No description available.

<p>Studying the ultrafast dynamics of small molecules can serve as the first step in understanding the dynamics in larger chemically and biologically relevant molecules. To make direct comparisons with existing computational techniques, the photons used in pump-probe spectroscopy must make perturbative transitions between the electronic states of isolated small molecules. In this dissertation experimental investigations of ultrafast dynamics in electronic excitations of neutral ethylene and carbon dioxide are discussed. These experiments are performed using VUV/XUV femtosecond pulses as pump and probe. </p><p> To make photons with sufficient energy for single photon transitions, VUV and XUV light is generated by high harmonic generation (HHG) using a high pulse energy (&ap;30&ndash;40 mJ) Ti:sapphire femtosecond laser. Sufficient flux must be generated to enable splitting of the HHG light into pump and probe arms. The system produces >10¹⁰ photons per shot, corresponding to nearly 10 MW of peak power in the XUV. Using a high flux of high energy photons creates a unique set of challenges when designing a detector capable of performing pump-probe experiments. A velocity map imaging (VMI) detector has been designed to address these challenges, and has become a successful tool facilitating studies into molecular dynamics that were not possible before its implementation. </p><p> The emphasis on using high energy, single photon transitions allowed theoretical calculations to be directly compared to experimental yields for the first time. This comparison resolved a long standing issue in the excited state lifetime of ethylene, and provided a confirmation of the branching ratio between the two nonadiabatic relaxation pathways that return ethylene back to its ground state from the &pi;*. The participation of the 3s Rydberg state has also been measured by collecting the time resolved photoelectron spectrum during the dynamics on ethylene&rsquo;s &pi;* excited state, confirming calculations predicting the effect of the 3s. </p><p> In carbon dioxide the first time resolved measurement in the lowest electronic excitation of carbon dioxide has been performed. A high kinetic energy release channel shows the signature of wavepacket dynamics within the excited state manifold. Deviation from the direct dissociation predicted for the pumped state provides experimental evidence confirming theoretical predictions of nonadiabatic transitions within the lowest lying electronically excited states. </p>

<p> Nucleic acids, despite being among the most important macromolecules involved in biological life, remain poorly understood in terms of atomistic resolution dynamics at biologically relevant timescales. Due to recent advances in computational power and high resolution structure elucidation we are able to investigate the dynamics of four important nucleic acid structures, namely 5'&ndash;<i>CGAT</i>₆<i>GGC</i>&ndash;3', 5'&ndash;<i> CGCGAT</i>₄<i>GGC</i>-3', 5' -<i>GCATCGAT</i>₂<i>GGC</i>&ndash; 3' (referred to as A₆, A₄, and A₂ DNA respectively) and the TAR HIV-1 RNA molecule on the nanosecond and microsecond timescales. The trajectories are numerically characterized by the NMR relaxation parameter S² which provides an established measure of motion comparable to experiment, from nanosecond based ensembles in the case of A₆, A₄, and A₂DNA, and microsecond based ensembles for A₆DNA and TAR RNA. Specifically, this comparison suggests that while DNA exhibits saturated motions at the nanosecond-microsecond timescale, HIV-1 TAR RNA exhibits motions seemingly correlated across timescales suggesting it has not yet fully saturated motion at the microsecond timescale. Effects of internally correlated, temporally correlated, and diffusively continual motions for nucleic acids are discussed. Finally, the potential of mean force (PMF) of one such smooth transition, the A / B transition, is reported in the presence of a Single Walled Carbon Nanotube (SWNT) for DNA of GC and AT rich sequences.</p>

Chloroplast protein degradation during senescence is delayed in autophagy mutants

<p> Regulation of gene expression is a fundamental biological process required to adapt the full set of hereditary information (i.e., the genome) to the varied environments that any organism encounters. Here, we elucidate two distinct forms of gene regulation &ndash; of endogenous genes by binding of transcription factors to information-containing genomic sequences and of selfish genes (&ldquo;transposons&rdquo;) by targeting of small RNAs to repetitive genomic sequences &ndash; using a wide array of approaches. </p><p> To study regulation by transcription factors, we used glucocorticoid receptor (GR), a hormone-activated, DNA-binding protein that controls inflammation, metabolism, stress responses and other physiological processes. <i>In vitro</i>, GR binds as an inverted dimer to two imperfectly palindromic &ldquo;half sites&rdquo; separated by a &ldquo;spacer&rdquo;. Moreover, GR binds different sequences with distinct conformations, as demonstrated by nuclear magnetic resonance spectroscopy (NMR) and other biophysical methods. </p><p> <i>In vivo</i>, GR employs different functional surfaces when regulating different genes. We investigated whether sequences bound by GR <i> in vivo</i> might be a composite of several motifs, each biased toward utilization of a particular pattern of functional surfaces of GR. Using microarrays and deep sequencing, we characterized gene expression and genomic occupancy by GR, with and without glucocorticoid treatment, of cells expressing GR alleles bearing differences in three known functional surfaces. We found a &ldquo;sub-motif&rdquo;, the GR &ldquo;half site&rdquo;, that relates to utilization of the dimerization interface and directs genomic binding by GR in a distinct conformation. </p><p> To study repression of tranposons, we characterized the production and function of small RNAs in the yeast <i>Cryptococcus neoformans</i>. We found that target transcripts are distinguished by suboptimal introns and inefficient splicing. We identified a complex, SCANR, required for synthesis of small RNAs and demonstrate that it physically associates with the spliceosome. We propose that recognition of gene products by SCANR is in kinetic competition with splicing, thereby further promoting small RNA production from target transcripts. </p><p> To achieve these results, we developed new bioinformatics tools: twobitreader, a small Python package for efficient extraction of genomic sequences; scripter, a flexible back-end for easily creating scripts and pipeline; and seriesoftubes, a pipeline built upon scripter for the analysis of deep sequencing data. </p>

<p> Members of the ANL superfamily of enzymes are involved in primary and secondary metabolism throughout all domains of life and identify key pathways that contribute to essential physiological reactions as well as defense mechanisms to evade competition. Specifically, acetyl-CoA synthetases are directly involved in energy metabolism, while NonRibosoaml Peptide Synthetases and some Aryl-CoA Ligases produce secondary natural products that confer virulence for the producing organism. Due to the ANL superfamily's ubiquitous involvement in primary and secondary metabolism, gaining an understanding of how these enzymes work and identifying ways to regulate them could provide an alternative route for antibiotic targets. It is well documented that domain alternation is paramount for the ANL superfamily of enzymes including the adenylation and thioester-forming reactions of NRPS adenylation domains. This thesis utilizes structural and functional analysis in conjunction with computational methods to further our understanding of these unique enzymes. </p><p> In chapter 2 we present the structure of an adenylation:Peptidyl Carrier Protein di-omain NRPS from the cryptic PA1221 biosynthetic operon from <i> Pseudomonas aeruginosa.</i> The PA1221 structure is the second example of an adenylation:PCP in the PDB and validates the chimeric fusion interactions of EntE-B. The similar interacting regions are between the 2^nd PCP helix and a helix in the N-terminal subdomain of the adenylation domain as well as the loop connecting the longest &beta;-strands of the C-terminal subdomains interacting with loop 1 of the PCP. </p><p> Chapter 3 presents the structure of an acetoacetatyl-CoA Synthetase that is a confirmed substrate for a protein acetyltransferase, PatA, for inactivation through acetylation of the catalytic A10 lysine. This <i>Streptomyces lividans</i> acetoacetyl-CoA synthetase is the first structure to fully resolve the loop connecting C-terminal extension helix to the C-terminal subdomain. The C-terminal extension is only present in ACS proteins revealing an interaction where the C-terminal extension stabilizes the dynamic P-loop in the adenylate forming conformation. </p><p> In chapter 4 we further explore the PA1221 operon by functionally identifying the substrate preference of PA1215, the hypothetical fatty-acyl-CoA Ligase, that is proposed to acylate the charge PCP of PA1221. We computationally validate the substrate preference with a homology model and AutoDock to gain insight into the proteins slow kinetics. We also provide further insight into the biochemistry of a subset of ANL superfamily members, the phenylacetic acid CoA ligases, involved in the utilization of aryl-carboxylic acids as a carbon source as well as the derivatization of penicillin. We analyze their unique dimeric structures identifying structural motifs that are contributed through the dimeric interface, but are otherwise located to different sides of the enzyme in a monomeric form. </p><p> Finally, to help identify how the protein moves between the two productive conformations we subject members of the superfamily to computational dynamic simulations including Anisotropic Network Modeling, Interpolative Elastic Network Modeling, all-atom molecular dynamics, and analyze the output from these methods with Principal Component and Normal Mode Analysis. We developed a method to visualize a dynamic reaction coordinate through measuring the Conformation Determining Angle (defined by structural motifs that are present in superfamily members) and use this metric to interrogate all ANL superfamily member PDB entries for domain organization. Finally, we test our hypothesis that domain alternation proceeds through an extended, open conformation with structural comparisons and MD. Here we report functional and structural analysis of ANL superfamily members that are related through bacterial cell metabolism and natural product biosynthesis.</p>

<p> Acidic aqueous-phase pretreatment is a promising approach that has been directed at maximizing intermediates yields (e.g. sugars, sugar degradation products, and lignin) from biomass for fuel and chemical production. This dissertation explores the kinetic fundamentals of biomass hydrolysis in acidic aqueous-phase with different catalysts (e.g. sulfuric acid, metal chlorides), operating conditions (e.g. temperature, time pressure), and equipment configurations (e.g. batch, flowthough). </p><p> The kinetic analysis revealed that crystalline cellulose is insusceptible to hydrolysis compared with agarose at low temperature (e.g.140 &deg;C), while it decomposed rapidly at elevated temperature (e.g. 220 &deg;C). Higher temperature with reduced time was desirable for glucose production whereas lower temperature with prolonged time was preferred for xylose generation. In acidic conditions, furfural and levulinic acid were stable whereas 5-hydroxymethylfurfural was susceptible to decomposition with high rate constant. MgCl₂ can promote the cleavage of C-O-C bond in polysaccharides (e.g. agarose) and enhance the subsequent dehydration reaction to 5-hydroxymethylfurfural. Unlike transition metal chlorides and H2SO₄, MgCl₂ has little ability to induce retro aldol and rehydration reactions to generate byproducts like lactic acid and levulinic acid. Mg²⁺ possessing hgiher activity than other alkali and alkaline earth metal chlorides (Na⁺ and Ca2⁺) resulted in 40.7% yield and 49.1% selectivity of 5-hydroxymethylfurfural. </p><p> Dissolution of biomass was significantly enhance using acidic hot water flowthrough pretreatment at 200&mdash;280&deg;C. Significant cellulose removal accompanied with the transformation of cellulose I to cellulose II and amorphous cellulose were observed when temperature was above 240 &deg;C for water-only and 220 &deg;C for dilute acid. Approximately100% of the xylan and &sim;90% of the cellulose were solubilized and recovered. Up to 15% of the lignin was solubilized, while the remaining lignin was insoluble. Over 90% sugar yields were obtained from pretreated whole slurries using less than 10 FPU/g cellulase plus hemicellulase enzyme. </p><p> A kinetic model was developed to depict the biomass degradation in flowthrough system. This model predicted the sugar generation more precisely than the conventional homogeneous first-order reaction models. Mass transfer limitations were minimized using 4mm biomass particle sizes with 4g biomass loading at 25mL/min flow rate, produced hydrolyzate slurries with 13g/L potential sugar concentrations.</p>

<p> Abstract not available.</p>

<p> These studies combine crossed laser-molecular beam scattering, velocity map imaging and computation to study the thermal decomposition mechanisms of geminal dinitro energetic materials. In our experimental studies, we photolytically generate two key intermediates in the decomposition of such energetic materials, 2-nitro-2-propyl radical and 2-nitropropene by photodissociating 2-bromo-2-nitropropane at 193 nm. These intermediates were produced at high internal energies and access a number of competing unimolecular dissociation channels. The scattering and imaging experiments provided evidence for four photodissociation pathways of 2-bromo-2-nitropropane along with a total of twelve subsequent dissociations of the resulting intermediate. In addition to the experimental studies, extensive calculations were conducted at the G4//B3LYP/6-311++g(3df,2p) level to compute the energetics of these dissociation events. Interestingly, a novel transition state (TS) for NO loss was discovered for the 2-nitro-2-propyl radical, which has a reaction barrier substantially lower than a traditional nitro-nitrite isomerization mechanism would suggest. This pathway is only available to nitroalkyl radicals and is extremely exothermic. To confirm the presence of this novel TS in a real-world system, the potential energy surface (PES) of a new energetic material, 1,1-diamino-2,2-dinitroethene (FOX-7), was calculated. This PES confirms the presence of the novel NO loss TS in FOX-7; this new TS allows for a low energy pathway to the production of NO in the unimolecular decomposition of FOX-7. In addition, these calculations demonstrate that initial loss of NO is competitive with simple C-N bond fission in unsaturated energetic materials. This low barrier and high exothermicity may be responsible for the extensive NO production and energy release observed in energetic materials with geminal dinitro groups.</p>

<p> G-quadruplex is the most widely known four-stranded nucleic acid structure which has shown to alter gene regulation both in vitro and in vivo. Under certain conditions, another four-stranded structure, i-motif, is also formed in the strand complementary to the G-quadruplex forming sequence. Recent studies suggest gene regulatory roles for the i-motif structure as well. Although there is substantial understanding on the folding topology of G-quadruplex and i-motif structures, their mechanical stability which determines the interaction with motor proteins, such as DNA/RNA polymerases, are poorly studied. Since DNA exists as a double stranded form in vivo, the investigation of i-motif becomes highly important to fully understand the biological functions of G-quadruplexes. Using laser tweezers based single-molecule study, we investigated the mechanical stability of an i-motif structure in the predominant variant of human ILPR fragment (5'-TGTC4ACAC4TGTC4ACAC4TGT). In addition, we have shown that a partially folded structure composed of only three tandem C-rich repeats coexists with the i-motif. Both structures share similar unfolding forces of 22-26 pN. Discovery of stable structures in less than four C-rich repeats suggested that the structure can serve as an intermediate during the i-motif folding/unfolding pathway. Using chemical footprinting and single-molecule approaches, we show that a dsDNA fragment in ILPR, 5'-(ACAG4TGTG4ACAG4TGTG4ACA), can fold into G-quadruplex or i-motif structure under specific conditions. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif, changes in free energy of unfolding provided compelling evidence that only one species is present in each dsDNA. Based on this observation, we propose that G-quadruplex and i-motif are mutually exclusive in human ILPR. Furthermore, we show that these two species have an unfolding force >17 pN. From mechanical perspective, this could justify the regulatory role a DNA tetraplex may play in the expression of human insulin inside cells in which dsDNA is the predominate form.</p>