M. Tech. (Department of Chemistry, Faculty of Applied and Computer Science): Vaal University of Technology / Here, we report the synthesis and characterization of silver (Ag) and silver selenide (Ag2Se) nanoparticles using the metal-organic route method. This method involves the reduction of selenium powder and silver nitrate in the presence of trioctylphosphine as a solvent. Tri-n-octylphosphine oxide (TOPO) and hexadecylamine (HDA) were used in the study as capping molecules. The optical properties of the as-prepared nanoparticles were studied using UV-Visible and photoluminescence spectroscopy (PL). Transmission electron microscopy (TEM) and X-ray powder diffraction (XRD) were used to study the structural properties. The effect of capping molecules and temperature were investigated on the growth of the nanoparticles. The prepared nanoparticles seem to depend on the reaction temperature were the increase in temperature led to an increase in particle sizes. The growth of the as-prepared TOPO-capped Ag2Se nanoparticles was influenced by temperature, this was evident when the temperature was increased, the nanoparticles evolved from sphere to hexagonal shape. TOPO-capped nanoparticles showed the tendency of agglomeration with increase in temperature compared to HDA-capped nanoparticles. The X-ray diffraction results showed peaks which were identified as due to α-Ag2Se body centered cubic compound for both TOPO/HDA-capped Ag2Se nanoparticles. Some evidence of impurities were observed in the XRD analysis and indexed to metallic silver. HDA-capped Ag nanoparticles were found to be affected by temperature variation. The prepared nanoparticles were characterized with UV-Vis spectroscopy and transmission electron microscopy. XRD analysis was not performed due to small yield obtained. The absorption spectra of HDA-capped Ag nanoparticles at different temperatures show a surface Plasmon resonance (SPR) band in the regions 418 - 428 nm. Uniform spherical shapes were obtained for both 130 and 190 °C and fewer particles were obtained at 160 °C. The synthesis of TOPO–capped Ag nanoparticles was unsuccessful since none of the particles were isolated from the solution due to its lower capping ability or it may be that TOPO is binding too strongly to Ag. The polymer nanofibres were electrospun using electrospinning technique. Parameters such as concentration and voltage were investigated. These parameters significantly affect the formation of fibre morphology. PVP and PMMA polymers were used for this study. The electrospun composite fibres were characterized using UV-Visible spectroscopy, scanning electron microscopy (SEM), Thermal gravimetric analysis (TGA), X-ray diffraction (XRD) and Fourier transformer infrared (FTIR) spectroscopy. The SEM results show that increasing the polymer concentration resulted in increased fibre diameters. Hence increasing the voltage decreases the fibre diameters. Ag2Se nanoparticles were incorporated into PVP and PMMA and electrospun using electrospinning to produce composite fibres. Their addition into PVP polymer fibres improved the fibre’s uniformity and further decreased their diameters. The SEM of composite fibres for PMMA is not shown. The absorption bands for PVP composites fibres show a blue shift from the pure Ag2Se nanoparticles, whereas the one for PMMA show a red shift from the pure Ag2Se nanoparticles. Both the composite fibres for PVP and PMMA show a blue shift from the bulk of Ag2Se. The XRD analysis of the composite fibres shows no significant effect upon addition of Ag2Se nanoparticles on the amorphous peak of the PVP polymer, whereas on the PMMA, it shows peaks which were due to the face centered cubic phase of Ag. The FTIR spectra of the composite fibres and pure polymers (PVP and PMMA) gave almost identical features. TGA curves show no significant effect on the thermal properties of the PVP polymer and its composites, however, on the PMMA composite fibres it show an increase in the thermal stability of the polymers upon addition of Ag2Se nanoparticles. The study was based on silver nanoparicles and its antibacterial activities. One of the synthetic challenges for silver nanoparticles is their solubility and yield. Selenide was introduced in the study to improve such shortcomings of silver nanoparticles and also for possible improved properties, chemical stability and increased activity against bacteria. The selenide group on the metal also provides stronger chemical interaction between the nanoparticles and the polymer. Therefore, the intension was to use these nanoparticles into polymer fibres for potential use in wound dressing.

Le coiffage pulpaire direct, dans des situations pathologiques critiques où la vitalité de la dent est menacée, vise à stimuler le potentiel de cicatrisation de la pulpe et induire une régénération dentinaire. Afin d'optimiser cette thérapeutique, le Laboratoire IMEB-ERT 30 a développé en partenariat avec la société SEPTODONT un nouveau matériau, le Biodentine™. Ce ciment, composé essentiellement de silicate tricalcique, possède des qualités physiques permettant son utilisation comme substitut dentinaire. Le premier objectif de notre travail a été d'évaluer les propriétés biologiques du Biodentine™ et ses interactions avec les cellules cibles en culture. La bioactivité du nouveau ciment a ensuite été étudiée à l'aide du modèle de culture de dents entières humaines ex vivo. Ce modèle expérimental original a été mis au point dans notre laboratoire et permet d'étudier les phases précoces de la régénération dentinaire lors du coiffage direct. Grâce à ce modèle, nous avons démontré l'activation, la prolifération et la migration de cellules progénitrices pulpaires périvasculaires en réponse à une lésion cavitaire profonde. Le coiffage direct avec les ciments de silicates tricalciques a induit la formation de foyers minéralisés à proximité de la lésion. La caractérisation moléculaire de ces foyers a montré qu'il s'agit d'une forme de dentine réparatrice synthétisée par des cellules odontoblast-like. Le deuxième objectif de notre travail a été d'étudier l'effet du nouveau biomatériau sur la sécrétion de certains facteurs de croissances, impliqués dans les phases précoces de la cicatrisation pulpaire, et de le comparer à celui d'autres matériaux de coiffage. / The objective of direct pulp capping is to stimulate the pulp healing potential and to induce dentin regeneration. This is of prime importance in critical pathologic situations compromising the tooth vitality. To improve the outcome of this treatment, the IMEB-ERT30 Laboratory, in collaboration with the SEPTODONT Company, has developed a new restorative material called Biodentine™. This cement, essentially composed of tricalcium silicates, has the required physical properties to be used as a dentin substitute. The first aim of this work was to evaluate the biological properties of Biodentine™ and its interactions with the target cells in cell culture. Then, the bioactivity of the new cement was studied using an entire human tooth culture model ex vivo. This original experimental model, developed in our Laboratory, is suitable in studying the early steps of dentin regeneration after direct pulp capping. With this model, we demonstrated the activation, proliferation, and migration of perivascular pulpal progenitor cells in response to pulp injury. The direct pulp capping with tricalcium silicate cements induced mineral foci formation in the vicinity of the pulp lesion. Molecular characterization of these foci confirmed it was of a reparative dentin type produced by odontoblast-like cells. The second objective of our work was to study the effect of the new cement on the secretion of some growth factors involved in the early steps of pulp wound healing, and compare it to that of other pulp capping materials. The results demonstrated an up-regulation of b-FGF, VEGF and PDGF-AB secretion in response to the target cells injuries, suggesting a stimulation of angiogenesis.

Este estudo translacional avaliou o efeito da aplicação direta de materiais capeadores à base de óleo de copaíba sobre a polpa dentária de ratos. O biomaterial foi analisado de acordo com os processos inflamatórios, reparadores e regenerativos. Cavidades classe I foram preparadas na face oclusal dos molares superiores e inferiores de ratos da linhagem Wistar (n=120). Posteriormente, uma cavidade padronizada foi realizada com broca carbide esférica ¼. Exposições pulpares foram obtidas e após a hemostasia com algodão estéril e água destilada, foi realizado o capeamento pulpar com aplicação de biomateriais diretamente no tecido pulpar. Os biomateriais utilizados foram: COP (Óleo de Copaíba); Ca(OH)2 (hidróxido de cálcio), Ca(OH)2+COP; MTA (agregado trióxido mineral); MTA+COP; BIODENTINA (Biodentine®); BIODENTINA+COP. Todas as cavidades foram restauradas com cimento provisório. Os animais foram sacrificados após o período de 7, 14, 28 dias de acordo com os princípios de ética e experimentação animal. Vinte e um grupos foram criados a partir da combinação de tempos e materiais, cada grupo foi constituído de 5 animais. As peças foram preparadas e processadas pela técnica histológica e coradas pela HE (hemotoxilina e eosina) realizando cortes aleatórios, sendo analisados ao microscópio óptico em 100X de aumento por 2 examinadores. Os critérios avaliados foram: respostas inflamatória e degenerativa, organização tecidual, dentina reacional e reparativa. No último tempo experimental (28 dias) de todos os grupos foi analisado a superfície (mm2) e o volume (mm3) da ponte de dentina formada pela técnica da micro-CT (microtomografia computadorizada) de alta resolução. Os dados foram estatisticamente analisados utilizando o teste T pareado, determinando a superfície e volume da dentina reparadora formada entre os grupos experimentais. Nas análises histológicas foi utilizado o teste não-paramétrico de Mann-Whitney realizando comparações entre os grupos (?=5%). A BIODENTINA demonstrou uma tendência de acelerar o processo de regeneração pulpar, iniciando a deposição de dentina reparativa ao sétimo dia do capeamento. Este material apresentou uma dentina mais espessa em comparação com os outros biomateriais. O MTA também demonstrou uma eficiência na formação da ponte dentinária, entretanto em 20% das amostras não foi observada a presença de dentina reparadora. Ao acrescentar o COP nestes materiais, a formação da ponte dentinária apresentava heterogênea e incompleta, além disso esses grupos continham um processo inflamatório aumentado. A dentina reparadora do grupo de Ca(OH)2 demonstrou presença de túnel, com característica heterogênea. A adição do COP ao Ca(OH)2 aumentou a formação de dentina nas paredes ao redor da câmara pulpar em 80% das amostras, porém esta dentina não apresentava melhora na qualidade. Desta forma, o capeamento pulpar com COP isolado ou associado a Ca(OH)2, MTA e BIODENTINA não formou uma ponte de dentina completa e homogênea no local da exposição pulpar. / This translational study evaluated the effect of the direct application of copaiba oil based capping materials on the rat pulp. The biomaterial was analyzed according to inflammatory, repairing and regenerative processes. Class I cavities were prepared on the occlusal surface of the upper and lower molars of Wistar rats (n = 120). Subsequently, standardized cavities was performed with ¼ spherical carbide drill. Pulp exposures were obtained and after their hemostasis with sterile cotton and distilled water, pulp capping was done with application of biomaterials directly on the pulp tissue. The biomaterials used were: COP (Copaíba oil); Ca(OH)2 (calcium hydroxide), Ca(OH)2+COP; MTA (aggregate mineral trioxide); MTA+COP; BIODENTINE (Biodentine®); BIODENTINA+COP. All cavities were restored with temporary cement. The animals were sacrificed after the period of 7, 14, 28 days according to the principles of ethics and animal experimentation. Twenty-one groups were created from the combination of times and materials, each group consisted of 5 animals. The specimens were prepared and processed by histological technique and stained by HE (haemotoxin and eosin); randomized cuts were examined under optical microscope with 100X enlargement by two different examiners. The evaluated criteria were inflammatory and degenerative response, tissue organization, reactive and reparative dentin. In the last experimental period (28 days) of all groups, the surface (mm2) and the volume (mm3) of the reactive and reparative dentin formed were analyzed by the high-resolution micro-CT (computerized microtomography) technique. The data were statistically analyzed using the paired T-test, determining the surface and volume of the reparative dentin formed between the experimental groups. In the histological analyzes, the non-parametric Mann-Whitney test was used, comparing groups (? = 5%). BIODENTINE demonstrated a tendency to accelerate the process of pulpal regeneration, initiating the deposition of reparative dentin on the seventh day of the capping. This material had a thicker dentin compared to the other biomaterials. The MTA also demonstrated an efficiency in the formation of the dentin bridge; however, 20% of the samples did not observe the presence of reparative dentin. When adding the COP in these materials, the formation of the dentin bridge was heterogeneous and incomplete, in addition these groups contained an increased inflammatory process. The repair dentin of the Ca(OH)2 group showed a tunnel with a heterogeneous characteristic. The addition of COP to Ca(OH)2 increased dentin formation in the walls around the pulp chamber in 80% of the samples, but this dentin did not show improvement in quality. Thus, pulp cap with COP isolated or associated with Ca(OH)2, MTA and BIODENTINE did not form a complete and homogeneous dentin bridge at the site of pulp exposure.

Cavalcanti, BN. Resposta de fibroblastos de polpa humanos frente a substâncias liberadas por capeadores pulpares diretos [Tese de Doutorado]. São Paulo: Faculdade de Odontologia da USP; 2003. RESUMO O objetivo do presente estudo foi o de avaliar os efeitos citotóxicos de substâncias liberadas durante a aplicação de materiais utilizados em capeamento pulpar direto, sobre fibroblastos de polpa dentária humana. Utilizou-se para o experimento meios condicionados pelas substâncias a serem testadas, divididas nos grupos a seguir: grupo I: controle (meio de cultivo sem condicionamento); grupo II: cimento de hidróxido de cálcio; grupo III: adesivo dentinário; grupo IV: ácido ortofosfórico a 37%. O condicionamento foi realizado, colocando-se meio de cultivo fresco sobre os materiais de modo que a presa (grupo II), polimerização (grupo III) ou o contato direto (grupo IV) liberassem substâncias para esse meio de cultivo. Esse meio era colocado sobre as células durante todo o experimento, excetuando-se o grupo IV, onde o contato foi feito por um período de 15 segundos, conforme recomendações clínicas. Posteriormente foram realizadas contagens em hemocitômetro pelo método de exclusão por azul de Trypan, que cora somente as células mortas. As contagens foram realizadas em períodos de 0, 6, 12 e 24 horas para o experimento de viabilidade celular (curto prazo), onde se avaliou o percentual de células vivas sobre o total de células, e em períodos de 1, 3, 5 e 7 dias para o experimento de sobrevivência celular, no qual se avaliou o número absoluto de células vivas. Observou-se que as substâncias liberadas pelo adesivo dentinário são citotóxicas em qualquer período, diminuindo consideravelmente a viabilidade celular e afetando suas curvas de crescimento. Aquelas liberadas pelo ácido ortofosfórico a 37% provocam diminuição da viabilidade somente nos primeiros momentos do contato com as células, enquanto as substâncias liberadas durante a presa do hidróxido de cálcio não são citotóxicas em nenhum momento. / The purpose of the present study was to evaluate the cytotoxic effects of substances leached during the use of direct pulp capping materials, on human pulp fibroblasts. There were used cell culture mediums conditioned by the test materials, as follows: group I: control (fresh medium without conditioning); group II: calcium hydroxide cement; group III: bonding system; group IV: 37% orthophosphoric acid. The medium conditioning was made, pouring the fresh conditioning medium on the materials, in order that its setting (group II), polymerization (group III) or the direct contact (group IV) would be able to leach substances to this culture medium. These conditioned mediums were put on the cells for the entire experiment, excepting the group IV, in which the mediums were put in contact with the cells for 15 seconds, following clinical recommendations. Cell counting was performed in hemocytometer, using the Trypan blue exclusion method, which mark only the dead cells. These counting was made at experimental times of 0, 6, 12 and 24 hours for the cell viability assay (short term), where it is evaluated the percentage of live cells on the total number of cells, and at experimental times of 1, 3, 5 and 7 days for the survival assay, in which is evaluated the absolute number of live cells. It was observed that the substances leached by the bonding system are cytotoxic at all experimental times, decreasing significantly the cell viability and affecting its growing rate. Those leached by the 37% orthophosphoric acid decreased the cell viability only at the first contact with the cells, and the substances leached during the setting of the calcium hydroxide cement are not cytotoxic.

Deponering är globalt sett det vanligaste sättet att hantera avfall, men i Sverige får sedan 2001 enbart avfall som inte kan återanvändas deponeras. Innan deponiförordningen trädde i kraft 2001 deponerades många olika typer av avfall. En deponi används idag som ett slutförvar för avfall och för att minimera infiltration av regnvatten och eventuell föroreningsspridning sluttäcks gamla deponier. Sluttäckningen består av flera skikt som tillsammans ska täta deponin och leda bort vatten. Att förvara avfall i en sluttäckt deponi är miljöfarlig verksamhet och det är därför av intresse att kontrollera omgivningarna under och efter sluttäckning för att säkerställa att eventuell föroreningsspridning minskar. På Hovgårdens avfallsanläggning i Uppsala kommun har en deponi som använts sedan 1971 sluttäckts under hösten 2018. I två provtagningsrör vid deponin, i vilka provtagning och analys skett regelbundet sedan 1991, har förhöjda halter av vissa ämnen kunnat uppmätas efter att arbetet med sluttäckningen påbörjades. Syftet med detta examensarbete var att få ökad förståelse för trender i ämneshalter samt att utreda hur sluttäckning påverkar grundvattennivåer och ämneskoncentrationer runt deponin. Uppmätta halter i provtagningsrören sammanställdes och trender analyserades med statistiska verktyg i mjukvaran R. Dessutom konstruerades en grundvattenmodell över avfallsanläggningen i MODFLOW och MT3DMS. Stationära flödessimuleringar och transportsimuleringar med klorid utfördes. Tolv av 33 analyserade ämnen och parametrar uppvisar statistiskt signifikanta ökningar i antingen ett av provtagningsrören, båda eller båda analyserade tillsammans. Dessa är alkalinitet, elektrisk konduktivitet, sulfat, klorid, kalcium, natrium, svavel, kalium, magnesium, strontium, barium, och nitratkväve. Sex av dem minskar statistiskt signifikant och dessa är pH-värdet, kadmium, zink, kvicksilver, bly och arsenik. Grundvattenmodellen visar att grundvattnet under etapp 1 flödar mot resten av anläggningen men att flöden ut från deponin finns i de sydvästra och sydöstra hörnen. Sluttäckningen leder till lägre nivåer och en förändrad grundvattendelare vilket kommer minska läckaget, främst i den sydvästra delen. Modellsimuleringar med olika grundvattenbildningsmängder och kloridkoncentrationer visade att ökningar av kloridhalter inte kan förklaras med minskad grundvattenbildning i kombination med förhöjda koncentrationer. Förklaringen bör därmed vara andra fysikaliska och eventuellt kemiska processer som skett samtidigt som sluttäckningen. De parametrar som påverkade simulerade ämneshalter mest var konduktansen i diken och dränering samt moränens hydrauliska konduktivitet. / Landfills are globally the most common waste treatment method but in Sweden the method is since 2001 used only for waste that cannot be reused in any way. Before 2001, almost any type of waste could be landfilled. Today a landfill is used as a final storage and old landfills are capped to minimize infiltration and contaminant transport. A capping consists of several layers which together seal and drain the landfill surface. Storing waste in a capped landfill is considered an environmentally hazardous activity, therefore it is of great interest to monitor the surrounding groundwater during and after capping to make sure that any contaminant concentrations decreases. At Hovgården waste treatment plant in Uppsala, Sweden, a landfill used since 1971 was capped during 2018. Elevated contaminant concentrations have been measured in two monitoring wells close to the landfill after the capping procedure started. The aim of this master thesis was to increase the understanding of trends in contaminant concentrations and to investigate how capping affects groundwater levels and concentrations. Concentration data were compiled and statistical tools in R were used to analyze trends. Additionally, a groundwater model of the area was created in MODFLOW and MT3DMS. The model was used to simulate different steady state scenarios with and without chloride transport. Twelve of 33 analyzed substances and parameters show a statistically significant increase. These are alkalinity, electrical conductance, sulphate, chloride, calcium, sodium, sulfur, potassium, magnesium, strontium, barium and nitrate. Six show a decrease, these are pH, cadmium, zinc, mercury, lead and arsenic. The groundwater model indicates that the groundwater flow in the landfill is directed towards the rest of the waste treatment plant except for two places where leakage occurs: the southwest and southeast corners of the landfill. The capping results in lower groundwater levels and an altered groundwater divide, which will reduce the contaminant leakage, particularly in the southwest part. Model simulations with varying recharge and chloride concentrations showed that the increase in chloride levels cannot be explained with only decreased infiltration combined with increased concentrations. Therefore, the explanation is assumed to be other physical processes and possibly chemical reactions due to or at the same time as the capping. The model parameters that effected the simulated concentrations to the greatest extent are the conductances in drains, and the hydraulic conductivity of the till soil.

The mRNA cap added to the 5’ end of nascent transcripts is required for the efficient gene expression in eukaryotes. In vertebrates, the guanosine cap is methylated at N7 position by RNMT, which is in complex with its activating subunit RAM. Additionally, the first and second transcribed nucleotides can be methylated at ribose O2 position by CMTR1 and CMTR2 respectively. The mRNA cap protects transcripts from degradation and recruits cap-binding factors to promote pre-mRNA processing, nuclear export and translation initiation. In mouse embryonic stem cells (mESCs), high levels of RAM maintain expression of pluripotency factors. Differentiation of mESCs to neural progenitors is accompanied by a suppression of RAM, resulting in downregulation of pluripotency factors and efficient formation of neural cells. Here, I demonstrated that the suppression of RAM during neural differentiation is promoted via ubiquitination and proteasomal degradation. Concurrently, neural differentiation is associated with an increase in CMTR1 expression, creating a developmental cap methyltransferase switch. Moreover, differentiation into endodermal and mesodermal lineages exhibited distinct changes in the mRNA capping enzymes expression. In mESCs, RAM promotes expression of translation-associated proteins and promotes global loading of mRNA on ribosomes. RAM contributes to the ESC-specific gene expression program, by maintaining optimal expression of pluripotency-associated transcripts and inhibiting expression of neural genes. Chromatin immunoprecipitation revealed that RAM, RNMT and CMTR1 promote binding of RNA polymerase II at gene loci. In RAM-repressed cells, RNA polymerase II binding was reduced at pluripotency-associated genes, but relatively increased at neural genes. Moreover, knock-down of RNMT or CMTR1 induced increased or decreased accumulation of RNA polymerase II at promoter proximal regions respectively. In naïve T cells, Rnmt or Cmtr1 conditional knock-outs caused downregulation of translation-related transcripts and upregulation of cell cycle transcripts. Furthermore, many transcripts were specifically dependent on RNMT or CMTR1 for expression, demonstrating distinct roles of these cap methyltransferases. Thus, the mRNA cap methylation emerges as an important regulator of pluripotency and differentiation, modulating gene expression at transcriptional and post-transcriptional levels.

In the design of rail track structures where the subgrade cannot achieve the desired capacity, enabling the required standard of track geometry to be maintained for the speed, axle load and tonnage to be hauled, a capping layer of granular material is placed between the natural ground or the embankment fill material and the ballast to protect the underlying weaker layers. In spite of the important role played by the capping layer, very little research has been carried out on its performance. The current practice of design of the capping layer, therefore, is based on working stress philosophy where reduced levels of stresses are assumed not to degrade the subgrade. Even on tracks containing a thick ballast layer that ensures allowable levels of working stress the subgrade has been found to have permanently deformed. Design of capping layers based on plastic deformation, therefore, appears appropriate. This thesis aims at determining the load levels that cause detrimental plastic deformation in the capping layer. The suite of material properties that characterise plastic deformations of capping layer is neither readily available nor easily determined. This thesis proposes a cheaper method of evaluating a range of capping layer material properties using penetration tests on specimens contained in California Bearing Ratio (CBR) test moulds coupled with a finite element modelling based back calculation technique. The suite of material properties thus determined are used for the simulation of the behaviour of capping layers under the boundary and loading conditions similar to those in practice. The predicted results are validated using laboratory experiments on large size capping layer specimens.

Unconventional nanofabrication techniques; both those which have been newly developed and those under development, had brought inexpensive, facile, yet high quality means to fabricate nanostructures that have feature sizes of less than 100 nm in industry and academia. This dissertation focuses on developing unconventional fabrication techniques, building studying platforms, and studying the mechanisms behind them. The studies are divided into two main facets and four chapters. The first facet, in Chapter II and Chapter III, deals with the research and development of different nanofabrication techniques and nanostructures. These techniques include litho-synthesis, colloidal lithography, and photolithography. The nanostructures that were fabricated by these techniques include the metal nanoparticle arrays, and the self-assembled CdSe nanoring arrays. At the same time, the dissertation provides mechanisms and models to describe the physical and chemical nature of these techniques. The second area of this study, in Chapter III to Chapter V, presents the applications of these nanostructures in fundamental studies, i.e. the mechanisms of plasmon enhanced fluorescence and photo-oxidation kinetics of CdSe quantum dots, and applications such as molecular sensing and material fabrication. More specifically, these applications include tuning the optical properties of CdSe quantum dots, biomodification of CdSe quantum dots, and copper ion detection using plasmon and photo enhanced CdSe quantum dots. We have successfully accomplished our research goals in this dissertation. Firstly, we were able to tune the emission wavelength of quantum dots, blue-shifted for up to 45 nm, and their surface functionalization with photo-oxidation. A kinetic model to calculate the photo-oxidation rates was established. Secondly, we established a simple mathematical model to explain the mechanism of plasmon enhanced fluoresce of quantum dots. Our calculation and experimental data support the fluorescence resonance energy transfer (FRET) mechanism between quantum dots and the metal nanoparticles. Thirdly, we successfully pattered the CdSe quantum dots (diameter ~4 nm) into nanorings with tunable diameters and annular sizes on different substrates. We also established a physical model to quantitatively explain the mechanism with the forces that involved in the formation of the nanorings.

Rinderpest virus (RPV) belongs to the order Mononegavirale which comprises non segmented negative sense RNA viruses including human pathogens such as Measles, Ebola and Marburg virus. RPV is the causative agent of Rinderpest disease in large ruminants, both domesticated and wild. The viral genome contains a non segmented negative sense RNA encapsidated by nucleocapsid protein (N-RNA). Viral transcription/replication is carried out by the virus encoded RNA dependent RNA polymerase represented by the large protein L and phosphoprotein P as (L-P) complex. Viral transcription begins at the 3’ end of the genome 3’le-N-P-M-F-H-N-tr-5’ with the synthesis of 55nt leader RNA followed by the synthesis of other viral mRNAs. A remarkable feature common to all members of Paramyxoviridae family is the gradient of transcription from 3’ end to the 5’ end of the genome due to attenuation of polymerase transcription at each gene junction. The present study aims at functional characterization of Rinderpest virus transcription and the associated activities required for viral mRNA capping. In addition, an attempt has been made to understand the novel role of a host factor, Ebp1, playing a key role in virus multiplication in infected cells. The specific aims of the study are presented in detail below. 1. Development of in vitro transcription system for RPV mRNA synthesis and role of phosphorylation of P protein in transcription. The transition of viral polymerase from transcription to replication in infected cells has been a long standing puzzle in all paramyxoviruses. Earlier work carried out using RPV minigenome with a CAT reporter gene and studies with phosphorylation null mutant P, has revealed the importance of P phosphorylation for viral transcription in vivo. However, the contribution of other cellular factors in the viral transcription/replication switch could not be ruled out in these assays. In order to understand the specific role of P protein in transcription/replication, it was necessary to develop a cell free transcription system for viral mRNA synthesis. Hence, viral genomic RNA (N-RNA) was purified from RPV infected cells using CsCl density gradient centrifugation. The viral RNA polymerase consisting of L-P complex was separately expressed in insect cells and partially purified by glycerol gradient centrifugation. Glycerol gradient fraction containing the L-P complex was found to be active in viral transcription. Notably, the gradient of transcription of viral mRNA was observed in vitro with the partially purified recombinant L-P complex similar to in vivo. However, the recombinant polymerase complex failed to synthesis the 55nt leader RNA, in agreement with the recent finding in VSV that the transcriptase complex was unable to synthesize leader RNA and viral transcription is initiated at the N gene start site unlike the conventional 3’ entry mode. The newly developed in vitro reconstituted transcription system was used to analyze the effect of P phosphorylation on viral transcription. The results presented in chapter 2, indicate that phosphorylated P supports transcription whereas unphosphorylated P transdominantly inhibits the transcription in vitro suggesting the possible role of the status of P protein phosphorylation in determining transcription/replication switch. 2. Enzymatic activities associated with RPV L protein- role in viral mRNA capping. Post transcriptional modification of mRNA such as capping and methylation determines the translatability of viral mRNA by cellular ribosome. In negative sense RNA viruses, synthesis of viral mRNA is carried out by the viral encoded RNA polymerase in the host cell cytoplasm. Since the host capping and methylation machinery is localized to the nucleus, viruses should either encode their own mRNA modification enzymes or adopt alternative methods as has been reported for orthomyxoviruses (cap snatching) and picornaviruses (presence of IRES element). In order to test, if RPV RNA polymerase possesses any of the capping and methylation activities, both virus as well as the RNP complex containing the viral N-RNA and RNA polymerase (L-P) were purified from infected cells. Using the purified virus and RNP complex, the first two activities required for mRNA capping vis-à-vis, RNA triphosphatase and guanylyltransferase were tested and the results are described in chapter 3 and 4. Purified virus as well as the RNP complex showed both RNA triphosphatase (RTPase) and Nucleotide triphosphatase activities. Neither purified N-RNA or recombinant P proteins show these activities suggesting that it is indeed mediated by viral L protein. By the metal dependency of the reaction and by the motif conservation with other reported RTPases, RPV L protein was assigned to the metal dependent RTPase tunnel family. Capping activity was also seen with the L protein present in RNP complex by its ability to form a covalent complex with GMP moiety of GTP. The specificity of the reaction with GTP, inhibition of Enzyme-GMP complex formation by the inorganic pyrophosphate and the susceptibility of Enzyme-GMP complex under acidic conditions clearly indicated that RPV L represents the viral guanylyl transferase. Further confirmation was obtained by the indirect capping assay in which Enzyme-GMP complex was formed when recombinant L protein was incubated with the cap labeled RNA due to the reversible nature of capping reaction. Owing to the large size of L protein (240 KDa), it is conceivable that the L protein functions in a modular fashion for different activities pertaining to RNA synthesis and modification. Sequence comparison of L proteins from different morbilliviruses revealed the presence of three conserved domains namely domain I (aa 1-606), domain II (aa 650-1694) and domain III (aa 1717-2183). Since domain II has already been assigned as the viral RNA dependent RNA polymerase, domain I and domain III were chosen for further characterization. Both domains were cloned, expressed and purified to homogeneity using recombinant baculovirus expression system. However, the recombinant domain III alone showed the NTPase activity where as neither domain I or III showed RTPase activity. This is expected since a part of the conserved RTPase motif was located in domain II in the multiple sequence alignment with other viral and yeast RTPases. In addition, the recombinant domain III also showed the characteristic enzyme-GMP complex formation but failed to be active in the indirect capping assay. Therefore, both domain II and domain III are likely to be involved in the co-transcriptional capping of viral mRNA. In support of this view, recent report in VSV suggests the presence of additional motif in domain II which is essential for viral mRNA capping. Preliminary evidence has been presented in the appendix section for the presence of N7 guanine methyl transferase activity with L protein although further experiments are needed to confirm this activity. 3. Role of host factor Ebp1 in negative sense RNA virus replication - a possible antagonist In recent years, many cellular factors such as actin, tubulin and profilin have been shown to be involved in viral transcription. Ebp1-ErbB3 binding protein was initially isolated as a cellular protein which binds to Influenza viral polymerase subunit PB1. Ebp1 selectively inhibits the influenza virus transcription in vitro whereas the cap binding and endonuclease activity of PB1 subunit of viral polymerase is unaffected. Till now there are no reports of the role of Ebp1 in non segmented negative sense RNA virus infection. The fifth chapter describes the role of Ebp1 in RPV infection and vice versa. RPV infection leads to down regulation of Ebp1 mRNA levels which in turn leads to decreased protein synthesis. Subsequently, it was found that Ebp1 interacts presumably with viral N protein, being a part of the viral RNP complex in both infected cells as well as in purified virion. Further, over expression of Ebp1 inhibits viral transcription and as a consequence the virus multiplication in vivo suggesting a mutual antagonism between virus and the host cell through Ebp1 protein.

Hydrophobic organic contaminants (HOCs) often reside in sediments sorbed to particles, most tightly to particles with high content of organic carbon. If persistent, such pollutants can accumulate in the sediment for many years and constitute a contamination risk for sediment-living organisms and organisms at higher trophic levels, including humans. Since traditional remediation techniques are associated with complications (e.g. release of contaminants during dredging operations, disturbance of benthic faunal communities), or constraints (handling of large amounts of contaminated sediment and water, limitations due to depth and size of the area, high costs), there is a need for new alternative methods. In situ remediation through thin-layer capping (a few centimeter cover) with a sorbing material such as activated carbon (AC) has been proposed as an alternative remediation method. Compared to traditional remediation techniques, AC amendment in a thin layer means less material handling and lower costs and is assumed to be less disruptive to benthic communities. The objectives of this thesis were to investigate the ecological effects from thin layer capping as well as the efficiency in contaminant retention. Thin layer capping amended with AC proved to reduce availability of HOCs to the tested organisms, the gastropod Nassarius nitidus (Paper II), the clam Abra nitida (Paper III) and to polychaete worms  (Paper II and III). The remediation technique also decreased the sediment-to-water fluxes of the contaminants (Paper II and III). However, AC amended thin-layer capping was also found to cause negative biological effects. In laboratory studies with only a few species the negative effects were minor, or difficult to discern with the endpoints used (Paper II and III). In a larger multi-species mesocosm (boxcore) study, on the other hand, the negative effects were more prominent (Paper I) and in a large scale field study the benthic community was found to be profoundly disturbed by the AC amendment, with the effects persisting or even worsening ca one year (14 months) post amendment (Paper IV). / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 3: Manuscript; Paper 4: Manuscript.</p> / Carbocap / Opticap / Thinc