Synthesis and Evaluation of a Novel Class of G-Quadruplex-Stabilizing Small Molecules Based on the 1,3-Phenylene-Bis(piperazinyl benzimidazole) System

Achieving stabilization of telomeric DNA in G-quadruplex conformation by Various organic compounds has been an important goal for the medicinal chemists seeking to develop new anticancer agents. Several compounds are known to stabilize G-quadruplexes. However, relatively few are known to induce their formation and/or alter the topology, of the preformed quadruplex DNA. Herein, four compounds having the 1,3-phenylene-bis(piperazinyl benzimidazole) unit as a basic skeleton have been synthesized, and their interactions with the 24-mer telomeric DNA sequences from Tetrahymena thermophilia d(T(2)G(4))(4) have been investigated using high-resolution techniques Such as circular dichroism (CD) spectropolarimetry, CD melting, emission spectroscopy, and polyacrylamide gel electrophoresis. The data obtained, in the presence of one of three ions (Li+, Na+, or K+), indicate that all the new compounds have a high affinity for G-quadruplex DNA, and the strength of the binding with G-quadruplex depends on (1) phenyl ring substitution, (ii) the piperazinyl side chain, and (iii) the type of monovalent cation present in the buffer. Results further Suggest that these compounds are able to abet the conversion of the Intramolecular quadruplex into parallel stranded intermolecular G-quadruplex DNA. Notably, these compounds are also capable of inducing and stabilizing the parallel stranded quadruplex from randomly structured DNA in the absence of any stabilizing cation. The kinetics of the structural changes Induced by these compounds could be followed by recording the changes in the CD signal as a function of time. The implications of the findings mentioned above are discussed in this paper.

Nephrin-associated molecules in human glomerular diseases

The glomerular epithelial cells and their intercellular junctions, termed slit diaphragms, are essential components of the filtration barrier in the kidney glomerulus. Nephrin is a transmembrane adhesion protein of the slit diaphragm and a signalling molecule regulating podocyte physiology. In congenital nephrotic syndrome of the Finnish type, mutation of nephrin leads to disruption of the permeability barrier and leakage of plasma proteins into the urine. This doctoral thesis hypothesises that novel nephrin-associated molecules are involved in the function of the filtration barrier in health and disease. Bioinformatics tools were utilized to identify novel nephrin-like molecules in genomic databases, and their distribution in the kidney and other tissues was investigated. Filtrin, a novel nephrin homologue, is expressed in the glomerular podocytes and, according to immunoelectron microscopy, localizes at the slit diaphragm. Interestingly, the nephrin and filtrin genes, NPHS1 and KIRREL2, locate in a head-to-head orientation on chromosome 19q13.12. Another nephrin-like molecule, Nphs1as was cloned in mouse, however, no expression was detected in the kidney but instead in the brain and lymphoid tissue. Notably, Nphs1as is transcribed from the nephrin locus in an antisense orientation. The glomerular mRNA and protein levels of filtrin were measured in kidney biopsies of patients with proteinuric diseases, and marked reduction of filtrin mRNA levels was detected in the proteinuric samples as compared to controls. In addition, altered distribution of filtrin in injured glomeruli was observed, with the most prominent decrease of the expression in focal segmental glomerulosclerosis. The role of the slit diaphragm-associated genes for the development of diabetic nephropathy was investigated by analysing single nucleotide polymorphisms. The genes encoding filtrin, densin-180, NEPH1, podocin, and alpha-actinin-4 were analysed, and polymorphisms at the alpha-actinin-4 gene were associated with diabetic nephropathy in a gender-dependent manner. Filtrin is a novel podocyte-expressed protein with localization at the slit diaphragm, and the downregulation of filtrin seems to be characteristic for human proteinuric diseases. In the context of the crucial role of nephrin for the glomerular filter, filtrin appears to be a potential candidate molecule for proteinuria. Although not expressed in the kidney, the nephrin antisense Nphs1as may regulate the expression of nephrin in extrarenal tissues. The genetic association analysis suggested that the alpha-actinin-4 gene, encoding an actin-filament cross-linking protein of the podocytes, may contribute to susceptibility for diabetic nephropathy.

Structure and Dynamics of Coil-like Molecules by Residual Dipolar Couplings

Nuclear magnetic resonance (NMR) spectroscopy provides us with many means to study biological macromolecules in solution. Proteins in particular are the most intriguing targets for NMR studies. Protein functions are usually ascribed to specific three-dimensional structures but more recently tails, long loops and non-structural polypeptides have also been shown to be biologically active. Examples include prions, -synuclein, amylin and the NEF HIV-protein. However, conformational preferences in coil-like molecules are difficult to study by traditional methods. Residual dipolar couplings (RDCs) have opened up new opportunities; however their analysis is not trivial. Here we show how to interpret RDCs from these weakly structured molecules. The most notable residual dipolar couplings arise from steric obstruction effects. In dilute liquid crystalline media as well as in anisotropic gels polypeptides encounter nematogens. The shape of a polypeptide conformation limits the encounter with the nematogen. The most elongated conformations may come closest whereas the most compact remain furthest away. As a result there is slightly more room in the solution for the extended than for the compact conformations. This conformation-dependent concentration effect leads to a bias in the measured data. The measured values are not arithmetic averages but essentially weighted averages over conformations. The overall effect can be calculated for random flight chains and simulated for more realistic molecular models. Earlier there was an implicit thought that weakly structured or non-structural molecules would not yield to any observable residual dipolar couplings. However, in the pioneering study by Shortle and Ackerman RDCs were clearly observed. We repeated the study for urea-denatured protein at high temperature and also observed indisputably RDCs. This was very convincing to us but we could not possibly accept the proposed reason for the non-zero RDCs, namely that there would be some residual structure left in the protein that to our understanding was fully denatured. We proceeded to gain understanding via simulations and elementary experiments. In measurements we used simple homopolymers with only two labelled residues and we simulated the data to learn more about the origin of RDCs. We realized that RDCs depend on the position of the residue as well as on the length of the polypeptide. Investigations resulted in a theoretical model for RDCs from coil-like molecules. Later we extended the studies by molecular dynamics. Somewhat surprisingly the effects are small for non-structured molecules whereas the bias may be large for a small compact protein. All in all the work gave clear and unambiguous results on how to interpret RDCs as structural and dynamic parameters of weakly structured proteins.

The Out-of-India hypothesis: What do molecules suggest?

The remarkable geological and evolutionary history of peninsular India has generated much interest in the patterns and processes that might have shaped the current distributions of its endemic biota. In this regard the Out of India hypothesis, which proposes that rafting peninsular India carried Gondwanan forms to Asia after the break-up of Gondwana super continent, has gained prominence. Here we have reviewed molecular studies undertaken on a range of taxa of supposedly Gondwanan origin to better understand the Out-of-India scenario. This re-evaluation of published molecular studies indicates that there is mounting evidence supporting Out-of-India scenario for various Asian taxa. Nevertheless, in many studies the evidence is inconclusive due to lack of information on the age of relevant nodes. Studies also indicate that not all Gondwanan forms of peninsular India dispersed out of India. Many of these ancient lineages are confined to peninsular India and therefore are relict Gondwanan lineages. Additionally for some taxa an Into India rather than Out-of-India scenario better explains their current distribution. To identify the Out-of-India component of Asian biota it is imperative that we understand the complex biogeographical history of India. To this end, we propose three oversimplified yet explicit phylogenetic predictions. These predictions can be tested through the use of molecular phylogenetic tools in conjunction with palaeontological and geological data.

Ratios of T-cell immune-effectors with tumour associated macrophages and PD-1/PD-L1 axis immune-checkpoint molecules, as prognosticators in diffuse large B cell lymphoma: A population-based study

Background Risk-stratification of diffuse large B-cell lymphoma (DLBCL) requires identification of patients with disease that is not cured despite initial R-CHOP. Although the prognostic importance of the tumour microenvironment (TME) is established, the optimal strategy to quantify it is unknown. Methods The relationship between immune-effector and inhibitory (checkpoint) genes was assessed by NanoString™ in 252 paraffin-embedded DLBCL tissues. A model to quantify net anti-tumoural immunity as an outcome predictor was tested in 158 R-CHOP treated patients, and validated in tissue/blood from two independent R-CHOP treated cohorts of 233 and 140 patients respectively. Findings T and NK-cell immune-effector molecule expression correlated with tumour associated macrophage and PD-1/PD-L1 axis markers consistent with malignant B-cells triggering a dynamic checkpoint response to adapt to and evade immune-surveillance. A tree-based survival model was performed to test if immune-effector to checkpoint ratios were prognostic. The CD4*CD8:(CD163/CD68)*PD-L1 ratio was better able to stratify overall survival than any single or combination of immune markers, distinguishing groups with disparate 4-year survivals (92% versus 47%). The immune ratio was independent of and added to the revised international prognostic index (R-IPI) and cell-of-origin (COO). Tissue findings were validated in 233 DLBCL R-CHOP treated patients. Furthermore, within the blood of 140 R-CHOP treated patients immune-effector:checkpoint ratios were associated with differential interim-PET/CT+ve/-ve expression.

Selective detection of single-enantiomer spectrum of chiral molecules aligned in the polypeptide liquid crystalline solvent: Transition selective one-dimensional H-1-H-1 COSY

One-dimensional (1D) proton NMR spectra of enantiomers are generally undecipherable in chiral orienting poly-gamma-benzyl-L-glutamate (PBLG)/CDCl3 solvent. This arises due to large number of couplings, in addition to superposition of spectra from both the enantiomers, severely hindering the H-1 detection. On the other hand in the present study the benefit is derived front the presence of several couplings among the entire network of interacting protons. Transition selective 1D H-1-H-1 correlation experiment (1D-COSY) which utilizes the Coupling assisted transfer of magnetization not only for unraveling the overlap but also for the selective detection of enantiopure spectrum is reported. The experiment is simple, easy to implement and provides accurate eanantiomeric excess in addition to the determination of the proton-proton couplings of an enantiomer within a short experimental time (few minutes). (C) 2009 Elsevier Inc. All rights reserved.

Diffusion of flexible, charged, nanoscopic molecules in solution: Size and pH dependence for PAMAM dendrimer

In order to understand self-diffusion (D) of a charged, flexible, and porous nanoscopic molecule in water, we carry out very long, fully atomistic molecular dynamics simulation of PAMAM dendrimer up to eight generations in explicit salt water under varying pH. We find that while the radius of gyration (R-g) varies as N-1/3, the self-diffusion constant (D) scales, surprisingly, as N-alpha, with alpha=0.39 at high pH and 0.5 at neutral pH, indicating a dramatic breakdown of Stokes-Einstein relation for diffusion of charged nanoscopic molecules. The variation in D as a function of radius of gyration demonstrates the importance of treating water and ions explicitly in the diffusion process of a flexible nanoscopic molecule. In agreement with recent experiments, the self-diffusion constant increases with pH, revealing the importance of dielectric friction in the diffusion process. The shape of a dendrimer is found to fluctuate on a nanosecond time scale. We argue that this flexibility (and also the porosity) of the dendrimer may play an important role in determining the mean square displacement of the dendrimer and the breakdown of the Stokes-Einstein relation between diffusion constant and the radius.

Two views on interstellar dust: near-infrared scattering and polarized thermal dust emission

Interstellar clouds are not featureless, but show quite complex internal structures of filaments and clumps when observed with high enough resolution. These structures have been generated by 1) turbulent motions driven mainly by supernovae, 2) magnetic fields working on the ions and, through neutral-ion collisions, on neutral gas as well, and 3) self-gravity pulling a dense clump together to form a new star. The study of the cloud structure gives us information on the relative importance of each of these mechanisms, and helps us to gain a better understanding of the details of the star formation process. Interstellar dust is often used as a tracer for the interstellar gas which forms the bulk of the interstellar matter. Some of the methods that are used to derive the column density are summarized in this thesis. A new method, which uses the scattered light to map the column density in large fields with high spatial resolution, is introduced. This thesis also takes a look at the grain alignment with respect to the magnetic fields. The aligned grains give rise to the polarization of starlight and dust emission, thus revealing the magnetic field. The alignment mechanisms have been debated for the last half century. The strongest candidate at present is the radiative torques mechanism. In the first four papers included in this thesis, the scattered light method of column density estimation is formulated, tested in simulations, and finally used to obtain a column density map from observations. They demonstrate that the scattered light method is a very useful and reliable tool in column density estimation, and is able to provide higher resolution than the near-infrared color excess method. These two methods are complementary. The derived column density maps are also used to gain information on the dust emissivity within the observed cloud. The two final papers present simulations of polarized thermal dust emission assuming that the alignment happens by the radiative torques mechanism. We show that the radiative torques can explain the observed decline of the polarization degree towards dense cores. Furthermore, the results indicate that the dense cores themselves might not contribute significantly to the polarized signal, and hence one needs to be careful when interpreting the observations and deriving the magnetic field.

Enhanced Tetrahedral Ordering of Water Molecules in Minor Grooves of DNA: Relative Role of DNA Rigidity, Nanoconfinement, and Surface Specific Interactions

Confinement and Surface specific interactions call induce Structures otherwise unstable at that temperature and pressure. Here we Study the groove specific water dynamics ill the nucleic acid sequences, poly-AT and poly-GC, in long B-DNA duplex chains by large scale atomistic molecular dynamics simulations, accompanied by thermodynamic analysis. While water dynamics in the major groove remains insensitive to the sequence differences, exactly the opposite is true for the minor groove water. Much slower water dynamics observed in the minor grooves (especially in the AT minor) call be attributed to all enhanced tetrahedral ordering (< t(h)>) of water. The largest value of < t(h)> in the AT minor groove is related to the spine of hydration found in X-ray Structure. The calculated configurational entropy (S-C) of the water molecules is found to be correlated with the self-diffusion coefficient of water in different region via Adam-Gibbs relation D = A exp(-B/TSC), and also with < t(h)>.

Studies of the star-forming structures in the dense interstellar medium: a view by dust extinction

New stars in galaxies form in dense, molecular clouds of the interstellar medium. Measuring how the mass is distributed in these clouds is of crucial importance for the current theories of star formation. This is because several open issues in them, such as the strength of different mechanism regulating star formation and the origin of stellar masses, can be addressed using detailed information on the cloud structure. Unfortunately, quantifying the mass distribution in molecular clouds accurately over a wide spatial and dynamical range is a fundamental problem in the modern astrophysics. This thesis presents studies examining the structure of dense molecular clouds and the distribution of mass in them, with the emphasis on nearby clouds that are sites of low-mass star formation. In particular, this thesis concentrates on investigating the mass distributions using the near infrared dust extinction mapping technique. In this technique, the gas column densities towards molecular clouds are determined by examining radiation from the stars that shine through the clouds. In addition, the thesis examines the feasibility of using a similar technique to derive the masses of molecular clouds in nearby external galaxies. The papers presented in this thesis demonstrate how the near infrared dust extinction mapping technique can be used to extract detailed information on the mass distribution in nearby molecular clouds. Furthermore, such information is used to examine characteristics crucial for the star formation in the clouds. Regarding the use of extinction mapping technique in nearby galaxies, the papers of this thesis show that deriving the masses of molecular clouds using the technique suffers from strong biases. However, it is shown that some structural properties can still be examined with the technique.

Synthesis and Characterization of a Class of Donor-Acceptor Conjugated Molecules: Experiments and Theoretical Calculations

A class of conjugated molecules containing donor (thiophene) and acceptor (malononitrile) is synthesized by Knoevenagel condensation reaction between 2-(2,6-dimethy1-4H-pyran-4-ylidene) malononitrile and thiophene carbaldehyde containing two and three thiophene units. The resulting molecules are characterized by H-1 and C-13 NMR. We have performed UV-vis absorption, fluorescence, and cyclic voltammetry measurements on these materials. The spectroscopic and electrochemical measurements proved beyond doubt that these materials possess lowexcitation gap and are suitable for being an active material in various electronic devices. We have also performed electronic structure calculations using density functional theory (DFT) and INDO/SCI methods to characterize the ground and excited states of this class of molecules. These donor-acceptor molecules show a strong charge transfercharacter that increases with the increase in the number of thiophene rings coupled to the malononitrile acceptor moiety. We have also calculated the pi-coherence length, Stoke's shift, and effect of solvents on excited states for this class of molecules, Our theoretical values agree well with experimental results.

Interactions of linear dicationic molecules with lipid A: structural requisites for optimal binding affinity

The structural determinants of the binding affinity of linear dicationic molecules toward lipid A have been examined with respect to the distance between the terminal cationic functions, the basicity, and the type of cationic moieties using a series of spermidine derivatives and pentamidine analogs by fluorescence spectroscopic methods, The presence of two terminal cationic groups corresponds to enhanced affinity, A distinct sigmoidal relationship between the intercationic distance and affinity was observed with a sharp increase at 11 Angstrom, levelling off at about 13 Angstrom. The basicity (pK) and nature of the cationic functions are poor correlates of binding potency, since molecules bearing primary amino, imidazolino, or guanido termini are equipotent, The interaction of pentamidine, a bisamidine drug, with lipid A, characterized in considerable detail employing the putative intermolecular excimerization of the drug, suggests a stoichiometry of 1:1 in the resultant complex, The binding is driven almost exclusively by electrostatic forces, and is dependent on the ionization states of both lipid A and the drug, Under conditions when lipid A is highly disaggregated, pentamidine binds specifically to bis-phosphoryl- but not to monophosphoryl-lipid A indicating that both phosphate groups of lipid A are necessary for electrostatic interactions by the terminal amidininium groups of the drug, Based on these data, a structural model is proposed for the pentamidine-lipid A complex, which may be of value in designing endotoxin antagonists from first principles.