We developed a method for fluorescence in situ identification of individual mRNA molecules, allowing quantitative and accurate measurement, in single cells, of allele-specific transcripts that differ by only a few nucleotides. By using a combination of allele-specific and non-allele-specific probe libraries, we achieved >95% detection accuracy. We used this technique to investigate the allele-specific stochastic expression of Nanog, which encodes a pluripotency factor, in murine embryonic stem cells. We find that Nanog does not switch between monoallelic and biallelic expression when culture conditions are altered. We next worked towards adapting our allele-specific single molecule mRNA fluorescent in situ hybridization technique to detect early expression of the immunoglobulin kappa gene in Pre-B cells. Mature B cells only express a single allele of the immunoglobulin kappa gene, and assaying allele-specific expression in single cells will allow the study of the mechanism behind this choice. We also developed a theoretical model of cell specification in the mammalian inner ear using single-molecule mRNA expression data. During mammalian hair cell development, prosensory cells acquire a spatial pattern of distinct cellular fates. This process is dependent upon the expression of the transcription factor Atoh1, and is mediated by Notch signaling between neighboring cells. We find that both the Notch ligand and transcription factor Atoh1 are expressed in an extended region before turning off in non-hair cells. Our model reveals that this extended pattern creates a system that can suppress extraneous expression over a large region and is robust to movement of prosensory cells as the cochlea extends, especially in the case of a limited time window for specification. Our model can also explain the two types of expression patterns of Atoh1 that are observed.

This work investigates the dynamics of gene regulatory network formed by Oct4, Sox2 and Nanog in embryonic stem cells (ESCs). Despite a large number of existing studies on stem cells, current technologies used often force a compromise between quantification of gene expression via bulk measurements and qualitative imaging of cell heterogeneity. There are few options that allow for accurate and quantitative single-cell analysis that is robust yet not associated with a high degree of technical difficulty or obscured by amplification. Here, we adapted a high resolution, single-molecule RNA fluorescent in situ hybridization technique (smFISH) to study gene expression of the core pluripotency circuit upon various types of perturbations such as differentiation, induction or knockdown of one of the three pluripotent factors. We used previously-published smFISH procedures as our initial template for investigating gene regulatory dynamics of the core pluripotency circuit during those perturbation assays. To obtain a more comprehensive picture of the regulatory circuit, we developed a modified smFISH strategy to measure mRNA and protein expression simultaneously in single ESCs. By incorporating a novel modification into the smFISH technique which allows accurate quantification of transcripts that differ by short sequences, we managed to identify a few interesting features of the core pluripotency circuit. Taken together, we demonstrated our ability to perform single-cell, single-molecule assays that reveal highly quantitative information in unprecedented detail.

<p> Understanding the physical basis of enzyme dynamics is a major challenge in biology. Although modeling the motion of individual atoms is straightforward, combining these movements into descriptions of macromolecular function proves more difficult. X-ray crystallography produces atomic-level visualizations of an ensemble of countless molecules; however, current methods capture only the average protein conformation and thus cannot completely describe the underlying dynamics. A parallel source of information, diffuse scattering, is present in diffraction images and directly reports on correlated atomic motions. </p><p> I created experimental and computational tools to measure macromolecular diffuse scattering and compare it against hypotheses of correlated motion. The first tool, <i>phenix.diffuse</i>, calculates diffuse scattering patterns from known structural ensembles. I applied this software to the refinement technique <i>Translation-Libration-Screw</i> and solved a pre-existing degeneracy within the predicted motion of glycerophosphodiesterase GpdQ. Surprisingly, I also uncovered a fundamental flaw in the implementation of TLS refinement in structural biology software, revealing unphysical motions to be present in nearly 25% of all known macromolecular structures. </p><p> Next, I developed the comprehensive pipeline <i>DIALS-LUNUS</i> for the measurement of macromolecular diffuse scattering. This system was applied to crystals of the proline isomerase cyclophilin A (CypA) and trypsin, ultimately producing high-resolution diffuse maps of both proteins. These maps were compared to several distinct models of motion that were previously indistinguishable to crystallographic techniques. By comparing the experimental data to each predicted diffuse scattering pattern, I was able to successfully identify the most probable mechanism of motion. Ultimately, these studies provide a new avenue of exploration in the pursuit of understanding molecules as dynamic entities.</p>

Note: / In section A a review has been made of single crystal studies done from 1960-1973. This review has been put into tabular form (see tables I, 2, 4-9). A critical analysis and INDO calculations of some of ther adicals are reported....

Thesis (PH.D.) -- Syracuse University, 2004. / "Publication number AAT 3132723."

Thesis (PH.D.) -- Syracuse University, 2006 / "Publication number AAT 3241852."

<p>One of the main challenges that biological oscillators face at the cellular level is maintaining coherence in the presence of molecular noise. Mechanisms of noise resistance have been proposed, however the findings are sometimes contradictory and not universal. Another challenge faced by biological oscillators is the proper timing of cellular events and effective distribution of cellular resources when there is more than one oscillator in the same cell. Biological oscillators are often coupled, however, the mechanisms and extent of these couplings are poorly understood. In this thesis, I describe three separate yet interconnected projects in an attempt to understand these biophysical phenomena.</p><p>I show that slow DNA unbinding rates are important in titration-based oscillators and can mitigate molecular noise. Multiple DNA binding sites can also increase the coherence of the oscillations through protected states, where the DNA binding/unbinding between these states has little effect on gene expression. I then show that experimental titration-based oscillator in budding yeast is innately coupled to the cell cycle. The oscillator and the cell cycle show 1:1 and 2:1 phase locking similar to what has been observed in natural systems. Finally, by studying the relationship between the circadian redox rhythm and genetic circadian clock in plants I show how perturbation of one of the coupled oscillators can be transformed into a reinforcement signal for the other one via a balanced network architecture.</p> / Dissertation

The term "binding hot spots" refers to regions of a protein surface with large contributions to the binding free energy. Computational solvent mapping serves as an analog to the major experimental techniques developed for the identification of such hot spots using X-ray and nuclear magnetic resonance (NMR) methods. Applications of the fast Fourier-transform-based mapping algorithm FTMap show that similar binding hot spots also occur in DNA molecules and interact with small molecules that bind to DNA with high affinity. Solvent mapping results on B-DNA, with or without Hoogsteen (HG) base pairing, have revealed the significance of "HG breathing" on the reactivity of DNA with formaldehyde. Extending the method to RNA molecules, I applied the FTMap algorithm to flexible structures of HIV-1 transactivation response element (TAR) RNA and Tau exon 10 RNA. Results show that despite the extremely flexible nature of these small RNA molecules, nucleic acid bases that interact with ligands consistently have high hit rates, and thus binding sites can be successfully identified. Based on this experience as well as the prior work on DNA, I extended the FTMap algorithm to mapping nucleic acids and implemented it in an automated online server available to the research community. FTSite, a related server for finding binding sites of proteins, was also extended to develop PeptiMap, an accurate and robust protocol that can determine peptide binding sites on proteins. Analyses of structural ensembles of ligand-free proteins using solvent mapping have shown that such ensembles contain pre-existing binding hot spots, and that such hot spots can be identified without any a priori knowledge of the ligand-bound structure. Furthermore, the structures in the ensemble having the highest binding-site hit rate are closest to the ligand-bound structure, and a higher hit rate implies improved structural similarity between the unbound protein and its bound state, resulting in high correlation coefficient between the two measures. These advances should greatly enhance researchers' ability to identify functionally important interactions among biomolecules in silico.

<p> Lipid membranes enclose cells and organelles, and actively participate in cellular processes. Their many functional roles require tight regulation of properties including structure and dynamics. Cells achieve this by producing and dynamically tuning the concentration and organization of hundreds of structurally different types of lipid molecules in the various cellular membranes. The cell-bounding plasma membranes of eukaryotes in particular, exhibit an actively maintained asymmetric lipid distribution across their two leaflets. In addition to exposing certain types of lipids to the extracellular space or intracellular milieu, this specialized transbilayer lipid arrangement also affects the properties of the membrane itself and its interactions with proteins, in ways that are difficult to explore and thus not understood. To address this problem and enable further advancements in the field, we have developed both <i> in vitro</i> and <i>in silico</i> protocols for building asymmetric model membranes with finely controlled lipid compositions. These protocols allowed us to investigate the dynamics, energetics and structural consequences of interleaflet communication: with small-angle scattering we uncovered asymmetry-mediated changes in the lipid packing of individual leaflets in free-floating liposomes; with electron spin resonance we revealed the ensuing trends in lipid order; and nuclear magnetic resonance helped us bring new appreciation of the interplay between asymmetric bilayers and transmembrane protein inclusions. To interpret and better understand the experimental observations, we developed a new <i> in silico</i> protocol for constructing atomistic models of tension-free asymmetric bilayers and used it to simulate the experimentally measured membranes and validate the simulation conditions. By devising a novel computational framework for calculating the compressibility of individual bilayer leaflets, we analyzed the energetics of protein interaction with the asymmetric membranes and obtained an estimate of the elastic energy of mixing the two leaflets. Together with additional experimental and computational studies of symmetric membrane systems, the results revealed fascinating ways in which cells can mediate the functional diversity of their membranes. The new methods and protocols leading to these insights generate previously unattainable opportunities for dissecting and exploring membrane-mediated cellular processes.</p><p>

abstract: Calcitonin Gene-Related Peptide (CGRP) is an intrinsically disordered protein that has no regular secondary structure, but plays an important role in vasodilation and pain transmission in migraine. Little is known about the structure and dynamics of the monomeric state of CGRP or how CGRP is able to function in the cell, despite the lack of regular secondary structure. This work focuses characterizing the non-local structural and dynamical properties of the CGRP monomer in solution, and understanding how these are affected by the sequence and the solution environment. The unbound, free state of CGRP is measured using a nanosecond laser-pump spectrophotometer, which allows measuring the end-to-end distance (a non-local structural property) and the rate of end-to-end contact formation (intra-chain diffusional dynamics). The data presented in this work show that electrostatic interactions strongly modulate the structure of CGRP, and that peptide-solvent interactions are sequence and charge dependent and can have a significant effect on the internal dynamics of the peptide. In the last few years migraine research has shifted focus to disrupting the CGRP-receptor pathway through the design of pharmacological drugs that bind to either CGRP or its receptor, inhibiting receptor activation and therefore preventing or reducing the frequency of migraine attacks. Understanding what types of intra- and inter-chain interactions dominate in CGRP can help better design drugs that disrupt the binding of CGRP to its receptor. / Dissertation/Thesis / Doctoral Dissertation Physics 2015