Synthesis, characterization and study of physical properties of novel M(n+1)AX(n) compounds

Mn+1AXn compounds, the ternary layered nanolaminates have gathered momentum in the last decade since its advent because of their unusual but exciting properties. These technologically important compounds combine some of the best properties of metals and ceramics. Like ceramics they are refractory, oxidation resistant, elastically stiff and relatively light. They also exhibit metallic properties like excellent machinability, thermal and electrical conductivity. This dissertation concentrates on the synthesis of germanium-based 211 Mn+1AXn compounds. The main objective of the research was to synthesize predominantly single phase samples of Cr2GeC, V2GeC and Ti2GeC. Another goal was to study the effect of solid substitutions on the M-site of Mn+1AXn compounds with Ge as an A-element. This study is in itself the first to demonstrate the synthesis of (Cr0.5V0.5)2GeC a novel Mn+1AXn compound. Scanning electron microscopy coupled with energy dispersive spectroscopy, x-ray diffraction and electron probe microanalysis were employed to confirm the presence of predominantly single phase samples of M2GeC compounds where M = Ti, V, Cr and (Cr 0.5V0.5). A large part of the dissertation also focuses on the effect of the compressibility on the Ge-based 211 Mn+1AXn compounds with the aid of diamond anvil cell and high energy synchrotron radiation. This study also concentrates on the stability of these compounds at high temperature and thereby determines its suitability as high temperature structural materials. In order to better understand the effect of substitutions on A-site of 211 Mn+1 AXn compounds under high pressure and high temperature, a comparison is made with previously reported 211 Mn+1AXn compounds with Al, Ga and S as A-site elements.

Characterization of recombinant chloroperoxidase, and F103A and C29H/C79H/C87H mutants

Mechanistically and structurally chloroperoxidase (CPO) occupies a unique niche among heme containing enzymes. Chloroperoxidase catalyzes a broad range of reactions, such as oxidation of organic substrates, dismutation of hydrogen peroxide, and mono-oxygenation of organic molecules. To expand the synthetic utility of CPO and to appreciate the important interactions that lead to CPO’s exceptional properties, a site-directed mutagenesis study was undertaken. ^ Recombinant CPO and CPO mutants were heterologously expressed in Aspergillus niger. The overall protein structure was almost the same as that of wild type CPO, as determined by UV-vis, NMR and CD spectroscopies. Phenylalanine103, which was proposed to regulate substrate access to the active site by restricting the size of substrates and to control CPO’s enantioselectivity, was mutated to Ala. The ligand binding affinity and most importantly the catalytic activity of F103A was dramatically different from wild type CPO. The mutation essentially eliminated the chlorination and dismutation activities but enhanced, 4-10 fold, the epoxidation, peroxidation, and N-demethylation activities. As expected, the F103A mutant displayed dramatically improved epoxidation activity for larger, more branched styrene derivatives. Furthermore, F103A showed a distinctive enantioselectivity profile: losing enantioselectivity to styrene and cis-β-methylstyrene; having a different configuration preference on α-methylstyrene; showing higher enantioselectivites and conversion rates on larger, more branched substrates. Our results show that F103 acts as a switch box that controls the catalytic activity, substrate specificity, and product enantioselectivity of CPO. Given that no other mutant of CPO has displayed distinct properties, the results with F103A are dramatic. ^ The diverse catalytic activity of CPO has long been attributed to the presence of the proximal thiolate ligand. Surprisingly, a recent report on a C29H mutant suggested otherwise. A new CPO triple mutant C29H/C79H/C87H was prepared, in which all the cysteines were replaced by histidine to eliminate the possibility of cysteine coordinating to the heme. No active form protein was isolated, although, successful transformation and transcription was confirmed. The result suggests that Cys79 and Cys87 are critical to maintaining the structural scaffold of CPO. ^ In vitro biodegradation of nanotubes by CPO were examined by scanning electron microscope method, but little oxidation was observed. ^

Molecular characterization of Cladium peat from the Florida Everglades: biomarker associations with humic fractions

The accumulation and preservation of peat soils in Everglades freshwater marshes and mangrove swamps is an essential process in the ecological functioning of these ecosystems. Human intervention and climate change have modified nutrient dynamics and hydroperiod in the Everglades and peat loss due to such anthropogenic activities is evident. However, not much is known on the molecular level regarding the biogeochemical characteristics, which allow peat to be preserved in the Everglades. Lipid biomarkers trapped within or bound to humic-type structures can provide important geochemical information regarding the origin and microbial transformation of OM in peat. Four lipid fractions obtained from a Cladium peat, namely the freely extractable fraction and those associated with humin, humic acid, and fulvic acid fractions, showed clear differences in their molecular distribution suggesting different OM sources and structural and diagenetic states of the source material. Both, higher plant derived and microbial lipids were found in association with these humic-type substances. Most biomarker distributions suggest an increment in the microbial/terrestrial lipid ratio from the free to humin to humic to fulvic fractions. Microbial reworking of lipids, and the incorporation of microbial biomarkers into the humic-type fractions was evident, as well as the preservation of diagenetic byproducts. The lipid distribution associated with the fulvic acids suggests a high degree of microbial reworking for this fraction. Evidence for this 3D structure was obtained through the presence of the relatively high abundance of α,ω-dicarboxylic acids and phenolic and benzenecarboxylic compounds. The increment in structural complexity of the phenolic and benzencarboxylic compounds in combination with the reduction in the carbon chain length of the dicarboxylic acids from the free to fulvic fraction suggests the latter to be structurally the most stable, compacted and diagenetically altered substrate. This analytical approach can now be applied to peat samples from other areas within the Everglades ecosystem, affected differently by human intervention with the aim to assess changes in organic matter preservation.

Isolation and characterization of the U1 and U2 snRNAs in the silk gland of the 5th instar larval stage silk moth Bombyx mori

In isolation and characterization studies, expression level U1 and U2 snRNA isoforms were obtained from the 5th instar larval stage silk gland (SG). The DNA content of the SG cells is approximately 200,000-fold higher compared to the usual (2N) somatic cells of B. mori due to endoreduplication. In this study, the existence of U1 and U2 snRNA isoforms in the SG of the organism is investigated. Bombyx mori U1 and U2-specific RT-PCR libraries from the silk gland were generated. Five U1 and eight U2 isoforms were isolated and characterized. Nucleotide differences, structural alterations, as well as protein and RNA interaction sites were analyzed in these variants. For the U1 snRNA variants, they were compared to the previously reported BmN isoforms. In all these U-snRNA variants, polymorphic sites do not predominate at the core of known functional sequences, which were interspecifically conserved. Variant sites and inter-species differences are located in moderately conserved regions. Free energy (ΔG) values for the entire U1 and U2 snRNA secondary structures and for the individual stem/loops domains of the isoforms were generated and compared to determine their structural stability. This will be the first time that U1 and U2 variants are shown specific for a development stage (larval) other than embryonic or adult. ^ Using phylogenetic analysis, evolutionary trees were generated for the U1 and U2 snRNAs using animal, plant, protista and fungal species. The resulting trees were boostrapped for robustness and rooted with the self-splicing RNA group II intron sequence from the cyanobacterium Calothrix. Using phylogenetic analyses, possible structural and functional evolutionary interdependence between the U1 and U2 snRNAs was investigated. ^

Characterization of Juvenile Hormone Biosynthetic Enzymes in the Mosquito, Aedes aegypti

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. They are synthesized and secreted by a pair of small endocrine glands, the corpora allata (CA), which are intimately connected to the brain. The enzymes involved in the biosynthesis of JH are attractive targets for the control of mosquito populations. This dissertation is a comprehensive functional study of five Aedes aegypti CA enzymes, HMG-CoA synthase (AaHMGS), mevalonate kinase (AaMK), phosphomevalonate kinase (AaPMK), farnesyl diphosphate synthase (AaFPPS) and farnesyl pyrophosphate phosphatase (AaFPPase). The enzyme AaHMGS catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to produce HMG-CoA. The enzyme does not require any co-factor, although its activity is enhanced by addition of Mg2+. The enzyme AaMK is a class I mevalonate kinase that catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate. Activity of AaMK is inhibited by isoprenoids. The enzyme AaPMK catalyzes the cation-dependent reversible reaction of phosphomevalonate and ATP to form diphosphate mevalonate and ADP. The enzyme AaFPPS catalyzes the condensation of isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form geranyl diphosphate (GPP) and farnesyl pyrophosphate (FPP). The enzyme AaFPPS shows an unusual product regulation mechanism, with chain length final product of 10 or 15 C depending on the metal cofactor present. The enzymes AaFPPase-1 and AaFPPase-2 efficiently hydrolyze FPP into farnesol, although RNAi experiments demonstrate that only AaFPPase-1 is involved in the catalysis of FPP into FOL in the CA of A. aegypti. This dissertation also explored the inhibition of the activity of some of the JH biosynthesis enzymes as tools for insect control. We described the effect of N-acetyl-S-geranylgeranyl-L-cysteine as a potent inhibitor of AaFPPase 1 and AaFPPase-2. In addition, inhibitors of AaMK and AaHMGS were also investigated using purified recombinant proteins. The present study provides an important contribution to the characterization of recombinant proteins, the analysis of enzyme kinetics and inhibition constants, as well as the understanding of the importance of these five enzymes in the control of JH biosynthesis rates.

Synthesis, crystal structure and spectroscopic characterization of a coordination polymer of copper(II) chloride with ethylenediamine and the 2-hydroxybenzoate ion

Peer reviewed