Drug cultures in Estonia : contexts, meanings and patterns of illicit drug use.

In Estonia, illicit drug use hardly existed before the social changes of the 1990s when, as a result of economic and cultural transformations, the country became part of a world order centred in the West. On the one hand, this development is due to the spread of international youth culture, which many young people have perceived as being associated with drugs; on the other hand, it results from the marginalisation of a part of the population. The empirical part of the study is based mostly on in-depth interviews with different drug users conducted during between 1998 and 2002. Complementary material includes the results of participant observations, interviews with key experts, and the results of previous quantitative studies and statistics. The young people who started experimenting with illicit drugs from the 1990s and onwards perceived them as a part of an attractive lifestyle - a Western lifestyle, a point which is worth stressing in the case of Estonia. Although the reasons for initiation into drug use were similar for the majority of young people, their drug use habits and the impact of the drug use on their lives began to differ. I argue that the potential pleasure and harm which might accompany drug use is offset by the meanings attached to drugs and the sanctions and rituals regulating drug use. In the study both recreational and problem use have been analysed from different aspects in seven articles. I have investigated different types of drug users: new bohemians, cannabis users, in whose case partying and restrictive drug use is positively connected to their lives and goals within established society; stimulant-using party people for whom drugs are a means of having fun but who do not have the same restrictive norms regulating their drug use as the former and who may get into trouble under certain conditions; and heroin users for whom the drug rapidly progressed from a means of having fun to an obligation due to addiction. The research results point at the importance not only of the drug itself and the socio-economic situation of the user, but also of the cultural and social context within which the drug is used. The latter may on occasions be a crucial factor in whether or not initial drug use eventually leads to addiction.

Mass spectrometry and n-in-one analytics in early drug discovery: Combinatorial chemistry libraries, lipophilicity and absorption screening

This thesis describes current and past n-in-one methods and presents three early experimental studies using mass spectrometry and the triple quadrupole instrument on the application of n-in-one in drug discovery. N-in-one strategy pools and mix samples in drug discovery prior to measurement or analysis. This allows the most promising compounds to be rapidly identified and then analysed. Nowadays properties of drugs are characterised earlier and in parallel with pharmacological efficacy. Studies presented here use in vitro methods as caco-2 cells and immobilized artificial membrane chromatography for drug absorption and lipophilicity measurements. The high sensitivity and selectivity of liquid chromatography mass spectrometry are especially important for new analytical methods using n-in-one. In the first study, the fragmentation patterns of ten nitrophenoxy benzoate compounds, serial homology, were characterised and the presence of the compounds was determined in a combinatorial library. The influence of one or two nitro substituents and the alkyl chain length of methyl to pentyl on collision-induced fragmentation was studied, and interesting structurefragmentation relationships were detected. Two nitro group compounds increased fragmentation compared to one nitro group, whereas less fragmentation was noted in molecules with a longer alkyl chain. The most abundant product ions were nitrophenoxy ions, which were also tested in the precursor ion screening of the combinatorial library. In the second study, the immobilized artificial membrane chromatographic method was transferred from ultraviolet detection to mass spectrometric analysis and a new method was developed. Mass spectra were scanned and the chromatographic retention of compounds was analysed using extract ion chromatograms. When changing detectors and buffers and including n-in-one in the method, the results showed good correlation. Finally, the results demonstrated that mass spectrometric detection with gradient elution can provide a rapid and convenient n-in-one method for ranking the lipophilic properties of several structurally diverse compounds simultaneously. In the final study, a new method was developed for caco-2 samples. Compounds were separated by liquid chromatography and quantified by selected reaction monitoring using mass spectrometry. This method was used for caco-2 samples, where absorption of ten chemically and physiologically different compounds was screened using both single and nin- one approaches. These three studies used mass spectrometry for compound identification, method transfer and quantitation in the area of mixture analysis. Different mass spectrometric scanning modes for the triple quadrupole instrument were used in each method. Early drug discovery with n-in-one is area where mass spectrometric analysis, its possibilities and proper use, is especially important.

Effect of dietary cholesterol and ubiquinone on isoprene synthesis in rat liver

The effect of dietary cholesterol and ubiquinone on the synthesis of isoprene compounds in the liver, as tested by the incorporation of acetate-1-14C and mevalonate-2-14C, was studied in rats. In cholesterol feeding, there appears to be a second site of inhibition after squalene in addition to the previously known primary site of inhibition at the β-hydroxy-β-methyl glutaryl-CoA reductase. Feeding ubiquinone inhibited at some common step between acetate and mevalonate in the synthesis of both cholesterol and ubiquinone, without affecting the acetate activation or fatty acid synthesis, and also at a step in the synthesis of ubiquinone not common with the synthesis of cholesterol. These results are suggestive of a role for ubiquinone in the regulation of isoprene synthesis.

Explorations in the distributional and semantic similarity of words

A straightforward computation of the list of the words (the `tail words' of the list) that are distributionally most similar to a given word (the `head word' of the list) leads to the question: How semantically similar to the head word are the tail words; that is: how similar are their meanings to its meaning? And can we do better? The experiment was done on nearly 18,000 most frequent nouns in a Finnish newsgroup corpus. These nouns are considered to be distributionally similar to the extent that they occur in the same direct dependency relations with the same nouns, adjectives and verbs. The extent of the similarity of their computational representations is quantified with the information radius. The semantic classification of head-tail pairs is intuitive; some tail words seem to be semantically similar to the head word, some do not. Each such pair is also associated with a number of further distributional variables. Individually, their overlap for the semantic classes is large, but the trained classification-tree models have some success in using combinations to predict the semantic class. The training data consists of a random sample of 400 head-tail pairs with the tail word ranked among the 20 distributionally most similar to the head word, excluding names. The models are then tested on a random sample of another 100 such pairs. The best success rates range from 70% to 92% of the test pairs, where a success means that the model predicted my intuitive semantic class of the pair. This seems somewhat promising when distributional similarity is used to capture semantically similar words. This analysis also includes a general discussion of several different similarity formulas, arranged in three groups: those that apply to sets with graded membership, those that apply to the members of a vector space, and those that apply to probability mass functions.

Understanding Human Drug Conjugating Enzymes; Regio- and Stereoselectivity in Sulfotransferase 1A3 and the UDP-Glucuronosyltransferases

Sulfotransferases (SULTs) and UDP-glucuronosyltransferases (UGTs) are important detoxification enzymes and they contribute to bioavailability and elimination of many drugs. SULT1A3 is an extrahepatic enzyme responsible for the sulfonation of dopamine, which is often used as its probe substrate. A new method for analyzing dopamine-3-O-sulfate and dopamine-4-O-sulfate by high-performance liquid chromatography was developed and the enzyme kinetic parameters for their formation were determined using purified recombinant human SULT1A3. The results show that SULT1A3 strongly favors the 3-hydroxy group of dopamine, which indicates that it may be the major enzyme responsible for the difference between the circulating levels of dopamine sulfates in human blood. All 19 known human UGTs were expressed as recombinant enzymes in baculovirus infected insect cells and their activities toward dopamine and estradiol were studied. UGT1A10 was identified as the only UGT capable of dopamine glucuronidation at a substantial level. The results were supported by studies with human intestinal and liver microsomes. The affinity was low indicating that UGT1A10 is not an important enzyme in dopamine metabolism in vivo. Despite the low affinity, dopamine is a potential new probe substrate for UGT1A10 due to its selectivity. Dopamine was used to study the importance of phenylalanines 90 and 93 in UGT1A10. The results revealed distinct effects that are dependent on differences in the size of the side chain and on the differences in their position within the protein. Examination of twelve mutants revealed lower activity in all of them. However, the enzyme kinetic studies of four mutants showed that their affinities were similar to that of UGT1A10 suggesting that F90 and F93 are not directly involved in dopamine binding in the active site. The glucuronidation of β-estradiol and epiestradiol (α-estradiol) was studied to elucidate how the orientation of the 17-OH group affects conjugation at the 3-OH or the 17-OH of either diastereomer. The results show that there are clear differences in the regio- and stereoselectivities of UGTs. The most active isoforms were UGT1A10 and UGT2B7 demonstrating opposite regioselectivity. The stereoselectivities of UGT2Bs were more complex than those of UGT1As. The amino acid sequences of the human UGTs 1A9 and 1A10 are 93% identical, yet there are large differences in their activity and substrate selectivity. Several mutants were constructed to identify the residues responsible for the activity differences. The results revealed that the residues between Leu86 and Tyr176 of UGT1A9 determine the differences between UGT1A9 and UGT1A10. Phe117 of UGT1A9 participated in 1-naphthol binding and the residues at positions 152 and 169 contributed to the higher glucuronidation rates of UGT1A10. In summary, the results emphasize that the substrate selectivities, including regio- and stereoselectivities, of UGTs are complex and they are controlled by many amino acids rather than one critical residue.

Glucose-Triggered Drug Delivery from Borate Mediated Layer-by-Layer Self-Assembly

In this study, we report a novel approach for glucose-triggered anticancer drug delivery from the self-assembly of neutral poly(vinyln alcohol) (PVA) and chitosan. In the present study, we have fabricated multilayer thin film of PVA-borate and chitosan on colloidal particle (MF particle) and monitored the layer-by-layer growth using Zetapotential measurements. Formation of multilayer membrane on MF particle has been further characterized with transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Subsequently,disintegration of multilayer thin film and microcapsules was observed in presence of glucose. We investigated the disassembly of PVA-borate and chitosan self-assembly under CLSM and atomic force microscopy. These results suggest that this multilayer thin film is very efficient for encapsulation and release of DOX molecules above certain concentration of glucose (25 mM). This glucose-sensitive self-assembly is relevant for the application of anticancer therapeutic drug delivery.

USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.

Beyond Words : The European Court of Justice and Legal Certainty in Multilingual EU Law

This study addresses the issue of multilingualism in EU law. More specifically, it explores the implications of multilingualism for conceptualising legal certainty, a central principle of law both in domestic and EU legal systems. The main question addressed is how multilingualism and legal certainty may be reconciled in the EU legal system. The study begins with a discussion on the role of translation in drafting EU legislation and its implications for interpreting EU law at the European Court of Justice (ECJ). Uncertainty regarding the meaning of multilingual EU law and the interrelationship between multilingualism and ECJ methods of interpretation are explored. This analysis leads to questioning the importance of linguistic-semantic methods of interpretation, especially the role of comparing language versions for clarifying meaning and the ordinary meaning thesis, and to placing emphasis on other, especially the teleological, purpose-oriented method of interpretation. As regards the principle of legal certainty, the starting-point is a two-dimensional concept consisting of both formal and substantive elements; of predictability and acceptability. Formal legal certainty implies that laws and adjudication, in particular, must be predictable. Substantive legal certainty is related to rational acceptability of judicial decision-making placing emphasis on its acceptability to the legal community in question. Contrary to predictability that one might intuitively relate to linguistic-semantic methods of interpretation, the study suggests a new conception of legal certainty where purpose, telos, and other dynamic methods of interpretation are of particular significance for meaning construction in multilingual EU law. Accordingly, the importance of purposive, teleological interpretation as the standard doctrine of interpretation in a multilingual legal system is highlighted. The focus on rational, substantive acceptability results in emphasising discourse among legal actors among the EU legal community and stressing the need to give reasons in favour of proposed meaning in accordance with dynamic methods of interpretation including considerations related to purposes, aims, objectives and consequences. In this context, the role of ideal discourse situations and communicative action taking the form of interaction among the EU legal community in an ongoing dialogue especially in the preliminary ruling procedure is brought into focus. In order for this dialogue to function, it requires that the ECJ gives persuasive, convincing and acceptable reasons in justifying its decisions. This necessitates transparency, sincerity, and dialogue with the relevant audience.

Words as Events : Cretan Mantinádes in Performance and Composition

Words as Events introduces the tradition of short, communicative rhyming couplets, the mantinádes, which are still sung and recited in a variety of performance situations on the island of Crete. The local focus on communicative economy and artistry is further examined in an in-depth analysis of the processes and ideals of composition. Short genres of oral poetry have been widely neglected in folklore research; however, their demand for structural and semantic coherence as well as their dialogic nature appeals to very different human needs from those of the longer poems. In contemporary Crete, poems also appear in written contexts, they are submitted to modern mass media, and people widely exchange them as text messages. By striving to understand the tradition during a period of change, this study analyzes the larger principles that ground the communication, self-expression and creativity in the genre. The aim of this research is thus twofold: to present this specific register of dialogic oral poetry as well as to create a theoretical approach sensitive to the creativity of such short registers. The study is based on long-term ethnographic fieldwork conducted in Crete between the years 1997 2009. An important aspect of the methodology was to create long-term ties with a number of key-informants who composed mantinádes. The interdisciplinary theoretical and methodological basis for this analysis is in the contemporary Finnish and international research on oral poetry and on anthropological research on communicative speech genres. These theoretical insights are extended by addressing questions of spontaneity and individual agency. Since mantinádes are at the same time a model for composition and a reserve of poems in fixed form, the aesthetics and objectives of performance and composition are also plural. In a traditional singing event, the basic motivation for the singers is to provide a meaningful contribution to a selected theme, which is the shared topic of the poetic dialogue. Consequently, similar topics giving rise to poetic associations can be encountered during various moments of everyday life: the dialogic nature is embodied in the tradition to such degree that it also arises in the performances and composition of single poems and outside of any institutionalized performance arenas. Therefore, as this study discusses in detail, even the apparently non-contextualized poems recited between locals or occurring in the contemporary mass media arenas, are understood and evaluated as utterances that provide an individual perspective within a certain dialogue.

Porphyrin synthesis in drug induced hepatic porphyria

Liver δ-aminolaevulate (ALA) synthetase and ALA dehydratase are induced to a greater extent in 3,5-diethoxy carbonyl-1,4-dihydrocollidine (DDC) injected mice as compared to the allyl isopropyl acetamide (AIA) injected rats. DDC treated mice do not show an increase in porphobilinogen (PEG) levels commensurate with the increase in ALA levels and the two enzyme activities, but accumulate enormous quantities of protoporphyrin in the liver. Normal mouse liver has an inherent greater capacity to convert PBG to porphyrins as compared to that of the rat. This together with the inhibition of iron incorporation into protoporphyrin in vivo at later stages of DDC administration can account for the large accumulation of protoporphyrin in these animals.

Fatty acid specificity for the esterification of vitamin A and cholesterol by intestinal and pancreatic enzymes in rats

VITAMIN A and cholesterol esters have been shown to undergo extensive hydrolysis in the lumen of the small intestine during the process of absorption; they are re-esterified to appear in the lymph mostly as esters1,2. However, the vitamin A esters of the lymph, blood and liver of the rat are formed by long-chain fatty acids3 and in the normal rat liver, probably as palmitates4. On the other hand, cholesterol esters are usually made up of poly-unsaturated fatty acids in the lymph and blood of rats5. For the absorption of the two lipid materials, the enzymes of the pancreas have been largely implicated, while not much attention has been paid to the possible role of the mucosal enzymes. From the behaviour of the mucosal enzymes, as presented here, it appears that probably these enzymes play a more important part in the re-esterification of the two lipid materials during their absorption.

High cholesterol diet and esterification of cholesterol by the intestinal mucosa of rats

Young male rats maintained on a diet containing 1% cholesterol were sacrificed at the end of 1st, 2nd, 3rd, 5th, and 7th week. Acetone powders prepared from their intestinal mucosa and pancreas were tested for the synthetic and hydrolytic activities for Vitamin A and cholesterol esters. The esterifying activity of the mucosal enzymes for both Vitamin A and cholesterol increased progressively up to the end of the 5th week; the increase in esterification of cholesterol was more marked with respect to saturated fatty acids, as compared to the unsaturated ones. The pancreatic enzymes remained unaffected. It is suggested that one of the reasons for the accumulation of cholesterol esters in animal tissues may be the increased esterification of the sterol in the mucosa induced by dietary cholesterol.