Single-particle tracking is a technique that allows for quantitative analysis of the localization and movement of particles. In this technique, trajectories are constructed by determining and connecting the positions of individual particles from consecutive images. Recent advances have made it possible to track hundreds of particles in an individual cell by labeling the particles of interest with photoactivatable or photoconvertible fluorescent proteins and tracking one or a few at a time. Single-particle tracking can be used to study the diffusion of particles. Here, we use intracellular single-particle tracking and trajectory simulations to study the diffusion of the fluorescent protein mEos2 in living Escherichia coli cells. Our data are consistent with a simple model in which mEos2 diffuses normally at 13 µm2 s−1 in the E. coli cytoplasm. Our approach can be used to study the diffusion of intracellular particles that can be labeled with mEos2 and are present at high copy numbers. Single-particle tracking can also be used to determine whether an individual particle is bound or free if the free particle diffuses significantly faster than its binding targets and remains bound or free for a long time. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that, unlike bound subunits, free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with co-transcriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

No description available.

[Truncated abstract] This study was carried out to investigate the teaching and learning processes occurring in the topic of reaction mechanisms in three tertiary level organic chemistry courses and focussed on investigating perceptions about the importance of teaching and learning about reaction mechanisms and about the difficult aspects of the topic .... In the organic chemistry courses under investigation, students achieved many of the explicitly stated aims that their lecturers identified. The students rarely achieved implicit outcomes anticipated by the lecturer. Lecturers demonstrate a tendency to use particular structural representations when discussing certain types of reaction process. The study identified that students commonly use these same types when working through particular reaction processes. In addition, it was found that the use of a particular structure could cue students into thinking about only one type of reaction process taking place in a given reaction. The use of language that is consistent with a consideration of only single reaction particles was also commonly observed in lectures. While this can be adequate in some circumstances, other aspects of reaction processes are better considered in terms of multiple reaction particles ... The project proposes an integrated model, which takes into account the many levels (macroscopic, single particle molecular, multiple particle molecular and intramolecular) involved when describing reaction processes. It is felt that a consideration of the levels discussed in this model is useful when teaching and learning about reaction mechanisms.

Septinas são GTPases consideradas como um novo componente do citoesqueleto. Essas proteínas interagem entre si para formar heterocomplexos filamentosos e estruturas de alta ordem que são importantes para a citocinese e uma variedade de outros processos celulares. Existem muitos aspectos mecânicos dessas proteínas que não são totalmente compreendidos, incluindo a forma como os heterocomplexos se agrupam corretamente. Em humanos, há 13 genes que codificam septinas, classificadas em quatro grupos quanto à similaridade em relação à estrutura primária. O complexo hexamérico SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 foi o melhor caracterizado, com uma estrutura cristalina resolvida à 4 Å. Segundo às Regras de Kinoshita, as septinas desse complexo podem ser substituídas nesse arranjo por outras pertencentes ao mesmo grupo. Neste trabalho utilizamos a técnica de microscopia eletrônica de transmissão com análise de partícula única para estudar dois complexos de septinas. Um dos complexos estudados neste projeto é formado por septinas humanas, para as quais atualmente não há informações estruturais disponíveis. O complexo SEPT5-SEPT6-SEPT7 foi expresso heterólogamente em E. coli e purificado em alta concentração salina para evitar a polimerização. A análise de partículas únicas de imagens por contrastação negativa mostrou a presença de partículas alongadas de aproximadamente 25 nm de comprimento, compostos por seis monômeros, como esperado. Com o objetivo de localizar a posição da SEPT5 no complexo, foi realizada uma fusão com MBP (Maltose Binding Protein) e imunomarcação com anticorpo monoclonal anti-SEPT5, concluindo que a SEPT5 está localizada na extremidade do complexo hexamérico. Porém, a SEPT5 pertence ao mesmo grupo da SEPT2, que foi relatada estar localizada no centro do hexâmero. Este resultado possibilitou uma nova discussão sobre a maneira que as septinas formam os complexos e, como a sensibilidade à concentração salina está relacionada com a fragilidade da interface NC, análogo ao observado em complexos de Saccharomyces cerevisiae. Um complexo de Ciona intestinalis incluindo a SEPT2, SEPT6, SEPT7 e SEPT9, também expresso heterólogamente em E. coli, foi preparado por contrastação negativa. A análise de partícula única das imagens coletadas mostrou um heterocomplexo aparentemente hexamérico, embora fosse esperado um octâmero devido à presença das quatro septinas diferentes, sendo uma pertencente à cada um dos quatro grupos. Os resultados deste trabalho proporcionaram um avanço na compreensão da formação de heterocomplexos de septinas e como essas proteínas interagem umas com as outras nesta montagem. / Septins are GTPases that appear to be a novel component of the cytoskeleton. These proteins interact with each other to form filamentous heterocomplexes and high order structures which are important for cytokinesis and a variety of other cellular processes. There are many mechanistic aspects of these proteins that are not fully understood, including how the heterocomplexes correctly assemble. In humans, there are 13 genes encoding septins, classified in four groups based on primary structure. The SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 hexameric complex was the best characterized, with a crystalline structure solved at 4 Å. According to Kinoshita´s Rules, the septins of this complex can be replaced in this arrangement by others belonging to the same group. In this work, we used transmission electron microscopy with single particle analysis to study two septin complexes. One of the complexes studied in this project is composed of three human septins, for which there is currently no structural information available. The SEPT5-SEPT6-SEPT7 complex was heterologously expressed in E. coli and purified at high salt concentration to avoid polymerization. Single particle analysis of negatively stained samples showed the presence of elongated particles of approximately 25 nm in length. To locate SEPT5 in the complex, a fusion with MBP (Maltose Binding Protein) and immunoblotting with anti-SEPT5 monoclonal antibody was performed, concluding that SEPT5 is located at the end of the hexameric complex. However, SEPT5 belongs to the same group as SEPT2, which was reported to be located in the center of the hexamer. This result allowed for a new discussion on the way that septins form heterocomplexes and also, on how the sensitivity of the NC interface in related to salt concentration, analogous to that observed in the heterocomplex of Saccharomyces cerevisiae. A Ciona intestinalis complex including SEPT2, SEPT6, SEPT7 and SEPT9, also expressed heterologously in E. coli, was prepared by negative staining. The single particle analysis of the collected images showed an apparently hexameric heterocomplex, although an octamer was expected due to the presence of the four different septins, one belonging to each of the four groups. The results of this work represent advances in the understanding of the formation of septin heterocomplexes and how these proteins interact with each other during assembly.

The determination of the isotopic composition of single uranium oxide particles, size 0.3-2 μm, for nuclear safeguards is current performed by either thermal ionisation mass spectrometry (TIMS) or Secondary Ion Mass Spectrometry (SIMS). Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS), a well-established analytical technique for determining the isotopic composition of solid materials, has the potential to be another method by which single uranium oxide particles can be analysed, complementing established protocol, but requires optimisation. In this study the ability of LA-ICP-MS to determine the isotopic composition, principally 234U/238U, 235U/238U and 236U/238U, of glass reference materials and sub-micron uranium oxide particles is investigated. To achieve the best detection efficiency a prototype high-speed ablation cell and injector design, designed previously at Loughborough University, was coupled to a high efficiency multi collector (MC-) ICP-MS. As a result an increase in signal-to-noise ratio and a measured detection efficiency of 5-7% was achieved for a LA-MC-ICP-MS system. The capability of the LA-MC-ICP-MS system, for the determination of the uranium isotopic composition of single particles was compared to a more established low-volume ablation cell. A source of additional uncertainty, blind time arising from incompatibilities with the mixed detector array of the MC-ICP-MS was identified. The impact of the additional uncertainty on isotope ratio analysis was modeled and a method developed to filter out affected data. LA-ICP-MS and LA-MC-ICP-MS were used to successfully determine the uranium isotopic compositions of sub-micron uranium oxide particles, of a known certified composition. A sample planchet containing particles of two distinct isotopic compositions was resolved. The utility of three data evaluation strategies to determine the isotopic composition of single uranium oxide particles was investigated. The necessity and advantages of calculating isotope ratios using the geometric mean is demonstrated, which has application for isotope ratio analysis performed on all forms of mass spectrometry. A novel approach to prepare particulate samples for laser ablation analysis, cytocentrifugation, is described. By using as the solvent, a mixture of nail polish and acetone, dispersed particles are held in a strong film layer thin enough to allow embedded particles to be imaged by SEM-EDX. A sample of uranium oxide particles in an environmental matrix prepared using cytocentrifugation is analysed by LA-MC-ICP-MS and their isotopic composition resolved.

A systematic study of the neutron single-particle states in N=51 isotones has been performed. Final states in 89Sr, 91Zr and 93Mo have been populated using the (d,p) and (alpha,3He) single-neutron adding reactions. The Yale tandem provided beams of 15-MeV deuterons and 50-MeV alpha particles with the outgoing ejectiles momentum analysed using an Enge split-pole spectrometer. A supplementary measurement of the d(86Kr,p)87Kr reaction, at a beam energy of 10 MeV/u, was made in inverse kinematics using the HELIOS spectrometer. Outgoing protons execute a helical orbit in a uniform field before detection, before they return to axis, using a position-sensitive silicon array. Absolute cross sections were measured for states up to 4 MeV in excitation. The transferred angular momentum was identified through a comparison of angular distributions and the ratio of cross section between reactions. Relative spectroscopic factors were extracted through a DWBA analysis.The measured centroids for the single-particle energies of the s1/2, d3/2, d5/2, g7/2 and h11/2 orbitals were compared to calculations using a Gaussian central potential with and without the addition of a tensor interaction. Through this comparison it was deduced that the inclusion of a tensor interaction is required to explain the observed shifts in the single-particle energies of the d3/2 and g7/2 orbitals. This system provided an observation of a switch in behaviour, from repulsive to attractive and vice versa, of the effect of the tensor interaction. This occurs due to a change from j> to j< proton occupancy at Z=40.

Galactic cosmic rays are the high-energy particles that stream into our solar system from distant corners of our Galaxy. The Earth's atmosphere serves as an ideal detector for the high energy cosmic rays which interact with the air molecule nuclei causing propagation of extensive air showers. The primary cosmic ray particles interact with the molecules in the atmosphere and produce showers of secondary particles (mainly pions) at about 15 km altitude. These pions decay into muons which are the dominant particles of radiation (about 80%) at the surface of the Earth. In recent years, there are growing interests in the applications of the cosmic ray measurements such as space/earth weather monitoring, homeland security activities based on the cosmic ray muon tomography, radiation effects on health via air travel, etc. A simulation program (based on the Geant4 software package developed at CERN) has been developed at Georgia State University for studying cosmic ray showers in the atmosphere. The results of this simulation study will provide unprecedented knowledge of geo-position-dependent cosmic ray shower profiles and will significantly advance cosmic ray applications. Simulation results are critically important for determining the temperature coefficients in every pressure layer in the atmosphere in order to calculate the temperature variations using the cosmic ray data. Using a single particle shower simulation, the weighted particle altitude distributions on a global scale are calculated with geomagnetic field implementation. The results of the simulation can aid the computation of the effective temperature in stratosphere.

This thesis is an experimental study of the fluidization of binary mixture in particulate flows. A fluidized bed with two distributors was built with water being used as carrying fluid. Three types of solid particles of nylon, glass and aluminum of the same size and different densities are used in the experiments. The wall effect on a single particle fluidization, the fluidization of binary mixture of large density difference (nylon and aluminum of density ratio of 0.42), and the fluidization of binary mixture of close density (glass and aluminum with density ratio of 0.91) were investigated. Also, the effect of distributors on mono-disperse and bi-disperse particle fluidization was investigated. Results show that the presence of narrow walls reduces the minimum fluidization velocity for a single particle by as much as nearly 40%. Also, in the case of binary mixture of close density particles, uniform mixing was easily achieved and no segregation was observed, but in the case of large density difference particles, there exists significant segregation and separation. At high velocity, the uniform distributor behaves like a transport bed. To achieve a full bed in the single jet, it requires 1.5 times velocity of the uniform distributor. This behavior determines their application in the industries.

Semester project deals with the processing of data from TEM and their analysis (to- mography, single particle analysis). The main aim of this work is to determine the 3D structure of the studied enzyme. As a test sample with low symmetry is used restriction endonuclease EcoR124I.

<p>The single particle model was extended to include electrode and particle volume expansion effects observed in high capacity alloying electrodes. The model was used to predict voltage profiles in sodium ion batteries with tin and tin-phosphide negative electrodes. It was seen that the profiles predicted by the modified model were significantly better than the classical model. A parametric study was done to understand the impact of properties such as particle radius, diffusivity, reaction rate etc on the performance of the electrode. The model was also modified for incorporating particles having a cylindrical morphology. For the same material properties, it was seen that cylindrical particles outperform spherical particles for large L/R values in the cylinder due to the diffusion limitations at low L/R ratios. A lattice spring-based degradation model was used to observe crack formation and creep relaxation within the particle. It was observed that the fraction of broken bonds increases with an increase in strain rate. At low strain rates, it was seen that there was a significant expansion in particle volumes due to creep deformation. This expansion helped release particle stresses subsequently reducing the amount of fracture.</p>