Human African trypanosomiasis (sleeping sickness) is a devastating disease which is endemic in parts of sub-Saharan Africa. It is caused by the protozoan parasite T. brucei, which are transmitted by the bite of infected tsetse flies. Although the disease is fatal if left untreated, there is a lack of safe, effective and affordable drugs available; therefore new drugs are urgently needed. The aim of the work presented in this thesis is to develop novel trypanocidal compounds. It is divided into two parts to reflect the two distinct strategies employed to achieve this aim. The first part focuses on the inhibition of glycophosphoinositol (GPI) anchor synthesis by inhibiting the Zn2+-dependent enzyme, GlcNAc-PI de-N-acetylase. Trypanosomes have a variable surface glycoprotein (VSG) coat, which allows them to evade the human immune system. The GPI anchor attaches the VSG to the cell membrane; therefore inhibiting GPI synthesis should expose the parasite to the immune system. Initially, large substrate analogues were synthesized. These showed weak inhibition of the enzyme. Zinc-binding fragments were screened, and small molecule inhibitors based on salicylhydroxamic acid were then synthesized. These compounds showed modest inhibition, but the excellent ligand efficiency of salicylhydroxamic acid indicates this may be a promising starting point for further inhibitors. The second part details the P2 strategy. The P2 transporter is a nucleoside transporter unique to T. brucei, which concentrates adenosine. The transporter also binds and selectively concentrates compounds that contain benzamidine and diaminotriazine P2 motifs, which can enhance the potency and selectivity of these compounds. The sleeping sickness drugs melarsoprol and pentamidine contain P2 motifs. Compounds comprising a P2 targeting motif, a linker and a trypanocidal moiety were synthesized. Initially, a diaminotriazine P2 motif was attached to a trypanocidal tetrahydroquinoline (THQ) protein farnesyl transferase (PFT) inhibitor, with limited success. The P2 strategy was also applied to a non-selective, trypanocidal, quinol moiety. The quinol moiety was attached to diaminotriazine and benzamidine P2 motifs, and an increase in selectivity for T. brucei over MRC5 cells was observed.

This dissertation consists of three empirical analyses examining the interactive and evolving nature of government regulations and how the regulated industries respond to the changes in the regulatory climate. Using the U.S. pulp and paper mills as an example, the three essays bring together a number of strands of literature in environmental economics and policy studies discussing how changes in the U.S. environmental policy are shaped by industry concerns and which strategies firms choose in order to adjust to the changes in policy. Essay 1 examines if, in addition to the standard input factors, indirect costs associated with tax and environmental policies affect papermakers’ ‘stay put’ investment decisions. The findings suggest that state environmental stringency has a negative impact on investments, but it is statistically insignificant and higher taxes do not deter investments. The Essay 2 studies whether voluntary abatement and prevention efforts at pulp and paper mills affects regulatory stringency they face. The analysis tests the hypotheses of ‘responsive regulation’ and whether regulators are driven by numerical pollution targets or budgetary constraints. The findings suggest that voluntary pollution abatement and prevention have greater impact on regulatory stringency than government budgets. Finally, Essay 3 analyzes the relationship between pollution prevention (P2) policy instruments and adoption of P2 modifications. The study tests the hypotheses of whether P2 policy instruments have positive impact on P2 adoptions. The results suggest that the policy instruments have different effects on different types of P2 modifications and that regulatory and political threat is a strong predictor of P2 adoptions.

No description available.

The control of phosphatidylinositol 3, 4, 5-trisphosphate [PtdIns(3,4,5)P3] level depends on the activities of both PI kinase and PtdIns(3,4,5)P3 phosphatases: 5-phosphatase like SHIP1 and SHIP2, and 3-phosphatase like PTEN. The ubiquitous SH2 domain containing inositol 5-phosphatase SHIP2 contains both a series of protein interacting domains and the ability to dephosphorylate PtdIns(3,4,5)P3. Previous reports obtained in SHIP2 deficient mice have shown that SHIP2 is involved in the control of insulin sensitivity and reducing weight gain on fatty diet. <p><p>Since SHIP2 is a lipid phosphatase as well as a docking protein, the initial aim that emerged in the lab was to measure the inositol lipid levels in SHIP2 +/+ and deficient cells and compare the levels of 3-phosphoinositides PtdIns(3,4,5)P3 and PtdIns(3,4)P2. At first, we developed mouse embryonic fibroblasts (MEF) as a cellular model. Amongst various stimuli tested, surprisingly, only serum showed an obvious difference in terms of PtdIns(3,4,5)P3 level. This lipid was significantly up regulated in SHIP2 -/- cells but only after short-term (i.e. 5-10 min) incubation with serum. The difference in PtdIns(3,4,5)P3 levels in heterozygous fibroblast cells was intermediate between the +/+ and -/- cells. Serum stimulated PI3K activity appeared to be comparable between +/+ and -/- cells. Moreover, PKB, but not MAP kinase activity, was also potentiated in SHIP2 deficient cells stimulated by serum. The up regulation of PKB activity in serum stimulated cells was totally reversed in the presence of the PI3K inhibitor LY-294002, in both +/+ and -/- cells.<p><p>Reactive oxygen species (ROS) have emerged as physiological mediators of many cellular responses. H2O2 mimics some effects of insulin in a number of cell culture systems. It also inactivates tyrosine phosphatase activities including PTEN. In addition, in Swiss 3T3 fibroblasts, Gray et al reported that exposure of the cells to H2O2 resulted a huge increase in PtdIns(3,4)P2 level. The authors suspected that the effect was attributed to a inositol 5-phosphatase activity. We thus exposed our cells to H2O2 in order to address the question of the role of SHIP2 in response to oxidative stress.<p><p>We worked on the same SHIP2 MEF model, stimulated by H2O2: at 15 min, PtdIns(3,4,5)P3 was markedly increased in SHIP2 -/- cells as compared to +/+ cells. In contrast, no significant increase in PtdIns(3,4)P2 could be detected at 15 or 120 min incubation of the cells with H2O2 (0.6 mM). PKB activity was upregulated in SHIP2 -/- cells in response to H2O2 and therefore follows the regulation of PtdIns(3,4,5)P3. As for serum, the PI3K activity appeared to be comparable between +/+ and -/- cells. The levels of PTEN and type I 4-phosphatase [an enzyme that acts on PtdIns(3,4)P2] remained unchanged between the two types of cells. SHIP2 add back experiments in SHIP2 -/- cells confirm its critical role in the control of PtdIns(3,4,5)P3 level in response to H2O2: the decrease in PtdIns(3,4,5)P3, observed in SHIP2 expressing cells, was no longer seen in cells infected with a catalytic mutant of this enzyme. <p> / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

<p>Peptides, specifically cell-penetrating peptides (CPP), have become wonderful research tools due to their enhanced stability, solubility, and ease of synthesis. They have been used for a wide range of biomedical applications, from insecticides to biosensors and drug-delivery scaffolds. The work presented in this dissertation characterizes the biophysical properties of two different CPPs. The first is the cationic amphiphilic polyproline helix (CAPH) peptide, P14LRR. In addition to cell penetration, this CPP has demonstrated broad spectrum antibacterial properties. Fluorescence polarization (FP) and SEC-MALS were conducted to understand the dissociation constant (KD) and oligomerization effects of P14LRR with respect to its putative molecular target in Staphylococcus aureus (S. aureus). A biotinylated derivative of this peptide was also used as a drug-delivery scaffold to transport fluorescently conjugated streptavidin into mammalian cells. A second CPP, DAN13, was also developed as a biosensor for phosphoinositide lipids, specifically PI(4,5)P2. This was effected through careful calibration using stacked supported lipid bilayers (SSLB) in combination with total internal reflection fluorescence (TIRF) microscopy. This was then used to determine the absolute densities and spatial distribution of PIP2 in live KRas mutant cells.</p>

Class I PI3Ks are a family (α, β, δ and γ) of ubiquitous lipid kinases that can be activated by cell surface receptors to 3-phosphorylate PI(4,5)P2 (phosphatidylinositol(4,5)-bisphosphate) and generate the signalling lipid PI(3,4,5)P3. The PI(3,4,5)P3 signal then activates a diverse collection of effector proteins involved in regulation of cell migration, metabolism and growth. The importance of this network is evidenced by the relatively high frequency with which cancers acquire gain-of-function mutations in this pathway and huge efforts to make PI3K inhibitors to treat cancer. The canonical model describing these events suggests class I PI3Ks are activated at the plasma membrane and generate PI(3,4,5)P3 in the inner leaflet of the plasma membrane where its effectors are activated. The PI(3,4,5)P3 signal can be terminated directly, by the tumour-suppressor and PI(3,4,5)P3-3-phosphatase PTEN, or modified to a distinct PI(3,4)P2 signal, by SHIP-family 5-phosphatases. The PI(3,4)P2 is removed by INPP4-family 4-phosphatases. Published work has shown that PI(3,4,5)P3 signalling can also occur in endosomes and nuclei, however, there is very little data defining the intracellular distribution of endogenous class I PI3Ks that supports these ideas; this is as a result of technical problems such as; their very low abundance, poor antibody-based tools and artefacts generated by overexpression of PI3Ks. Past work has indicated that, in PTEN-null mouse models of prostate tumour progression, either PI3Kβ or PI3Ks α and β, have important roles. Furthermore, the cell types and mechanism involved remained unclear. Recent published work in the host laboratory had indicated that there is an unexpectedly large accumulation of PI(3,4)P2 in PTEN-null cells that might be an important part of its status as a major tumour suppressor. The explanation and prevalence of this observation was unclear but potentially a result of PTEN also acting as a PI(3,4)P2 3-phosphatase in vivo. MEFs were derived from genetically-modified mice expressing endogenous, AviTagged class I PI3K subunits and used in experiments to define the subcellular localisation of class I PI3Ks. We found that following stimulation with PDGF, class IA PI3K subunits were unexpectedly depleted from the adherent basal membrane, in contrast, p85α and p110α, but not p85β and p110β, accumulated transiently in the nucleus. Interestingly, p110β, but none of the other subunits, was constitutively localised in the nucleus. These results support the idea that class I PI3K and PI(3,4,5)P3 signalling occurs in the nucleus. In organoids derived from WT, PI3Kγ-null or PTEN-null mouse prostate, application of PI3K-selective inhibitors revealed that PI3Kα had a dominant role in generating PI(3,4,5)P3 in prostate epithelial cells. The levels of PI(3,4)P2 were also elevated substantially in PTEN-null, compared to WT prostate organoids, use of PI3K-selective inhibitors suggested that it was also generated by PI3Kα. These data were consistent with the idea that PTEN can act as a PI(3,4)P2 3-phosphatase. Surprisingly, raising the pH of the organoids medium dramatically increased accumulation of PI(3,4,5)P3 and PI(3,4)P2, although the cause of this effect was unclear, we hypothesised the pH of the local environment may influence signalling via class I PI3Ks.

Approved for public release, distribution is unlimited / This thesis presents Concept of Operations (CONOPS) for two specific automated language translation (ALT) devices, the P2 Phraselator and the Voice Response Translator (VRT). The CONOPS for each device are written as Appendix A and Appendix B respectively. The body of the thesis presents a broad introduction to the present state of ALT technology for the reader who is new to the general subject. It pursues this goal by introducing the human language translation problem followed by nine characteristic descriptors of ALT technology devices to provide a basic comparison framework of existing technologies. The premise is that ALT technology is presently in a state where it is tackled incrementally with various approaches. Two tables are provided that illustrate six commercially available devices using the descriptors. A scenario is then described in which the author observed the two subject ALT devices (depicted in the CONOPS in the Appendices) being employed within an international military exercise. Some unique human observations associated with the use of these devices in the exercise are discussed. A summary is provided of the Department of Defense (DOD) process that is exploring ALT technology devices, specifically the Language and Speech Exploitation Resources (LASER) Advanced Concept Technology Demonstration ACTD. / Lieutenant Commander, United States Navy

This thesis is a study of the problem of subjectivity and prostitution in the Middle Ages. Three legal case studies of unpublished archival material and one chapter focussing on fictional texts from late mediaeval Germany and Switzerland are used to investigate the conditions of prostitutes' subjectification in law and literature. The thesis takes impetus from Ruth Karras's recent articulation of the problem of prostitution and sexuality, seeking to engage critically with her notion of “prostitute” as a medieval sexual identity that might be applied to any woman who had extra-marital sex. In dealing with trial records, it also aims to make a methodological contribution to the study of crime and the problem of locating the individual. Chapters I-III examine the records of criminal cases featuring the testimony of prostitutes, or women who risked such categorisation, to consider the available subject positions both within and outwith the context of municipal regulation. Whilst acknowledging the force of normative ideas about prostitutes as lustful women, these chapters argue that prostitutes' subject positions in legal cases were adopted according to local conditions, and depended upon the immediate circumstances of the women involved. They also consider trial records as a form of masculine discourse, arguing that an anxious masculine subject can be seen to emerge in response to the phenomenon of prostitution. Chapter IV expands this discussion by drawing on literary texts showing how prostitutes prompted concern on the part of male poets and audiences, for whom their sexual agency was a threat which belied their theoretical status as sexual objects. Note: Transcriptions of the legal cases making up chapters I-III are provided in Appendices A, B, and C.

Ce travail est une contribution à l'étude numérique de la propagation des ondes sismiques dans les formations géologiques superficielles. Dans la première partie, un programme de calcul combinant les méthodes d'Aki-Larner et de Thomson-Haskell a été développé. Il permet de calculer la réponse de configurations géologiques géométriquement complexes à des ondes SH planes. Une étude systématique de quelques configurations géologiques-type montre que les effets dûs aux hétérogénéités verticales (contraste d'impédance: amplification) et latérales (irrégularités géométriques: diffraction) sont fortement couplés : il est le plus souvent impossible de décomposer le calcul d'une structure complexe en 2 sous-problèmes, plus simples. En génie parasismique, il est donc nécessaire de traiter chaque site cas par cas pour les études détaillées. Dans la deuxième partie, on étudie l'influence de la saturation en eau dans les formations poreuses très perméables. La représentation du milieu saturé est celle de Biot, avec quelques compléments issus de la théorie de l'homogénéisation. Ce modèle est appliqué au calcul de la réponse sismique de structures simples tricouches (sol poreux sec sur sol poreux saturé sur bedrock élastique imperméable) en 1 D ou 2D, dans la gamme 0 - 25 HZ. On montre des cas théoriques pour lesquels l'onde P2 (générée aux interfaces du milieu poreux, puis très atténuée et non observable en surface) intervient dans le mécanisme d'atténuation des ondes de compression. On insiste sur l'importance de l'interaction fluide-solide aux frontières du milieu poreux: dès que la perméabilité est supérieure à 10-10 m2 environ, il s'avère nécessaire de prendre en compte explicitement la présence de fluide. En particulier, il est alors impossible (sous peine d'erreurs supérieures à 100 %) d'approximer le matériau poreux par un modèle monophasique viscoélastique équivalent.

This doctoral thesis addresses in a sequence of five essays the question how fiscal policy and economic output are interrelated in emerging Europe and how this relationship is shaped by the respective politico-economic environment and the individual-level support for economic reforms. Following main findings can be highlighted: (1) Countries in Central, Eastern and Southeastern Europe (CESEE) respond to a fiscal expansion in the euro area with fiscal easing at home, while the GDP response is mixed across countries.(2)Automatic fiscal stabilizers are comparatively small and discretionary fiscal policy has been largely pro-cyclical in CESEE. (3) The public spending and revenue structure is more "growth-friendly" in CESEEthan in the EU-15. (4) In transition economies with more democratic institutions and a better quality of governance, individuals with high market-relevant skills show a significantly larger support of the privatization status quo than individuals with low market skills. (5) The society in Russia - triggered by a lack of social capital - chooses to demand more state regulationand tolerate corruption to reduce negative externalities imposed by private business.(author's abstract)