Introduction of high-content screening method for studying drug-induced mitochondrial dysfunction in hepatocytes

Drug induced liver injury is one of the frequent reasons for the drug removal from the market. During the recent years there has been a pressure to develop more cost efficient, faster and easier ways to investigate drug-induced toxicity in order to recognize hepatotoxic drugs in the earlier phases of drug development. High Content Screening (HCS) instrument is an automated microscope equipped with image analysis software. It makes the image analysis faster and decreases the risk for an error caused by a person by analyzing the images always in the same way. Because the amount of drug and time needed in the analysis are smaller and multiple parameters can be analyzed from the same cells, the method should be more sensitive, effective and cheaper than the conventional assays in cytotoxicity testing. Liver cells are rich in mitochondria and many drugs target their toxicity to hepatocyte mitochondria. Mitochondria produce the majority of the ATP in the cell through oxidative phosphorylation. They maintain biochemical homeostasis in the cell and participate in cell death. Mitochondria is divided into two compartments by inner and outer mitochondrial membranes. The oxidative phosphorylation happens in the inner mitochondrial membrane. A part of the respiratory chain, a protein called cytochrome c, activates caspase cascades when released. This leads to apoptosis. The aim of this study was to implement, optimize and compare mitochondrial toxicity HCS assays in live cells and fixed cells in two cellular models: human HepG2 hepatoma cell line and rat primary hepatocytes. Three different hepato- and mitochondriatoxic drugs (staurosporine, rotenone and tolcapone) were used. Cells were treated with the drugs, incubated with the fluorescent probes and then the images were analyzed using Cellomics ArrayScan VTI reader. Finally the results obtained after optimizing methods were compared to each other and to the results of the conventional cytotoxicity assays, ATP and LDH measurements. After optimization the live cell method and rat primary hepatocytes were selected to be used in the experiments. Staurosporine was the most toxic of the three drugs and caused most damage to the cells most quickly. Rotenone was not that toxic, but the results were more reproducible and thus it would serve as a good positive control in the screening. Tolcapone was the least toxic. So far the conventional analysis of cytotoxicity worked better than the HCS methods. More optimization needs to be done to get the HCS method more sensitive. This was not possible in this study due to time limit.

Trends in Pharmaceutical Product Development 2000-2010

Merkittävä osa alkuperäislääkevalmistajien tutkimus- ja tuotekehityskuluista näyttää olevan suunnattu olemassa olevien lääkkeiden kehittämiseen. Tämä voi oletettavasti johtaa kiinnostaviin formulaatiokehitysstrategioihin. Tutkimuksen tarkoituksena oli selvittää, voidaanko farmaseuttisen tuotekehityksen trendejä havaita myönnettyjen myyntilupien perusteella. Tutkimuksen mielenkiinnon kohteena olivat myös suurimpien lääkeyritysten käyttämät elinkaaren hallinnan keinot, joilla suojataan myyvimpiä tuotteita geneeriseltä kilpailulta ja varmistetaan markkinaosuus. Tutkimuksen painopiste oli kiinteissä oraalisissa lääkevalmisteissa. Laadullisten ja määrällisten menetelmien yhdistelmää käytettiin laajan näkökulman saamiseksi tutkittavaan aiheeseen. Suomalaisten myyntilupaviranomaisten haastatteluja käytettiin keräämään taustatietoa tutkimuksen määrällistä osaa varten. Määrällinen osa koostui myyntilupatietokannoista, jotka käsittivät kaikkien menettelyjen kautta Suomessa myönnetyt myyntiluvat, keskitetyn menettelyn kautta EU:ssa myönnetyt myyntiluvat ja maailman kymmenen suurinta lääkeyritystä USA:ssa. Tutkimustulosten perusteella rinnakkaislääkkeiden määrässä tapahtui merkittävä nousu Suomessa kaikkien menettelyjen kautta myönnetyissä myyntiluvissa ja EU:ssa keskitetyn menettelyn kautta myönnetyissä myyntiluvissa vuosina 2000-2010. Tämä muutos saattaa ainakin osaksi johtua lainsäädännöllisistä muutoksista, joilla luotiin kannustimia rinnakkaislääkkeiden käyttöön ja valmistukseen, kuten lääkevaihto ja viitehintajärjestelmä. USA:n tiedot osoittivat suurten lääkevalmistajien kiinnostuksen elinkaaren hallintaan: suurin osa maailman kymmenelle suurimmalle lääkeyritykselle myönnetyistä myyntiluvista vuosina 2005-2010 oli tähän tarkoitukseen. Elinkaaren hallinnan suhde uusiin lääkeaineisiin oli lähes 4:1. Kiinteä oraalinen lääkemuoto on kiistatta kaikista suosituin tapa annostella lääke, minkä vahvistivat sekä arvioijien haastattelut että myyntilupatiedot. Kiinteiden oraalisten rooli oli entistäkin korostuneempi rinnakkaislääkkeiden kohdalla. Kun innovatiivisuutta mitattiin epätyypillisten annosmuotojen määrällä, USA:n tiedot kiinteistä oraalisista lääkemuodoista osoittivat vahvaa innovatiivisuutta Suomen ja EU:n tietoihin verrattuna. Tämä saattaa heijastaa suurten lääkeyritysten innovatiivista tuotevalikoimaa. Epätyypillisten kiinteiden oraalisten annosmuotojen osuus oli huomattavasti pienempi rinnakkaislääkkeissä kuin alkuperäislääkkeissä kaikilla alueilla. Elinkaaren hallinnassa käytetyimmät strategiat olivat uusi formulaatio, uusi vahvuus ja uusi yhdistelmä olemassa olevasta valmisteesta. Kiinteiden oraalisten lääkemuotojen osalta kaksi kolmasosaa uusista elinkaaren hallinnan formulaatioista oli säädellysti vapauttavia valmisteita. Elinkaaren hallinta on olennainen osa suurten lääkeyritysten liiketoimintastrategiaa, ja sen tärkeyttä havainnollistettiin Coreg-tablettien tapausesimerkillä.

Development of radiosynthesis methods for 18F-labelled radiopharmaceuticals : General considerations and production of a dopamine transporter radioligand

Positron emission tomography (PET) is a molecular imaging technique that utilises radiopharmaceuticals (radiotracers) labelled with a positron-emitting radionuclide, such as fluorine-18 (18F). Development of a new radiotracer requires an appropriate radiosynthesis method: the most common of which with 18F is nucleophilic substitution with [18F]fluoride ion. The success of the labelling reaction is dependent on various factors such as the reactivity of [18F]fluoride, the structure of the target compound in addition to the chosen solvent. The overall radiosynthesis procedure must be optimised in terms of radiochemical yield and quality of the final product. Therefore, both quantitative and qualitative radioanalytical methods are essential in developing radiosynthesis methods. Furthermore, biological properties of the tracer candidate need to be evaluated by various pre-clinical studies in animal models. In this work, the feasibility of various nucleophilic 18F-fluorination strategies were studied and a labelling method for a novel radiotracer, N-3-[18F]fluoropropyl-2beta-carbomethoxy-3beta-4-fluorophenyl)nortropane ([18F]beta-CFT-FP), was optimised. The effect of solvent was studied by labelling a series of model compounds, 4-(R1-methyl)benzyl R2-benzoates. 18F-Fluorination reactions were carried out both in polar aprotic and protic solvents (tertiary alcohols). Assessment of the 18F-fluorinated products was studied by mass spectrometry (MS) in addition to conventional radiochromatographic methods, using radiosynthesis of 4-[18F]fluoro-N-[2-[1-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-2-pyridinyl-benzamide (p-[18F]MPPF) as a model reaction. Labelling of [18F]beta-CFT-FP was studied using two 18F-fluoroalkylation reagents, [18F]fluoropropyl bromide and [18F]fluoropropyl tosylate, as well as by direct 18F-fluorination of sulfonate ester precursor. Subsequently, the suitability of [18F]beta-CFT-FP for imaging dopamine transporter (DAT) was evaluated by determining its biodistribution in rats. The results showed that protic solvents can be useful co-solvents in aliphatic 18F-fluorinations, especially in the labelling of sulfonate esters. Aromatic 18F-fluorination was not promoted in tert-alcohols. Sensitivity of the ion trap MS was sufficient for the qualitative analysis of the 18F-labelled products; p-[18F]MPPF was identified from the isolated product fraction with a mass-to-charge (m/z) ratio of 435 (i.e. protonated molecule [M+H]+). [18F]beta-CFT-FP was produced most efficiently via [18F]fluoropropyl tosylate, leading to sufficient radiochemical yield and specific radioactivity for PET studies. The ex vivo studies in rats showed fast kinetics as well as the specific uptake of [18F]beta-CFT-FP to the DAT rich brain regions. Thus, it was concluded that [18F]beta-CFT-FP has potential as a radiotracer for imaging DAT by PET.

Phonological Mean Length of Utterance as an indicator of typical and impaired phonological development in children acquiring Finnish

The aim of this study was to examine the applicability of the Phonological Mean Length of Utterance (pMLU) method to the data of children acquiring Finnish, for both typically developing children and children with a Specific Language Impairment (SLI). Study I examined typically developing children at the end of the one-word stage (N=17, mean age 1;8), and Study II analysed children s (N=5) productions in a follow-up study with four assessment points (ages 2;0, 2;6, 3;0, 3;6). Study III was carried out in the form of a review article that examined recent research on the phonological development of children acquiring Finnish and compared the results with general trends and cross-linguistic findings in phonological development. Study IV included children with SLI (N=4, mean age 4;10) and age-matched peers. The analyses in Studies I, II and IV were made using the quantitative pMLU method. In the pMLU method, pMLU values are counted for both the words that the children targeted (so-called target words) and the words produced by the children. When the child s average pMLU value was divided with the average target word pMLU value, it is possible to examine that child s accuracy in producing the words with the Whole-Word Proximity (PWP) value. In addition, the number of entirely correctly produced words is counted to obtain the Whole-Word Correctness (PWC) value. Qualitative analyses were carried out in order to examine how the children s phoneme inventories and deficiencies in phonotactics would explain the observed pMLU, PWP and PWC values. The results showed that the pMLU values for children acquiring Finnish were relatively high already at the end of the one-word stage (Study I). The values were found to reflect the characteristics of the ambient language. Typological features that lead to cross-linguistic differences in pMLU values were also observed in the review article (Study III), which noted that in the course of phonological acquisition there are a large number of language-specific phenomena and processes. Study II indicated that overall the children s phonological development during the follow-up period was reflected in the pMLU, PWP and PWC values, although the method showed limitations in detecting qualitative differences between the children. Correct vowels were not scored in the pMLU counts, which led to some misleadingly high pMLU and PWP results: vowel errors were only reflected in the PWC values. Typically developing children in Study II reached the highest possible pMLU results already around age 3;6. At the same time, the differences between the children with SLI and age-matched peers in the pMLU values were very prominent (Study IV). The values for the children with SLI were similar to the ones reported for two-year-old children. Qualitative analyses revealed that the phonologies of the children with SLI largely resembled the ones of younger, typically developing children. However, unusual errors were also witnessed (e.g., vowel errors, omissions of word-initial stops, consonants added to the initial position in words beginning with a vowel). This dissertation provides an application of a new tool for quantitative phonological assessment and analysis in children acquiring Finnish. The preliminary results suggest that, with some modifications, the pMLU method can be used to assess children s phonological development and that it has some advantages compared to the earlier, segment-oriented approaches. Qualitative analyses complemented the pMLU s observations on the children s phonologies. More research is needed in order to verify the levels of the pMLU, PWP and PWC values in children acquiring Finnish.

Development of tissue culture and virus-induced gene silencing for Calendula officinalis

Calendula officinalis is grown widely as an ornamental plant across Europe. It belongs to the large. Asteraceae family. In this study, the aim was to explore the possibilities to use Calendula officinalis as a new model organism for flower development and secondary mechanism studies in Asteraceae. Tissue culture of Calendula officinalis was established using nine different cultivars. Murashige & Skoog (MS) medium with four different combinations of plant growth regulators were tested. Of all these combinations, the medium containing 1mg/l BAP, 0.1 mg/l IAA, and 1mg/l Zeatin achieved highest frequency of adventitious shoot regeneration from hypocotyl and cotyledon explants. Virus-induced gene silencing is a recent developed genetic tool for charactering the gene functions in plants, and extends the range of host plants that are not accessible for Agrobacterium transformation. Here, tobacco rattle virus (TRV)-based VIGS technique was tested in calendula (cv. Single Orange). We used TRV carrying Gerbera hybrid phytoene desaturase (PDS) gene fragment to induce PDS silencing in calendula. Vacuum infiltration and syringe infiltration methods both resulted in photo-bleaching phenotypes in leaves, bracts and petals. Loss-of-function phenotypes occurred on calendula 13 days post-infiltration. In conclusion, the data indicates that calendula explants can be regenerated through tissue culture which is a prerequisite for development of stable transformation methods. However, further optimization is still needed to improve the frequency. In addition, VIGS was applied to silence PDS marker gene expression indicating that this method has potential for gene functional studies in future.

Lean Thinking in Software Development : Impacts of Kanban on Projects

The history of software development in a somewhat systematical way has been performed for half a century. Despite this time period, serious failures in software development projects still occur. The pertinent mission of software project management is to continuously achieve more and more successful projects. The application of agile software methods and more recently the integration of Lean practices contribute to this trend of continuous improvement in the software industry. One such area warranting proper empirical evidence is the operational efficiency of projects. In the field of software development, Kanban as a process management method has gained momentum recently, mostly due to its linkages to Lean thinking. However, only a few empirical studies investigate the impacts of Kanban on projects in that particular area. The aim of this doctoral thesis is to improve the understanding of how Kanban impacts on software projects. The research is carried out in the area of Lean thinking, which contains a variety of concepts including Kanban. This article-type thesis conducts a set of case studies expanded with the research strategy of quasi-controlled experiment. The data-gathering techniques of interviews, questionnaires, and different types of observations are used to study the case projects, and thereby to understand the impacts of Kanban on software development projects. The research papers of the thesis are refereed, international journal and conference publications. The results highlight new findings regarding the application of Kanban in the software context. The key findings of the thesis suggest that Kanban is applicable to software development. Despite its several benefits reported in this thesis, the empirical evidence implies that Kanban is not all-encompassing but requires additional practices to keep development projects performing appropriately. Implications for research are given, as well. In addition to these findings, the thesis contributes in the area of plan-driven software development by suggesting implications both for research and practitioners. As a conclusion, Kanban can benefit software development projects but additional practices would increase its potential for the projects.

Income inequality in the process of economic development : An empirical approach

This thesis studies the effect of income inequality on economic growth. This is done by analyzing panel data from several countries with both short and long time dimensions of the data. Two of the chapters study the direct effect of inequality on growth, and one chapter also looks at the possible indirect effect of inequality on growth by assessing the effect of inequality on savings. In Chapter two, the effect of inequality on growth is studied by using a panel of 70 countries and a new EHII2008 inequality measure. Chapter contributes on two problems that panel econometric studies on the economic effect of inequality have recently encountered: the comparability problem associated with the commonly used Deininger and Squire s Gini index, and the problem relating to the estimation of group-related elasticities in panel data. In this study, a simple way to 'bypass' vagueness related to the use of parametric methods to estimate group-related parameters is presented. The idea is to estimate the group-related elasticities implicitly using a set of group-related instrumental variables. The estimation results with new data and method indicate that the relationship between income inequality and growth is likely to be non-linear. Chapter three incorporates the EHII2.1 inequality measure and a panel with annual time series observations from 38 countries to test the existence of long-run equilibrium relation(s) between inequality and the level of GDP. Panel unit root tests indicate that both the logarithmic EHII2.1 inequality measure and the logarithmic GDP per capita series are I(1) nonstationary processes. They are also found to be cointegrated of order one, which implies that there is a long-run equilibrium relation between them. The long-run growth elasticity of inequality is found to be negative in the middle-income and rich economies, but the results for poor economies are inconclusive. In the fourth Chapter, macroeconomic data on nine developed economies spanning across four decades starting from the year 1960 is used to study the effect of the changes in the top income share to national and private savings. The income share of the top 1 % of population is used as proxy for the distribution of income. The effect of inequality on private savings is found to be positive in the Nordic and Central-European countries, but for the Anglo-Saxon countries the direction of the effect (positive vs. negative) remains somewhat ambiguous. Inequality is found to have an effect national savings only in the Nordic countries, where it is positive.

'South-South' Remittance - Poverty reduction nexus. The Case of 'Hadiya' immigrants in South Africa, as Agents of 'Constituence development' in Ethiopia, and the policy implications of their 'remittance instrument'

This thesis explores the link between South-South remittance and development. It attempts to establish improved understanding about the role of immigrants as agents of constituency growth and development. By doing so, it illuminates the dark corners of the policy implications that the unconventional development agency of immigrants might have for countries in the Organization ft Economic Cooperation and Development (OECD). The thesis problematises the existence of state-centric international cooperation as providing the recipe for failed Aid in the face of global poverty menace. In the last half a century, the relative shi' of focus to non-state actors brought about the proliferation of NGOs. That, intrun, helped improve international access to crisis situations; however, their long-term remedial impacts on poverty and development have been contested. Major misgivings for non-governmental organizations (NGOs) are, on one hand, low level goal-bound expenditures and lack of independence from influence of the state, on the other. Therefore, the thesis enterprises to empirically verify its fundamental question whether remitting immigrants constitute an alternative development agency to the traditional players: the State and NGOs. Its main arguments are: due to state's failures in bringing sustainable development in many countries of the South, the future of poverty reduction and development also rests in immigrants' remittances. Nonetheless, in the last decade, remittance security-nexus dominated its discourse. Because of that remittance was viewed as something requiring global regime and restrictions. These temptations to tightly regulate remittance flows carry the danger of overlooking its trans-boundary nature and its strong link with livelihood of the poor. Therefore, to avoid unintended consequences of interventions, there need to be clear policy that bases itself on a discursive knowledge on the issues of North-South and South-South remittances The study involved both literature based and empirical research. It employed Discourse Analysis (C as main method for the former and snow-balling as its approach for the latter. For the first part the thesis constructed three conceptual models, these are: metrological model, police model and ecological model on remittance development-nexus. Through this modeling, the thesis achieved better deconstruction on the concepts remittance, immigrants and development agency. The protagonists of each model, the values and interests they represent, and their main arguments along various lines of dichotomies have been discussed. For instance, the main treats of meteorological model include: it sees remittance as transitional economic variable which require constant speculations and global management; it acts as meteorological station for following up or predicting the level, direction, flow and movement of global remittance. It focuses on official lines and considers the state as legitimate recipient of advic and positive consequence of remittance. On the other hand, police model views remittance as beir at best, development neutral or as an illicit activity requiring global regulations and tight control. Both immigrants and remittance viewed as subversive to establishments. It gives primacy to state stable agent of development and a partner for international cooperation. The anti-thesis to the police model is supplied by ecological model, which this thesis is a part. Ecological model on remittance and immigrants argues that, tight global regulations alone cannot be a panacea for possible abuse of informal remittance system. Ecological model, not only links remittance to poverty reduction, the main trust of development, but also considers the development agency of immigrants as critical factor for 21st century north-south development intervention. It sees immigrants as development conscious and their remittance instrument as most stable flow of finance to the developing countries. Besides, it sees remittance as effective poverty solutions than Foreign Direct Investment and international AID. This thesis focuses on the significance of South-South remittance and investigates the South Africa - Ethiopia remittance corridor, as case study; and empirically verifies the role of Ethiopian (Kembata and Hadiya) immigrants in South Africa as agents of local development back home. The study involved techniques of interview, group discussions, observations and investigative study. It also looked into the determinants of their migration to South Africa, and their remittance to Ethiopia. The theoretical models in the first part of the thesis have been operationalised throughout the empirical part to verify if the Kembata and Hadiya immigrants played the crucial role in their household poverty and local development in comparison with the Ethiopian state and the NGOs involved in the system. As evidenced by the research the thesis has made three distinct contributions to the discourse of remittance development-nexus. Fist, it systematized the debate about linkages between remittance, immigrants, development agency and policy of international cooperation by creating three conceptual models (school of thoughts); second, it singled out remitting immigrants as new agents of development in the South; third, it deconstructed concept of remittance and established South¬South remittance as additional sphere of academic investigation. In addition to the above contributions, the thesis finds that Kembata and Hadiya immigrants have engaged in various developmental activities in their locality than usually anticipated. Hence, it concludes that Ethiopian immigrants constitute an alternative development agency to the state and other non-state actors in their country, and the lesson can be applied to poverty reduction strategies in most developing countries.