Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

The Politics of Pulp Investment and the Brazilian Landless Movement (MST)

The Politics of Pulp Investment and the Brazilian Landless Movement (MST) The paper industry has been moving more heavily to the global South at the beginning of the 21st century. In a number of cases the rural populations of the global South have engaged in increasingly important resistance in their scuffle with the large-scale tree plantation-relying pulp investment model. The resistance had generally not yet managed to slow down Southern industrial tree plantation expansion until 2004. After all, even the MST, perhaps the strongest of the Southern movements, has limited power in comparison to the corporations pushing for plantation expansion. This thesis shows how, even against these odds, depending on the mechanisms of contention and case-specific conflict dynamics, in some cases the movements have managed to slow and even reverse plantation expansion. The thesis is based on extensive field research in the Brazilian countryside. It outlines a new theory of contentious agency promotion, emphasizing its importance in the shaping of corporate resource exploitation. The thesis includes a Qualitative Comparative Analysis of resistance influence on the economic outcomes of all (14) Brazilian large-scale pulp projects between 2004-2008. The central hypothesis of the thesis is that corporate resource exploitation can be slowed down more effectively and likely when the resistance is based on contentious agency. Contentious agency is created by the concatenation of five mutually supporting mechanisms of contention: organizing and politicizing a social movement; heterodox framing of pulp projects; protesting; networking; and embedding whilst maintaining autonomy. The findings suggest that contentious agency can slow or even reverse the expansion of industrial plantations, whereas when contentious agency promotion was inactive, fast or even unchecked plantation expansion was always the outcome. The rule applied to all the assessed 14 pulp conflict cases. The hypothesis gained strong support even in situations where corporate agency promotion was simultaneously active. In previous studies on social movements, there has been a lack of contributions that help us understand the causal mechanisms of contention influencing economic outcomes. The thesis answers to the call by merging a Polanyian analysis of the political economy with the Dynamics of Contention research program and making a case for the impact of contentious agency on capital accumulation. The research concludes that an efficient social movement can utilize mechanisms of contention to promote the potential of activism among its members and influence investment outcomes. Protesting, for example via pioneering land occupations, seemed to be particularly important. Until now, there has been no comprehensive theory on when and how contentious agency can slow down or reverse the expansion of corporate resource exploitation. The original contribution of this research is to provide such a theory, and utilize it to offer an extensive explanation on the conflicts over pulp investment in Brazil, the globalization of the paper industry, and slowing of industrial plantation expansion in the global South.

Pulp and Paper Fiction: On the Discursive Legitimation of Global Industrial Restructuring

Despite the central role of legitimacy in social and organizational life, we know little of the subtle meaning-making processes through which organizational phenomena, such as industrial restructuring, are legitimated in contemporary society. Therefore, this paper examines the discursive legitimation strategies used when making sense of global industrial restructuring in the media. Based on a critical discourse analysis of extensive media coverage of a revolutionary pulp and paper sector merger, we distinguish and analyze five legitimation strategies: (1) normalization, (2) authorization, (3) rationalization, (4) moralization, and (5) narrativization. We argue that while these specific legitimation strategies appear in individual texts, their recurring use in the intertextual totality of the public discussion establishes the core elements of the emerging legitimating discourse.

Wood and fiber properties of Norway spruce and its suitability for thermomechanical pulping.

In the first part of the study, the selected wood and fiber properties were investigated in terms of their occurrence and variation in wood, as well as their relevance from the perspective of thermomechanical pulping process and related end-products. It was concluded that the most important factors were the fiber dimensions, juvenile wood content, and in some cases, the content of heartwood being associated with extremely dry wood with low permeability in spruce. With respect to the above properties, the following three pulpwood assortments of which pulping potential was assumed to vary were formed: wood from regeneration cuttings, first-thinnings wood, and sawmill chips. In the experimental part of the study the average wood and fiber characteristics and their variation were determined for each raw material group prior to pulping. Subsequently, each assortment - equaling about 1500 m3 roundwood - was pulped separately for a 24 h period, at constant process conditions. The properties of obtained newsgrade thermomechanical pulps were then determined. Thermomechanical pulping (TMP) from sawmill chips had the highest proportion of long fibers, smallest proportion of fines, and had generally the coarsest and longest fibers. TMP from first-thinnings wood was just the opposite, whereas that from regeneration cuttings fell in between the above two extremes. High proportion of dry heartwood in wood originating from regeneration cuttings produced a slightly elevated shives content. However, no differences were found in pulp specific energy consumption. The obtained pulp tear index was clearly best in TMP made from sawmill chips and poorest in pulp from first-thinnings wood, which had generally inferior strength properties. No dramatical differences in any of the strength properties were found between pulp from sawmill residual wood and regeneration cuttings. Pulp optical properties were superior in TMP from first-thinnings. Unexpectedly, no noticeable differences, which could be explained with fiber morphology, were found in sheet density, bulk, air permeance or roughness between the three pulps. The most important wood quality factors in this study were the fiber length, fiber cross-sectional dimensions and percentage juvenile wood. Differences found in the quality of TMP manufactured from the above spruce assortments suggest that they could be segregated and pulped separately to obtain specific product characteristics, i.e., for instance tailor-made end-products, and to minimize unnecessary variation in the raw material quality, and hence, pulp quality.

Harvesting undelimbed Scots pine (Pinus sylvestris L.) from first thinnings for integrated production of kraft pulp and energy

The present study evaluates the feasibility of undelimbed Scots pine (Pinus sylvestris L.) for integrated production of pulp and energy in a kraft pulp mill from the technical, economic and environmental points of view, focusing on the potential of bundle harvesting. The feasibility of tree sections for pulp production was tested by conducting an industrial wood-handling experiment, laboratory cooking and bleaching trials, using conventional small-diameter Scots pine pulpwood as a reference. These trials showed that undelimbed Scots pine sections can be processed in favourable conditions as a blend with conventional small-diameter pulpwood without reducing the pulp quality. However, fibre losses at various phases of the process may increase when using undelimbed material. In the economic evaluation, both pulp production and wood procurement costs were considered, using the relative wood paying capability of a kraft pulp mill as a determinant. The calculations were made for three Scots pine first-thinning stands with the breast-height diameter of the removal (6 12 cm) as the main distinctive factor. The supply chains included in the comparison were based on cut-to-length harvesting, whole-tree harvesting and bundle harvesting (whole-tree bundling). With the current ratio of pulp and energy prices, the wood paying capability declines with an increase in the proportion of the energy fraction of the raw material. The supply system based on the cut-to-length method was the most efficient option, resulting in the highest residual value at stump in most cases. A decline in the pulp price and an increase in the energy price improved the competitiveness of the whole-tree systems. With short truck transportation distances and low pulp prices, however, the harvesting of loose whole trees can result in higher residual value at stump in small-diameter stands. While savings in transportation costs did not compensate for the high cutting and compaction costs by the second prototype of the bundle harvester, an increase in transportation distances improved its competitiveness. Since harvesting undelimbed assortments increases nutrient export from the site, which can affect soil productivity, the whole-tree alternatives included in the present study cannot be recommended on infertile peatlands and mineral soils. The harvesting of loose whole trees or bundled whole trees implies a reduction in protective logging residues and an increase in site traffic or payloads. These factors increase the risk of soil damage, especially on peat soils with poor bearing capacity. Within the wood procurement parameters which were examined, the CO2 emissions of the supply systems varied from 13 27 kg m3. Compaction of whole trees into bundles reduced emissions from transportation by 30 39%, but these reductions were insufficient to compensate for the increased emissions from cutting and compaction.

Determination of intestinal permeability in healthy horses using the contrast medium iohexol

Tämä lisensiaatin tutkielma koostuu kolmesta osasta; kirjallisuuskatsauksesta, kokeellisesta osasta ja liitteistä. Iohexol on ionisoitumaton, trijodattu ja vesiliukoinen röntgenvarjoaine. Iohexolia on hyödynnetty lääketieteessä useita vuosia. Iohexolia on käytetty muun muassa angio- ja myelografiassa, lisäksi iohexolia on hyödynnetty arvioitaessa munuaiskerästen suodattumisnopeutta sekä suoliston läpäisevyyden muutoksia. Hevosen tulehduksellisessa suolistosairaudessa (Inflammatory bowel disease, IBD) suoliston rakenne ja sen läpäisevyys muuttuu; tyypillistä on tulehdussolujen kertyminen suoliston seinämään ja myös sidekudosmuodostusta saattaa esiintyä. Suolisto muutoksia saatetaan havaita sekä ohut- että paksusuolessa. IBD aiheuttaa hevoselle laihtumista, johtuen ravintoaineiden puutteellisesta imeytymisestä ja proteiinien menetyksestä suoleen suoliston häiriötilan yhteydessä. Tällä hetkellä IBD:n diagnostiikka perustuu tyypillisiin oireisiin, kliiniseen tutkimukseen, verinäytteisiin, glukoosin imeytymistestiin ja peräsuolesta otettuun koepalaan. IBD:n diagnostiikka on kuitenkin erittäin haastavaa ja tutkimusmenetelmiin liittyy lukuisia ongelmia, jotka vähentävät niiden luotettavuutta IBD:n diagnostiikassa. Tutkimuksemme tarkoituksena on kehittää hevosen IBD:n diagnostiikkaa entistä helpompaan, luotettavampaan ja turvallisempaan suuntaan. Tämän alustavan tutkimuksen tavoitteet olivat: (1) tutkia voidaanko iohexol havaita hevosen seerumissa oraalisen annostelun jälkeen ja (2) muodostaa iohexolin pitoisuuskuvaaja ajan funktiona terveillä hevosilla. Materiaalimme koostui kymmenestä terveestä hevosesta, joilla ei ollut havaittu laihtumista tai ripulia. Ennen iohexolin annostelua hevosille suoritettiin kliininen tutkimus ja verinäytteet otettiin maha-suolikanavan sairauden poissulkemiseksi. Hevosille suoritettiin myös mahalaukun tähystys. 16 tunnin paaston jälkeen 1 ml/kg Iohexolia annosteltiin 10 % -liuoksena nenämahaletkulla suoraan mahaan ja verinäytteet otettiin 0, 30, 60, 120, 180, 240, 300 ja 360 minuuttia annostelun jälkeen. Iohexolin pitoisuus määritettiin käyttämällä korkean erotuskyvyn nestekromatografiaa. Iohexolin pitoisuuksista tietyillä ajanhetkillä muodostettiin kuvaaja. Hevosilla ei havaittu maha-suolikanavan sairauksia. Kaikki hevoset olivat hyvässä kuntoluokassa ja mahalaukun tähystyksessä ei havaittu merkittäviä muutoksia. Verinäytteiden tulokset olivat viiterajoissa. Kaikki hevoset sietivät iohexolia hyvin ja haittavaikutuksia ei havaittu. Iohexol oli havaittavissa seerumissa 60 minuutin kuluttua annostelusta. Kuvaajassa voitiin havaita kaksi huippua. Statistiset menetelmät tukivat löydöksiä. Iohexol testi oli yksinkertainen suorittaa ja siihen ei liittynyt haittavaikutuksia. Annos 1ml/kg oli havaittavissa seerumissa. Iohexolin pitoisuuskuvaaja muodosti kaksi huippua, ja tämänkaltainen ilmiö on kuvattu kirjallisuudessa aikaisemmin useiden lääkkeiden tapauksessa. Hevosella ilmiö liittyy todennäköisesti maha-suolikanavan rakenteellisiin ja fysiologisiin eroavaisuuksiin ja lisätutkimuksia ilmiön varmistamiseksi tarvitaan. Iohexol näyttää olevan potentiaalinen merkkiaine suoliston läpäisevyyden arviointiin ja lisätutkimuksia IBD:tä sairastavien hevosten seerumin iohexolin pitoisuuksista verrattuna terveiden hevosten seerumin iohexolin pitoisuuksiin on suunnitteilla.