Thesis (MIng)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

abstract: In eukaryotes, DNA is packed in a highly condensed and hierarchically organized structure called chromatin, in which DNA tightly wraps around the histone octamer consisting of one histone 3-histone 4 (H3-H4) tetramer and two histone 2A- histone 2B (H2A-H2B) dimers with 147 base pairs in an almost two left handed turns. Almost all DNA dependent cellular processes, such as DNA duplication, transcription, DNA repair and recombination, take place in the chromatin form. Based on the critical importance of appropriate chromatin condensation, this thesis focused on the folding behavior of the nucleosome array reconstituted using different templates with various controllable factors such as histone tail modification, linker DNA length, and DNA binding proteins. Firstly, the folding behaviors of wild type (WT) and nucleosome arrays reconstituted with acetylation on the histone H4 at lysine 16 (H4K16 (Ac)) were studied. In contrast to the sedimentation result, atomic force microscopy (AFM) measurements revealed no apparent difference in the compact nucleosome arrays between WT and H4K16 (Ac) and WT. Instead, an optimal loading of nucleosome along the template was found necessary for the Mg2+ induced nucleosome array compaction. This finding leads to the further study on the role of linker DNA in the nucleosome compaction. A method of constructing DNA templates with varied linker DNA lengths was developed, and uniformly and randomly spaced nucleosome arrays with average linker DNA lengths of 30 bp and 60 bp were constructed. After comprehensive analyses of the nucleosome arrays' structure in mica surface, the lengths of the linker DNA were found playing an important role in controlling the structural geometries of nucleosome arrays in both their extended and compact forms. In addition, higher concentration of the DNA binding domain of the telomere repeat factor 2 (TRF2) was found to stimulate the compaction of the telomeric nucleosome array. Finally, AFM was successfully applied to investigate the nucleosome positioning behaviors on the Mouse Mammary Tumor Virus (MMTV) promoter region, and two highly positioned region corresponded to nucleosome A and B were identified by this method. / Dissertation/Thesis / Ph.D. Chemistry 2010

Glutamate is the major excitatory neurotransmitter in the mammalian brain. It binds to three different subclasses of ionotropic glutamate receptors (iGluRs): AMPA, kainate and NMDA receptors, and triggers a cation influx that generates synaptic currents crucial to brain function. Significantly, iGluRs are implicated in various neurological disorders, such as depression, schizophrenia, Alzheimer’s and Parkinson’s diseases, autism, seizure, and stroke. Several crystal structures for intact iGluRs in various functional states (i.e. closed, activated and desensitized) have now been reported. The receptors have also been studied using single-particle cryo-electron microscopy. Together, these studies provide fascinating ‘snap-shots’ of the receptors as they transition between different states. What is lacking, so far, is information about the kinetics underlying these structural transitions, because the techniques used lack time resolution. I have used fast-scan atomic force microscopy (AFM), in some cases in combination with UV photolysis of caged L-glutamate, to study activation-induced structural changes in GluK2 kainate receptors and GluA2 AMPA receptors. AFM provides single-molecule resolution under fluid, permitting the imaging of proteins ‘in action’. Receptors were purified from transfected cells by immunoaffinity chromatography and imaged after integration into supported lipid bilayers. Activation of both receptors caused a rapid ~1-nm vertical compression of the receptor. In both cases, the height reduction did not occur in the presence of receptor antagonists. Further, the D776K mutant of the kainate receptor, which does not desensitize, did not undergo the height change, and cyclothiazide, which blocks desensitization of the AMPA receptor, also blocked the height change. I conclude, therefore, that the vertical compression is associated with receptor desensitization, and suggest that it may reflect a weakening of the interaction between receptor subunits at the LBD dimer interface. When imaged from the ‘top’ by AFM, the receptors appeared as double-blob structures, with each blob representing a pair of ATDs. By measuring the distance between the centres of the blobs in successive AFM images, I was able to monitor the mobility of the ATDs relative to each other before and during receptor stimulation. I found that for both kainate and AMPA receptors, the relative mobility of the ATDs became greater after stimulation. Further, at low glutamate concentrations, the ATDs of the (rapidly desensitizing) flop splice variant of the AMPA receptor were more mobile than those of the (more slowly desensitizing) flip splice variant. I suggest that the greater mobility of the flop splice variant might be connected with its more short-lived functional response to activation. In a final series of experiments, in collaboration with two other groups, I used AFM to measure conformational changes induced by allosterically-bound halide ions. We found that anion substitution (i.e. chloride to bromide, or chloride to iodide) produced vertical compression of AMPA receptors prior to agonist binding, and also (in electrophysiological experiments conducted by collaborators) altered the duration of agonist-evoked channel activity. The anion binding site was identified (in X-ray crystal structures obtained by collaborators) within the ligand binding domain, where flip-flop alternative splicing occurs. Interestingly both anion effects were isoform-dependent.

Made available in DSpace on 2014-06-11T19:28:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-11-10Bitstream added on 2014-06-13T19:58:06Z : No. of bitstreams: 1 rodrigues_ks_me_bauru.pdf: 3671952 bytes, checksum: 95922ebe5feb1ccd5d65c466e158d7a8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Desde 1986, quando Binnig et al (1986) criaram o microscópio de força atômica (AFM), esse aparelho se tornou um dos mais importantes microscópios de varredura (SPM), sendo usado para análise de DNA, nanotubos, etc. (Rützel et al, 2006). O AFM tem como componente principal uma microviga, com uma ponteira em uma das extremidades, que vibra próximo de sua frequencia de ressonância para mandar sinais a um fotodetector que traduz esse sinal e gera as imagens da superfície da amostra. O modo de operação tapping é o mais usado, e o comportamento caótico é muito comum nesse modo de operação, por esse motivo, AFM se tornou um assunto muito importante no mundo científico. Nesse trabalho, a microviga é modelada com o uso das equações de Bernoulli, as interações entre ela e a amostra são modeladas usando o potencial de Lennard Jones. Simulações numéricas detectam movimento caótico no sistema, a necessidade de estabilizá-lo nos leva a usar os seguintes métodos: Método do Balanço Harmônico, sincronização de Sistemas Não Lineares, Método das Equações de Estado Dependentes de Riccati (SDRE), Método de Realimentação de Sinal Atrasado. Por fim, a aplicação dos métodos se mostra eficiente, com pequeno erro e fácil implementação / Since 1986, when Binnig et al (1986) created the atomic force microscope (AFM), this unit became one of the most important scanning probe microscopes (SPM) being used for DNA analysis, nano tubes, etc. (Rutzel et al, 2006). The AFM has as a main component, a micro cantilever, with a tip at its free end, which vibrates near its resonance frequency to send signals to a photo detector that translates the signal and generates images of the sample surface. The tapping mod of operation is the most widely used and chaotic behavior is very common in this mode, therefore, AFM has become a very interesting subject in the scientific world. In this work, the micro cantilever is modeled using Bernoulli's equation and the interactions between the tip and the sample are modeled using the Lennard Jones potential. Numerical simulations detect chaotic motion in the system and the need to stabilize it leads us to use the following methods, Harmonic Balance Method; Synchronization of Nonlinear Systems; the State Dependent Riccati Equation control method (SDRE); the Method of Feedback Delay. Finally, the application of the methods proved to be effective, with small error and easy implementation

Background and aims: It is known that smooth muscle possesses substantial mechanical plasticity in that it is able to adapt to large changes in length without compromising its ability to generate force. It is believed that structural malleability of the contractile apparatus underlies this plasticity. There is strong evidence suggesting that myosin thick filaments of the muscle are relatively labile and their length in vivo is determined by the equilibrium between monomeric and filamentous myosin. The equilibrium in turn is governed by the state of phosphorylation of the 20-kD regulatory myosin light chain (MLC20, or RLC). It is known that phosphorylation of the myosin light chain favors formation of the filaments; it is not known how the light chain phosphorylation affects the lability of the filaments. The major aim of this thesis was to measure the mechanical integrity of the filaments formed from purified myosin molecules from bovine airway smooth muscle, and to determine whether the integrity was influenced by phosphorylation of the myosin light chain. Methods: Myosin was purified from bovine trachealis to form filaments, in ATP containing zero-calcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of RLC within 20-40 s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy. Results: RLC phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated. Conclusion: Our results suggest that RLC phosphorylation is a key regulatory step in modifying the structural properties of myosin filaments in smooth muscle, where formation and dissolution of the filaments are required in the cells’ adaptation to different cell length. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Doctor of Philosophy / Department of Chemical Engineering / Mary E. Rezac / Characterizing a catalytic metal surface during heterogeneous hydrogenation is an enabling area of catalysis research. Available technology, however, often requires ultra-high vacuum or other limiting conditions which prohibit in operando research. Atomic force microscopy (AFM) can provide direct observations of fluid/solid interfaces at atmospheric conditions and in real time. Tapping-mode AFM can examine chemical and physical phenomena on surfaces in addition to topography. The work here describes using phase-angle information from tapping-mode AFM to observe liquid/solid interfaces in real time during the hydrogenation of styrene. Through optimized tuning and scanning procedures, it was possible to observe the onset of hydrogenation on the surface of palladium immersed in liquid in real time and with the topographic resolution inherent to AFM. This opens new avenues for in operando research on heterogeneous catalysis, a field that is of great fundamental and industrial importance. For reference, a catalytic membrane reactor (CMR) was used to observe the hydrogenation of phenylacetylene over a palladium layer as a batch process. It was determined that with a H2 diffusion rate of 3.7·10-9 mol/s and a theoretical, calculated H2 demand of at least 2.3·10-7 mol/s, the reaction would be hydrogen starved and would not progress at a realistic timescale for observation by AFM. By instead using either ethylbenzene (EB) or styrene (St) as the liquid in a solvent-free approach and injecting a small volume of the other liquid into the system mid-scan, the effects of changes in chemistry on tip-surface interactions were observable. EB injections in both EB and St-immersed scans showed no significant change in phase angle. Injecting St into an EB-immersed scan environment, however, caused an increase in phase which remained relatively constant for the remaining duration of the scan, demonstrating for the first time that a liquid-phase hydrogenation reaction can be observed in operando through the phase shift of tapping mode AFM.

This thesis describes a novel reconstitution of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) for the purposes of structural characterization by atomic force microscopy (AFM) and functional characterization by electrical recordings of lipid bilayers. AMPARs are glutamate gated ion channels, ubiquitous in the vertebrate central nervous system where they mediate fast excitatory neurotransmission. In a healthy brain, AMPARs are involved in memory formation and learning and their dysfunction has been related to numerous neurological disorders such as Alzheimer's disease, epilepsy, schizophrenia and many others. AMPARs were reconstituted at high and low densities. Densely reconstituted samples contained >100 receptors per μm2, a value comparable to the AMPAR density at synapses. This allowed, for the first time, the imaging of full length tetrameric AMPARs in native-like conditions and with clearly assigned domains: the extracellular domains extended 14 nm above the membrane in agreement with electron microscopy (EM) and X-ray crystallography data. Lipid-protein interactions were studied in samples with low protein density with the receptors showing preference for lipids in the liquid crystalline phase. The activity of the reconstituted receptors was confirmed through single-channel recordings. This is the first case in which an AMPAR has been reconstituted and given (a) single-channel recordings with (b) physiologically plausible conductance levels and (c) pharmacological and no-protein controls and (d) structure. As a result, previously reported biochemistry and EM are now for the first time available in concert with AFM and single-channel recordings for a purified AMPAR of known composition.

Several members of the ABC-6 zeolite family, namely offretite, zeolite T, ZSM-34 and zeolite L, were synthesised with a view to studying the effect of synthetic parameters on the resulting crystal properties. A range of procedures for producing offretite and ZSM-34 were tested, and crystals with the optimal morphology, purity, crystallinity and size identified. A standard procedure for synthesising zeolite T was used, before the effect of systematically altering the cation concentration, Si/Al ratio and water content in the synthesis mixture was studied, and the optimal composition identified. Ex-situ AFM was used to study the morphology of the resulting crystals, as well as to determine information about surface features, such as the shape and height of terraces. This information could be used to infer the growth mechanism for each crystal. The AFM studies of zeolite L were compared with those of previous studies and found to agree. For offretite, zeolite T and ZSM-34, 1.2 nm high terraces elongated along the length of the {100} face of the crystals were observed. Where the crystals exhibited a micro-crystalline morphology without distinct faces HR-SEM was used to study their morphology and terracing. In-situ AFM was used to study the dissolution of the crystals in basic media. In each case the terraces were observed to dissolve primarily length-wise. The dissolution of terraces on zeolite T was considered in particular detail. The terrace height was measured during dissolution, and three distinct measurements were observed, 1.2, 0.8 and 0.2 nm. These heights were related to framework features. Lateral force AFM measurements were used to show the relationship between decreased terrace height and increased lateral deflection, indicating that dissolving areas of crystal exhibited increased tip-sample friction. The rate of dissolution of terraces on zeolite T was also considered. It was found that dissolution rate increased with increasing NaOH concentration, and with increasing tip-sample force in the AFM. These observations allowed order of reaction for the dissolution process of zeolite T to be estimated at 2.54. A magnesium substituted aluminophosphate ABC-6 family zeotype material, MgAPO-CJ60, was also synthesised and analysed. Solid-state NMR was used to determine the distribution of aluminium and phosphorous within the framework, which was found to be non-random.

In this report we detail our work fabricating and measuring graphene quantum dots. We investigate a technique, relatively widely used in several other materials but not yet well investigated in graphene, known as Atomic Force Microscope Lithography (AFML). We then use AFML to fabricate graphene quantum dot systems. Transport measurements are carried out on our graphene quantum dots at low temperatures and high parallel magnetic fields and we try to understand the behaviour of spins in graphene. In our initial investigations into AFML we use graphene samples electrically contacted using standard electron-beam lithography. We were able to cut the graphene lattice by applying a negative voltage to the AFM tip and moving the tip across a grounded graphene surface. We have shown, by measuring the current through the AFM tip during lithography, that cutting of graphene is not current driven. Using a combination of transport measurements and scanning electron microscopy we show that , while indentations accompanied by tip current appear in the graphene lattice for a range of tip voltages, real cuts are characterized by a strong reduction of the tip current above a threshold voltage. The flexibility of the technique was then demonstrated by the fabrication, measurement, modification and re-measurement of graphene nanodevices with resolution down to 15 nm. We subsequently developed a shadow-masking technique to electrically contact graphene samples thus eliminating the use of chemical resists and the associated contamination of the graphene surface. With these pristine samples we were able to oxidise and hydrogenate the graphene using AFML. A graphene quantum dot was then fabricated using AFML oxidation. We also fabricated a graphene quantum dot using e-beam lithography in combination with oxygen plasma etching. We studied electron spin physics in these structures by J:1pplying large parallel magnetic fields at low temperatures and performing electrical transport measurements. We do not find an ordered filling sequence of spin states, which we assign to edge disorder and surface charge impurities.

A significant body of literature has reported strategies and techniques to assess the mechanical properties of biological samples such as proteins, cellular and tissue systems. Atomic force microscopy has been used to detect elasticity changes of cancer cells. However, only a few studies have provided a detailed and complete protocol of the experimental procedures and data analysis methods for non-adherent blood cancer cells. In this work, the elasticity of NB4 cells derived from acute promyelocytic leukemia (APL) was probed by AFM indentation measurements to investigate the effects of the disease on cellular biomechanics. Understanding how leukemia influences the nanomechanical properties of cells is expected to provide a better understanding of the cellular mechanisms associated to cancer, and promises to become a valuable new tool for cancer detection and staging. In this context, the quantification of the mechanical properties of APL cells requires a systematic and optimized approach for data collection and analysis, in order to generate reproducible and comparative data. This Thesis elucidates the automated data analysis process that integrates programming, force curve collection and analysis optimization to assess variations of cell elasticity in response to processing criteria. A processing algorithm was developed by using the IGOR Pro software to automatically analyze large numbers of AFM data sets in an efficient and accurate manner. In fact, since the analysis involves multiple steps that must be repeated for many individual cells, an automated and un-biased processing approach is essential to precisely determine cell elasticity. Different fitting models for extracting the Young’s modulus have been systematically applied to validate the process, and the best fitting criteria, such as the contact point location and indentation length, have been determined in order to obtain consistent results. The designed automated processing code described in this Thesis was used to correlate alterations in cellular biomechanics of cancer cells as they undergo drug treatments. In order to fully assess drug effects on NB4 cells, viability assays were first performed using Trypan Blue staining for primary insights before initiating thorough microplate fluorescence intensity readings using a LIVE/DEAD viability kit involving ethidium and calcein AM labelling components. From 0 to 24 h after treatment using 30 µM arsenic trioxide, relative live cell populations increased until 36 h. From 0 to 12 h post-treatment, relative populations of dead cells increased until 24 h post-treatment. Furthermore, a drastic drop in dead cell count has been observed between 12 and 24 h. Additionally, arsenic trioxide drug induced alterations in elasticity of NB4 cells can be correlated to the cell viability tests. With respect to cell mechanics, trapping of the non-adherent NB4 cells within fabricated SU8-10 microwell arrays, allowed consistent AFM indentation measurements up to 48 h after treatment. Results revealed an increase in cell elasticity up to 12 h post-treatment and a drastic decrease between 12 and 24 h. Furthermore, arsenic trioxide drug induced alterations in elasticity of NB4 cells can be correlated to the cell viability tests. In addition to these indentation and viability testing approaches, morphological appearances were monitored, in order to track the apoptosis process of the affected cells. Relationships found between viability and elasticity assays in conjunction with morphology alterations revealed distinguish stages of apoptosis throughout treatment. 24 h after initial treatment, most cells were observed to have burst or displayed obvious blebbing. These relations between different measurement methods may reveal a potential drug screening approach, for understanding specific physical and biological of drug effects on the cancer cells.