The method of dipole orientation of protein complexes using electric fields plays a key role in the development of single particle imaging, since it enables orientation of the protein in vacuum. In the orientation process the protein is exposed to an external electric field along which the dipole axis of the protein will eventually align. Earlier studies using molecular dynamics simulations have implemented a constant electric field to examine the dipole orientation process. However, when injected into the electric field the protein experiences a gradually increasing field strength converging to some terminal field strength rather than a constant electric field. In order to examine the effects of the time-dependant nature of the electric field, in comparison to a constant one, fields with different time dependances were implemented in molecular dynamics simulations in vacuum performed with GROMACS. Ubiquitin was chosen as a model protein. The results of the study show time-increasing fields tend to result in slower orientation, but preserve the structure of the protein better than for a constant field. It was also shown that after 10 ns electric field exposure, with terminal field strengths greater  or equal to 0.6Vnm^-1, there was no apparent difference of the average degree of orientation of proteins within the time-increasing fields and the constant one. However, for fields of greater or equal to 1.5Vnm^-1 the constant field tended to result in a larger change of the protein structure.

The aim of this study is the three-dimensional structural characterization of the genome packaging inside viral capsids via cryo-electron microscopy and three-dimensional reconstruction. The genome of some single-stranded viruses can be densely packaged within their capsid shells. Several stretches of the genome are known to adopt stable secondary structures, however, to date, little is known about the three-dimensional organization of the genome inside their capsid shells. Two techniques have been developed to facilitate the structural elucidation of genome packaging: the asymmetric random-model method, and the symmetry-mismatch, random model method. Both techniques were successfully tested with model and experimental data. The new algorithms were applied to study the genome structure of poliovirus and satellite tobacco mosaic virus. We have not yet found a consistent structure for the two genomes. Nevertheless, we have found that the genome of satellite tobacco mosaic genome is very stable, supporting a model where the RNA acts as a scaffold, with potential implications in capsid stability and assembly.

Nanoscale structures play a fundamental role in diverse scientific areas, including biology and information technology. It is necessary to develop methods that can observe nanoscale structures and dynamic processes that involve them. Colloidal plasmonic nanoparticles (plasmonic “atoms”) and their clusters (plasmonic “molecules”) are nanoscale objects with remarkable optical properties that provide new opportunities for sensing and imaging on the relevant length and time scales. Many biology questions require optically monitoring of the dynamic behavior of biological systems on single molecule level. In contrast to the commonly used fluorescent probes which have the problem of bleaching, blinking and relatively weak signals, plasmonic probes display superb brightness, persistency and photostability, thus enable long observation time and high temporal and spacial resolutions. When plasmonic atoms are clustered together, their resonances redshift while the intensities increase as a result of plasmon coupling. These optical responses are dependent on the interparticle gaps and the overall geometry, which makes plasmonic molecules capable of detecting biomolecule clustering and measuring nanometer scale distance fluctuations. In this dissertation, individual plasmonic atoms are firstly evaluated as imaging probe and their interactions with lipid membrane are tested on a newly developed on-chip black lipid membrane system. Subsequently, plasmonic dimers (plasmon rulers) prepared through DNA-programmed self-assembly are monitored to detect the mechanical properties of single biopolymers. Measurement of the spring constant of short (tens of nucleotides or base pairs) DNAs is demonstrated through plasmon coupling microscopy. Colloidal plasmonic atoms of various materials, sizes and shapes scatter vivid colors in the full-visible range. Assembling them into plasmonic molecules provides additional degrees of freedom for color manipulation. More importantly, the electric field in the gaps of plasmonic molecules can be enhanced by several orders of magnitude, which is highly desirable in single molecule sensing applications. In this dissertation, the fundamentals of plasmonic coupling are investigated through one-dimensional gold nanosphere chains. Using the directed self-assembly approach, multichromatic color-switchable plasmonic nanopixels composed of plasmonic atoms and molecules of various materials, sizes, shapes and geometries are integrated in one image with nanometer precision, which facilitates the encoding of complex spectral features with high relevance in security tagging and high density optical data storage. / 2017-01-01T00:00:00Z

Studies of the interaction of the relevant metal, metalloid or nanoparticulate species with biological systems are underpinned by the provision of reliable information about chemical composition of the relevant materials. Often, no methods of chemical analysis are available. The work described in this dissertation centers on developing methods to help with studies for a variety of analytes and samples. A method was developed for the determination of 11 trace elements (As, Cd, Co, Cr, Cu, Fe, Mg, Mn, Pb, Ti, and Zn) in human breast milk and infant formulas by inductively coupled plasma optical emission spectrometry (ICP-OES) following microwave-assisted digestion. A method was established for the determination of trace elements, with an emphasis on titanium as titanium dioxide, in snack foods and consumer products. The interactions of some dissolved metals, including rare earth elements, and metallo-nanoparticles (silver, gold, titanium dioxide, aluminum oxide, and iron) with aquatic plants were studied. After exposure in a variety of mesocosms, the partitioning of the elemental species between various compartments was quantified by ICP-MS and ICP-OES following microwave-assisted digestion. An ICP mass spectrometry (MS) method has also been developed to quantify the uptake of gold and silver nanoparticles by C. elegans. Uptake of gold nanoparticles was size dependent, suggesting increased ingestion efficiency with increased particle diameter. The feasibility of discriminating between suspended TiO2 nanoparticles and dissolved titanium by the analysis of the rapid transient signal events obtained from the ICP-MS instrument operated in a rapid response mode was also developed. Data handling parameters were established that allowed a distinction in the signals for nanoparticulate and standard solutions. Spikes in the signal were defined by distinct parameters using the mean and standard deviation, where a spike in the signal was defined as a signal > + ks (k =3). This approach however did lead to a statistical difference in the spike signal events for solutions and nanosuspensions.

Trigger Factor (TF) is a prokaryotic chaperone protein that exerts its major chaperone activity while associated with translating ribosomes, assisting de novo folding of the emerging nascent chain. Although much is known about the kinetics behind TF-ribosome binding, most results are based on in vitro experiments which fail to mimic the cellular environment. Single-particle approaches have gained increasing power for studying binding kinetics of biomolecules in living cells. One such method is single-particle tracking by super-resolution fluorescence microscopy, where the position of a fluorescently labelled particle is recorded over time, giving information about the movement of the particle inside the cell. Changes in diffusion behaviour is then used as an indicator of changes in biological activities. In this work, a diffusion model that qualitatively and quantitatively describes TF’s binding to ribosomes is presented. The model was obtained by single-particle tracking of TF labelled with HaloTag. Particle movements were analysed with a Hidden Markov Model-based algorithm that fit the trajectories to a defined set of different diffusion states, where fast diffusion could be related to free TF and slow diffusion to a ribosome-bound state. Moreover, the model could distinguish between two types of ribosome interactions: TF’s stable binding to ribosomes and a faster sampling behaviour. The average time spent stably bound to ribosomes is 670 ms and these interactions account for 53% of TF’s activity. TF is one of many processing proteins that interact with the emerging peptide chain during translation. By using the same approach on more of these factors, the interplay between them and the growing nascent chain can be characterized, giving an increased understanding of the highly complex translation machinery.

<p>Bid is a member of the Bcl-2 family of proteins, which are known as the regula- tors of apoptosis. Bid recruits Bax, another Bcl-2 family protein, which forms large oligomers that permeabilize the mitochonrdial outer membrane during apoptosis. In this thesis, Bid complexes embedded in a mitochondria-like supported lipid bilayer were investigated using single molecule fluorescence techniques. The bilayer, con- taining a lipophilic tracer, was formed on a mica surface and ATTO647 labelled Bid was added to it. For experiments where the effect of Bax on Bid complexes was investigated, a wild type Bax or a HiLyte488 labelled Bax was added as well. The protein-bilayer sample was imaged using total internal reflection fluorescence (TIRF). The formation of a fluid bilayer was confirmed by the observation of the lateral diffusion of DiD. Single particle tracking of the lipid molecules was used to measure the diffusion coefficent of DiD which was determined to be 2.2μm2 /s. The TIRF images also revealed two populations of Bid complexes, immobile and mobile. The diffusion coefficient of the observed Bid complexes was determined to be about three times slower than that of DiD (0.8±0.5μm2 /s). This provides evidence that mobile Bid is embedded in the bilayer. Image analysis of immobile Bid complexes showed a step-wise decrease in the fluorescence intensity due to photobleaching. The oligomeric distribution of the immobile Bid complexes was determined from the num- ber of steps, which corresponds to the number of particles in each complex. From these distributions it was concluded that the imaged immobile Bid existed mainly as monomers. However dimer and trimer complexes of Bid were also observed. The detected oligomeric distribution was not affected by the presence of either wild type Bax or Hilyte488 Bax. However Bid was imaged for the first time participating in Bax complexes. The acquired results somewhat differ from what had been observed in confocal imaging of the same samples, where mostly larger Bid complexes (dimers and up) were detected. We attribute the difference to the superior sensitivity of the TIRF method presented here.</p> / Master of Science (MSc)

Among the central constituents of the innate immune system are macrophages, which are known for phagocytosis or ‘eating’ foreign particles or pathogens. Macrophages express several cell-surface proteins including transmembrane and membrane-anchored receptors, which play a vital role in their response to pathogenic stimuli. The plasma membrane is a highly fluid and dynamic environment, which facilitates the diffusion of lipids and proteins within the plane of the membrane. This study aims to measure the lateral diffusion of two types of plasma membrane receptors on macrophages, toll-like receptor II (TLR2) and cluster of differentiation 14 (CD14), to answer three main research questions: 1) Which type of fluorescence-based microscopy techniques is best suited for measuring the lateral diffusion of TLR2 and CD14 on macrophage plasma membrane? 2) Does culturing macrophages on different surface topographies impact the diffusion of TLR2 in the plasma membrane and its pro-inflammatory response, along with morphological changes? 3) Does aging alter the lateral diffusion of TLR2 in the plasma membrane of macrophages? To date, a variety of fluorescence-based methods have been developed to study the dynamics of cell membrane constituents. These techniques are based on either ensemble or single particle measurements. We have used single particle tracking methods to track the mobility of fluorescently labeled membrane receptors on murine bone marrow-derived macrophages. Total internal reflection fluorescence microscopy (TIRF) was used to visualize and capture the dynamics in live cells. Using a custom routine algorithm we detected, localized, and tracked the particles to calculate their diffusion coefficient, extracted from the mean-squared displacement as the most common measure of diffusion. We also measured the diffusion coefficient using an ensemble-based technique known as Raster Image Correlation Spectroscopy (RICS) with a confocal laser-scanning microscope. The use of confocal eliminates the out-of-focus signal and enables measurements that are confined to a narrow plane in the cell. Also, the ability of RICS to separate the slow and immobile fractions of particles makes it possible to detect heterogeneities in diffusion. To our knowledge, this is the first study that has utilized both SPT and RICS to directly compare receptors’ diffusion in different membrane sections. Moreover, this is the first study that has examined the diffusion of receptors on macrophages adhered to different surface topographies, and the first that has investigated the receptors’ diffusion in young and old macrophages. / Thesis / Doctor of Philosophy (PhD) / The immune system is highly dependent on a specialized subset of white blood cells known as macrophages that are capable of clearing damaged and dead cells as well as a wide range of invading micro-organisms. Specific receptor proteins present on the membrane of macrophages are involved in the recognition of particles and subsequent signaling to recruit other immune cells or to promote healing and wound repair. To date, a variety of fluorescence-based microscopy methods have been used to study the dynamics of cell membrane components. The mobility of several membrane receptors in macrophages has been studied using microscopy techniques, which have provided valuable insights into their function. However, there is still insufficient information about the behavior of two key receptors (TLR2 and CD14) that participate in signaling in response to bacterial products. This thesis aims to answer three major questions with regard to receptor mobility (i.e., diffusion) within macrophage membrane: 1) Which type of fluorescence-based microscopy technique is more suitable for measuring the mobility of TLR2 and CD14 receptors on macrophage membranes? 2) What is the impact of different surface topographies on TLR2 diffusion in adhered macrophages, as well as cell shape, and the ability of macrophages to internalize particles? 3) Does aging alter TLR2 mobility in the membrane of macrophages? The following chapters provide detailed answers to these questions. In brief, we have demonstrated that TLR2 and CD14 diffusion measurements in adhered macrophages highly depend on the membrane section chosen. In addition, our results show that micro- and nanostructured surface topographies alter the shape of adhered macrophages and yield higher bacteria internalization, while the diffusion of TLR2 is not changed. When comparing macrophages derived from young and old mice, we find similar diffusion rate of TLR2 in macrophages of the two age groups.

Der eukaryotische Zellzyklus ist auf allen Ebenen der Genexpres- sion reguliert. Sowohl breit angelegte genetische Screens als auch funktionale Studien zu den beteiligten Proteinen haben unser Ver- ständnis dieses fundamentalen Prozesses geprägt. In dieser Arbeit behandle ich räumliche Aspekte der post-transkriptionalen Regulation des Zellzyklus, die mit lichtmikroskopischen Einzelzell- und Einzel- molekülmethoden experimentell zugänglich werden. Insbesondere untersuchte ich die subzelluläre Lokalisierung der messenger RNA von CLB2, einem zentralen Regulator der Mitose im eukaryotischen Modellorganismus Saccharomyces cerevisiae (Bierhefe). Frühere Studien zeigten, dass diese RNA sich im Laufe des vegetativen Zellwachstums in der entstehenden Tochterzelle, der Knospe, anreichert. Mithilfe modernster Fluoreszenzmikroskopie charakterisierte ich die Bewe- gung und Verteilung einzelner CLB2 messenger RNA-Moleküle auf Zeitskalen von Millisekunden bis hin zur Generationszeit dieser He- fen. Ich zeigte, dass sich mit Hilfe von Multifokusmikroskopie unter Verwendung optimierter Fluoreszenzmarker und der Entwicklung objektiver Analysemethoden die Bewegung einzelner RNA-Moleküle zwischen Mutterzelle und Knospe nachvollziehen lässt. Dazu präsen- tiere ich eine Methode um die beobachteten Trajektorien der messenger RNA mathematischen Analysen der Systembiologie zugänglich zu machen. Weiterhin gab die Beobachtung der Verteilung einzelner CLB2 messenger RNA Moleküle über den Zellzyklus hinweg mittels einer neuartigen Lichtblattmikroskopie (Lattice Light Sheet Microscopy) Hinweise auf eine bisher unbekannte Dynamik in der Lokalisierung dieser messenger RNA. Die hier entwickelten Methoden ermöglichen eine quantitative Untersuchung räumlicher Aspekte der posttranskrip- tionalen Zellzyklusregulation. / The eukaryotic cell cycle is regulated on all levels of gene expression. Genetic screens and functional studies of the involved proteins have shaped our understanding of this fundamental process. In this thesis I use single cell and single molecule light microscopy methods to investigate spatial aspects of post-transcriptional cell cycle regulation. I investigated the subcellular localization of CLB2 mRNA, a central regulator of mitosis in the eukaryotic model organism Saccharomyces cerevisiae (baker’s yeast). Previous studies have shown that that this messenger RNA is enriched in the emerging daughter cell, the bud, during vegetative growth. Using pre-commercial fluorescence micro- scopes I characterized the dynamics and partitioning of single CLB2 mRNA on time scales from milliseconds to the generation time of this yeast. I demonstrate that using aberration corrected multifocus mi- croscopy, optimized fluorescent markers, and here developed objective analysis methods, the translocation of single mRNA molecules be- tween mother and bud can be observed. In addition, I report a method to make these trajectories available for the mathematical approaches of Systems Biology. Further, the observation of single CLB2 mRNA partitioning throughout the cell cycle with the use of lattice light sheet microscopy suggested a previously unknown localization behavior of the transcript. The methods developed here enable a quantitative analysis of spatial aspects of post-transcriptional cell cycle regulation.

Thesis (MSc (Physics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Non-commutative quantum mechanics is a generalisation of quantum mechanics which incorporates the notion of a fundamental shortest length scale by introducing non-commuting position coordinates. Various theories of quantum gravity indicate the existence of such a shortest length scale in nature. It has furthermore been realised that certain condensed matter systems allow effective descriptions in terms of non-commuting coordinates. As a result, non-commutative quantum mechanics has received increasing attention recently. A consistent formulation and interpretation of non-commutative quantum mechanics, which unambiguously defines position measurement within the existing framework of quantum mechanics, was recently presented by Scholtz et al. This thesis builds on the latter formalism, extends it to many-particle systems and links it up with non-commutative quantum field theory via second quantisation. It is shown that interactions of particles, among themselves and with external potentials, are altered as a result of the fuzziness induced by non-commutativity. For potential scattering, generic increases are found for the differential and total scattering cross sections. Furthermore, the recovery of a scattering potential from scattering data is shown to involve a suppression of high energy contributions, disallowing divergent interaction forces. Likewise, the effective statistical interaction among fermions and bosons is modified, leading to an apparent violation of Pauli’s exclusion principle and foretelling implications for thermodynamics at high densities. / AFRIKAANSE OPSOMMING: Nie-kommutatiewe kwantummeganika is ’n veralgemening van kwantummeganika wat die idee van ’n fundamentele kortste lengteskaal invoer d.m.v. nie-kommuterende ko¨ordinate. Verskeie teorie¨e van kwantum-grawitasie dui op die bestaan van so ’n kortste lengteskaal in die natuur. Dit is verder uitgewys dat sekere gekondenseerde materie sisteme effektiewe beskrywings in terme van nie-kommuterende koordinate toelaat. Gevolglik het die veld van nie-kommutatiewe kwantummeganika onlangs toenemende aandag geniet. ’n Konsistente formulering en interpretasie van nie-kommutatiewe kwantummeganika, wat posisiemetings eenduidig binne bestaande kwantummeganika raamwerke defineer, is onlangs voorgestel deur Scholtz et al. Hierdie tesis brei uit op hierdie formalisme, veralgemeen dit tot veeldeeltjiesisteme en koppel dit aan nie-kommutatiewe kwantumveldeteorie d.m.v. tweede kwantisering. Daar word gewys dat interaksies tussen deeltjies en met eksterne potensiale verander word as gevolg van nie-kommutatiwiteit. Vir potensiale verstrooi ¨ıng verskyn generiese toenames vir die differensi¨ele and totale verstroi¨ıngskanvlak. Verder word gewys dat die herkonstruksie van ’n verstrooi¨ıngspotensiaal vanaf verstrooi¨ıngsdata ’n onderdrukking van ho¨e-energiebydrae behels, wat divergente interaksiekragte verbied. Soortgelyk word die effektiewe statistiese interaksie tussen fermione en bosone verander, wat ly tot ’n skynbare verbreking van Pauli se uitsluitingsbeginsel en dui op verdere gevolge vir termodinamika by ho¨e digthede.

Foamyviren (FV) gehören zur Familie der Retroviridae, werden aber aufgrund besonderer Eigenschaften in eine eigene Unterfamilie, die Spumaretrovirinae, eingeordnet. FV besitzen vor allem in vitro einen sehr breiten Tropismus, so dass bisher keine Zelllinie bekannt war, die nicht durch FV infiziert werden konnte. Obwohl diese Besonderheit darauf schließen lässt, dass ein sehr ubiquitäres Molekül auf der Wirtszelloberfläche für die FV-Bindung verwendet wird, ist der Rezeptor für die Virus-Aufnahme noch nicht bekannt. Dass FV einen pH-abhängigen Aufnahmemechanismus verwenden, lässt eine endozytotische Aufnahme vermuten. Dennoch sind die frühen Replikationsschritte, die zur Fusion der viralen und Wirtszellmembran führen, nur unzureichend charakterisiert. Deswegen wurden in der vorliegenden Arbeit funktionelle autofluoreszierende FV hergestellt, um die Bindung und Aufnahmemechanismen foamyviraler Partikel in Wirtszellen mit fluoreszenzmikroskopischen Analysen zu untersuchen. Mit diesen Methoden konnten erstmalig vier Zelllinien identifiziert werden, die nicht mit FV infizierbar sind, und damit mögliche Kandidaten für die Identifizierung des unbekannten FV Rezeptors darstellen. Des Weiteren wurden die fluoreszierenden FV erfolgreich eingesetzt, um die Fusionsereignisse zwischen viraler und zellulärer Membran in Echtzeit in lebenden Zellen zu untersuchen. Die durchgeführte „Single Virus Tracking“-Analyse zeigte, dass PFV (Prototype FV) Env-tragende Partikel sowohl an der Plasmamembran als auch in vermeintlichen Endosomen fusionieren können, wohingegen SFV (Simian FV) Env-tragende Partikel die Fusion wahrscheinlich nur in Endosomen auslösen können. Hinweis zur Nutzung der Filmdateien: Öffnen mit QuickTimePlayer bzw. ImageJ