Understanding solid-state transformations during dehydration: new insights using vibrational spectroscopy and multivariate modelling

Many active pharmaceutical ingredients (APIs) have both anhydrate and hydrate forms. Due to the different physicochemical properties of solid forms, the changes in solid-state may result in therapeutic, pharmaceutical, legal and commercial problems. In order to obtain good solid dosage form quality and performance, there is a constant need to understand and control these phase transitions during manufacturing and storage. Thus it is important to detect and also quantify the possible transitions between the different forms. In recent years, vibrational spectroscopy has become an increasingly popular tool to characterise the solid-state forms and their phase transitions. It offers several advantages over other characterisation techniques including an ability to obtain molecular level information, minimal sample preparation, and the possibility of monitoring changes non-destructively in-line. Dehydration is the phase transition of hydrates which is frequently encountered during the dosage form production and storage. The aim of the present thesis was to investigate the dehydration behaviour of diverse pharmaceutical hydrates by near infrared (NIR), Raman and terahertz pulsed spectroscopic (TPS) monitoring together with multivariate data analysis. The goal was to reveal new perspectives for investigation of the dehydration at the molecular level. Solid-state transformations were monitored during dehydration of diverse hydrates on hot-stage. The results obtained from qualitative experiments were used to develop a method and perform the quantification of the solid-state forms during process induced dehydration in a fluidised bed dryer. Both in situ and in-line process monitoring and quantification was performed. This thesis demonstrated the utility of vibrational spectroscopy techniques and multivariate modelling to monitor and investigate dehydration behaviour in situ and during fluidised bed drying. All three spectroscopic methods proved complementary in the study of dehydration. NIR spectroscopy models could quantify the solid-state forms in the binary system, but were unable to quantify all the forms in the quaternary system. Raman spectroscopy models on the other hand could quantify all four solid-state forms that appeared upon isothermal dehydration. The speed of spectroscopic methods makes them applicable for monitoring dehydration and the quantification of multiple forms was performed during phase transition. Thus the solid-state structure information at the molecular level was directly obtained. TPS detected the intermolecular phonon modes and Raman spectroscopy detected mostly the changes in intramolecular vibrations. Both techniques revealed information about the crystal structure changes. NIR spectroscopy, on the other hand was more sensitive to water content and hydrogen bonding environment of water molecules. This study provides a basis for real time process monitoring using vibrational spectroscopy during pharmaceutical manufacturing.

Contributions to the Theory of Finite-State Based Grammars

This dissertation is a theoretical study of finite-state based grammars used in natural language processing. The study is concerned with certain varieties of finite-state intersection grammars (FSIG) whose parsers define regular relations between surface strings and annotated surface strings. The study focuses on the following three aspects of FSIGs: (i) Computational complexity of grammars under limiting parameters In the study, the computational complexity in practical natural language processing is approached through performance-motivated parameters on structural complexity. Each parameter splits some grammars in the Chomsky hierarchy into an infinite set of subset approximations. When the approximations are regular, they seem to fall into the logarithmic-time hierarchyand the dot-depth hierarchy of star-free regular languages. This theoretical result is important and possibly relevant to grammar induction. (ii) Linguistically applicable structural representations Related to the linguistically applicable representations of syntactic entities, the study contains new bracketing schemes that cope with dependency links, left- and right branching, crossing dependencies and spurious ambiguity. New grammar representations that resemble the Chomsky-Schützenberger representation of context-free languages are presented in the study, and they include, in particular, representations for mildly context-sensitive non-projective dependency grammars whose performance-motivated approximations are linear time parseable. (iii) Compilation and simplification of linguistic constraints Efficient compilation methods for certain regular operations such as generalized restriction are presented. These include an elegant algorithm that has already been adopted as the approach in a proprietary finite-state tool. In addition to the compilation methods, an approach to on-the-fly simplifications of finite-state representations for parse forests is sketched. These findings are tightly coupled with each other under the theme of locality. I argue that the findings help us to develop better, linguistically oriented formalisms for finite-state parsing and to develop more efficient parsers for natural language processing. Avainsanat: syntactic parsing, finite-state automata, dependency grammar, first-order logic, linguistic performance, star-free regular approximations, mildly context-sensitive grammars

Irish Folklore Commission 1935-1970 : history, ideology, methodology

Between 1935 and 1970 the state-funded Irish Folklore Commission (Coimisiún Béaloideasa Éireann) assembled one of the great folklore collections of the world under the direction of Séamus Ó Duilearga (James Hamilton Delargy). The aim of this study is to recount and assess the work and achievement of this commission. The cultural, linguistic, political and ideological factors that had a bearing on the establishment and making permanent of the Commission and that impinged on many aspects of its work are here elucidated. The genesis of the Commission is traced and the vision and mission of Séamus Ó Duilearga are outlined. The negotiations that preceded the setting up of the Commission in 1935 as well as protracted efforts from 1940 to 1970 to place it on a permanent foundation are recounted and examined at length. All the various collecting programmes and other activities of the Commission are described in detail and many aspects of its work are assessed. This study also deals with the working methods and conditions of employment of the Commission s field and Head Office staff as well as with Séamus Ó Duilearga s direction of the Commission. In executing this work extensive use has been made of primary sources in archives and libraries in Ireland, Sweden, Finland, Estonia, and North America. This is the first major study of this world-famous institute, which has been praised in passing in numerous publications, but here for the first time its work and achievement are detailed comprehensively and subjected to scholarly scrutiny. This study should be of interest not only to students of Irish oral tradition but to folklorists everywhere. The history of the Irish Folklore Commission is a part of a wider history, that of the history of folkloristics in Europe and North America in particular. Moreover, this work has relevance for many areas of the developing world today, where conditions are not dissimilar to those that pertained in Ireland in the 1930's when this great salvage operation was funded by the young, independent Irish state. It is also hoped that this work will be of practical assistance to scholars and the general public when utilising these collections, and that furthermore it will stimulate research into the assembling of other national collections of folklore as well as into the history of folkloristics in other countries, subjects which in recent years are beginning to attract more and more scholarly attention.

Viemärin päässä Kotkan kaupunki : Vesiensuojelun kehitys Kymijoen itäisen päähaaran suulla 1945-1970

The focus of this thesis is the marine environmental history of the eastern part and the estuary of the Kymi river from 1945 to 1970. There is no previous research on this area from an environmentally historical perspective, nor have many of the sources here discussed been previously used. Therefore the thesis expands academic understanding of local environmental processes and protection in and around the city of Kotka and the Kymi river. The thesis falls within the methodological field of socio-political history, as the research focus is centered on the local process of establishing the nature of environmental problems and solving them. The principal assumption has been that the city of Kotka, due to its ongoing expansion, was slow to respond to environmental hazards. The Kymi river was among the most degraded bodies of water during this period. Kotka on the other hand was a major center of wood processing industry and one of Finlands major industrial ports. In the past the river and its estuary had provided ample resources for fishers. It is this contradictory use of the environment that allows one to discuss the local struggle for the correct use of the environment. Primary sources include local and city archives, environmental studies, and legal documents linked with the above. The archives of the city of Kotka and of various private associations form the core sources. Environmental studies from the research period have been dealt with as sources to the local political power struggle. Alongside with current environmental research they also provide insight into the state of the environment. Another goals has been to accumulate environmental research for a future multidisciplinary study in this area. As a final conclusion it can be said that environmental degradation was widely understood as a problem only in the 1960s. The influential role of the city of Kotka however determined the pace with which these problems were then solved.

Biophysical Characterization of Oxidized Phospholipids : Implications for Altered Membrane Structure and Function

The present study aims to elucidate the modifications in the structure and functionality of the phospholipid matrix of biological membranes brought about by free radical-mediated oxidative damage of its molecular constituents. To this end, the surface properties of two oxidatively modified phospholipids bearing an aldehyde or carboxyl function at the end of truncated sn-2 acyl chain were studied using a Langmuir balance. The results obtained reveal both oxidized species to have a significant impact on the structural dynamics of phospholipid monolayers, as illustrated by the progressive changes in force-area isotherms with increasing mole fraction of the oxidized lipid component. Moreover, surface potential measurements revealed considerable modifications in the electric properties of oxidized phospholipid containing monolayers during film compression, suggesting a packing state-controlled reorientation of the intramolecular electric dipoles of the lipid headgroups and acyl chains. Based on the above findings, a model describing the conformational state of oxidized phospholipid molecules in biological membranes is proposed, involving the protrusion of the acyl chains bearing the polar functional groups out from the hydrocarbon phase to the surrounding aqueous medium. Oxidative modifications alter profoundly the physicochemical properties of unsaturated phospholipids and are therefore readily anticipated to have important implications for their interactions with membrane-associating molecules. Along these lines, the carboxyl group bearing lipid was observed to bind avidly the peripheral membrane protein cytochrome c. The binding was reversed following increase in ionic strength or addition of polyanionic ATP, thus suggesting it to be driven by electrostatic interactions between cationic residues of the protein and the deprotonated lipid carboxyl exposed to the aqueous phase. The presence of aldehyde function bearing oxidized phospholipid was observed to enhance the intercalation of four antimicrobial peptides into phospholipid monolayers and liposomal bilayers. Partitioning of the peptides to monolayers was markedly attenuated by the aldehyde scavenger methoxyamine, revealing it to be mediated by the carbonyl moiety possibly through efficient hydrogen bonding or, alternatively, formation of covalent adduct in form of a Schiff base between the lipid aldehydes and primary amine groups of the peptide molecules. Lastly, both oxidized phospholipid species were observed to bind with high affinity three small membrane-partitioning therapeutic agents, viz. chlorpromazine, haloperidol, and doxorubicin. In conclusion, the results of studies conducted using biomimetic model systems support the notion that oxidative damage influences the molecular architecture as well as the bulk physicochemical properties of phospholipid membranes. Further, common polar functional groups carried by phospholipids subjected to oxidation were observed to act as molecular binding sites at the lipid-water interface. It is thus plausible that oxidized phospholipid species may elicit cellular level effects by modulating integration of various membrane-embedded and surface-associated proteins and peptides, whose conformational state, oligomerization, and functionality is known to be controlled by highly specific lipid-protein interactions and proper physical state of the membrane environment.

Candida proteinases in the degradation of oral mucosal tissue components associated with Candida invasion

The aim of this thesis was to compare the degradation of human oral epithelial proteins by proteinases of different Candida yeast species. We focused on proteins associated with Candida invasion in the cell-to-cell junction, the basement membrane zone, the extracellular matrix, and local tissue inflammatory regulators. Another main objective was to evaluate the effect of the yeast/hyphal transition and pH on the degradative capability of Candida. The enzymatic activity of the Candida proteinases was verified by gelatin zymography. Laminins-332 (Lm-322) and -511(Lm-511) produced by human oral keratinocytes were gathered from the growth media, and E-cadherin (E-Cad) was isolated from the cell membrane of the keratinocytes by immunoprecipitation. The proteins were incubated with Candida cells and cell-free fractions, and degradation was detected by fluorography. Fibronectin degradation was visualised by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Matrix metalloproteinase-9 (MMP-9) activation and tissue inhibitor of metalloproteinase-1 (TIMP-1) fragmentation was detected by using the Western blot and enhanced chemoluminescence (ECL) techniques. Residual activity of TIMP-1 was evaluated by a casein degradation assay. A fluorimetric assay was used to detect and compare Candida proteinase activities with MMP-9. These studies showed that the ability of the different Candida yeast species to degrade human Lm-332, fibronectin, and E-Cad vary from strain to strain and that this degradation is pH-dependent. This indicates that local acidic pH in tissue may play a role in tissue destruction by activating Candida proteinases and aid invasion of Candida into deeper tissue. A potential correlation exists between the morphological form of the yeasts and the degradative ability; the C. albicans yeast form seems to be related to superficial infections, and hyphal forms can apparently invade deeper tissues between the epithelial cells by degradation of E-Cad. Basement membrane degradation is possible, especially in the junctional epithelium, which contains only Lm-332 as a structural component. Local tissue host inflammatory mediators, such as MMP-9, were activated, and TIMP-1 was degraded by certain Candida species, thus indicating the possibility of a weakened host tissue defence mechanism in vivo.

Women, environmental changes and forestry-related development : Gender-affected roles of rural people in land degradation and environmental rehabilitation in a dry region of Sudan

The purpose of the present study was to increase understanding of the interaction of rural people and, specifically, women with the environment in a dry area in Sudan. The study that included both nomadic pastoralists and farmers aimed at answering two main research questions, namely: What kinds of roles have the local people, and the women in particular, had in land degradation in the study area and what kinds of issues would a gender-sensitive, forestry-related environmental rehabilitation intervention need to consider there? The study adopted the definition of land degradation as proposed by the United Nations Convention to Combat Desertification (UNCCD), which describes land degradation as reduction or loss the biological or economic productivity and complexity of land in arid, semi-arid and dry sub-humid areas. The Convention perceives desertification as land degradation. The dry study area in Sudan, South of the Sahara, has been the subject of land degradation or desertification discussions since the 1970s, and other studies have been also conducted to assess the degradation in the area. Nevertheless, the exact occurrence, scale and local significance of land degradation in the area is still unclear. This study explored how the rural population whose livelihood depended on the area, perceived environmental changes occurring there and compared their conceptions with other sources of information of the area such as research reports. The main fieldwork methods included interviews with open-ended questions and observation of people and the environment. The theoretical framework conceptualised the rural population as land users whose choices of environmental activities are affected by multiple factors in the social and biophysical contexts in which they live. It was emphasised that these factors have their own specific characteristics in different contexts, simultaneously recognising that there are also factors that generally affect environmental practices in various areas such as the land users' environmental literacy (conceptions of the environment), gender and livelihood needs. The people studied described that environmental changes, such as reduced vegetation cover and cropland production, had complicated the maintenance of their livelihoods in the study area. Some degraded sites were also identified through observations during the fieldwork. Whether a large-scale reduction of cropland productivity had occurred in the farmers' croplands remained, however, unclear. The study found that the environmental impact of the rural women's activities varied and was normally limited. The women's most significant environmental impact resulted from their cutting of trees, which was likely to contribute, at least in some places, to land degradation, affecting the environment together with climate and livestock. However, when a wider perspective is taken, it becomes questionable whether the women have really played roles in land degradation, since gender, poverty and the need to maintain livelihood had caused them to conduct environmentally harmful activities. The women have had, however, no power to change the causes of their activities. The findings further suggested that an inadequate availability of food was the most critical problem in the study area. Therefore, an environmental programme in the area was suggested to include technical measures to increase the productivity of croplands, opportunities for income generation and readiness to co-operate with other programmes to improve the local people's abilities to maintain their livelihoods. In order to protect the environment and alleviate the women's work burden, the introduction of fuel-saving stoves was also suggested. Furthermore, it was suggested that increased planting of trees on homesteads would be supported by an easy availability of tree seedlings. Planting trees on common property land was, however, perceived as extremely demanding in the study area, due to scarcity of such land. In addition, it became apparent that the local land users, and women in particular, needed to allocate their labour to maintain the immediate livelihood of their families and were not motivated to allocate their labour solely for environmental rehabilitation. Nonetheless, from the point of view of the existing social structures, women's active participation in a community-based environmental programme would be rather natural, particularly among the farmer women who had already formed a women's group and participated in communal decision making. Forming of a women group or groups was suggested to further support both the farmer women's and pastoral women's active participation within an environmental programme and their general empowerment. An Environmental programme would need to acknowledge that improving rural people's well-being and maintaining their livelihood in the study area requires development and co-operation with various sectors in Sudan.

Monitoring of Fungal Growth and Degradation of Wood

Valko- ja ruskolahosienet tunnetaan luonnossa tehokkaimpina puun ja karikkeen lignoselluloosan lahottajina. Valkolahosienet pystyvät hajottamaan kaikkia puun osia: ligniiniä, selluloosaa ja hemiselluloosaa. Selektiivisesti ligniiniä hajottavat sienet lahottavat puusta suhteessa enemmän vaikeasti hajoavaa ligniiniä kuin selluloosaa tai hemiselluloosaa, jolloin jäljelle jää valkoista ja miltei puhdasta selluloosaa. Bioteknisissä sovelluksissa juuri selektiviiviset valkolahottajat ovat kiinnostavia. Niiden avulla voidaan puuhaketta esikäsitellä esimerkiksi paperinvalmistuksessa haitallisen ligniinin poistamiseksi. Ruskolahosienet ovat huomattavia puun, puutavaran ja puisten rakenteiden lahottajia, kuten tässä työssä käytetty Gloeophyllum trabeum (saunasieni ) ja Poria (Postia) placenta (istukkakääpä). Ruskolahosienet hajottavat puusta hemiselluloosan lisäksi selluloosaa, jolloin jää jäljelle ruskea ja jauhomaiseksi mureneva ligniini. Ruskolahosienet muovaavat ligniiniä jonkin verran. Kahden ruskolahosienen G. trabeumin ja P. placentan lisäksi tutkittiin valkolahosieniä, joista Ceriporiopsis subvermispora (karstakääpä) ja harvinainen Physisporinus rivulosus -sieni (talikääpä) hajottavat ligniiniä erittäin selektiivisesti. Phanerochaete chrysosporium on kaikkialla paljon tutkittu sieni, ja Phlebia radiata valkolahosientä (rusorypykkä) on tutkittu paljon mikrobiologian osastolla. Lisäksi tutkittiin Phlebia tremellosa -sienten (hytyrypykkä) ligninolyyttisten entsyymien tuottoa ja 14C-leimatun synteettisen ligniinin (DHP) hajotusta. P. radiata ja P. tremellosa -sienten on todettu aiemmin hajottavan ligniiniä selektiivisesti. Työssä selvitettiin miten sienten kasvua voi mitata, miten vertailukelpoisia eri mittaamismenetelmillä saadut tulokset ovat ja ilmenevätkö sienten aktiivisimmat kasvuvaiheet samaan aikaan eri menetelmillä mitattuna. Tärkeimmät tulokset olivat seuraavat havainnot: (i) P. radiata ja P. tremellosa -sienikannat tuottivat ligniini- ja mangaaniperoksidaasientsyymejä (LiP ja MnP) sekä lakkaasia, ja sienistä puhdistettiin 2-3 LiP- ja P. radiatasta yksi MnP-entsyymi; (ii) P. tremellosa -sienet hajottivat leimattua synteettistä ligniiniä (DHP) yhtä hyvin kuin paljon tutkitut P. chrysosporium ja P. radiata -sienet; (iii) puu, sienen luonnollinen kasvualusta, lisäsi valkolaho- ja ruskolahosienten demetoksylaatiota [O14CH3]-leimatusta ligniinin malliyhdisteestä 14CO2:ksi ilman puuta olleeseen alustaan verrattuna; (iv) demetoksylaatio (14CO2:n tuotto) oli normaalissa ilma-atmosfäärissä useimmiten parempi happeen verrattuna; (v) hapessa paras 14CO2:n tuotto saatiin puupalakasvatuksissa, joihin oli lisätty ravinnetyppeä tai typen lisäksi glukoosia sekä valkolaho- että ruskolahosienillä; (vi) ilmassa 14CO2:n tuotto oli puulla voimakkainta valkolahosienillä ilman lisäravinteita, kun taas G. trabeum -sienellä se oli yhtä hyvä eri alustoissa; (vii) biomassan muodostuminen rihmastojen ergosterolipitoisuuksista mitattuna oli ruskolahosienillä parempi kuin valkolahosienillä; (viii) ja biomassojen huippupitoisuudet olivat 6:lla sienellä eri suuruisia ja niiden maksimimäärien ajankohdat vaihtelivat viiden viikon kasvatusten kuluessa. Mikrobiologian osastolla Viikissä eristetty ja paljon tutkittu P. radiata -valkolahosieni oli mukana kaikissa tehdyissä kokeissa. Sienen LiP-aktiivisuus ja 14CO2:n tuotto 14C-rengas-leimatusta synteettisestä ligniinistä (DHP) korreloivat erittäin hyvin. Biomassan muodostuminen ergosterolilla määritettynä tuki hyvin entsyymiaktiivisuusmittauksilla ja isotooppikasvatuksilla saatuja tuloksia.

Bioavailability aspects of hydrophobic contaminant degradation in soils

This thesis concentrates on bioavailability of organic soil contaminants in the context of bioremediation of soil contaminated with volatile or non-volatile hydrophobic pollutants. Bioavailability and biodegradation was studied from four viewpoints: (i) Improvement of bioavailability and biodegradation of volatile hydrocarbons in contained bioremediation systems at laboratory - and pilot-scale. (ii) Improvement of bioavailability of non-volatile, hydrophobic compounds in such systems. (iii) Biodegradation of a non-volatile hydrophobic compound in soil organic matter in microcosms. (iiii) Bioavailability of nitrogen in an open, full-scale bioremediation system. It was demonstrated that volatility of organic compounds can be controlled by amending the soil with adsorbents. The sorbed hydrocarbons were shown to be available to soil microbiota. As the result, biodegradation of the volatile hydrocarbons was greatly favored at the expense of volatilization. PAH compounds were shown to be mobilized and their bioavailability improved by a hydrophobic, non-toxic additive, vegetable oil. Bioavailability of the PAHs was recorded as an increased toxicity of the soil. In spite of the increased bioavailability, biodegradation of the PAHs decreased. In microcosms simulating boreal forest organic surface soil, PAH-compound (pyrene) was shown to be removed from soil biologically. Therefore hydrophobicity of the substrate does not necessarily mean low availability and biodegradation in organic soil. Finally, in this thesis it was demonstrated that an unsuitable source of nitrogen or its overdose resulted in wasteful spending of this nutrient and even harmful effects on soil microbes. Such events may inhibit rather than promote the bioremediation process in soil.

Prolamin degradation in sourdoughs

This thesis examines protein behaviours that occur during cereal fermentations. The focus is on the prolamin degradation in sourdoughs. The thesis also looks at what happens to the oat globulins during an oat bran acidification process. The cereal prolamins are unique proteins in many respects. The wheat prolamins (glutenins and gliadins) are responsible for the formation of the gluten that provides the viscoelastic properties to wheat doughs whereas the rye prolamins (secalins) are unable to develop gluten-like structures. In addition, many baking technological features, such as flavour, shelf-life and dough properties are affected by the protein degradation that might occur during processing. On the other hand, the prolamins contain protein structures that are harmful to gluten sensitive people. It is thus evident that the degradation of the prolamins in sourdough processes may be approached from various aspects. This thesis describes some of these approaches. Four different cereal fermentations were carried out. Wheat sourdough (WSD) and rye sourdough (RSD) fermentations represented traditional sourdoughs. A germinated-wheat sourdough (GWSD) was a novel sourdough type that was prepared using germinated wheat grains that had high and diverse proteolytic activities. The oat bran fermentation (OBF) represented a fermentation system that lacked functional cereal proteases. The high molecular weight glutenins and rye secalins were degraded during the WSD and RSD fermentations, respectively. It was noteworthy that in WSD only a very limited degradation of the gliadins occurred. The gliadins were, however, hydrolysed very extensively during the GWSD fermentation. No protein degradation was observable in the OBF system. Instead the acidification altered the solubility of the oat globulins and this finally led to their aggregation. This thesis confirms that the endogenous proteases of cereals hydrolyse cereal prolamins in sourdoughs. The thesis also shows that the proteolytic activity of the used cereal raw material determines the extent of proteolysis that occurs in sourdough. This means that bakers may adjust the protein degradation in their sourdoughs by selecting the raw material based on its proteolytic activity. The thesis also demonstrates that by using germinated grains, with high and diverse proteolytic activity in sourdough preparations, the prolamins can be extensively degraded. Whether such highly proteolytic food technology could be used to manufacture new gluten-free cereal-based products for gluten sensitive people remains to be solved.