 000 individuals. Although the majority of pituitary adenomas are sporadic, a minority occur as components of familial syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 syndrome is caused by germ-line mutations in the MEN1 gene, whereas most of the CNC patients carry the mutated protein kinase A (PKA) regulatory subunit-1-�� (PRKAR1A) gene. Recently, other conditions predisposing to endocrine tumors have been identified: Pituitary Adenoma Predisposition (PAP) and MEN type 4 (MEN4). PAP was originally identified in a genetically homogeneous Finnish population. In a population based cohort from Northern Finland, aryl hydrocarbon receptor-interacting protein (AIP) gene mutations were found in 16% of all patients diagnosed with growth hormone (GH) producing pituitary adenoma, and in 40% of the subset of patients who were diagnosed under the age of 35 years. Since AIP mutations were originally described in a defined, homogeneous population from Northern Finland, it was relevant to study whether mutations also occur in more heterogeneous populations. In patient cohorts with different ethnic origins and variable clinical phenotypes, germ-line AIP mutations were detectable at low frequencies (range 0.8-7.4%). AIP mutation-positive patients were often diagnosed with a GH-producing adenoma at a young age, and usually had no family history of endocrine tumors. The low frequency of AIP mutations in randomly selected patients, and the lack of any family history of pituitary adenomas create a challenge for the identification of PAP patients. Our preliminary study suggests that AIP immunohistochemistry may serve as a pre-screening tool to distinguish between the AIP mutation-negative and the mutation-positive tumors. Tumors of various endocrine glands are components of MEN1 and CNC syndromes. Somatic MEN1 and PRKAR1A mutations in sporadic pituitary adenomas are rare, but occur in some of the other tumors related to these syndromes. The role of AIP mutations in endocrine neoplasia was studied and our results indicated that somatic AIP mutations are rare or non-existent in sporadic tumors of endocrine glands (0 of 111). Furthermore, germ-line AIP mutations in prolactin producing adenomas (2 of 9) confirmed the role of this pituitary tumor type in the PAP phenotype. Thyroid disorders are common in the general population, and the majority of them are sporadic. Interestingly, it has been suggested that thyroid disorders might be more common in PAP families. For this reason we studied germ-line AIP mutations in 93 index cases from familial non-medullary thyroid cancer (NMTC) families. The underlying gene or genes for familial NMTC have not been identified yet. None of the patients had any potentially pathogenic AIP mutation. This suggests that AIP is unlikely to play a role in familial NMTCs. A novel multiple endocrine syndrome was originally described in rats with phenotypic features of human MEN type 1 and 2. Germ-line mutations of cyclin-dependent kinase inhibitor 1B (CDKN1B also known as p27Kip1) gene were reported later in these rats and a germ-line mutation was also identified in one human family with MEN1-like phenotype (later named MEN4). To confirm the importance of this gene���s mutations in humans, we performed a mutation screening in MEN-like patients and in patients with pituitary adenoma. Our results indicate that CDKN1B/p27Kip1 mutations appear in a small portion of MEN1-like patients (one of 36), and that such mutations are rare or non-existent in both familial (0 of 19) and sporadic pituitary adenoma patients (0 of 50). In conclusion, this work strengthens the tumor susceptibility role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia. Clarifying the PAP phenotype facilitates the identification of potential AIP mutation carriers. Genetic counseling can be offered to the relatives and follow-up of the mutation carriers can be organized, hence an earlier diagnosis is feasible.

Isotopic fingerprints in surficial waters : Stable isotope methods applied in hydrogeological studies

The driving force behind this study has been the need to develop and apply methods for investigating the hydrogeochemical processes of significance to water management and artificial groundwater recharge. Isotope partitioning of elements in the course of physicochemical processes produces isotopic variations to their natural reservoirs. Tracer property of the stable isotope abundances of oxygen, hydrogen and carbon has been applied to investigate hydrogeological processes in Finland. The work described here has initiated the use of stable isotope methods to achieve a better understanding of these processes in the shallow glacigenic formations of Finland. In addition, the regional precipitation and groundwater records will supplement the data of global precipitation, but as importantly, provide primary background data for hydrological studies. The isotopic composition of oxygen and hydrogen in Finnish groundwaters and atmospheric precipitation was determined in water samples collected during 1995 2005. Prior to this study, no detailed records existed on the spatial or annual variability of the isotopic composition of precipitation or groundwaters in Finland. Groundwaters and precipitation in Finland display a distinct spatial distribution of the isotopic ratios of oxygen and hydrogen. The depletion of the heavier isotopes as a function of increasing latitude is closely related to the local mean surface temperature. No significant differences were observed between the mean annual isotope ratios of oxygen and hydrogen in precipitation and those in local groundwaters. These results suggest that the link between the spatial variability in the isotopic composition of precipitation and local temperature is preserved in groundwaters. Artificial groundwater recharge to glaciogenic sedimentary formations offers many possibilities to apply the isotopic ratios of oxygen, hydrogen and carbon as natural isotopic tracers. In this study the systematics of dissolved carbon have been investigated in two geochemically different glacigenic groundwater formations: a typical esker aquifer at Tuusula, in southern Finland and a carbonate-bearing aquifer with a complex internal structure at Virttaankangas, in southwest Finland. Reducing the concentration of dissolved organic carbon (DOC) in water is a primary challenge in the process of artificial groundwater recharge. The carbon isotope method was used to as a tool to trace the role of redox processes in the decomposition of DOC. At the Tuusula site, artificial recharge leads to a significant decrease in the organic matter content of the infiltrated water. In total, 81% of the initial DOC present in the infiltrated water was removed in three successive stages of subsurface processes. Three distinct processes in the reduction of the DOC content were traced: The decomposition of dissolved organic carbon in the first stage of subsurface flow appeared to be the most significant part in DOC removal, whereas further decrease in DOC has been attributed to adsorption and finally to dilution with local groundwater. Here, isotope methods were used for the first time to quantify the processes of DOC removal in an artificial groundwater recharge. Groundwaters in the Virttaankangas aquifer are characterized by high pH values exceeding 9, which are exceptional for shallow aquifers on glaciated crystalline bedrock. The Virttaankangas sediments were discovered to contain trace amounts of fine grained, dispersed calcite, which has a high tendency to increase the pH of local groundwaters. Understanding the origin of the unusual geochemistry of the Virttaankangas groundwaters is an important issue for constraining the operation of the future artificial groundwater plant. The isotope ratios of oxygen and carbon in sedimentary carbonate minerals have been successfully applied to constrain the origin of the dispersed calcite in the Virttaankangas sediments. The isotopic and chemical characteristics of the groundwater in the distinct units of aquifer were observed to vary depending on the aquifer mineralogy, groundwater residence time and the openness of the system to soil CO2. The high pH values of > 9 have been related to dissolution of calcite into groundwater under closed or nearly closed system conditions relative to soil CO2, at a low partial pressure of CO2.