Afin de se répliquer, les rétrovirus doivent disposer à la fois d’ARN épissés et non épissés. Chez le virus du Sarcome de Rous (RSV), l’accumulation de l’ARN non épissé dépend de l’élément NRS (Negative Regulator of Splicing). L’élément NRS est un élément bipartite. Sa région 5’ est assimilée à une séquence ESE (Exon Splicing Enhancer) à laquelle se fixent de nombreuses protéines SR tandis que sa région 3’ contient un pseudo-site 5’ non fonctionnel qui constitue un leurre qui est responsable de l’inhibition de l’épissage à l’unique site 5’ fonctionnel du virus. Seule la structure 3D de la partie 3’ du NRS qui contient le pseudo-site a été expérimentalement établie. Dans ce travail, nous avons déterminé la structure 2D de la totalité de l’élément NRS à l’aide de sondes chimiques et enzymatiques. La comparaison de cette structure expérimentale à celles que nous avons établies pour d’autres éléments NRS mutants fonctionnels et non fonctionnel ainsi qu’à celles théoriques de la totalité des virus aviaires séquencés argumente en faveur de la forte signification biologique de notre modèle. Des expériences d’épissage in vitro réalisées sur l’élément NRS sauvage ainsi que ses formes tronquées ont permis de mettre en évidence le rôle crucial de deux structures tige-boucles dans la fonction du NRS. Les expériences de purification de complexes formés avec un extrait nucléaire de cellules HeLa sur ces différents éléments NRS par des techniques chromatographie d’affinité ont permis de démontrer l’importance de l’association de ces deux structures tige-boucles avec les protéines SR et la snRNP U1. Nous avons défini un nouvel élément NRS minimal fonctionnel capable d’inhiber l’épissage et nous avons démontré l’activation de l’inhibition de l’épissage de l’élément NRS par la protéine 9G8 in vitro et in cellulo / Retroviruses require both spliced and unspliced RNAs for productive replication. Accumulation of unspliced RNA in Rous Sarcoma Virus (RSV) depends on the NRS element, (Negative Regulator of Splicing). The NRS element is bipartite. Its 5’ terminal part is considered as an ESE that binds SR proteins and its 3’ part contains a decoy 5’-splice site (ss), which inhibits splicing at the bona fide 5’ ss. Only the 3D structure of a small NRS fragment including the decoy 5’ ss had been experimentally studied. Here, by chemical and enzymatic probing of entire RSV NRS, we determine its 2D structure. By comparative analysis of 2D structures of functional and non-functional avian NRS variants and of all sequenced avian NRSs, we bring strong arguments for a biological significance of the established structure. By in vitro splicing assays, we show a crucial role of two of the established stem-loop structures and by affinity purification of complexes formed by WT and truncated NRSs in HeLa cell nuclear extract, we demonstrate their importance for SR protein and U1 snRNP association. We define a new small NRS element retaining splicing inhibitory properties and finally demonstrate the capability of the SR protein 9G8 to increase NRS activity in vitro and in cellulo

Sr-90 and Cs-137 are two key fission products that are important in a number of radioactive waste or contamination clean-up scenarios. This thesis investigates the sorptiondesorption behaviour of these two radionuclides with a number of sorbents relevant to one of the legacy waste storage ponds on the Sellafield site. Sorption-desorption studies were undertaken on brucite (Mg(OH)2), UO2 and UO3 powders to represent components of the fine particulate sludge that has accumulated on the bottom of the pond, resulting from corrosion of the Magnox fuel elements. Brucite was found not to have a significant interaction with Sr-90 or Cs-137. However, organic molecules in the form of humic acid and Pseudanabaena catenata cyanobacterial growth supernatant were both found to enhance sorption interactions of Sr-90 with brucite. The effect of humic acid was pH dependent and appeared to control both sorption and desorption behaviour at pH 11.5. Uranium oxides had the most significant effect on controlling Sr-90 and Cs-137 sorption-desorption behaviour. In this work X-Ray Absorption Spectroscopy (XAS) showed that 90Sr sorbed strongly to both uranium oxides as a bidentate inner-sphere complex. Humic acid did not appear to alter the interaction of Sr-90 with UO2. For Cs-137 there was a significant, reversible interaction with both oxides, although the interaction was far stronger with UO3. Successful detection of stable Sr and Cs isotopes on uranium oxides was achieved with Laser Induced Breakdown Spectroscopy (LIBS).

Live cell imaging with FTIR spectroscopy offers a high throughput, non-damage and lab-free method to study the cells in vivo which has significant advantages in the field of cancer diagnosis and drug screening. However, due to the strong absorbance of water, using infrared spectroscopy in such field remains to be an underdeveloped topic. This project demonstrates a novel method to perform IR imaging of cells in solution. A novel water correction method, which avoids the using of water combination band, is proposed. A buffer reference and a cell reference spectra were introduced to fitting the contribution based on protein bands. This method was implemented on three types of IR spectrometers, namely conventional FTIR spectrometer, synchrotron-based FTIR spectrometer and quantum cascade laser (QCL) microscope. To date, most of the live cell imaging carried out with IR sources utilise synchrotron radiation. Recently, a new bench top system, QCL microscope, has been developed. It incorporates four tunable QCL laser sources covering the wavenumber range 900-1800 cm-1 which are many orders of magnitude brighter than conventional sources. The proposed water correction method is, therefore, capable of processing the data recorded by all three types of IR spectrometers. Three prostate cancer cell lines were employed to evaluate the water correction method and the performance of three spectrometers on imaging of cell in solution. The obtained spectra was analysed with multivariate analysis, PCA and PC-LDA which shows good separation between cell lines. The data was also examined with Random Forest algorithm to establish a classifier and the diagnostic capability of the water corrected spectra was proven.

Tese de Mestrado Integrado. Engenharia Electrotécnica e de Computadores. Faculdade de Engenharia. Universidade do Porto. 2012

The first bimonthly resolved, paired δ18O and Sr/Ca time series from the slow-growing, tropical western Atlantic coral, Siderastrea siderea, from the Dry Tortugas, Florida has been generated and used to document that robust proxy climate records can be produced from this heretofore underutilized massive coral. The coral time series contains a 20-year long calibration window (1973-1992) for both δ18O and Sr/Ca and a 73-year long verification window (1900-1972) for Sr/Ca. These time series permits both the quantification of the relationship between coral δ18O-SST and Sr/Ca-SST using an augmented, 1° x 1° gridded SST record and the assessment of the stability of the proxy relationships over time. Both coral geochemical records are highly correlated with the augmented instrumental SST record through the calibration period and Sr/Ca remains highly correlated through the verification period both at the bimonthly (r = -0.97) and annual average level (r = -0.72). Additionally, both coral δ18O and Sr/Ca are highly reproducible within the same core, and Sr/Ca exhibits no extension-related vital effects. Sr/Ca-SST anomalies are also significantly correlated to the augmented SST anomalies, despite the removal of the serial autocorrelation. The skill of this proxy demonstrates its potential as a continuously growing, long-lived recorder of climate variability for the tropical Atlantic and Intra-American Seas. The relatively slow extension rate of the coral (~5 mm yr-1 during the 20th century) also suggests the potential for long records of climate variability (~200 years) of the region to be extracted from even modest-sized colonies (~1 m in height). The results of this study are important because relatively few century-long, sub-annually resolved time series of climate variability from massive Atlantic corals have been published, despite the significance of the tropical Atlantic climate modes of variability.

This study focuses on how the construction of a paleoclimate time series influences the interpretation in the frequency domain. Three time series are examined: a New Caledonian coral (Amedee Island), a Chinese speleothem (Dongge Cave), and New Mexican trees (El Malpais). This study presents a monthly resolved coral Sr/Ca time series from New Caledonia that reconstructs sea surface temperature (SST; 1648-1999). The chronology is based on density-band counting, cross-correlating Sr/Ca, and ²³°Th dating. The intracolony coral Sr/Ca variations are coherent on interannual to centennial time scales and are reproducible for >300 years. The SST reconstruction reveals estimated cooling trend (~0.4°C) from 1741-1815, a colder nineteenth century (~0.6°C), and a warming trend (~0.6°C) in the twentieth century. Spectral and wavelet analysis reveals significant inter-decadal periodicities (~14-21 years/cycle) that modulate with time, and nearly persistent multi-decadal periodicities (~25-33 years/cycle) that do not exhibit coherence with the Inter-decadal Pacific Oscillation. The multi-decadal periodicities may be a harmonic of the inter-decadal periodicities or may represent an independent mode not previously recognized. The Dongge Cave time series is based on uneven time intervals between data points (∆t) requiring interpolation to a constant ∆t for analysis with traditional spectral methods. A comparison of the even and uneven ∆t spectra using the Lomb-Scargle transform reveals the interpolated spectrum contains suppressed periodicities (<20 years/cycle), in contrast to the uninterpolated spectrum, resulting in a steeper slope in the red noise model thus influencing significance testing. The El Malpais time series is an average of tree-ring width series. Spectral analysis of the entire time series identified significant periodicities. However, significance varies between three temporal subsets, in which the number of series varies; therefore, these periodicities may be a function of the number of series or may represent a real temporal variability. Cross-spectral analysis of the El Malpais and Dongge Cave time series reveals significant coherence; however, cross-wavelet analysis, which examines localized frequencies in the time domain, reveals a lack of correlation; therefore, coherence in the frequency domain does not indicate correlation in the time domain.

The bacterial meningitis caused by Neisseria meningitidis is responsible for considerable morbidity and mortality throughout the world. Given the limitations of existing diagnostic tests and the severity of the illness associated with the disease, there is a clear requirement for a rapid and specific diagnostic assay. This thesis describes the development of nanoparticle based tests for the detection of Neisseria meningitidis specific cell surface markers. As an initial target antigen, a recombinant form of highly conserved outer membrane protein, OMP85 was used. Within the OMP85 protein sequence, a predicted antigenic sequence between residues 720 and 745 was identified and found to be unique to this organism. This amino acid sequence was synthesised as peptide (SR1) with a gly-gly-cysteine spacer sequence at the N-terminus using t-boc chemistry. Also, the major virulence factor, capsular polysaccharide of N. meningitidis serogroup B bacteria was purified. Polyclonal antibodies were raised against purified OMP85 antigen in rabbits and against SR1 peptide and also against formalin inactivated N. meningitidis serogroup B whole cell bacteria in sheep. This panel of different antibodies including the commercial anti-capsular monoclonal antibodies were examined for cross reactivity against a range of closely related Gram negative bacteria. Based on these cross-reactivity studies, a highly specific anti-NM antibody was developed following purification of the anti-SR1 antiserum by immuno-affinity chromatography. Purified OMP85 antigen and anti-OMP85 antibody were successfully conjugated on 13, 30, 40, 50 and 60 nm gold nanoparticles by an electrostatic adsorption method. Coupling of the gold nanoparticles results in a shift of the respective surface plasmon peak toward longer wavelengths (in the range of 600-800 nm) resulting in a change of the colour of the colloidal suspension from red to purple to blue. An attempt was made to develop a rapid diagnostic assay based on gold nanoparticle induced colour shift assay for N. meningitidis by utilising the specific interaction of OMP85 and anti-OMP85 antibody conjugated to gold nanoparticles as a model system. However, this system was not reproducible and is likely to be due to problems with stability of gold nanoparticles during the conjugation process. As an alternative approach, a highly selective quartz crystal microbalance (QCM)-based immunosensor was designed using the same OMP85/anti-OMP85 antibody system. A method was developed using polyvinylidene fluoride (PVDF) coated QCM crystals with protein A for the directional orientation of the antibodies. To further enhance the sensitivity of the test, OMP85-conjugated gold nanoparticles were used as signal amplification probes for the reproducible detection of the target down to 300 ng/mL, corresponding to a five fold increase in sensitivity compared to detection of OMP85 antigen alone. Also, this sensor has successfully been employed to detect whole cell bacteria at a concentration as low as 100 cfu/mL. Thus, in this study using the real-time QCM measurements, a novel strategy has been developed for the sensitive detection of both N. meningitidis bacteria and the protein antigen at very low concentrations, using gold nanoparticles as signal amplification probes.

Les séquences argileuses peu perméables telles que les argilites callovo-oxfordiennes du bassin de<br />Paris ou les siltites vraconiennes du Gard, ont été choisies pour l'implantation éventuelle d'un<br />laboratoire souterrain destiné à tester les propriétés physico-chimiques de ces formations dans<br />l'éventualité d'un stockage profond de déchets nucléaires. La connaissance et la compréhension des<br />modifications post-sédimentaires sont fondamentales dans la définition de ces propriétés. Le but du<br />présent travail visait donc à évaluer et quantifier ces changements par l'étude de ces roches, en<br />particulier des minéraux argileux.<br />Les échantillons proviennent de deux forages (HTM102 et MAR501). Les principaux minéraux<br />argileux dans le forage HTM102 sont l'illite et les interstratifiés illite/smectite. Les observations au<br />MET et au MEB couplées aux analyses isotopiques K-Ar et Rb-Sr mettent en évidence la néoformation de carbonates (calcite, dolomite) et d'argiles. Celles-ci apparaissent au MET comme des particules authigènes lattées en croissance sur des particules détritiques. La durée et l'extension de ces événements diagénétiques sont difficiles à évaluer du fait d'une contamination systématique des<br />échantillons par une contribution détritique, et ce même dans les fractions les plus fines. Cependant,<br />l'étude d'un niveau de bentonite dans la séquence permet de caler l'époque et la durée de la<br />diagenèse. En assimilant cette bentonite à un pôle authigène, il a été possible de reconstruire les<br />variations des valeurs K-Ar de chaque fraction argileuse dans la séquence. Ainsi, la corrélation<br />observée entre les variations du niveau marin relatif et la formation d'un matériel argileux riche en<br />smectite d'une part, et des chimies de fluides différentes d'autre part, sont autant d'arguments en<br />faveur de réactions diagénétiques en système sinon clos du moins restreint.<br />Les travaux sur le forage MAR501 sont comparables à ceux réalisés sur le forage HTM102, avec<br />également un mélange entre deux populations argileuses d'âge différent. Les glauconites<br />diagénétiques ont permis de caler le Vraconien au voisinage de 93,7 ± 0,3 Ma, en accord avec les<br />données stratigraphiques. L'étude d'une fracture colmatée par une matrice essentiellement argileuse<br />confirme le caractère confiné de cette séquence. Aucune migration d'éléments, et en particulier de<br />TR, n'a été mise en évidence lors de la circulation de paléofluides, indiquant que les minéraux<br />argileux ont conservé leurs propriétés géochimiques.<br />L'ensemble de ces résultats est en faveur de propriétés de confinement fiables dans le cas des deux séquences argileuses.

<p>I banksektorn är ekonomiska frågor betydligt mer institutionaliserade än miljöfrågor som bara varit aktuella i tiotalet år. Det leder till en viss osäkerhet att hantera miljöfrågor. Banksektorn försöker via legitimitetssökande standarder, ofta med start i bankkontorens direkta miljöpåverkan, att hantera miljöfrågorna. Bankers överlägset största miljöpåverkan ligger dock i den indirekta miljöpåverkan via kreditgivning och investeringar till kunder. Banker kan inte ta hela ansvaret utan det måste delas med kunderna som måste ställa krav på bankerna att erbjuda miljöansvarstagande tjänster. Bankernas ansvar ligger i att erbjuda miljöansvarstagande tjänster och aktivt presentera och förklara dem för kunderna. Enligt omvärldsanalysen har Marginalen goda förutsättningar för att initiera ett aktivt miljöarbete. Det är stor vikt att Marginalen inte bara hanterar den direkta miljöpåverkan utan även den mer miljöbelastande indirekta påverkan.</p> / <p>The world´s economic development during the past 100 years has led to improvements for millions of people. But it has also led to enormous environmental problems like global warming, species extinction and loss of biodiversity. Every human being and every sector in society are responsible to solve these grave environmental problems. One of those sectors is the banking sector. At first glance, the banking sector may look like an environmentally clean sector, especially when you compare the banking sector´s direct environmental impact with for example manufacturing industries. But when you look at the banking sector´s indirect environmental impact through loans and investments, you can envision the sectors potential to steer the society towards a sustainable development.</p><p>This thesis has shown this a complex matter though, since banks and their customers share responsibility to elevate environmental issues. Customers need to understand that banks indirect environmental impact is big and then put pressure on banks to provide environmental responsible services and products. Banks, on the other hand, offer quite complex services and products which mean that banks have a responsibility to explain services and products different environmental effects. Banks have handled environmental risks for a long time in credit grants, but banks are now starting to feel that customers demand for environmental products and services is on a rise. The thesis has also found out that different types of environmental standards are very important for a credible environmental work in the banking sector.</p>

<p>In the mid-1990’s, the global computer network known as the Internet was introduced in Sweden. With this study, we are shining a light on the entrance of Swedish Public Service media on the Internet. The two major Swedish Public Service companies Sveriges Television (“Swedish Television”, also called SVT) and Sveriges Radio (“Swedish Radio”, also called SR) are included in this study.</p><p>SR published its first website in 1994 and the site featured a presentation of the results from the Swedish parliament elections of that year. SVT published its first website in 1995, when the company wanted to find a way to develop the classic teletext.</p><p>The reason why we are studying these companies is that we wanted to find out why two publically funded companies decided to enter the new and relatively unknown Internet platform at a time when only three percent of the Swedish population had Internet access at home.</p><p>We have interviewed technicians and board members working for either SR or SVT at the time surrounding their entry on the Internet and we have also accessed board documents and other written information from the companies.</p><p>The study shows that both SR and SVT had an early presence on the Internet but without really investing. The respective boards were unsure about the usefulness of the new platform and the technicians were working without getting, according to themselves, enough time and money. The situation changed at the beginning of the 21^st century, after the timeframe of our study.</p>