This work reports on the construction of a 408 nm laser system designed to drive stimulated Raman transitions between the F = 4 and F = 5 2 S 1/2 states of 87 Sr + using the 2 P 3/2 state as the intermediate state. This laser system will be used as part of a 87 Sr + ion interferometer. This work also includes a discussion of relevant theory describing the interaction of the ions and laser, along with a calculation of the transition rates as a function of laser power and detuning.

The level structures of some single closed shell nuclei (⁸⁷₃₇Rb₅₀ and ⁸⁸₃₈Sr₅₀) and nearly closed shell nuclei (⁸⁷₃₈Sr₄₉ and ⁸⁸₃₇Rb₅₁) have been determined by means of beta decay processes of ⁸⁷Kr, ⁸⁸Kr and ⁸⁸Rb to final states ⁸⁷Rb, ⁸⁸Rb and ⁸⁸Sr respectively, and by means of the thermal neutron capture reaction on strontium (⁸⁶Sr(n,ɣ)⁸⁷Sr and ⁸⁷Sr(n,ɣ)⁸⁸Sr). The gamma radiation was studied using a Ge(Li) spectrometer and a Ge(Li)-NaI(Tl) coincidence spectrometer. Beta groups were identified using a plastic scintillator and a plastic-NaI(Tl) coincidence spectrometer. Spins and parities were determined for some of the levels from the deduced decay schemes. / Thesis / Doctor of Philosophy (PhD)

The underlying pathological process of coronary artery disease (CAD) is the development of coronary artery atherosclerotic occlusions and associated myocardial infarction. Both increased chronic inflammation and plasma low-density lipoprotein (LDL) cholesterol levels promote atherosclerosis. Inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9) is widely known for its role in enhancing LDL receptor (LDLR)-mediated cholesterol lowering when the LDLR-apolipoprotein E (APOE) axis is intact and protecting against atherosclerosis progression by reducing plasma cholesterol levels. In this thesis, we sought to test the effects of PCSK9 inhibition mediated cholesterol lowering on pre-existing CAD as well as the plasma cholesterol independent effects of PCSK9 inhibition on CAD by utilizing different mouse models. One year old scavenger receptor class B type I (Sr-b1) knockout (KO) mice which have an intact LDLR-APOE axis, develop coronary artery atherosclerosis and myocardial fibrosis induced by a high fat, high cholesterol and cholate containing (HFCC) diet. Weekly anti-PCSK9 antibody treatment initiated one week before switching to an HFCC diet increased hepatic LDLR protein levels, and reduced plasma cholesterol levels and the progression of atherosclerosis in both the aortic sinus and coronary arteries in one year old Sr-b1 KO mice (maintained on an HFCC diet for 7 weeks). Weekly anti-PCSK9 antibody treatment initiated 7 weeks after switching to an HFCC diet also increased hepatic LDLR protein levels and reduced plasma cholesterol levels in one year old Sr-b1 KO mice (maintained on an HFCC diet for 12 weeks). More importantly, anti-PCSK9 antibody treatment during the last 5 weeks of the 12-week HFCC diet feeding period also slowed down the growth of pre-existing atherosclerosis in both the aortic sinus and coronary arteries and reduced myocardial fibrosis and damage. Mice deficient in both Sr-b1 and ApoE (Sr-b1/ApoE double KO (dKO) mice) spontaneously and rapidly develop features reminiscent of human CAD. Whole body Pcsk9 genetic KO in both female and male Sr-b1/ApoE dKO mice did not affect plasma cholesterol levels despite increased hepatic LDLR protein levels, presumably due to the lack of APOE. However, genetic Pcsk9 inactivation significantly attenuated atherosclerosis in both the aortic sinus and coronary arteries, myocardial fibrosis and damage, left ventricle (LV) dysfunction and cardiac enlargement in both female and male Sr-b1/ApoE dKO mice. Restoring circulating PCSK9 by a recombinant adeno associated virus 8 (AAV8)-mediated hepatic expression of a Pcsk9 cDNA in Pcsk9/Sr-b1/ApoE triple KO mice reversed the plasma cholesterol independent protective effects of genetic PCSK9 KO on aortic sinus and coronary artery atherosclerosis and myocardial fibrosis and damage in both females and males. Treatment of Sr-b1/ApoE dKO mice with an anti-PCSK9 antibody which disrupts the interaction between the LDLR and PCSK9 protected against aortic sinus and coronary artery atherosclerosis in males but not in females and did not protect either males or females against myocardial fibrosis and damage, LV dysfunction or cardiac enlargement. My thesis demonstrates that anti-PCSK9 antibody mediated plasma cholesterol lowering delays the continued development of pre-existing CAD. My thesis also demonstrates that liver-derived, circulating PCSK9 promotes CAD in a plasma cholesterol independent manner in Sr-b1/ApoE dKO mice and these effects appear to be largely independent of the PCSK9-LDLR interaction, particularly in females. / Thesis / Doctor of Philosophy (PhD)

Plants employ sophisticated molecular machineries to fine-tune their responses to growth, developmental, and stress cues. Plants cellular response influences gene expression through regulating processes like transcription and splicing. To increase the genome coding potential and further regulate the expression, pre-mRNA is alternatively spliced. Serine/Arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize splicing cis-elements and regulate both constitutive and alternative splicing. Recent studies reported only 22 SR proteins encoded in the genome of rice (Oryza sativa), which are classified into 6 subfamilies. Oryza s. SC subfamily 106 kDa (Os-Sc106) locus is homologous to the human SR protein SFSR11 (SRp54). Os-Sc106 contains SR proteins characteristics, and was not included among the rice SR proteins. The clustered regularly interspaced short palindromic repeats (CRISPR) and its associated protein 9 (Cas9) system, an RNA-guided endonuclease complex that introduces a double-strand break (DSB) into the DNA. Innovative scientific advances in genome engineering have made CRISPR/Cas9 an excellent system to conduct functional knockout studies of genes in most biological systems including plants. In this study, I targeted the rice Os-Sc106 locus at exon1, and 3 via CRISPR/Cas9 system. Genotyping analyses revealed the recovery of Os-Sc106 mutants including complete functional knockouts such as sf11h-2, sf11h-8, and sf11h-55. Phenotypic analyses show that Os-Sc106 mutants (sf11h-2, 8, 55, and 57) are oversensitive under abiotic stress in comparison to WT plants, suggesting that Os-Sc106 locus encodes a protein that is important for regulating plant stress responses.

No description available.

No description available.

<p> Gene-targeted studies in mice have shown that the murine scavenger receptor class B type I (mSR-BI) is atheroprotective and plays a key role in the clearance of high density lipoprotein (HDL) cholesterol by the liver. We focused on the analysis of human SR-BI (hSR-BI) and the role of its C-terminal cytoplasmic tail on its localization, lipid uptake activity, and regulation in hepatocytes both in vitro and in vivo. Full length hSRBI and hSR-BI lacking its C-terminal cytoplasmic tail (hSR-BI-DM) localized to vesiclelike structures in the cytoplasm, to juxtanuclear regions and to the cell surface in HepG2 cells. Similar cytoplasmic punctate distribution was observed in transfected human and mouse aortic endothelial cells. </p> <p> In HepG2 cells both hSR-BI and hSR-BI-DM mediated HDL-lipid uptake; however, the truncation mutant displayed only half ofthe activity, suggesting that removal ofthe C-terminal cytoplasmic tail reduced but did not eliminate SR-BI's activity. In HepG2 cells treated with the PKC inhibitor, calphostin C, hSR-BI or hSR-BI-DM mediated HDL-lipid uptake was decreased by 40 and 50%, respectively, indicating that this activity is regulated by PKC. </p> <p> In order to determine the effects of hSR-BI and hSR-BI-DM in vivo, we set out to generate transgenic mice with hepatic overexpression ofeach protein using a bipartite expression system requiring driver and responder transgenes. Mice expressing the responder transgenes, PTREhSR-BI and PTREhSR-BI-DM, as well as a reporter transgene (PTRdacZ), driven by the same bi-directional promoter, were generated and mated to mice with a liver-specific driver trans gene, PMuptTA. The mice were analyzed and showed the presence of a reporter protein, ~-galactosidase, in their livers, but not in other tissues tested. Total and HDL cholesterol levels were not altered in PMuPtTA I PrREhSRBI or PMuptTA I PrREhSR-BI-DM transgenic mice. Further characterization ofthe double transgenic mice revealed that hSR-BI m.RNA transcripts were detected in the livers of PMuPtTA I PrREhSR-BI mice, but not in those ofPMuPtTA I PrREhSR-BI-DM mice. However, neither PMuptTA I PrREhSR-BI nor PMuptTA I PrREhSR-BI-DM mice showed increased expression of SR-BI in their livers. </p> / Thesis / Master of Science (MSc)

La télomérase, composée de l‘ARN TR et de la protéine TERT, responsable du maintien de la longueur des télomères est surexprimée dans la majorité des cellules cancéreuses. La dynamique de la régulation post-transcriptionnelle de TERT sur l‘activation de la télomérase a été étudiée dans le modèle de lymphomagenèse induite par l‘herpesvirus oncogène aviaire de la maladie de Marek. L‘augmentation de l‘activité télomérase des TCD4+ lors de l‘apparition des lymphomes résulte d‘une hausse du transcrit constitutif et de celle des transcrits cibles de la voie de dégradation du « non-sense mediated decay » (NMD) alors que l‘activité télomérase basale des TCD4+ non infectés est contrôlée par les isoformes dominantes négatives. La caractérisation de la protéine virale ICP27 de MDV-1, régulateur potentiel de l‘épissage des gènes, qui s‘exprime pendant la phase de réplication lytique du virus a complété cette étude. ICP27 est capable de co-localiser et d‘interagir avec les protéines SR du splicéosome ainsi que de réguler négativement l‘épissage des gènes cellulaire TERT et viral vIL8 de manière similaire à ICP27 de l‘herpesvirus simplex 1. Le modèle naturel de lymphomagenèse induite par MDV-1 a permis d‘établir pour la première fois un lien entre l‘activation de la télomérase in vivo et la régulation de l‘épissage de TERT, à laquelle pourrait participer la protéine virale ICP27. / The telomerase, consisting of an RNA template (TR) and a reverse transcriptase (TERT) maintains telomere length and is highly expressed in the majority of cancer cells. The splicing regulation of TERT was studied in Marek‘s disease (MD), a natural lymphoma induced by MDV-1, the avian MD herpesvirus. Telomerase activation observed in TCD4+ cells at the onset of MD lymphoma was due to an increase of constitutively spliced and « non-sense mediated decay » (NMD) while basal telomerase activity of non infected TCD4+ cells was controlled by dominant negative isoforms. In addition, the viral protein ICP27, a putative regulator of splicing, expressed during MDV-1 lytic infection was characterised. ICP27 co-localized and interacted with spliceosome SR proteins and negatively controlled splicing of TERT and vIL8 viral gene in a way similar to that of ICP27 of herpesvirus simplex 1. The MD model provides the only data on the in vivo regulation of TERT splicing, possibly mediated by ICP27, and telomerase activation during lymphomagenesis induced by a herpesvirus in its natural host.

La protéine EB2 du virus d'Epstein-Barr (EBV) est une protéine du cycle réplicatif du virus indispensable à la production de particules virales. Elle permet l’accumulation dans le cytoplasme de certains ARNm viraux issus de gènes dépourvus d’intron. Pour mettre en évidence les mécanismes qui permettent à EB2 d’exporter ses ARNm cibles dans le cytoplasme, nous avons identifié différents partenaires cellulaires d’EB2 et nous avons étudié certaines de ces interactions d’un point de vue fonctionnel. Nous avons pu montrer qu’EB2 recrute directement le facteur général d'export des ARNm, TAP/NXF1, ce qui lui permet d’être exportée du noyau vers le cytoplasme. Puis nous avons montré qu’EB2 interagit avec SRp20, une protéine impliquée notamment dans la régulation de l'épissage et l'export des ARNm cellulaires. Cette interaction entre EB2 et SRp20 est indispensable pour l’accumulation dans le cytoplasme de certains ARNm cibles d’EB2, notamment parce que SRp20 semble permettre le recrutement d'EB2 sur ces ARNm. Enfin, nous avons montré qu’EB2 forme un dimère et nous avons caractérisé le domaine de la protéine responsable de cette interaction. La dimérisation d'EB2 semble essentielle pour que la protéine interagisse avec certains de ses partenaires comme SRp20 ou encore REF. / The Epstein-Barr virus (EBV) protein EB2 is an early protein essential for the production of infectous virions. EB2 allows the cytoplasmic accumulation of a subset of viral mRNAs derived from intronless genes. To highlight the mecanisms by which EB2 exports his targets mRNA, we identified cellular partners and studied the functional role of some of these interactions. We showed that EB2 recruits directly the cellular mRNA export factor TAP/NXF1 and this interaction allows EB2’s shuttling between the nucleus and the cytoplasm. The we showed that EB2 interacts with SRp20, a cellular protein implicated in splicing regulation and mRNA export. This interaction is essential for the efficient cytoplasmic accumulation of some EB2 target mRNAs, partly because SRp20 appears to be able to recruit EB2 on these mRNAs. Then we showed that EB2 dimerises and we characterized the domain necessary for this interaction. This dimerisation appears to be essential for EB2’s interaction with several partners, including SRp20 and REF.

Der Scavenger Receptor BI (SR-BI) vermittelt den selective lipid transfer von Cholesterol und Vitamin E aus HDL in die Leber. Die zelluläre Aufnahme verschiedener Lipide aus HDL über den selben Mechanismus, vermittelt durch den selben Rezeptor wirft die Frage auf, ob diese Aufnahmeprozesse einander beeinflussen. Aktuelle Forschungsergebnisse zeigen, daß die Aufnahme von neutralen Lipiden (Cholesterolester, Triacylglycerol) aus HDL in die Zelle von der Lipidzusammensetzung der Donorpartikel abhängen könnte. Wir untersuchten, ob der Vitamin-E-Gehalt von HDL die Aufnahme und den Efflux von Cholesterol in und aus HepG2-Zellen beeinflußt. Die Inkubation von HepG2-Zellen mit [3H]Cholesterol-markiertem HDL mit ansteigendem Vitamin-E-Gehalt ergab eine steigende Aufnahme von Vitamin E, während sich die Cholesterolaufnahme nicht veränderte. Der erhöhte zelluläre Gehalt an Vitamin E bewirkte eine Reduktion der PKC-Alpha- und SR-BI-Expression in Verbindung mit einem erniedrigten Cholesterolefflux aus HepG2-Zellen zu nativem HDL als Akzeptorpartikel. Die Verarmung der Zellen an Cholesterol führte zu einer erniedrigten PKC-Alpha- und SR-BI-Expression. Hingegen veränderte die Erhöhung des zellulären Cholesterolgehalts von HepG2-Zellen die PKC-Alpha- und SR-BI-Expression nicht, während der Cholesterolefflux aus HepG2-Zellen zu HDL im Vergleich zur Kontrolle gesteigert war. Wir schließen aus diesen Ergebnissen, daß Vitamin E und Cholesterol die SR-BI-Expression modulieren können und daß SR-BI den Efflux und möglicherweise auch den Influx von Cholesterol in HepG2-Zellen vermittelt. / The scavenger receptor BI (SR-BI) mediates selective lipid transfer of cholesterol and vitamin E from HDL to the liver. Cellular uptake of different lipids from HDL by the same mechanism, mediated by the same receptor rise the question, whether these uptake processes affect each other. Recent results show that the cellular uptake of neutral lipids (cholesterol ester, triacylglycerol) from HDL may depend on the lipid composition of the donor particles. We investigated whether the vitamin E-content of HDL affects cholesterol uptake and efflux by HepG2 cells. Incubation of HepG2 cells with [3H]cholesterol-labeled HDL, which contained increasing vitamin E-concentrations resulted in an increased uptake of vitamin E, whereas the cholesterol uptake did not change. The increased cellular content of vitamin E caused a decreased PKCalpha and SR-BI-expression combined with a decreased cholesterol-efflux from HepG2 cells to native HDL as acceptor. Depletion of cellular cholesterol decreased PKC-alpha and SR-BI-expression in HepG2 cells. Increase of cellular cholesterol of HepG2 cells, however, did not change PKCalpha and SR-BI-expression, whereas cholesterol-efflux from HepG2 cells to HDL increased. We conclude that vitamin E and cholesterol can modulate the SR-BI-expression and that SR-BI mediates the efflux and possibly also the influx of cholesterol by HepG2 cells.