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.

Application of Fermentation Techniques in the Utilization of Prawn Shell Waste

The present study aimed at the utlisation of microbial organisms for the production of good quality chitin and chitosan. The three strains used for the study were Lactobacillus plantarum, Lactobacililus brevis and Bacillus subtilis. These strains were selected on the basis of their acid producing ability to reduce the pH of the fermenting substrates to prevent spoilage and thus caused demineralisation of the shell. Besides, the proteolytic enzymes in these strains acted on proteinaceous covering of shrimp and thus caused deprotenisation of shrimp shell waste. Thus the two processes involved in chitin production can be affected to certain extent using bacterial fermentation of shrimp shell.Optimization parameters like fermentation period, quantity of inoculum, type of sugar, concentration of sugar etc. for fermentation with three different strains were studied. For these, parameters like pH, Total titrable acidity (TTA), changes in sugar concentration, changes in microbial count, sensory changes etc. were studied.Fermentation study with Lactobacillus plantarum was continued with 20% w/v jaggery broth for 15 days. The inoculum prepared yislded a cell concentration of approximately 108 CFU/ml. In the present study, lactic acid and dilute hydrochloric acid were used for initial pH adjustment because; without adjusting the initial pH, it took more than 5 hours for the lactic acid bacteria to convert glucose to lactic acid and during this delay spoilage occurred due to putrefying enzymes active at neutral or higher pH. During the fermentation study, pH first decreased in correspondence with increase in TTA values. This showed a clear indication of acid production by the strain. This trend continued till their proteolytic activity showed an increasing trend. When the available sugar source started depleting, proteolytic activity also decreased and pH increased. This was clearly reflected in the sensory evaluation results. Lactic acid treated samples showed greater extent of demineralization and deprotenisation at the end of fermentation study than hydrochloric acid treated samples. It can be due to the effect of strong hydrochloric acid on the initial microbial count, which directly affects the fermentation process. At the end of fermentation, about 76.5% of ash was removed in lactic acid treated samples and 71.8% in hydrochloric acid treated samples; 72.8% of proteins in lactic acid treated samples and 70.6% in hydrochloric acid treated samples.The residual protein and ash in the fermented residue were reduced to permissible limit by treatment with 0.8N HCI and 1M NaOH. Characteristics of chitin like chitin content, ash content, protein content, % of N- acetylation etc. were studied. Quality characteristics like viscosity, degree of deacetylation and molecular weight of chitosan prepared were also compared. The chitosan samples prepared from lactic acid treated showed high viscosity than HCI treated samples. But degree of deacetylation is more in HCI treated samples than lactic acid treated ones. Characteristics of protein liquor obtained like its biogenic composition, amino acid composition, total volatile base nitrogen, alpha amino nitrogen etc. also were studied to find out its suitability as animal feed supplement.Optimization of fermentation parameters for Lactobacillus brevis fermentation study was also conducted and parameters were standardized. Then detailed fermentation study was done in 20%wlv jaggery broth for 17 days. Also the effect of two different acid treatments (mild HCI and lactic acid) used for initial pH adjustment on chitin production were also studied. In this study also trend of changes in pH. changes in sugar concentration ,microbial count changes were similar to Lactobacillus plantarum studies. At the end of fermentation, residual protein in the samples were only 32.48% in HCI treated samples and 31.85% in lactic acid treated samples. The residual ash content was about 33.68% in HCI treated ones and 32.52% in lactic acid treated ones. The fermented residue was converted to chitin with good characteristics by treatment with 1.2MNaOH and 1NHCI.Characteristics of chitin samples prepared were studied and extent of Nacetylation was about 84% in HCI treated chitin and 85%in lactic acid treated ones assessed from FTIR spectrum. Chitosan was prepared from these samples by usual chemical method and its extent of solubility, degree of deacetylation, viscosity and molecular weight etc were studied. The values of viscosity and molecular weight of the samples prepared were comparatively less than the chitosan prepared by Lactobacillus plantarum fermentation. Characteristics of protein liquor obtained were analyzed to determine its quality and is suitability as animal feed supplement.Another strain used for the study was Bacillus subtilis and fermentation was carried out in 20%w/v jaggery broth for 15 days. It was found that Bacillus subtilis was more efficient than other Lactobacillus species for deprotenisation and demineralization. This was mainly due to the difference in the proteolytic nature of the strains. About 84% of protein and 72% of ash were removed at the end of fermentation. Considering the statistical significance (Pand deproteinisation, we have taken 0.8N HCI for the demineralization study and 0.6M NaOH for deprotenisation study. Properties of chitin and chitosan prepared were analyzed and studied.

A brackishwater isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaeid and non-penaeid rearing systems

A Pseudomonas sp PS-102 recovered from Muttukkadu brackish water lagoon, situated south of Chennai, showed significant activity against a number of shrimp pathogenic vibrios. Out of the 112 isolates of bacterial pathogens comprising Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, and Aeromonas spp, 73% were inhibited in vitro by the cell-free culture supernatant of Pseudomonas sp PS-102 isolate. The organism produced yellowish fluorescent pigment on King's B medium, hydrolysed starch and protein, and produced 36.4% siderophore units by CAS assay and 32 μM of catechol siderophores as estimated by Arnow's assay. The PS-102 isolate showed wide ranging environmental tolerance with, temperatures from 25 to 40 °C, pH from 6 to 8, salinity from 0 to 36 ppt, while the antagonistic activity peaked in cultures grown at 30 °C, pH 8.0 and at 5 ppt saline conditions. The antagonistic activity of the culture supernatant was evident even at 30% v / v dilution against V. harveyi. The preliminary studies on the nature of the antibacterial action indicated that the antagonistic principle as heat stable and resistant to proteolytic, lipolytic and amylolytic enzymes. Pseudomonas sp PS 102 was found to be safe to shrimp when PL-9 stage were challenged at 107 CFU ml−1 and by intramuscular injection into of ∼5 g sub-adults shrimp at 105 to 108 CFU. Further, its safety in a mammalian system, tested by its pathogenicity to mice, was also determined and its LD50 to BALB/c mice was found to be 109 CFU. The results of this study indicated that the organism Pseudomonas sp PS 102 could be employed as a potential probiont in shrimp and prawn aquaculture systems for management and control of bacterial infections

Establishment and characterization of India's first marine fish cell line (SISK) from the kidney of sea bass (Lates calcarifer)

A continuous cell line (SISK) from kidney of sea bass, Lates calcarifer, has been established and characterized. The cell line was maintained in Leibovitz' L-15 supplemented with 15% fetal bovine serum. This cell line has been subcultured more than 100 times over a period of 2 years. The SISK cell line consists of predominantly of epithelial-like cells. These cells showed strong positive for epithelial markers such as cytokeratin 19 and pancytokeratin. The cells were able to grow at temperature between 25 and 32 °C with optimum temperature of 28 °C. The growth rate of sea bass kidney cells increased as the FBS proportion increased from 2% to 20% at 28 °C with optimum growth at the concentrations of 15% or 20% FBS. The distribution of chromosome number was 30 to 56 with a modal peak at 48 chromosomes. Polymerase chain reaction products were obtained from SISK cells and tissues of sea bass with primer sets of microsatellite markers of sea bass. Five fish viruses were tested on this cell line to determine its susceptibility to these viruses and this was found to be susceptible to MABV NC1 and nodavirus, and the infection was confirmed by RT-PCR and CPE. This suggests that the SISK cell line has good potential for the isolation of various fish viruses. This cell line has been shown to be susceptible to bacterial extracellular products. The SISK cell line is the India's first marine fish cell line.

Microwave Dielectric Properties of BaO -2CeO2-nTiO2 Ceramics

The BaO-2CeO2-nTiO2 ceramics with n = 3, 4 and 5 have been prepared with CeO2 as starting material . The ceramics have been characterized using scanning electron microscopy , X-ray diffraction , Raman and X-ray photoelectron spectroscopy techniques. The microwave dielectric properties have been measured using standard dielectric resonator techniques . BaO-2CeO2-3TiO2 (123), BaO-2CeO2-4TiO2 ( 124) and BaO-2CeO2-5TiO2 ( 125) ceramics showed dielectric constants of 38, 27 and 32, respectively . All the ceramics showed fairly good unloaded Q - factors . 124 and 125 compounds exhibited low tf values, while 123 showed a high rf value

Farm Programmes of Electronic Media: A Comparative Study of Audience Perception in Kerala

Farm communication and extension programs are vital part of the farm development attempts. Electronic media plays a major role in farm extension activities. Kerala, the consumer state, which was a complete agricultural state in pre-independence period, is the sprouting land of agricultural extension and publication activities in print media. Later AIR (All India Radio) farm programs and farm broadcasting of Doordarshan enriched the role of electronic media in farm extension activities. The media saturated southern state of India received this new electronic media farm communication revolution whole heartedly. However, after 1990, Kerala witnessed a flood of private T V channels and currently there are 24 channels in this regional language, named Malayalam. All major news and entertainment channels are broadcasting farm programs. Farm programs of AIR and Doordarshan, broadcasted in Malayalam language, have been well accepted to the farmers‘ in Kerala. However, post-independence period, witnessed the formation of Kerala state in Indian Union and the first ballot-elected communist Government started its administration. After the land reform bills, the state witnessed a gradual decrease in agricultural production. Even if it is not reflected much in the attitude and practices of farm community and farm broadcast of traditional electronic broadcasting, a change is observable after the post-liberalization era of India. Private Television channels, which were focused on entertainment value of programs, started broadcasting farm programs and the parameters of program production went through certain changes. In this situation, there is ample relevance for a study about the farm programs of electronic media in terms of a comparative study of audience perception. The study is limited in the state of Kerala as it is the most media saturated state in India. The study analyzes the rate, nature and scope of adoption of farming methods transmitted through electronic media (T.V. and Radio) in Malayalam language.All kinds of Farm programs including comprehensive program serials, success stories, seasonal cropping methods, experts opinion, been analyzed on the basis of the following objectives.  To find whether propagating new farm methods through farm programs in electronic media or the availability of adequate infrastructure and economic factors make a farmer to adopt a new farming method.  To find which electronic media has more influence on farmers to adopt agricultural programs.  To find which form of electronic media gets better feedback from farmers  To find out whether the programs of T.V. or Radio is more acceptable to farmers than the print media.  To find whether farmers gets the message through their preferred medium for the message. The researcher recorded opinions from a panel of agricultural officers, farm Information officers, agro extension researchers and experts. According to their opinions and guidelines, a pilot study is designed and conducted in Kanjikuzhy Panchayath, in Alappuzha district, Kerala. The Panchayath is selected by considering its ideal nature of being the sample for a social Science research. Besides, the nature of farming in the Panchayath, which devoid of the cultivation of cash crops also supported its sample value. As per the observations from the pilot study, researcher confirmed the Triangulation method as the methodology of research. The questionnaire survey, being the primary part contained 42 Questions with 6 independent and 32 dependent variables. The survey is conducted among 400 respondents in Idukki, Alappuzha and Pathanamthitta districts considering geographical differences and distribution of different types of crops. The response from a total of 360 respondents, 120 from each district, finally selected for tabulation and data analysis.The data analysis, based on percentage analysis, along with the results from focus group discussion among a selected group of 20 farmers, together produced the results as follows. Farmers, who are the audience of farm programs, have a very serious approach towards the medium. They are maintaining a critical point of view towards the content of the programs. Farmers are reasonably aware about the financial side of the programs and the monitory aspirations of both private and Government owned Television channels. Even though, the farmers are not aware on the technical terminology and jargons, they have ideas about success stories, program serials and they are even informed about channels are not maintaining an audience research section like AIR. Though the farmers accept Doordarshan as the credential source of farm information and methods, they are inclined to the entertainment value of programs too. They prefer to have more entertainment value for the programs of Doordarshan. Surprisingly, they have very solid suggestions on even about the shots which add entertainment value to the farm broadcasting methods of Doordarshan. Farmers are very much aware about the fact that media is just an instrument for inspiration and persuasion. They strongly believe that the source of information and new methods is agricultural research and an effective change happens only when there are adequate infrastructure and marketing facilities, along with the proper support from Government agricultural guideline and support systems like Krishi Bhavans. They strongly believe that media alone cannot create any magic in increasing agricultural production. Farmers are pointing out the lack of response to the feedback and queries of farmers on farming methods, as an evidence for the difference in levels of commitment of Government and private owned Television channels.Farmers are still perceiving AIR farm programs are far more committed to farmers and farming than any other electronic medium. However, they are seriously lacking Radio receivers with medium wave reception facility. Farmers perceive that the farming methods on new crops are more adoptable than the farming methods of traditional crops in both private and Government owned Television channels. There are multiple factors behind this observation from farmers. Farmers changed in terms of viewing habits and they prefer success stories, which are totally irrelevant and they even think that such stories encourage people to go for farming and they opined that such stories are good sources of inspiration. However, they are all very much sure about the importance and particular about the presence of entertainment factor even in farm programs. Farmers expect direct interaction of any expert of the new farming method to implement the method in their agriculture practices. Though introduction of a new idea in the T.V. is acceptable, farmers need the direct instruction of expert on field to start implementing the new farming practices Farmers still have an affinity towards print media reports and agricultural pages and they have complaints to print media on the removal of agricultural information pages from news papers. They prefer the reports in print media as it facilitates them to collect and refer articles when they need it. Farmers are having an eye of doubt about the credibility of farm programs by private T.V. channels. Even if they prefer private Television channels for listening and adopting new farming methods and other farm information, they scrutinize programs to know whether they are sponsored programs by agrochemical or agro-fertilizer manufacturer.

Gill Nets of Kerala

The fishing industry the world over is passing through a critical situation.The landings of marine fishes seemed to have reached saturation in major fishing areas of the world.As a general rule fisheries employing fishing gear and techniques used by small scale and artisanal fishermen either from shore or from onboard small fishing craft come under small-scale fisheries.This study on gill nets of Kerala, the fishing method depended upon by maximum fishermen of the state focuses on the importance of this selective and low energy fishing method in the marine fishing sector of the state.The study opens with the conceptual framework by briefly reviewing the crisis in the marine fisheries sector. Maximum fishermen depend upon gill net, which is, an important selective and low energy fishing gear. A review of relevant literature on aspects such as material, selectivity and techno-economic efficiency together with scope and main objectives of the study form the major part of the compass of the introductory chapter.This survey provided the inputs for selection of centres. The chapter presents the basis for selection of sample centres, sample units and methodology for field and experimental study.The subject matter of the fourth chapter is a basic study on gear aterials. The weathering resistance, which is an important criterion to assess the material performance, was studied for polyamide monofilament in comparison to polyamide multifilament and polyethylene twisted monofilament.The study provides supporting evidence of oxidation and characteristic C-O stretching in polyethylene and cyclic lactam .formation and presence of OH in polyamide.The study indicates that small mesh gill netting can be encouraged as a selective fishing method in the inshore waters with restrained use of 30 and 32 mm mesh sizes. The economic efficiency was assessed using standard indices such as rate of return, internal rate of return, pay back period, fishery income, energy efficiency and factor productivity. The effect of size and cost of capital and cost of production on the economics of operation is also discussed in this chapter. It was observed that level of technology did not have direct effect on economic performance.

Studies on the Back Water Oyster, grassostrea madra— sensis (Preston) of Cochin Harbour

A detailed study of the hydrography of the Cochin Backwaters, the habitat off crassostrea madrasensis has been carried out. Data pertaining to air temperature, water temperature, salinity, dissolved oxygen, turbidity and rainfall have been collected and presented. The temperature fluctuation was in the range of 5°C only and that of salinity between 1.1%o and 32.9%o. Fairly steady salinity has been recorded during the pre-monsoon period (February to May) and drastic declension during the monsoon period (June-September).Dissolved oxygen varied between 2.5 ml/l and 6.5 ml/l. Turbidity was highest in June (27.9 p.p.m.) and minimum (10.2 p.p.m.) in February. A detailed study on marine biofouling in the Cochin Backwaters has been made with special reference to primary film, settlement and growth of the fouling organisms such as hydroids, bryozoans, tube-dwelling polychaetes, barnacles and modiolus