Studies on cyclic AMP phosphodiesterases

The second messenger cyclic AMP plays a pivotal role in the control of cell function through the transduction of extracellular signals into intracellular activation. However, this cyclic nucleotide negatively modulates the synthesis and release of inflammatory mediators and the proliferation of lymphocytes. The sole mechanism for degradation of cAMP is achieved through the action of cyclic AMP phosphodiesterses (PDEs). This activity is provided by a multigene family which produces a variety of enzymes. The PDE4 isoforms appear to be the predominant cAMP-metabolising enzymes in essentially all pro-inflammatory and immune cells implicated in the pathogenesis of asthma, arthritis and atopic dermatitis. With respect to the development of novel drugs to treat such diseases, an understanding of the regulation and the function of PDE4 enzymes has been identified as crucial. In this thesis, three different, but related, studies were carried out. The first study was an investigation of the expression and the regulation of cAMP phosphodiesterases in human Jurkat T lymphocytes. In the second study, the properties of the proteins encoded by five different mRNA transcripts from the human PDE4D gene were analysed after transient expression in COS7 cells. These included the PDE4D1 and PDE4D2 forms which I had shown to become induced in Jurkat T-cells with chronically elevated cAMP levels. Finally, the role of the N- and C-terminal regions of the inactive human PDE4A splice variant, 2EL originally cloned from a Jurkat T-cell library, was investigated. The Jurkat T-cell PDE activity was determined using selective PDE activators and inhibitors. Although a small amount of PDEl, PDE2 and PDE7 enzymes were present, PDE3 and PDE4 enzymes were shown to provide the major Jurkat T-cell PDE activity. Chronic elevation of inti'acellular cAMP levels achieved either by direct stimulation of adenylate cyclase or via stimulation of Gs or using a cell-permanent cAMP analogue increased PDE3 and PDE4 activities but did not affect PDE1, PDE2 and PDE7 activities. Despite the fact that the major PDE activity in quiescent Jurkat T-cells was contributed by PDE3 enzymes, PDE4 activity appeared to play a key role in the control of intracellular cAMP levels. Analyses done using RT-PCR and immunoblotting demonstrated that forskolin-elevated intracellular cAMP levels induced the 'short' form of PDE4D isoforms, PDE4D1 and PDE4D2, but did not induce any other PDE4 isoforms. However, chronic elevation of cAMP levels also led to the down-regulation of the novel 118 kDa PDE4A species found in resting Jurkat T-cells. The two human 'short' forms (PDE4D1 and PDE4D2) and the three 'long' forms (PDE4D3, PDE4D4 and PDE4D5) of human PDE4D isoforms were transiently expressed in COS7 cells. The 'short' PDE4D forms exhibited similar properties and were exclusively expressed in cytosol. In contrast, the 'long' forms showed different enzyme kinetics and were found in both particulate and cytosol fractions. It was suggested that the unique N-terminal regions of the PDE4D3, PDE4D4 and PDE4D5 proteins, which are derived from alternatively spliced regions of their mRNAs, are important in determining their enzyme activity, inhibitor sensitivity and subcellular localisation. The inactive human PDE4A species 2EL was cloned from resting Jurkat T-cells. However, it was not known that the inactivity is due to the premature truncation of either N- or C-terminal putative catalytic domain. This was investigated using chimeras and truncated PDE forms. It was shown that the C-terminal premature truncation terminates the 2EL's catalytic activity

Problems encountered in conventional HIV 1/2 Algorithms: lack of necessity for immunoblot assays to confirm repeated ELISA reactive results

Background: The use of conventional (serologically based) HIV 1/2 diagnostic algorithms has become controversial in recent years.Objectives: Sera from patients who underwent verification tests were evaluated because repeated ELISA-reactive results demonstrated a HIV1+HIV2 positive band pattern.Methods: The line immunoassay (LIA) test was used for repeated HIV enzyme immunoassays (EIA)-reactive sera in patients at three centers. The Bio-Rad Geenius™ HIV 1/2 and the HIV-1 RNA tests were used. HIV-1 and RNA HIV-2 were investigated using PCR.Results: LIA was used to evaluate 3,224 out of 10,591 samples with repeated ELISA reactivity (30%). We found that 32 (1%) of the sera, along with HIV1 bands and HIV2 gp36 bands, were positive. Only 28 of the 32 verified serum samples with gp36 bands were repeated, and no gp36 band positivity was detected using the Bio-Rad Geenius™ HIV-1/2 confirmatory assay in these serum samples. The HIV-2 proviral DNAs were also negative. Therefore, we excluded the possibility of HIV1+2 co-infection. All samples from the 32 patients were positive for HIV-1 RNA.Conclusion: Our findings highlight the need to exclude confirmatory tests like the LIA test from the current diagnostic HIV algorithm and replace it with rapid HIV-1 and HIV-2 confirmatory immunochromotographic tests.Keywords: HIV, AIDS, HIV-2

Problems encountered in conventional HIV 1/2 Algorithms: lack of necessity for immunoblot assays to confirm repeated ELISA reactive results

Background: The use of conventional (serologically based) HIV 1/2 diagnostic algorithms has become controversial in recent years. Objectives: Sera from patients who underwent verification tests were evaluated because repeated ELISA-reactive results demonstrated a HIV1+HIV2 positive band pattern. Methods: The line immunoassay (LIA) test was used for repeated HIV enzyme immunoassays (EIA)-reactive sera in patients at three centers. The Bio-Rad Geenius\u2122 HIV 1/2 and the HIV-1 RNA tests were used. HIV-1 and RNA HIV-2 were investigated using PCR. Results: LIA was used to evaluate 3,224 out of 10,591 samples with repeated ELISA reactivity (30%). We found that 32 (1%) of the sera, along with HIV1 bands and HIV2 gp36 bands, were positive. Only 28 of the 32 verified serum samples with gp36 bands were repeated, and no gp36 band positivity was detected using the Bio-Rad Geenius\u2122 HIV-1/2 confirmatory assay in these serum samples. The HIV-2 proviral DNAs were also negative. Therefore, we excluded the possibility of HIV1+2 co-infection. All samples from the 32 patients were positive for HIV-1 RNA. Conclusion: Our findings highlight the need to exclude confirmatory tests like the LIA test from the current diagnostic HIV algorithm and replace it with rapid HIV-1 and HIV-2 confirmatory immunochromotographic tests

Penilaian Kinerja Keuangan Koperasi di Kabupaten Pelalawan

This paper describe development and financial performance of cooperative in District Pelalawan among 2007 - 2008. Studies on primary and secondary cooperative in 12 sub-districts. Method in this stady use performance measuring of productivity, efficiency, growth, liquidity, and solvability of cooperative. Productivity of cooperative in Pelalawan was highly but efficiency still low. Profit and income were highly, even liquidity of cooperative very high, and solvability was good

Juxtaposing BTE and ATE – on the role of the European insurance industry in funding civil litigation

One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI

Search for stop and higgsino production using diphoton Higgs boson decays

Results are presented of a search for a "natural" supersymmetry scenario with gauge mediated symmetry breaking. It is assumed that only the supersymmetric partners of the top-quark (stop) and the Higgs boson (higgsino) are accessible. Events are examined in which there are two photons forming a Higgs boson candidate, and at least two b-quark jets. In 19.7 inverse femtobarns of proton-proton collision data at sqrt(s) = 8 TeV, recorded in the CMS experiment, no evidence of a signal is found and lower limits at the 95% confidence level are set, excluding the stop mass below 360 to 410 GeV, depending on the higgsino mass

Severe early onset preeclampsia: short and long term clinical, psychosocial and biochemical aspects

Preeclampsia is a pregnancy specific disorder commonly defined as de novo hypertension and proteinuria after 20 weeks gestational age. It occurs in approximately 3-5% of pregnancies and it is still a major cause of both foetal and maternal morbidity and mortality worldwide1. As extensive research has not yet elucidated the aetiology of preeclampsia, there are no rational preventive or therapeutic interventions available. The only rational treatment is delivery, which benefits the mother but is not in the interest of the foetus, if remote from term. Early onset preeclampsia (<32 weeks’ gestational age) occurs in less than 1% of pregnancies. It is, however often associated with maternal morbidity as the risk of progression to severe maternal disease is inversely related with gestational age at onset2. Resulting prematurity is therefore the main cause of neonatal mortality and morbidity in patients with severe preeclampsia3. Although the discussion is ongoing, perinatal survival is suggested to be increased in patients with preterm preeclampsia by expectant, non-interventional management. This temporising treatment option to lengthen pregnancy includes the use of antihypertensive medication to control hypertension, magnesium sulphate to prevent eclampsia and corticosteroids to enhance foetal lung maturity4. With optimal maternal haemodynamic status and reassuring foetal condition this results on average in an extension of 2 weeks. Prolongation of these pregnancies is a great challenge for clinicians to balance between potential maternal risks on one the eve hand and possible foetal benefits on the other. Clinical controversies regarding prolongation of preterm preeclamptic pregnancies still exist – also taking into account that preeclampsia is the leading cause of maternal mortality in the Netherlands5 - a debate which is even more pronounced in very preterm pregnancies with questionable foetal viability6-9. Do maternal risks of prolongation of these very early pregnancies outweigh the chances of neonatal survival? Counselling of women with very early onset preeclampsia not only comprises of knowledge of the outcome of those particular pregnancies, but also knowledge of outcomes of future pregnancies of these women is of major clinical importance. This thesis opens with a review of the literature on identifiable risk factors of preeclampsia

Search for anomalous production of events with three or more leptons in pp collisions at √s = 8TeV

Published by the American Physical Society under the terms of the Creative Commons Attribution 3.0 License. Further distribution of this work must maintain attribution to the author(s) and the published articles title, journal citation, and DOI.A search for physics beyond the standard model in events with at least three leptons is presented. The data sample, corresponding to an integrated luminosity of 19.5fb-1 of proton-proton collisions with center-of-mass energy s=8TeV, was collected by the CMS experiment at the LHC during 2012. The data are divided into exclusive categories based on the number of leptons and their flavor, the presence or absence of an opposite-sign, same-flavor lepton pair (OSSF), the invariant mass of the OSSF pair, the presence or absence of a tagged bottom-quark jet, the number of identified hadronically decaying τ leptons, and the magnitude of the missing transverse energy and of the scalar sum of jet transverse momenta. The numbers of observed events are found to be consistent with the expected numbers from standard model processes, and limits are placed on new-physics scenarios that yield multilepton final states. In particular, scenarios that predict Higgs boson production in the context of supersymmetric decay chains are examined. We also place a 95% confidence level upper limit of 1.3% on the branching fraction for the decay of a top quark to a charm quark and a Higgs boson (t→cH), which translates to a bound on the left- and right-handed top-charm flavor-violating Higgs Yukawa couplings, λtcH and λctH, respectively, of |λtcH|2+|λctH|2<0.21

Measurement of associated W plus charm production in pp collisions at √s=7 TeV

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