Lipid assisted membrane entry in specific phospholipid targeted protein systems

The purpose of this thesis project is to study changes in the physical state of cell membranes during cell entry, including how these changes are connected to the presence of ceramide. The role of enzymatical manipulation of lipids in bacterial internalization is also studied. A novel technique, where a single giant vesicle is chosen under the microscope and an enzyme coupled-particle attached to the micromanipulator pipette towards the vesicle, is used. Thus, the enzymatic reaction on the membrane of the giant vesicle can be followed in real-time. The first aim of this study is to develop a system where the localized sphingomyelinase membrane interaction could be observed on the surface of the giant vesicle and the effects could be monitored with microscopy. Domain formation, which resembles acid sphingomyelinase (ASMase), causes CD95 clustering in the cell membrane due to ceramide production (Grassmé et al., 2001a; Grassmé et al., 2001b) and the formation of small vesicles inside the manipulated giant vesicle is observed. Sphingomyelinase activation has also been found to be an important factor in the bacterial and viral invasion process in nonphagocytic cells (Grassmé et al., 1997; Jan et al., 2000). Accordingly, sphingomyelinase reactions in the cell membrane might also give insight into bacterial or viral cellular entry events. We found sphingomyelinase activity in Chlamydia pneumonia elementarybodies (EBs). Interestingly, the bacterium enters host cells by endocytosis but the internalization mechanism of Chlamydia is unknown. The hypothesis is that sphingomyelin is needed for host cell entry in the infection of C. pneumonia. The second project focuses on this subject. The goal of the third project is to study a role of phosphatidylserine as a target for a membrane binding protein. Phosphatidylserine is chosen because of its importance in fusion processes. This will be another example for the importance of lipids in cell targeting, internalization, and externalization.

Synthesis, characterization, thermal degradation, and comparative chain dynamics studies of weak-link polysulfide polymers

Synthesis, spectroscopic and thermal characterization of two new classes of polysulfide polymers: poly[1(phenoxymethyl) ethylene polysulfide] (PPMEP), and poly [1-(phenoxy) ethylene polysulfide] (PPEP) is presented. The direct pyrolysis mass spectrometry (DP-MS) technique, used to study the thermal degradation behavior of these polysulfide polymers, indicated that the polymers underwent degradation through the weak-links scission. The thermal stability of the polysulfide polymers decreased as the ``rank'' (number of sulfur atoms in the polysulfide linkage; n=1, 2, 4) increased. The main-chain flexibility of these polysulfide polymers in terms of their C-13 NMR spinlattice relaxation time (T-1) measurements on the backbone methine (-CH-) and methylene (-CH2-) carbons are reported here for the first time. A comparative study of the solution chain dynamics indicated that it increased as ``rank'' of the polysulfide linkages decreased as well as by introducing side chain spacers such as, ether (-O-) or methyleneoxy (-CH2O-) groups.

Hybrid Sol-Gel Combustion Synthesis of Nanoporous Anatase

Nanoporous anatase with a thin interconnected filmlike morphology has been synthesized in a single step by coupling a nonhydrolytic condensation reaction of a Ti precursor with a hybrid sol-gel combustion reaction. The method combines the advantages of a conventional sol-gel method for the formation of porous structures with the high crystallinity of the products obtained by combustion methods to yield highly crystalline, phase-pure nanoporous anatase. The generation of pores is initiated by the formation of reverse micelles in a polymeric polycondensation product, which expand during heating, leading to larger pores. A reaction scheme involving a complex formation and nonhydrolytic polycondensation reaction with ester elimination leads to the formation of ail extended Ti-O-Ti network. The effect of process parameters, such as temperature and relative ratio of cosurfactants, on phase formation has been studied. The possibility of band gap engineering by controlled doping during synthesis and the possibility of attachment of molecular/nanoparticle sensitizers provide opportunities for easy preparation of photoanodes for solar cell applications.

De novo synthesis of acetylcholinesterase in roots of Pisum sativum

Pisum sativum seeds contain a conserved acetylcholinesterase (AChE) which is active during the early stages of germination. The enzyme activity soon disappears and reappears after 72 hr of germination. A protein devoid of catalytic ability, but exhibiting similar chromatographic and electrophoretic properties as the active AChE, could be detected after 24 hr of germination. The pattern of incorporation of labelled amino acids into AChE and the influence of cycloheximide revealed that the AChE found in the roots from 72 hr onwards was entirely new. During this period of growth, the AChE protein accounts for 4–10% of the total proteins in the root tissue.

One-Pot Synthesis of beta-Amino/beta-Hydroxy Selenides and Sulfides from Aziridines and Epoxides

Diaryl disulfides and diselenides undergo facile cleavage on treatment with rongalite (sodium hydroxymethanesulfinate) to generate the corresponding thiolate and selenolate species in Situ, which effect the ring opening of aziridines and epoxides in a regioselective manner. A simple, mild, cost-effective protocol has been developed to prepare beta-amino and beta-hydroxy sulfides and selenides in a one-pot operation.

A green protocol for room temperature synthesis of silver nanoparticles in seconds

We describe here a rapid, energy-efficient, green and economically scalable room temperature protocol for the synthesis of silver nanoparticles. Tannic acid, a polyphenolic compound derived from plant extracts is used as the reducing agent. Silver nanoparticles of mean size ranging from 3.3 to 22.1 nm were synthesized at room temperature by the addition of silver nitrate to tannic acid solution maintained at an alkaline pH. The mean size was tuned by varying the molar ratio of tannic acid to silver nitrate. We also present proof of concept results demonstrating its suitability for room temperature continuous flow processing.

Synthesis, structural and ferromagnetic properties of La1-xKxMnO3 (0.0 <= x <= 0.25) phases by solution combustion method

We describe the solution combustion synthesis and characterization of La1-xKxMnO3 (0.0 <= x <= 0.25) perovskite phases, which is a low temperature initiated, rapid route to prepare metal oxides. As-synthesized compounds are amorphous in nature; crystallinity was observed on heating at 800 degrees C for 5 min. Structural parameters were determined by the Rietveld refinement method using powder XRD data. Parent LaMnO3 compound crystallizes in the orthorhombic structure (space group Pbnm, No. 62). Potassium substituted compounds were crystallized with rhombohedral symmetry (space group R-3c, No. 167). The ratio of the Mn3+/Mn4+ was determined by the iodometric titration. The Fourier transform infrared spectrum (FTIR) shows two absorption bands for Mn-O stretching vibration (v, mode), Mn-O-Mn deformation vibration (v(b) mode) around 600 cm(-1) and 400 cm(-1) for the compositions, x = 0.0, 0.05 and 0-10. Four-probe electrical resistivity measurements reveal a composition controlled metal to insulator transition (TM-1), the maximum TM-1 was observed for the composition La0.85K0.15MnO3 at 287 K. Room temperature vibrating sample magnetometer data indicate that for the composition up to x = 0-10, the compounds are paramagnetic whereas composition with x = 0.15, 0.20 and 0.25 show magnetic moments of 27, 29 and 30 emu/g, respectively.

Molecular details of phage phi6 RNA-dependent RNA synthesis

For most RNA viruses RNA-dependent RNA polymerases (RdRPs) encoded by the virus are responsible for the entire RNA metabolism. Thus, RdRPs are critical components in the viral life cycle. However, it is not fully understood how these important enzymes function during viral replication. Double-stranded RNA (dsRNA) viruses perform the synthesis of their RNA genome within a proteinacous viral particle containing an RdRP as a minor constituent. The phi6 bacteriophage is the best-studied dsRNA virus, providing an excellent background for studies of its RNA synthesis. The purified recombinant phi6 RdRP is highly active in vitro and it possesses both RNA replication and transcription activities. The crystal structure of the phi6 polymerase, solved in complex with a number of ligands, provides a working model for detailed in vitro studies of RNA-dependent RNA polymerization. In this thesis, the primer-independent initiation of the phi6 RdRP was studied in vitro using biochemical and structural methods. A C-terminal, four-amino-acid-long loop protruding into the central cavity of the phi6 RdRP has been suggested to stabilize the incoming nucleotides of the initiation complex formation through stacking interactions. A similar structural element has been found from several other viral RdRPs. In this thesis, this so-called initiation platform loop was subjected to site-directed mutagenesis to address its role in the initiation. It was found that the initiation mode of the mutants is primer-dependent, requiring either an oligonucleotide primer or a back-priming initiation mechanism for the RNA synthesis. The crystal structure of a mutant RdRP with altered initiation platform revealed a set of contacts important for primer-independent initiation. Since phi6 RdRP is structurally and functionally homologous to several viral RdRPs, among them the hepatitis C virus RdRP, these results provide further general insight to understand primer-independent initiation. In this study it is demonstrated that manganese phasing could be used as a practical tool for solving structures of large proteins with a bound manganese ion. The phi6 RdRP was used as a case study to obtain phases for crystallographic analysis. Manganese ions are naturally bound to the phi6 RdRP at the palm domain of the enzyme. In a crystallographic experiment, X-ray diffraction data from a phi6 RdRP crystal were collected at a wavelength of 1.89 Å, which is the K edge of manganese. With this data an automatically built model of the core region of the protein could be obtained. Finally, in this work terminal nucleotidyl transferase (TNTase) activity of the phi6 RdRP was documented in the isolated polymerase as well as in the viral particle. This is the first time that such an activity has been reported in a polymerase of a dsRNA virus. The phi6 RdRP used uridine triphosphates as the sole substrate in a TNTase reaction but could accept several heterologous templates. The RdRP was able to add one or a few non-templated nucleotides to the 3' end of the single- or double-stranded RNA substrate. Based on the results on particle-mediated TNTase activity and previous structural information of the polymerase, a model for termination of the RNA-dependent RNA synthesis is suggested in this thesis.

In situ synthesis and characterization of polyaniline-CaCu3Ti4O12 nanocrystal composites

High dielectric constant (ca. 2.4 x 10(6) at 1 kHz) nanocomposite of polyaniline (PANI)/CaCu3Ti4O12 (CCTO) was synthesized using a simple procedure involving in situ polymerization of aniline in dil. HCl. The PANI and the composite were subjected to X-ray diffraction, Fourier transform infrared, thermo gravimetric, scanning electron microscopy and transmission electron microscopy analyses. The presence of the nanocrystallites of CCTO embedded in the nanofibers of PANI matrix was established by TEM. Frequency dependent characteristics of the dielectric constant. dielectric loss and AC conductivity were studied for the PANI and the composites. The dielectric constant increased as the CCTO content increased in PANI but decreased with increasing frequency (100 Hz-1 MHz) of measurement. The dielectric loss was two times less than the value obtained for pure PANI around 100 Hz. The AC conductivity increased slightly up to 2 kHz as the CCTO content increased in the PANI which was attributed to the polarization of the charge carriers.

Palladium(II) and platinum(II) complexes of beta-functionalized ethyl selenolates: Effect of substitution on synthesis, reactivity, spectroscopy, structures and thermal behavior

Sodium ethylselenolates with functional groups X (where X = -OH, -COOH, -COOMe and -COOEt) at beta-carbon were prepared in situ by reductive cleavage of corresponding diselenide with NaBH4 either in methanol or aqueous ammonia. Treatment of these selenolates with [M2Cl2(mu-Cl)(2)(PR'(3))(2)] (M = Pd or Pt; PR'(3) = PMePh2, PnPr(3)) in different stoichiometry yielded various bi- and tri-nuclear complexes. The homoleptic hexanuclear complexes [Pd(mu-SeCH2CH2X)(2)](6) (X = OH, COOH, COOEt), were obtained by reacting Na2PdCl4 with NaSeCH2CH2X. All these complexes have been fully characterized. Molecular structures of ethylselenolates containing hydroxyl and carboxylic acid groups revealed solid state associated structures through inter-molecular hydrogen bond interactions. Trinuclear complex, [Pd3Cl2(mu-SeCH2CH2COOH)(4)(PnPr(3))(2)] (3a), was disposed in a boat form unlike chair conformation observed for the corresponding methylester complex. The effect of beta-functionality in ethylselenolate ligands towards reactivity, structures and thermal properties of palladium and platinum complexes has been extensively Studied.

An expeditious, bidirectional synthesis of furofuranones: a new application of Morita-Baylis-Hillman adducts

A concise, flexible approach of general utility to the furo[3,2-b]furanones frorn readily available Morita Baylis-Hillman adducts is delineated In an expeditious variant of this approach, a four-step cascade process is executed in a one-pot operation to generate the furofuranoid framework containing two quaternary centers .

Ruthenium(II)-CO complexes of N-[(2-pyridyl)methyliden]-alpha(or beta)-aminonaphthalene: Synthesis, spectral studies, crystal structure, redox properties and DFT calculation

The characterization and properties of trans-(X)-[RuX2(CO)(2)(alpha/beta-NaiPy)] (1, 2) (alpha-NaiPy (a), beta-NaiPy (b); X = Cl (1), I (2)) are described in this work. The structures are confirmed by single crystal X-ray diffraction studies. Reaction of these compounds with Me3NO in MeCN has isolated monocarbonyl trans-(X)-RuX2(CO)(MeCN)(alpha/beta-NaiPy)] (3, 4). The complexes show intense emission properties. Quantum yields of 1 and 2 (phi= 0.02-0.08) are higher than 3 and 4 (phi = 0.006-0.015). Voltammogram shows higher Ru(III)/Ru(II) (1.3-1.5 V) potential of 1 and 2 than that of 3 and 4 (0.8-0.9 V) that may be due to coordination of two pi-acidic CO groups in former. The electronic spectra and redox properties of the complexes are compared with the results obtained by density functional theory (DFT) and time-dependent density functional theory (TD-DFT) using polarizable continuum model (CPCM).

Synthesis of new 4-isopropylthiazole hydrazide analogs and some derived clubbed triazole, oxadiazole ring systems - A novel class of potential antibacterial, antifungal and antitubercular agents

In the present study a series of 4-isopropylthiazole-2-carbohydrazide analogs, derived clubbed oxadiazole-thiazole and triazole-thiazole derivatives have been synthesized and characterized by IR, H-1 NMR, C-13 NMR, elemental and mass spectral analyses. The synthesized compounds were evaluated for their preliminary in vitro antibacterial, antifungal and antitubercular activity against Mycobacterium tuberculosis H(37)Rv strain by broth dilution assay method. The synthesized compounds 7a, 7b, 7d and 4 showed an antitubercular efficacy considerably greater than that of the parent 4-isopropyl-1,3-thiazole-2-carbohydrazide 1, suggesting that the substituted 4-isopropylthiazole-2-carbohydrazide moiety plays an important role in enhancing the antitubercular properties of this class of compounds. Compounds 2c, 3, 4, 6d, 7a and 7b exhibited good or moderate antibacterial and antifungal activity. Compounds 4 and 7b showed appreciable cytotoxicity at a concentration of 250 mu M.

Synthesis of ZnO Nanoneedles on Flexible Polymer Substrates at Room Temperature by Activated Reactive Evaporation

ZnO nanoneedles were successfully deposited on flexible polymer substrates at room temperature by activated reactive evaporation. Neither a catalyst nor a template was employed in this synthesis. These synthesized needles measured 500 - 600 nm in length and its diameter varied from 30 - 15 nm from the base to the tip. The single-crystalline nature of the nanoneedle was observed by high-resolution transmission electron microscopy studies. The Raman studies on these nanoneedles had shown that they are oxygen deficient in nature. A possible growth mechanism has been proposed here, in which the nanoneedles nucleate and grow in the gas phase by vapor-solid mechanism.