Les peptides vecteurs riches en Arginine (Arg) ont la faculté de transporter des molécules à travers les membranes cellulaires, d'une manière récepteur- et énergie indépendante, sans toxicité envers la cellule et présentent ainsi un fort potentiel pour la libération de molécules thérapeutiques ou diagnostiques. La compréhension du mécanisme d'internalisation cellulaire et de l'interaction membranaire de ces peptides vecteurs est donc primordiale pour leur développement pharmaceutique. Dans cette étude, deux peptide svecteurs riches en Arg et dérivés de la pénétratine ont été étudiés : les peptides RW16(RRWRRWWRRWWRRWRR) et RW9 (RRWWRRWRR). Dans un premier temps,l'analyse biophysique complète de l'interaction peptide/lipide (P/L) a été réalisée pour le peptide RW16 et une interaction favorisée en présence de lipides anioniques a été révélée.Dans un second temps, des peptides dérivés de RW9 ont été synthétisés dans lesquels chaque tryptophane a été systématiquement remplacé par une phenylalanine. L'internalisation cellulaire et les interactions P/L de RW9 ont été étudiées, et l'importance de la position et du nombre de tryptophane dans la séquence peptidique a été mise en évidence. / Cell-penetrating peptides (CPPs) are able to efficiently transport cargos acrosscell membranes in a receptor- and energy-independent manner, without being cytotoxic to cells and thus present a great potential in drug delivery and diagnosis. The understanding of the cellular internalization and membrane interaction mechanisms is thus fundamental for their pharmaceutical development. In this study, two Arginine-rich CPPs derived from penetratin have been investigated: the peptides RW16 (RRWRRWWRRWWRRWRR) andRW9 (RRWWRRWRR). Firstly, a complete biophysical study of the peptide/lipid (P/L)interactions of RW16 has been accomplished and a preferential interaction for anionic lipids was demonstrated. Secondly, peptides derived from RW9 have been synthesized where tryptophan residues have been systematically replaced by phenylalanine. Cellular internalization and P/L interactions have been characterized, and the importance of the number and position of tryptophan has been highlighted.

L'expérience AMS sera mise en orbite à partir de 2006 pour une durée de 3 ans afin d'étudier le rayonnement cosmique et d'ouvrir de nouvelles perspectives pour la recherche d'antimatière et de matière noire. Cette thèse présente d'abord le travail mené pour les développements du détecteur Tcherenkov (RICH) d'AMS qui conduira à une mesure précise de la vitesse et de la charge des particules le traversant. Nous exposons le choix des photodétecteurs, les tests de l'électronique, les caractéristiques générales du compteur ainsi que la mise en oeuvre de l'analyse des données obtenues avec deux prototypes (incluant des mesures au CERN). La seconde partie du travail est consacrée à l'étude théorique du rayonnement cosmique et d'un signal exotique potentiel pour AMS : les trous noirs en évaporation. Les conséquences astrophysiques, cosmologiques et gravitationnelles de l'existence de ces objets sont considérées en détails.

<p>The aquatic plant <i>Ipomoea aquatica</i> is a popular vegetable in Southeast Asia, often cultivated in nutrient rich and polluted waters. The overall aim of this thesis was to estimate potential risks for human health and reduced plant growth due to accumulation and toxicity of total-Hg, methyl-Hg, Cd and Pb.</p><p>In plants from cultivations in Thailand, the concentrations of Cd and Pb in the shoots were well beneath recommended maximum values for human consumption, but at some sites the Hg concentrations were high. It was demonstrated that <i>I. aquatica</i> has the capacity to accumulate much higher Cd and Pb concentrations in the shoots than found in field-cultivations, before exhibiting toxic symptoms. The Hg concentrations, however, occasionally reached levels that are toxic for the plant. Up to11% of total-Hg was methyl-Hg, the most toxic Hg species, though at one site it was 50-100%. To study if methyl-Hg is formed in <i>I.</i> <i>aquatica</i>, plants were exposed to inorganic Hg through the roots. Of the Hg that reached the young, metabolically active parts of the shoots, a part was transformed to methyl-Hg. A major proportion of absorbed metals was retained in the roots, which had a high tolerance for high internal metal concentrations. </p><p>The nutrient level did influence accumulation and effects of Hg, Cd and Pb in<i> I. aquatica</i>. Low external nutrient levels resulted in increased metal accumulation in the shoots and in metal-induced toxic effects in the plant at low external metal levels. A generous supply of sulphur or nitrogen induced formation of thiol-rich peptides in <i>I. aquatica</i>, compounds that have a metal detoxifying effect in plants. </p><p>To conclude, the levels of Cd and Pb in field cultivated <i>I. aquatica</i> do not pose any apparent threat to human health or risk for reduced plant growth. The levels of Hg however, were high at some sites and could be a health threat, for children and foetuses in particular, and especially considering the presence of methyl-Hg. The use of fertilizers is favourable as it reduces the risk for increased metal concentrations in <i>I.</i> <i>aquatica</i> and for reduced crop yields. </p>

<p>Virus mRNAs can be divided into functional regions. The focus of this thesis will be to investigate the function of one of these regions, the 3’ untranslated region (UTR). The 3’UTR of HCV contains a U-rich element and the late 3’UTR of HPV-1 contains an AU-rich element. The roles of these regions in translation and stability of HCV and HPV have been studied. </p><p>A method was established for studying translation of HCV mRNA in living cells. Noninfectious minivirus clones were synthesised <i>in vitro </i>and were transfected into cells by electroporation. This made it possible to bypass the nucleus and to transfer RNA directly into the cell cytoplasm. We found that HCV mRNAs that are translated from the HCV internal ribosome entry site (IRES) are inefficiently translated in comparison to capped and polyadenylated cellular mRNAs. Interestingly, the addition of a cap and a poly(A) tail resulted in a tremendous increase in the initiation of translation at the HCV IRES. This was the result of a discontinuous scanning or shunting mechanism. We also found that the 3’UTR had a small but not significant effect on the virus mRNA translation. Next, we set up an <i>in vitro </i>stability assay to investigate if HCV 3’UTR affects the stability of the virus mRNA. We found that the HCV 3’UTR is very unstable but interaction with the cellular La protein protects the mRNA from premature degradation.</p><p>In parallel experiments, we studied translation and stability of the HPV-1 late mRNAs. By studying an AU-rich sequence in the 3’UTR, we mapped two minimal inhibitory sequence elements, UAUUUAU and UAUUUUUAU that reduced mRNA half-life. We found that the same motifs in the AU-rich element inhibit mRNA translation, demonstrating that the AU-rich element acts via a bimodal mechanism to reduce mRNA stability and inhibit translation.</p>

<p>This thesis is based on work done in two different experimental projects. </p><p>The first project, the Tau RICH, is a previously proposed τ-neutrino appearance experiment for the CERN neutrino beam at the Gran Sasso laboratory in Italy. The proposed experimental concept is based on the use of focusing RICH detectors with liquid radiator (C₆F₁₄). Simulations made with a Geant4 code show that in the proposed experimental set-up, Cherenkov light from delta electrons will constitute a severe background that in practice would render the experiment unfeasible.</p><p>The second project, ATLAS, is a general purpose detector at the CERN 14 TeV proton-proton collider LHC which will start operation in 2007. To make the reconstruction and selection of the events in ATLAS more accurate, complete and up-to-date information on the interaction of the produced particles with the detector is needed. A service program code, the Material Integration Service (MIS), has been developed which makes use of the detector descriptions already available in a Geant4 code and which uses a novel algorithm, based on line integrals evaluated within small volume elements that build up the detector. This code is demonstrated to constitute a practically useful tool of satisfactory performance and accuracy.</p><p>The charged Higgs boson production in the gluon-bottom quark mode, gb → tH^±, followed by charged Higgs decays into a chargino and a neutralino, is studied for a specific choice of values for the Minimal Supersymmetric Standard Model (MSSM) parameters. It is shown, using a Monte Carlo code to simulate the ATLAS detector and the assumed MSSM physics model, that for an integrated luminosity of 300 fb^-1, in the intermediate region 4 < tanβ < 10 where H^± decays to SM particles cannot be used for H^± discovery, charged Higgs decays to non-SM particles can be used for Higgs discovery at the 5 σ significance level.</p>

Virus mRNAs can be divided into functional regions. The focus of this thesis will be to investigate the function of one of these regions, the 3’ untranslated region (UTR). The 3’UTR of HCV contains a U-rich element and the late 3’UTR of HPV-1 contains an AU-rich element. The roles of these regions in translation and stability of HCV and HPV have been studied. A method was established for studying translation of HCV mRNA in living cells. Noninfectious minivirus clones were synthesised in vitro and were transfected into cells by electroporation. This made it possible to bypass the nucleus and to transfer RNA directly into the cell cytoplasm. We found that HCV mRNAs that are translated from the HCV internal ribosome entry site (IRES) are inefficiently translated in comparison to capped and polyadenylated cellular mRNAs. Interestingly, the addition of a cap and a poly(A) tail resulted in a tremendous increase in the initiation of translation at the HCV IRES. This was the result of a discontinuous scanning or shunting mechanism. We also found that the 3’UTR had a small but not significant effect on the virus mRNA translation. Next, we set up an in vitro stability assay to investigate if HCV 3’UTR affects the stability of the virus mRNA. We found that the HCV 3’UTR is very unstable but interaction with the cellular La protein protects the mRNA from premature degradation. In parallel experiments, we studied translation and stability of the HPV-1 late mRNAs. By studying an AU-rich sequence in the 3’UTR, we mapped two minimal inhibitory sequence elements, UAUUUAU and UAUUUUUAU that reduced mRNA half-life. We found that the same motifs in the AU-rich element inhibit mRNA translation, demonstrating that the AU-rich element acts via a bimodal mechanism to reduce mRNA stability and inhibit translation.

This thesis is based on work done in two different experimental projects. The first project, the Tau RICH, is a previously proposed τ-neutrino appearance experiment for the CERN neutrino beam at the Gran Sasso laboratory in Italy. The proposed experimental concept is based on the use of focusing RICH detectors with liquid radiator (C6F14). Simulations made with a Geant4 code show that in the proposed experimental set-up, Cherenkov light from delta electrons will constitute a severe background that in practice would render the experiment unfeasible. The second project, ATLAS, is a general purpose detector at the CERN 14 TeV proton-proton collider LHC which will start operation in 2007. To make the reconstruction and selection of the events in ATLAS more accurate, complete and up-to-date information on the interaction of the produced particles with the detector is needed. A service program code, the Material Integration Service (MIS), has been developed which makes use of the detector descriptions already available in a Geant4 code and which uses a novel algorithm, based on line integrals evaluated within small volume elements that build up the detector. This code is demonstrated to constitute a practically useful tool of satisfactory performance and accuracy. The charged Higgs boson production in the gluon-bottom quark mode, gb → tH±, followed by charged Higgs decays into a chargino and a neutralino, is studied for a specific choice of values for the Minimal Supersymmetric Standard Model (MSSM) parameters. It is shown, using a Monte Carlo code to simulate the ATLAS detector and the assumed MSSM physics model, that for an integrated luminosity of 300 fb-1, in the intermediate region 4 &lt; tanβ &lt; 10 where H± decays to SM particles cannot be used for H± discovery, charged Higgs decays to non-SM particles can be used for Higgs discovery at the 5 σ significance level.

The aquatic plant Ipomoea aquatica is a popular vegetable in Southeast Asia, often cultivated in nutrient rich and polluted waters. The overall aim of this thesis was to estimate potential risks for human health and reduced plant growth due to accumulation and toxicity of total-Hg, methyl-Hg, Cd and Pb. In plants from cultivations in Thailand, the concentrations of Cd and Pb in the shoots were well beneath recommended maximum values for human consumption, but at some sites the Hg concentrations were high. It was demonstrated that I. aquatica has the capacity to accumulate much higher Cd and Pb concentrations in the shoots than found in field-cultivations, before exhibiting toxic symptoms. The Hg concentrations, however, occasionally reached levels that are toxic for the plant. Up to11% of total-Hg was methyl-Hg, the most toxic Hg species, though at one site it was 50-100%. To study if methyl-Hg is formed in I. aquatica, plants were exposed to inorganic Hg through the roots. Of the Hg that reached the young, metabolically active parts of the shoots, a part was transformed to methyl-Hg. A major proportion of absorbed metals was retained in the roots, which had a high tolerance for high internal metal concentrations. The nutrient level did influence accumulation and effects of Hg, Cd and Pb in I. aquatica. Low external nutrient levels resulted in increased metal accumulation in the shoots and in metal-induced toxic effects in the plant at low external metal levels. A generous supply of sulphur or nitrogen induced formation of thiol-rich peptides in I. aquatica, compounds that have a metal detoxifying effect in plants. To conclude, the levels of Cd and Pb in field cultivated I. aquatica do not pose any apparent threat to human health or risk for reduced plant growth. The levels of Hg however, were high at some sites and could be a health threat, for children and foetuses in particular, and especially considering the presence of methyl-Hg. The use of fertilizers is favourable as it reduces the risk for increased metal concentrations in I. aquatica and for reduced crop yields.

A whole-cell DHA-rich algal product (A-DHA, provided by Evonik Industries) that is rich in DHA (125 mg DHA/g dry matter) is a possible replacement for fish oil in salmonid diets. The nutrient digestibilities of the algal product were measured in rainbow trout in freshwater and in Atlantic salmon in saltwater (32-33 ppm). In experiment 1, rainbow trout (initial weight ~ 300g) were randomly assigned to 12 x 120 L tanks (n = 10 per tank). A reference diet containing 1% Celite as an indigestible marker and three test diets with increasing percentage of A-DHA substitution (6.67%, 13.33% and 20%) were fed. Feces were collected using a settling column and feed and feces analyzed for digestible dry matter (DM), gross energy (GE), ash, crude protein (CP), essential amino acids and total lipid. The digestibility of six long-chain fatty acids including 18:1n-9 (OA), 18:2n-6 (LA), 18:3n-3 (ALA), 20:4n-6 (ARA), 20:5n-3 (EPA) and 22:6n-3 (DHA) was measured. In experiment 2, Atlantic salmon (~170g) were randomly distributed to 12 fiberglass tanks (600L) with 106 fish per tank. The fish were assigned to four diets with the same levels of A-DHA inclusion as for rainbow trout and yttrium oxide (Y2O3) was used as an inert marker. Feces were collected by stripping and the digestibilities of DM, CP and lipid as well as OA, LA, ALA, ARA, EPA and DHA were determined. In experiment 1, the apparent digestibility of dietary DM, GE and lipid in rainbow trout declined significantly with increasing inclusion of A-DHA (P < 0.01). The inclusion of A-DHA had no effect on the digestibility of CP and ash as well as the availability of essential amino acids (P > 0.05). Furthermore, increased inclusion of A-DHA resulted in significantly lower digestibility of ARA, EPA and DHA (P < 0.05). A similar pattern was seen in the digestibility of OA, LA and ALA, although the effect of A-DHA inclusion was not statistically significant. Regression analysis revealed that nutrient contribution from A-DHA had significantly negative linear and quadratic effects on the apparent digestibility of DM, GE, and lipid. The inclusion levels of A-DHA had both significantly negative linear and quadratic effects on digestibility of LA and ALA, whereas only significantly negative linear effect was found on OA. Significantly negative linear and quadratic regressions were observed for the digestibility of ARA, EPA and DHA. The linear regression for CP was significantly negative and the regressions for the individual amino acids were not significant (P > 0.05). In experiment 2, dietary inclusion of A-DHA had a significantly negative effect on lipid digestibility in Atlantic salmon, at all inclusion rates whereas the significant negative effect on digestibilities of DM and CP was only observed in fish fed 20% A-DHA. The digestibilities of OA, LA, ALA and EPA were greater than 91%. In contrast, the apparent digestibilities of ARA and DHA decreased significantly with increasing substitution of A-DHA (P < 0.01). Significantly negative linear and quadratic regressions were found between nutrient contribution from A-DHA to the diets and apparent digestibility of DM, CP and lipid, so were LA, EPA and DHA. However, there were only significant quadratic regressions for OA, ALA and ARA, but not significant linear effects. Subsequently, a twelve-week feeding trial in rainbow trout was conducted to investigate the impact of replacing fish oil with A-DHA in canola-oil-based diets on the growth performance and fatty acid composition and retention. Four experimental diets containing only canola oil (CO; 13.5%), fish oil (FO; 13.5%), canola oil and fish oil (C+F; 7.4% and 6.1%, respectively) or canola oil and A-DHA (C+A; 15.5% and 6%, respectively) were formulated to contain 386.2 g/kg digestible crude protein and 17.58 MJ/kg digestible energy. In addition, the C+A diet was formulated to have the same DHA concentration as in the C+F diet. Each diet was fed to three tanks of rainbow trout (average initial weight of 70g; n = 17/tank) and the fish were fed to apparent satiation 2 times daily. At the end of the growth trial, all fish approximately tripled their weight. No significant differences were noted between the dietary treatments in growth performance as measured by final weight, average weight gain, feed intake, specific growth rate (SGR) and feed conversion ratio (FCR). Although FO and C+A fed fish tended to accumulate more lipids, final whole body lipid content did not differ significantly between dietary treatments (P = 0.11). The concentrations of EPA, DHA as well as total n-3 fatty acid were significantly higher in fish fed the FO diet than fish fed the other 3 diets. The C+A fed fish had lower EPA and higher DHA concentrations compared with the CO and C+F fed fish; however, the differences were not significant. Apparent retention of total lipid in the trout was not significantly influenced by treatments (P > 0.05). Similarly, dietary treatments had no significant effect on the apparent retention of total saturated fatty acids, total mono-unsaturated fatty acids, n-3 polyunsaturated fatty acids and n-6 polyunsaturated fatty acids. The retention of 18:4n-3 (SDA) was significantly higher (> 100%) in fish fed CO and C+A compared with fish fed FO and C+F (< 51%), indicating greater bioconversion of ALA to SDA in the CO and C+A fed fish than in FO and C+F fed fish. The retention of EPA in the CO and C+A fed fish was over 100%, suggesting a net synthesis of EPA in these treatment groups. In contrast, the EPA retention in the FO and C+F fed fish was 55 and 21%, respectively, which showed a tendency to be significantly lower than that in the other two groups (P = 0.09). The CO fed fish had significantly higher DHA retention than fish fed the other 3 diets. The DHA retention in the FO fed fish (112%) was numerically but not significantly higher than in the C+F (66%) and C+A fed fish (73%). Thus, feeding the C+A to rainbow trout resulted in DHA retention equal to feeding the C+F.

A novel calmodulin (CaM)-binding cysteine-rich protein from Dictyostelium, cyrA, with epidermal growth factor-like (EGFL) repeats was discovered and characterized. Calcium-dependent and –independent CaM-binding was verified. Western blots show that full length cyrA is detected constitutively throughout development. Analyses of the extracellular medium reveal that cyrA is cleaved and that the fragments containing the N-terminus are secreted early in development, while those containing the C-terminus are secreted later. In support of this, GFP and immunohistochemistry studies reveal that cyrA localizes to the endoplasmic reticulum and secretory vesicles of vegetative cells, and to the extracellular matrix (slime sheath) of migrating slugs. The addition of EGFL1 peptides enhanced cell motility and cAMP-mediated chemotaxis. Finally, cyrA cleavage is regulated by extracellular Dictyostelium CaM and by the extracellular EGFL repeats. In total the data suggest that cyrA is a true matricellular protein that mediates cell motility during multicellular development.