Design and study of self-assembled functional organic and hybrid systems for biological applications

The focus of self-assembly as a strategy for the synthesis has been confined largely to molecules, because of the importance of manipulating the structure of matter at the molecular scale. We have investigated the influence of temperature and pH, in addition to the concentration of the capping agent used for the formation of the nano-bio conjugates. For example, the formation of the narrower size distribution of the nanoparticles was observed with the increase in the concentration of the protein, which supports the fact that γ-globulin acts both as a controller of nucleation as well as stabiliser. As analyzed through various photophysical, biophysical and microscopic techniques such as TEM, AFM, C-AFM, SEM, DLS, OPM, CD and FTIR, we observed that the initial photoactivation of γ-globulin at pH 12 for 3 h resulted in small protein fibres of ca. Further irradiation for 24 h, led to the formation of selfassembled long fibres of the protein of ca. 5-6 nm and observation of surface plasmon resonance band at around 520 nm with the concomitant quenching of luminescence intensity at 680 nm. The observation of light triggered self-assembly of the protein and its effect on controlling the fate of the anchored nanoparticles can be compared with the naturally occurring process such as photomorphogenesis.Furthermore,our approach offers a way to understand the role played by the self-assembly of the protein in ordering and knock out of the metal nanoparticles and also in the design of nano-biohybrid materials for medicinal and optoelectronic applications. Investigation of the potential applications of NIR absorbing and water soluble squaraine dyes 1-3 for protein labeling and anti-amyloid agents forms the subject matter of the third chapter of the thesis. The study of their interactions with various proteins revealed that 1-3 showed unique interactions towards serum albumins as well as lysozyme. 69%, 71% and 49% in the absorption spectra as well as significant quenching in the fluorescence intensity of the dyes 1-3, respectively. Half-reciprocal analysis of the absorption data and isothermal titration calorimetric (ITC) analysis of the titration experiments gave a 1:1 stoichiometry for the complexes formed between the lysozyme and squaraine dyes with association constants (Kass) in the range 104-105 M-1. We have determined the changes in the free energy (ΔG) for the complex formation and the values are found to be -30.78, -32.31 and -28.58 kJmol-1, respectively for the dyes 1, 2 and 3. Furthermore, we have observed a strong induced CD (ICD) signal corresponding to the squaraine chromophore in the case of the halogenated squaraine dyes 2 and 3 at 636 and 637 nm confirming the complex formation in these cases. To understand the nature of interaction of the squaraine dyes 1-3 with lysozyme, we have investigated the interaction of dyes 1-3 with different amino acids. These results indicated that the dyes 1-3 showed significant interactions with cysteine and glutamic acid which are present in the side chains of lysozyme. In addition the temperature dependent studies have revealed that the interaction of the dye and the lysozyme are irreversible. Furthermore, we have investigated the interactions of these NIR dyes 1-3 with β- amyloid fibres derived from lysozyme to evaluate their potential as inhibitors of this biologically important protein aggregation. These β-amyloid fibrils were insoluble protein aggregates that have been associated with a range of neurodegenerative diseases, including Huntington, Alzheimer’s, Parkinson’s, and Creutzfeldt-Jakob diseases. We have synthesized amyloid fibres from lysozyme through its incubation in acidic solution below pH 4 and by allowing to form amyloid fibres at elevated temperature. To quantify the binding affinities of the squaraine dyes 1-3 with β-amyloids, we have carried out the isothermal titration calorimetric (ITC) measurements. The association constants were determined and are found to be 1.2 × 105, 3.6× 105 and 3.2 × 105 M-1 for the dyes, 1-3, respectively. To gain more insights into the amyloid inhibiting nature of the squaraine dyes under investigations, we have carried out thioflavin assay, CD, isothermal titration calorimetry and microscopic analysis. The addition of the dyes 1-3 (5μM) led to the complete quenching in the apparent thioflavin fluorescence, thereby indicating the destabilization of β-amyloid fibres in the presence of the squaraine dyes. Further, the inhibition of the amyloid fibres by the squaraine dyes 1-3, has been evidenced though the DLS, TEM AFM and SAED, wherein we observed the complete destabilization of the amyloid fibre and transformation of the fibre into spherical particles of ca. These results demonstrate the fact that the squaraine dyes 1-3 can act as protein labeling agents as well as the inhibitors of the protein amyloidogenesis. The last chapter of the thesis describes the synthesis and investigation of selfassembly as well as bio-imaging aspects of a few novel tetraphenylethene conjugates 4-6.Expectedly, these conjugates showed significant solvatochromism and exhibited a hypsochromic shift (negative solvatochromism) as the solvent polarity increased, and these observations were justified though theoretical studies employing the B3LYP/6-31g method. We have investigated the self-assembly properties of these D-A conjugates though variation in the percentage of water in acetonitrile solution due to the formation of nanoaggregates. Further the contour map of the observed fluorescence intensity as a function of the fluorescence excitation and emission wavelength confirmed the formation of J-type aggregates in these cases. To have a better understanding of the type of self-assemblies formed from the TPE conjugates 4-6, we have carried out the morphological analysis through various microscopic techniques such as DLS, SEM and TEM. 70%, we observed rod shape architectures having ~ 780 nm in diameter and ~ 12 μM in length as evidenced through TEM and SEM analysis. We have made similar observations with the dodecyl conjugate 5 at ca. 70% and 50% water/acetonitrile mixtures, the aggregates formed from 4 and 5 were found to be highly crystalline and such structures were transformed to amorphous nature as the water fraction was increased to 99%. To evaluate the potential of the conjugate as bio-imaging agents, we have carried out their in vitro cytotoxicity and cellular uptake studies though MTT assay, flow cytometric and confocal laser scanning microscopic techniques. Thus nanoparticle of these conjugates which exhibited efficient emission, large stoke shift, good stability, biocompatibility and excellent cellular imaging properties can have potential applications for tracking cells as well as in cell-based therapies. In summary we have synthesized novel functional organic chromophores and have studied systematic investigation of self-assembly of these synthetic and biological building blocks under a variety of conditions. The investigation of interaction of water soluble NIR squaraine dyes with lysozyme indicates that these dyes can act as the protein labeling agents and the efficiency of inhibition of β-amyloid indicate, thereby their potential as anti-amyloid agents.

OPTIMAL UTILIZATION OF SERVICE FACILITY FOR A k-OUT-OF-n SYSTEM WITH REPAIR BY EXTENDING SERVICE TO EXTERNAL CUSTOMERS IN A RETRIAL QUEUE

In this paper, we study a k-out-of-n system with single server who provides service to external customers also. The system consists of two parts:(i) a main queue consisting of customers (failed components of the k-out-of-n system) and (ii) a pool (of finite capacity M) of external customers together with an orbit for external customers who find the pool full. An external customer who finds the pool full on arrival, joins the orbit with probability and with probability 1− leaves the system forever. An orbital customer, who finds the pool full, at an epoch of repeated attempt, returns to orbit with probability (< 1) and with probability 1 − leaves the system forever. We compute the steady state system size probability. Several performance measures are computed, numerical illustrations are provided.

Studies on ecophysiology of pfnafus indicus h. milne edwards in the grow-out system

In the present study, the main emphasis was to find out it seasonal and ecosystem specific variations of calcium magnesium, phosphorus, copper and zinc in the water and the sediment has any role to play in altering the concentration of these elements in different tissues of intermoult p.indicus as evidenced by the seasonal studies in the grow out and the marine ecosystems

Bioremediation In Prawn Grow Out Systems

Winter School on 'RECENT ADVANCES IN DIAGNOSIS AND MANAGEMENT OF DISEASES IN MARICULTURE' 7-27 November, 2002 Course Manual,CMFRI

Soft Computing Approach for Optimization of siRNA Efficiency Prediction for Post-transcriptional Gene Silencing

Post-transcriptional gene silencing by RNA interference is mediated by small interfering RNA called siRNA. This gene silencing mechanism can be exploited therapeutically to a wide variety of disease-associated targets, especially in AIDS, neurodegenerative diseases, cholesterol and cancer on mice with the hope of extending these approaches to treat humans. Over the recent past, a significant amount of work has been undertaken to understand the gene silencing mediated by exogenous siRNA. The design of efficient exogenous siRNA sequences is challenging because of many issues related to siRNA. While designing efficient siRNA, target mRNAs must be selected such that their corresponding siRNAs are likely to be efficient against that target and unlikely to accidentally silence other transcripts due to sequence similarity. So before doing gene silencing by siRNAs, it is essential to analyze their off-target effects in addition to their inhibition efficiency against a particular target. Hence designing exogenous siRNA with good knock-down efficiency and target specificity is an area of concern to be addressed. Some methods have been developed already by considering both inhibition efficiency and off-target possibility of siRNA against agene. Out of these methods, only a few have achieved good inhibition efficiency, specificity and sensitivity. The main focus of this thesis is to develop computational methods to optimize the efficiency of siRNA in terms of “inhibition capacity and off-target possibility” against target mRNAs with improved efficacy, which may be useful in the area of gene silencing and drug design for tumor development. This study aims to investigate the currently available siRNA prediction approaches and to devise a better computational approach to tackle the problem of siRNA efficacy by inhibition capacity and off-target possibility. The strength and limitations of the available approaches are investigated and taken into consideration for making improved solution. Thus the approaches proposed in this study extend some of the good scoring previous state of the art techniques by incorporating machine learning and statistical approaches and thermodynamic features like whole stacking energy to improve the prediction accuracy, inhibition efficiency, sensitivity and specificity. Here, we propose one Support Vector Machine (SVM) model, and two Artificial Neural Network (ANN) models for siRNA efficiency prediction. In SVM model, the classification property is used to classify whether the siRNA is efficient or inefficient in silencing a target gene. The first ANNmodel, named siRNA Designer, is used for optimizing the inhibition efficiency of siRNA against target genes. The second ANN model, named Optimized siRNA Designer, OpsiD, produces efficient siRNAs with high inhibition efficiency to degrade target genes with improved sensitivity-specificity, and identifies the off-target knockdown possibility of siRNA against non-target genes. The models are trained and tested against a large data set of siRNA sequences. The validations are conducted using Pearson Correlation Coefficient, Mathews Correlation Coefficient, Receiver Operating Characteristic analysis, Accuracy of prediction, Sensitivity and Specificity. It is found that the approach, OpsiD, is capable of predicting the inhibition capacity of siRNA against a target mRNA with improved results over the state of the art techniques. Also we are able to understand the influence of whole stacking energy on efficiency of siRNA. The model is further improved by including the ability to identify the “off-target possibility” of predicted siRNA on non-target genes. Thus the proposed model, OpsiD, can predict optimized siRNA by considering both “inhibition efficiency on target genes and off-target possibility on non-target genes”, with improved inhibition efficiency, specificity and sensitivity. Since we have taken efforts to optimize the siRNA efficacy in terms of “inhibition efficiency and offtarget possibility”, we hope that the risk of “off-target effect” while doing gene silencing in various bioinformatics fields can be overcome to a great extent. These findings may provide new insights into cancer diagnosis, prognosis and therapy by gene silencing. The approach may be found useful for designing exogenous siRNA for therapeutic applications and gene silencing techniques in different areas of bioinformatics.