Metaconcrete: Engineered aggregates for enhanced dynamic performance

This work presents the development and investigation of a new type of concrete for the attenuation of waves induced by dynamic excitation. Recent progress in the field of metamaterials science has led to a range of novel composites which display unusual properties when interacting with electromagnetic, acoustic, and elastic waves. A new structural metamaterial with enhanced properties for dynamic loading applications is presented, which is named metaconcrete. In this new composite material the standard stone and gravel aggregates of regular concrete are replaced with spherical engineered inclusions. Each metaconcrete aggregate has a layered structure, consisting of a heavy core and a thin compliant outer coating. This structure allows for resonance at or near the eigenfrequencies of the inclusions, and the aggregates can be tuned so that resonant oscillations will be activated by particular frequencies of an applied dynamic loading. The activation of resonance within the aggregates causes the overall system to exhibit negative effective mass, which leads to attenuation of the applied wave motion. To investigate the behavior of metaconcrete slabs under a variety of different loading conditions a finite element slab model containing a periodic array of aggregates is utilized. The frequency dependent nature of metaconcrete is investigated by considering the transmission of wave energy through a slab, which indicates the presence of large attenuation bands near the resonant frequencies of the aggregates. Applying a blast wave loading to both an elastic slab and a slab model that incorporates the fracture characteristics of the mortar matrix reveals that a significant portion of the supplied energy can be absorbed by aggregates which are activated by the chosen blast wave profile. The transfer of energy from the mortar matrix to the metaconcrete aggregates leads to a significant reduction in the maximum longitudinal stress, greatly improving the ability of the material to resist damage induced by a propagating shock wave. The various analyses presented in this work provide the theoretical and numerical background necessary for the informed design and development of metaconcrete aggregates for dynamic loading applications, such as blast shielding, impact protection, and seismic mitigation.

Sulfur-Cycling in Methane-Rich Ecosystems: Uncovering Microbial Processes and Novel Niches

Microbial sulfur cycling communities were investigated in two methane-rich ecosystems, terrestrial mud volcanoes (TMVs) and marine methane seeps, in order to investigate niches and processes that would likely be central to the functioning of these crucial ecosystems. Terrestrial mud volcanoes represent geochemically diverse habitats with varying sulfur sources and yet sulfur-cycling in these environments remains largely unexplored. Here we characterized the sulfur-metabolizing microorganisms and activity in 4 TMVs in Azerbaijan, supporting the presence of active sulfur-oxidizing and sulfate-reducing guilds in all 4 TMVs across a range of physiochemical conditions, with diversity of these guilds being unique to each TMV. We also found evidence for the anaerobic oxidation of methane coupled to sulfate reduction, a process which we explored further in the more tractable marine methane seeps. Diverse associations between methanotrophic archaea (ANME) and sulfate-reducing bacterial groups (SRB) often co-occur in marine methane seeps, however the ecophysiology of these different symbiotic associations has not been examined. Using a combination of molecular, geochemical and fluorescence in situ hybridization coupled to nano-scale secondary ion mass spectrometry (FISH-NanoSIMS) analyses of in situ seep sediments and methane-amended sediment incubations from diverse locations, we show that the unexplained diversity in SRB associated with ANME cells can be at least partially explained by preferential nitrate utilization by one particular partner, the seepDBB. This discovery reveals that nitrate is likely an important factor in community structuring and diversity in marine methane seep ecosystems. The thesis concludes with a study of the dynamics between ANME and their associated SRB partners. We inhibited sulfate reduction and followed the metabolic processes of the community as well as the effect of ANME/SRB aggregate composition and growth on a cellular level by tracking 15N substrate incorporation into biomass using FISH-NanoSIMS. We revealed that while sulfate-reducing bacteria gradually disappeared over time in incubations with an SRB inhibitor, the ANME archaea persisted in the form of ANME-only aggregates, which are capable of little to no growth when sulfate reduction is inhibited. These data suggest ANME are not able to synthesize new proteins when sulfate reduction is inhibited.

Synthesis and potency of long end-associative polymers for mist control

Long linear polymers that are end-functionalized with associative groups were studied as additives to hydrocarbon fluids to mitigate the fire hazard associated with the presence of mist in a crash scenario. These polymers were molecularly designed to overcome both the shear-degradation of long polymer chains in turbulent flows, and the chain collapse induced by the random placement of associative groups along polymer backbones. Architectures of associative groups on the polymer chain ends that were tested included clusters of self-associative carboxyl groups and pairs of hetero-complementary associative units.

Linear polymers with clusters of discrete numbers of carboxyl groups on their chain ends were investigated first: an innovative synthetic strategy was devised to achieve unprecedented backbone lengths and precise control of the number of carboxyl groups on chain ends (N). We found that a very narrow range of N allows the co-existence of sufficient end-association strength and polymer solubility in apolar media. Subsequent steady-flow rheological study on solution behavior of such soluble polymers in apolar media revealed that the end-association of very long chains in apolar media leads to the formation of flower-like micelles interconnected by bridging chains, which trap significant fraction of polymer chains into looped structures with low contribution to mist-control. The efficacy of very long 1,4-polybutadiene chains end-functionalized with clusters of four carboxyl groups as mist-control additives for jet fuel was further tested. In addition to being shear-resistant, the polymer was found capable of providing fire-protection to jet fuel at concentrations as low as 0.3wt%. We also found that this polymer has excellent solubility in jet fuel over a wide range of temperature (-30 to +70°C) and negligible interference with dewatering of jet fuel. It does not cause an adverse increase in viscosity at concentrations where mist-control efficacy exists.

Four pairs of hetero-complementary associative end-groups of varying strengths were subsequently investigated, in the hopes of achieving supramolecular aggregates with both mist-control ability and better utilization of polymer building blocks. Rheological study of solutions of the corresponding complementary associative polymer pairs in apolar media revealed the strength of complementary end-association required to achieve supramolecular aggregates capable of modulating rheological properties of the solution.

Both self-associating and complementary associating polymers have therefore been found to resist shear degradation. The successful strategy of building soluble, end-associative polymers with either self-associative or complementary associative groups will guide the next generation of mist-control technology.

Intramolecular conflict : conformation and self-assembly of architecturally complex macromolecules in solution

The solution behavior of linear polymer chains is well understood, having been the subject of intense study throughout the previous century. As plastics have become ubiquitous in everyday life, polymer science has grown into a major field of study. The conformation of a polymer in solution depends on the molecular architecture and its interactions with the surroundings. Developments in synthetic techniques have led to the creation of precision-tailored polymeric materials with varied topologies and functionalities. In order to design materials with the desired properties, it is imperative to understand the relationships between polymer architecture and their conformation and behavior. To meet that need, this thesis investigates the conformation and self-assembly of three architecturally complex macromolecular systems with rich and varied behaviors driven by the resolution of intramolecular conflicts. First we describe the development of a robust and facile synthetic approach to reproducible bottlebrush polymers (Chapter 2). The method was used to produce homologous series of bottlebrush polymers with polynorbornene backbones, which revealed the effect of side-chain and backbone length on the overall conformation in both good and theta solvent conditions (Chapter 3). The side-chain conformation was obtained from a series of SANS experiments and determined to be indistinguishable from the behavior of free linear polymer chains. Using deuterium-labeled bottlebrushes, we were able for the first time to directly observe the backbone conformation of a bottlebrush polymer which showed self-avoiding walk behavior. Secondly, a series of SANS experiments was conducted on a homologous series of Side Group Liquid Crystalline Polymers (SGLCPs) in a perdeuterated small molecule liquid crystal (5CB). Monodomain, aligned, dilute samples of SGLCP-b-PS block copolymers were seen to self-assemble into complex micellar structures with mutually orthogonally oriented anisotropies at different length scales (Chapter 4). Finally, we present the results from the first scattering experiments on a set of fuel-soluble, associating telechelic polymers. We observed the formation of supramolecular aggregates in dilute (≤0.5wt%) solutions of telechelic polymers and determined that the choice of solvent has a significant effect on the strength of association and the size of the supramolecules (Chapter 5). A method was developed for the direct estimation of supramolecular aggregation number from SANS data. The insight into structure-property relationships obtained from this work will enable the more targeted development of these molecular architectures for their respective applications.

Structure-, stereochemistry-, and metal-regulated DNA binding/cleaving molecules

In the cell, the binding of proteins to specific sequences of double helical DNA is essential for controlling the processes of protein synthesis (at the level of DNA transcription) and cell proliferation (at the level of DNA replication). In the laboratory, the sequence-specific DNA binding/cleaving properties of restriction endonuclease enzymes (secreted by microorganisms to protect them from foreign DNA molecules) have helped to fuel a revolution in molecular biology. The strength and specificity of a protein:DNA interaction depend upon structural features inherent to the protein and DNA sequences, but it is now appreciated that these features (and therefore protein:DNA complexation) may be altered (regulated) by other protein:DNA complexes, or by environmental factors such as temperature or the presence of specific organic molecules or inorganic ions. It is also now appreciated that molecules much smaller than proteins (including antibiotics of molecular weight less than 2000 and oligonucleotides) can bind to double-helical DNA in sequence-specific fashion. Elucidation of structural motifs and microscopic interactions responsible for the specific molecular recognition of DNA leads to greater understanding of natural processes and provides a basis for the design of novel sequence-specific DNA binding molecules. This thesis describes the synthesis and DNA binding/cleaving characteristics of molecules designed to probe structural, stereochemical, and environmental factors that regulate sequence-specific DNA recognition.

Chapter One introduces the DNA minor groove binding antibiotics Netropsin and Distamycin A, which are di- and tri(N-methylpyrrolecarboxamide) peptides, respectively. The method of DNA affinity cleaving, which has been employed to determine DNA binding properties of designed synthetic molecules is described. The design and synthesis of a series of Netropsin dimers linked in tail-to-tail fashion (by oxalic, malonic, succinic, or fumaric acid), or in head-to-tail fashion (by glycine, β-alanine, and γ-aminobutanoic acid (Gaba)) are presented. These Bis(Netropsin)s were appended with the iron-chelating functionality EDTA in order to make use of the technique of DNA affinity cleaving. Bis(Netropsin)-EDTA compounds are analogs of penta(N-methylpyrrolecarboxamide)-EDTA (P5E), which may be considered a head-to-tail Netropsin dimer linked by Nmethylpyrrolecarboxamide. Low- and high-resolution analysis of pBR322 DNA affinity cleaving by the iron complexes of these molecules indicated that small changes in the length and nature of the linker had significant effects on DNA binding/cleaving efficiency (a measure of DNA binding affinity). DNA binding/cleaving efficiency was found to decrease with changes in the linker in the order β-alanine > succinamide > fumaramide > N-methylpyrrolecarboxamide > malonamide >glycine, γ-aminobutanamide > oxalamide. In general, the Bis(Netropsin)-EDTA:Fe compounds retained the specificity for seven contiguous A:T base pairs characteristic of P5E:Fe binding. However, Bis(Netropsin)Oxalamide- EDTA:Fe exhibited decreased specificity for A:T base pairs, and Bis(Netropsin)-Gaba-EDT A:Fe exhibited some DNA binding sites of less than seven base pairs. Bis(Netropsin)s linked with diacids have C₂-symmmetrical DNA binding subunits and exhibited little DNA binding orientation preference. Bis(Netropsin)s linked with amino acids lack C₂-symmetrical DNA binding subunits and exhibited higher orientation preferences. A model for the high DNA binding orientation preferences observed with head-to-tail DNA minor groove binding molecules is presented.

Chapter Two describes the design, synthesis, and DNA binding properties of a series of chiral molecules: Bis(Netropsin)-EDTA compounds with linkers derived from (R,R)-, (S,S)-, and (RS,SR)-tartaric acids, (R,R)-, (S,S)-, and (RS,SR)-tartaric acid acetonides, (R)- and (S)-malic acids, N ,N-dimethylaminoaspartic acid, and (R)- and (S)-alanine, as well as three constitutional isomers in which an N-methylpyrrolecarboxamide (P1) subunit and a tri(N-methylpyrrolecarboxamide)-EDTA (P3-EDTA) subunit were linked by succinic acid, (R ,R)-, and (S ,S)-tartaric acids. DNA binding/cleaving efficiencies among this series of molecules and the Bis(Netropsin)s described in Chapter One were found to decrease with changes in the linker in the order β-alanine > succinamide > P1-succinamide-P3 > fumaramide > (S)-malicamide > N-methylpyrrolecarboxamide > (R)-malicamide > malonamide > N ,N-dimethylaminoaspanamide > glycine = Gaba = (S,S)-tartaramide = P1-(S,S)-tanaramide-P3 > oxalamide > (RS,SR)-tartaramide = P1- (R,R)-tanaramide-P3 > (R,R)-tartaramide (no sequence-specific DNA binding was detected for Bis(Netropsin)s linked by (R)- or (S)-alanine or by tartaric acid acetonides). The chiral molecules retained DNA binding specificity for seven contiguous A:T base pairs. From the DNA affinity cleaving data it could be determined that: 1) Addition of one or two substituents to the linker of Bis(Netropsin)-Succinamide resulted in stepwise decreases in DNA binding affinity; 2) molecules with single hydroxyl substituents bound DNA more strongly than molecules with single dimethylamino substituents; 3) hydroxyl-substituted molecules of (S) configuration bound more strongly to DNA than molecules of (R) configuration. This stereochemical regulation of DNA binding is proposed to arise from the inherent right-handed twist of (S)-enantiomeric Bis(Netropsin)s versus the inherent lefthanded twist of (R)-enantiomeric Bis(Netropsin)s, which makes the (S)-enantiomers more complementary to the right-handed twist of B form DNA.

Chapter Three describes the design and synthesis of molecules for the study of metalloregulated DNA binding phenomena. Among a series of Bis(Netropsin)-EDTA compounds linked by homologous tethers bearing four, five, or six oxygen atoms, the Bis(Netropsin) linked by a pentaether tether exhibited strongly enhanced DNA binding/cleaving in the presence of strontium or barium cations. The observed metallospecificity was consistent with the known affinities of metal cations for the cyclic hexaether 18-crown-6 in water. High-resolution DNA affinity cleaving analysis indicated that DNA binding by this molecule in the presence of strontium or barium was not only stronger but of different sequence-specificity than the (weak) binding observed in the absence of metal cations. The metalloregulated binding sites were consistent with A:T binding by the Netropsin subunits and G:C binding by a strontium or barium:pentaether complex. A model for the observed positive metalloregulation and novel sequence-specificity is presented. The effects of 44 different cations on DNA affinity cleaving by P5E:Fe were examined. A series of Bis(Netropsin)-EDTA compounds linked by tethers bearing two, three, four, or five amino groups was also synthesized. These molecules exhibited strong and specific binding to A:T rich regions of DNA. It was found that the iron complexes of these molecules bound and cleaved DNA most efficiently at pH 6.0-6.5, while P5E:Fe bound and cleaved most efficiently at pH 7.5-8.0. Incubating the Bis(Netropsin) Polyamine-EDTA:Fe molecules with K₂PdCl₄ abolished their DNA binding/cleaving activity. It is proposed that the observed negative metalloregulation arises from kinetically inert Bis(Netropsin) Polyamine:Pd(II) complexes or aggregates, which are sterically unsuitable for DNA complexation. Finally, attempts to produce a synthetic metalloregulated DNA binding protein are described. For this study, five derivatives of a synthetic 52 amino acid residue DNA binding/cleaving protein were produced. The synthetic mutant proteins carried a novel pentaether ionophoric amino acid residue at different positions within the primary sequence. The proteins did not exhibit significant DNA binding/cleaving activity, but they served to illustrate the potential for introducing novel amino acid residues within DNA binding protein sequences, and for the development of the tricyclohexyl ester of EDTA as a superior reagent for the introduction of EDT A into synthetic proteins.

Chapter Four describes the discovery and characterization of a new DNA binding/cleaving agent, [SalenMn(III)]OAc. This metal complex produces single- and double-strand cleavage of DNA, with specificity for A:T rich regions, in the presence of oxygen atom donors such as iodosyl benzene, hydrogen peroxide, or peracids. Maximal cleavage by [SalenMn(III)]OAc was produced at pH 6-7. A comparison of DNA singleand double-strand cleavage by [SalenMn(III)]⁺ and other small molecules (Methidiumpropyl-EDTA:Fe, Distamycin-EDTA:Fe, Neocarzinostatin, Bleomycin:Fe) is presented. It was found that DNA cleavage by [SalenMn(III)]⁺ did not require the presence of dioxygen, and that base treatment of DNA subsequent to cleavage by [SalenMn(III)]⁺ afforded greater cleavage and alterations in the cleavage patterns. Analysis of DNA products formed upon DNA cleavage by [SalenMn(III)] indicated that cleavage was due to oxidation of the sugar-phosphate backbone of DNA. Several mechanisms consistent with the observed products and reaction requirements are discussed.

Chapter Five describes progress on some additional studies. In one study, the DNA binding/cleaving specificities of Distamycin-EDTA derivatives bearing pyrrole N-isopropyl substituents were found to be the same as those of derivatives bearing pyrrole N-methyl substituents. In a second study, the design of and synthetic progress towards a series of nucleopeptide activators of transcription are presented. Five synthetic plasmids designed to test for activation of in vitro run-off transcription by DNA triple helix-forming oligonucleotides or nucleopeptides are described.

Chapter Six contains the experimental documentation of the thesis work.

Biochemical and Biophysical Characterization of Huntingtin

Huntington’s disease (HD) is a fatal autosomal dominant neurodegenerative disease. HD has no cure, and patients pass away 10-20 years after the onset of symptoms. The causal mutation for HD is a trinucleotide repeat expansion in exon 1 of the huntingtin gene that leads to a polyglutamine (polyQ) repeat expansion in the N-terminal region of the huntingtin protein. Interestingly, there is a threshold of 37 polyQ repeats under which little or no disease exists; and above which, patients invariably show symptoms of HD. The huntingtin protein is a 350 kDa protein with unclear function. As the polyQ stretch expands, its propensity to aggregate increases with polyQ length. Models for polyQ toxicity include formation of aggregates that recruit and sequester essential cellular proteins, or altered function producing improper interactions between mutant huntingtin and other proteins. In both models, soluble expanded polyQ may be an intermediate state that can be targeted by potential therapeutics.

In the first study described herein, the conformation of soluble, expanded polyQ was determined to be linear and extended using equilibrium gel filtration and small-angle X-ray scattering. While attempts to purify and crystallize domains of the huntingtin protein were unsuccessful, the aggregation of huntingtin exon 1 was investigated using other biochemical techniques including dynamic light scattering, turbidity analysis, Congo red staining, and thioflavin T fluorescence. Chapter 4 describes crystallization experiments sent to the International Space Station and determination of the X-ray crystal structure of the anti-polyQ Fab MW1. In the final study, multimeric fibronectin type III (FN3) domain proteins were engineered to bind with high avidity to expanded polyQ tracts in mutant huntingtin exon 1. Surface plasmon resonance was used to observe binding of monomeric and multimeric FN3 proteins with huntingtin.

Proton magnetic resonance studies of ribonucleic acid complexes. I. Complexes of biological bases and oligonucleotides with RNA. II. Template recognition and the degeneracy of the genetic code

Part I. Complexes of Biological Bases and Oligonucleotides with RNA

The physical nature of complexes of several biological bases and oligonucleotides with single-stranded ribonucleic acids have been studied by high resolution proton magnetic resonance spectroscopy. The importance of various forces in the stabilization of these complexes is also discussed.

Previous work has shown that purine forms an intercalated complex with single-stranded nucleic acids. This complex formation led to severe and stereospecific broadening of the purine resonances. From the field dependence of the linewidths, T₁ measurements of the purine protons and nuclear Overhauser enhancement experiments, the mechanism for the line broadening was ascertained to be dipole-dipole interactions between the purine protons and the ribose protons of the nucleic acid.

The interactions of ethidium bromide (EB) with several RNA residues have been studied. EB forms vertically stacked aggregates with itself as well as with uridine, 3'-uridine monophosphate and 5'-uridine monophosphate and forms an intercalated complex with uridylyl (3' → 5') uridine and polyuridylic acid (poly U). The geometry of EB in the intercalated complex has also been determined.

The effect of chain length of oligo-A-nucleotides on their mode of interaction with poly U in D₂0 at neutral pD have also been studied. Below room temperatures, ApA and ApApA form a rigid triple-stranded complex involving a stoichiometry of one adenine to two uracil bases, presumably via specific adenine-uracil base pairing and cooperative base stacking of the adenine bases. While no evidence was obtained for the interaction of ApA with poly U above room temperature, ApApA exhibited complex formation of a 1:1 nature with poly U by forming Watson-Crick base pairs. The thermodynamics of these systems are discussed.

Part II. Template Recognition and the Degeneracy of the Genetic Code

The interaction of ApApG and poly U was studied as a model system for the codon-anticodon interaction of tRNA and mRNA in vivo. ApApG was shown to interact with poly U below ~20°C. The interaction was of a 1:1 nature which exhibited the Hoogsteen bonding scheme. The three bases of ApApG are in an anti conformation and the guanosine base appears to be in the lactim tautomeric form in the complex.

Due to the inadequacies of previous models for the degeneracy of the genetic code in explaining the observed interactions of ApApG with poly U, the "tautomeric doublet" model is proposed as a possible explanation of the degenerate interactions of tRNA with mRNA during protein synthesis in vivo.