No description available.

Synchrotron Radiation (SR) X-ray solution scattering/diffraction and cryo-electron microscopy have been used to characterise the structure of tubulin polymers assembled in a variety of biochemical conditions. Cryo-microscopy has been used to image individual structures and to interpret the X-ray diffraction profiles from the protein structures in solution. The time-resolved X-ray data provided by SR has been analysed using correlation techniques in order to elucidate the structural pathway of reactions triggered by temperature changes. The results can be summarised as follows: • The structure and temperature-induced assembly of microtubules polymerised from microtubule protein (MTP), and purified tubulin (TB) preparations are very similar. Differences in the assembly process concern the existence of prenucleation events, linked to the presence of cold structured aggregates, only present in the MTP solutions. The only structural difference is the presence of MAPs attached to the wall of MTP microtubules. When TB microtubules were assembled in the presence of taxol the surface lattice was preserved but the diameter was smaller, corresponding to an average loss of one proto filament. • The vinblastine-induced self-assembly of TB can give rise to both, double-coiled spirals that aggregate in a non-ordered fashion, and paracrystalline structures where the repeating motif is a single-coiled spiral. The type of polymer is mainly detennined by the amount of magnesium ion present in the preparation. Concentrations higher than 7.5 mM result in double coils similar to those found in MAPs-containing preparations, pointing to a similarity of effect possibly due to the positive charge of both MAPs and magnesium ions. At any magnesium concentration, a temperature change induced a reversible change in the pitch of the spirals, reflecting a temperature-induced confonnational change in the tubulin subunit.

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<p> Chromatin conformation and dynamics remains unsolved despite the critical role of the chromatin in fundamental genetic functions such as transcription, replication, and repair. At the molecular level, chromatin can be viewed as a linear array of nucleosomes, each consisting of 147 base pairs (bp) of double-stranded DNA (dsDNA) wrapped around a protein core and connected by 10 to 90 bp of linker dsDNA. </p><p> Using small-angle X-ray scattering (SAXS), we investigated how the conformations of model nucleosome arrays in solution are modulated by ionic condition as well as the effect of linker histone proteins. To facilitate ensemble modeling of these SAXS measurements, we developed a simulation method that treats coarse-grained DNA as a Markov chain, then explores possible DNA conformations using Metropolis Monte Carlo (MC) sampling. This algorithm extends the functionality of SASSIE, a program used to model intrinsically disordered biological molecules, adding to the previous methods for simulating protein, carbohydrates, and single-stranded DNA. Our SAXS measurements of various nucleosome arrays together with the MC generated models provide valuable solution structure information identifying specific differences from the structure of crystallized arrays.</p>

<p> Past studies have shown that tumor growth generally follows an exponential growth function or, with a limiting growth constraint, the sigmoid Gompertzian function, where a terminal tumor size is reached at late times. The classical Gompertzian description of tumor growth applies in the case of two-dimensional (2D) <i>in vitro</i> cell studies due to the effect of physical limitations on possible growth area. This project asked whether Gompertzian form applies to the <i>in vitro</i> growth of multifocal 3D tumor nodules, whose size is determined by aggregation events as well as cell proliferation. Previous reports have indicated that these three-dimensional (3D) spheroids appear to reach a terminal size, even though the full available 3D volume is not occupied. In this scenario it is not immediately obvious if individual nodules are growth-constrained by nutrient or oxygen diffusion, or rather if the ensemble of all nodules exhibits Gompertzian form. 3D <i>in vitro </i> ovarian cancer cells were chosen as the population to be studied. The ovarian cancer cells were grown in overlay on a laminin-rich extracellular matrix (ECM). This model system is a common and widely used cell culture platform in cancer cell research. Using this system, division of the ovarian cancer cells into heterogeneous clusters that aggregate into larger clusters, and then reach a steady bimodal distribution of small and large aggregates, was observed. The average volume as well as the total volume of these two cell aggregate groups were measured over time to determine the nodules&rsquo; growth behavior would plateau without a growth area limitation. Biological processes may limit the size and behavior of cells within sphere-like multicellular nodules differently than a simple layer of cells on a petri dish. The standard deviation of the rapidly growing nodule volume population within a 3D <i> in vitro</i> ovarian cancer sample was shown to grow according to a quadratic function, while the population of small nodules stays constant over time. The overall growth behavior of the total volume of the rapidly growing nodules was Gompertzian. The spread between the increasing average size of the large and growing nodule population and the constant average size of the population of small nodules increased exponentially. A particle velocity tracking program was used to search for a relationship between the lateral velocity of the nodules within the field of view and the average size of the rapidly growing nodules. The average lateral velocity of all nodules was shown to weakly decrease over time. This indicates that the behavior of 3D grown ovarian cancer cells follow a dissemination pattern in which small cells or nodules of ovarian cancer cells demonstrate higher dissemination than large nodules. The motion of smaller cell nodules or single cells may be advantageous in the <i> in vivo,</i> as well as the in vitro settings. This advantage may produce the bimodal distribution of mobile small aggregates and large slow-moving and growing aggregates, and in turn, this behavior may demonstrate that dissemination of small aggregates of ovarian cancer cells occurs in a 3D environment.</p>

<p>Cell membranes are dynamic structures with complex organization. The complexity of the cell membrane arises from intrinsic membrane structure, membrane microdomains within the plasma membrane and the membrane cytoskeleton. Plasma membrane receptors are integral membrane proteins with diverse structures and functions which bind specific ligands to trigger cellular responses. Due to compartmentalization of the plasma membrane and the formation of membrane microdomains, receptors are distributed non-homogeneously in the cell membrane bilayer. Both lateral and rotational diffusion of membrane receptors reflects different kinds of intermolecular interactions within the plasma membrane environment. Understanding protein diffusion within the membrane is very important to further understanding biomolecular interactions in vivo during complex biological processes including receptor-mediated signaling. </p><p> Rotational diffusion depends linearly on the in-membrane volume of the rotating proteins. Relative to lateral diffusion, rotational diffusion is a more sensitive probe of an individual molecule&rsquo;s size and local environment. We have used asymmetric quantum dots (QD) to conduct imaging measurements of individual 2H3 cell Type I Fc&epsiv; receptor rotation on timescales down to 10 msec per frame. We have also used time-tagged single photon counting measurements of individual QD to examine &micro;sec timescales, although rapid timescales are limited by QD emission rates. In both approaches, decays of time-autocorrelation functions (TACF) for fluorescence polarization fluctuations extend into the millisecond timescale, as implied by time-resolved phosphorescence anisotropy results. Depending on instrumental parameters used in data analysis, polarization fluctuation TACFs can contain a contribution from the intensity fluctuation TACF arising from QD blinking. Such QD blinking feed-through is extremely sensitive to these analysis parameters which effectively change slightly from one measurement to another. We discuss approaches based on the necessary statistical independence of polarization and intensity fluctuations to guarantee removal of a blinking-based component from rotation measurements. Imaging results demonstrate a range of rotational behavior among individual molecules. Such slow motions, not observable previously, may occur with large signaling complexes, which are important targets of study in cell biology. These slow motions appear to be a property of the membrane itself, not of the receptor state. Our results may indicate that individual mesoscale membrane regions rotate or librate with respect to the overall cell surface. </p><p> The luteinizing hormone receptor (LHR) is a seven transmembrane domain receptor and a member of the GPCR family. It is located on luteal cells, granulosa and theca cells in females. Understanding how these protein receptors function on the plasma membrane will lead to better understanding of mammalian reproduction. LHR becomes aggregated upon binding hCG when receptors are expressed at physiological numbers. Binding of hormone to LHR leads to activation of adenylate cyclase (AC) and an increase in intracellular cyclic AMP (cAMP). ICUE3 is an Epac-based cAMP sensor with two fluorophores, cyan fluorescent protein (CFP) and the YFP variant, cpVenus, and a membrane-targeting motif which can be palmitoylated. Upon binding cAMP, ICUE3 undergoes a conformational change that separates CFP and YFP, significantly reducing FRET and thus increasing the ratio of CFP to YFP fluorescence upon excitation with an arc lamp or 405nm laser source. Hence we have investigated hLHR signal transduction using the cyclic AMP reporter probe, ICUE3. A dual wavelength emission ratio (CFP/YFP) imaging method was used to detect a conformational change in ICUE3 upon binding cAMP. This technique is useful in understanding the sequence of intercellular events following hormone binding to receptor and in particular, the time course involved in signal transduction in a single cell. Our data suggested that CHO cells expressing ICUE3 and directly treated with different concentrations of cAMP with saponin can provide a dose-dependent relationship for changes in intracellular cAMP levels. Forskolin (50&micro;M) causes maximal activation of the intracellular cAMP and an increase in the CFP/YFP emission ratio. In CHO cells expressing both ICUE3 and hLHR-mCherry, the CFP/YFP ratio increased in cells treated with forskolin and in hCG- treated cells. In flow cytometry studies, similar results were obtained when CHO cells expressed &lt; 60k LHR-mCherry per cell. Our results indicate that ICUE3 can provide real time information on intracellular cAMP levels, and the ICUE3 is a reliable cAMP reporter can be used to examine various aspects of LH receptor-mediated signaling. </p>

<p>Photosynthesis&mdash;the conversion of sunlight to chemical energy&mdash;is fundamental for supporting life on our planet. Despite its importance, the physical principles that underpin the primary steps of photosynthesis, from photon absorption to electronic charge separation, remain to be understood in full. Electronic coherence within tightly-packed light-harvesting (LH) units or within individual reaction centers (RCs) has been recognized as an important ingredient for a complete understanding of the excitation energy transfer (EET) dynamics. However, the electronic coherence across units&mdash;RC <i> and</i> LH or LH <i>and</i> LH&mdash;has been consistently neglected as it does not play a significant role during these relatively slow transfer processes. Here, we turn our attention to the absorption process, which, as we will show, has a much shorter built-in timescale. We demonstrate that the&mdash;often overlooked&mdash;spatially extended but short-lived excitonic delocalization plays a relevant role in general photosynthetic systems. Most strikingly, we find that absorption intensity is, quite generally, redistributed from LH units to the RC, increasing the number of excitations which can effect charge separation without further transfer steps. A biomemetic nano-system is proposed which is predicted to funnel excitation to the RC-analogue, and hence is the first step towards exploiting these new design principles for efficient artificial light-harvesting. </p>

This thesis describes techniques for performing calculations of absorbed dose conversion factors for therapeutic kilovoltage and megavoltage x-ray beams, by application of the Monte Carlo method. These factors are low-energy x-ray backscatter factors, B, low- and medium-energy x-ray mass energy absorption coefficient ratios, water-to-air, We,IP)w.a,r ' and megavoltage x-ray stopping power ratios, water-to-air, s. The EGS4 and ITS Monte Carlo systems were used to model the relevant particle transport. Consistent theoretical expressions for absorbed dose in kilovoltage x-ray beams have been proposed. For low-energy x-rays, the expression for absorbed dose to water, D, requires values of defined as a water kerma ratio at the surface of a water phantom, and also of [(Jie,,/P)w.,r]p, evaluated over the primary spectrum (free-in-air). For medium-energy O x-rays, values of (Pen(Z,f)I'P)w,air are necessary, which are dependent on depth, z, and field-size,f. Bragg-Gray cavity theory entails values of SWthI(z) to convert readings from recommended ionisation chambers, calibrated in terms of air-kerma or exposure, into D. Bremsstrahlung spectra have been calculated by detailed Monte Carlo simulations of the NPL standard accelerator, a Philips SL series linac and a Siemens Stabilipan x-ray unit. Values of (i1 ,,/P)w air and B' have been calculated for therapeutic polyenergetic kilovoltage beams, over a comprehensive range of field sizes and beam qualities, following the Monte Carlo calculation of photon fluence in water and by a kerma-weighted averaging technique utilising primary fluence spectra and pre-calculated values of monoenergetic Values of Spencer-Attix s,, have similarly been calculated for the NPL standard and therapeutic linac beams following the Monte Carlo calculation of electron fluence in water and by a dose-weighted averaging technique utilising primary fluence spectra and pre-calculated monoenergetic dose distributions. Accurate TPR values, which account for linac spectrum quality shift off-axis, have been acquired by convolving water terma distributions with point-energy-deposition kernels. The beam quality dependence of N, the NFL absorbed dose-to-water calibration factor, has subsequently been investigated. Data appropriate for insertion into recommended expressions for D is presented and compared to data provided in the literature.

No description available.

No description available.