Open Access Repository of Indian Theses

SABNE, MADHUKAR

STUDIES IN CELLULOSE : MODIFICATION OF CELLULOSE ESTER FOR USE IN DESALINATION

NAIR, SREELATA

Summary 
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SUMMARY 
Approximately 448 million new cases of curable STIs (Chlamydia infection, 
gonorrhoea, syphilis and trichominiasis) are reported annually with 75% to 80% occur 
in developing countries. Neisseria gonorrhoeae and Chlamydia trachomatis are two 
major STD causing pathogens. Signs and symptoms of the disease include 
conjunctivitis, urethritis, epididymitis, proctitis, and salpingitis. If untreated, infection 
leads to infertility, low fetal birth weight, premature delivery and pelvic inflammatory 
disorder. The infection can also disseminate via blood stream to other organs leading 
to pain in joints (arthritis), degeneration of heart valve, gonococcal meiningitis etc. It 
is also a leading cause of neonatal blindness. Infection due Neisseria gonorrhoeae and 
Chlamydia trachomatis also facilitates acquisition of HIV and HPV. Approximately 
70% to 80% women and 50% men remain asymptomatic. Co-infection of Chlamydia 
and Neisseria is readily reported and infection rates are higher in women in 
adolescents. Although commercial kits are available, their high cost and requirement 
of technical expertise make them far from use in developing countries like India. 
Culture method and gram stain remains the method of choice, but culture method 
takes 2-3 days for results to come before treatment could be recommended. This leads 
in persistence and spread of disease in the society. Thus WHO has recommended 
syndromic management of treatment of Chlamydia and Neisseria. There occurs 
overlap in the spectrum of symptoms due to infection by Chlamydia and Neisseria 
and with various other STI causing pathogens. Discharge and pain in lower abdomen 
are also common during pregnancy. The guidelines for syndromic management issued 
by WHO are neither specific nor sensitive for the two pathogens. Also infections by 
Chlamydia and Neisseria are highly asymptomatic thus treatment only on the basis of 
vaginal discharge syndrome not only misses out asymptomatic infections but may 
also leads to overtreatment. This further leads to increased drug costs, change in 
pattern of endogenous flora, side effects of multiple drugs and emergence of antibiotic 
resistant strains by lateral gene transfer. 
Penicillin and tetracycline were widely used antibiotics till 1980’s, but due to emergence 
of resistant strains of Neisseria, these were discontinued in most of the countries. Thus, 
newer β-lactam antibiotics like ceftriaxone, cefixime, fluoroquinolones, ciprofloxacin, 
Summary 
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ofloxacin, and levofloxacin are recommended for treatment. However, Neisseria with 
reduced susceptibility towards fluoroqiuninolones, ceftriaxone and cefixime are reported 
from different parts of the world. South East Asia is reported to be the centre of origin of 
several resistant strains probably due to syndromic management of gonorrhea. 
Continuous appearance of multi drug resistant strains as well as strains resistant to new 
drugs is alarming. Thus, there is an urgent need for economic, rapid, point of care test for 
co-detection of Chlamydia and Neisseria as well as an indepth understanding of 
gonococcal antibiotic resistance mechanisms is essential for the development of new and 
effective antimicrobial agents.  
The major objective of this work was to develop an in-house PCR assay that is cost 
effective and has high sensitivity and specificity and rapid so that it can be used for 
co-detection of C. trachomatis and N. gonorrhoeae infection. Patients (n=812) having 
symptoms and expecting treatment were enrolled in the study. Using uPCR, dPCR 
and commercial assays our study suggests high prevalence of Neisseria (27.8%) and 
Chlamydia (26.3%) amongst symptomatic women seeking treatment.  Most of the 
women were from age group of 18-30 years. In-house uniplex PCR (uPCR) was 
developed and evaluated against commercially available, Roche Amplicor MWP kit 
for detection of Neisseria gonorrhoeae using unique sequence of orf1 gene as target. 
Our method was found to be 95.8% sensitive and 97.9% specific. Once uPCR was 
evaluated, dPCR was developed and standardized for co detection of Neisseria 
gonorrhoeae and Chlamydia trachomatis. Uniplex PCR for Chlamydia trachomatis 
was already established in lab against gyrA gene. Samples (n=533) were evaluated for 
dPCR against uPCR. Discrepant samples (n=24) were resolved using Roche Amplicor 
MWP Kit. Based on the concordance between uPCR and Roche assay, these samples 
were scored positive for either Chlamydia or Neisseria. As per these results the 
sensitivity of in house dPCR was found to be 85.7% (95 CI: 77.1%-92.8 %) with 
specificity of 97.3% (95 CI:  95.3%-98.5 %). 
Lack of resources and technical expertise makes diagnosis of STDs difficult in rural 
areas of India. Thus, to make our method as test of choice, PCR premix-reagents (2X) 
have been stabilized  such that assay reaction mixture can be kept at 4ºC for upto 4 
months,  making it an easy assay to be carried out in peripheral laboratories where 
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deep freezers are not  available. Further PCR amplification using gDNA isolated from 
dry swabs as mode of sample collection and transportation was also standardized. We 
found that dry swabs work equally good for collection of cervical swab samples as 
wet swabs (swabs in PBS with 1mM). Since dry swabs could be transported at room 
temperature making the method of collection and transport of the samples easy from 
remote areas to the nearest laboratory, the assay so designed will be of great use for 
easy, rapid diagnosis and surveillance programs in developing countries.  
To further enhance the sensitivity and specificity of our test, molecular beacon were 
designed against the amplified region of orf1 and gyrA gene. Since molecular beacons 
can detect single nucleotide change, it thus also enhances the specificity of the test. 
Many commercial kits like Roche MWP kit uses labeled probe as the detection 
method. Their detection method is based on indirect ELISA and thus is time 
consuming (3hrs), cumbersome and requires technical expertise. Various other 
detection assays also use molecular beacons but most of these involve automated real 
time technologies. These kits generally remain out of reach in developing countries. 
In the present study, detection of amplified PCR product is done directly in PCR tube 
using a simple to use, indigenous fluorescent based dark reader for detection. To look 
for relative load of infection, the intensity of flourescence can be measured by ELISA 
reader. Looking for flourescence by directly putting PCR tubes on dark reader is far 
less time consuming than running agarose gel and/or PCR-ELISA and requires no 
technical expertise. The prime advantage of using closed tube is it minimizes chances 
of cross contamination and carry-over contamination. The present study offers an 
easy, rapid and economical NAAT-based assay to study the burden and pattern of 
infection by Neisseria gonorrhoeae and Chlamydia trachomatis.  
Although multidrug resistance strains have been reported from Delhi and other parts of 
India, there is paucity of information on the mutational patterns of the above mentioned 
genes in these isolates. In the next objective of the work, we tried to envisage the 
plausible mechanism of antibiotic resistance in clinical isolates of Neisseria gonorrhoeae. 
Resistance could be plasmid mediated or chromosomal mediated. Plasmid mediated 
resistance in confined to penicillin and tetracycline. Mutations in various genes (gyrA, 
parC, penA, mtrR, mtrE, porB etc.) act synergistically for providing chromosomal 
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mediated resistance to Neisseria. Therefore, to understand how mutations in these genes 
contribute to resistance, we in present study explored the co-relation between mutations in 
various genes (penA, mtrR, mtrE and porB) responsible for antibiotic resistance with the 
change in MIC values for penicillin, tetracycline and ceftriaxone. Fluorescence assay and 
electrophoretic mobility shift assay were carried out to study the effect of mutations in 
MtrR on its biological activity. Attempts have been made to in-silico model the structure 
of MtrR to understand how mutations affect its interaction with DNA. Protein structure of 
wild type and mutant MtrR was compared to understand the structural effect of 
mutations.  We also studied the binding of wild type and mutant MtrR with penicillin as 
well as DNA using both in silico approach and fluorimetric assay. 
Plasmid mediated resistance to penicillin is one of the major mechanism and in India, we 
found African type of plasmid in all the resistant isolates. Multi drug chromosomal 
mediated resistance was found to be highly prevalent in clinical isolates. All the clinical 
isolates (n=28) studied carried mutation in one or more genes and played role in 
imparting resistance. PIB phenotype (88.88%; 24/27) was found to be far more prevalent 
than PIA phenotype for porB gene. None of the resistant isolate carried mutations in PIA 
phenotype (11.11%; 3/27). The mutations at G120 and A121 position of loop3 of the 
mature PorB protein has been documented for increased resistance to several antibiotics. 
Mutations either at G120/ A121 or in both amino acids was reported both in isolates 
resistant as well as reduced susceptible/susceptible for penicillin as well as tetracycline. 
A121S mutation was present in five resistant isolates only. We here report a novel 
mutation of N122K (present always along with A121S) residing in loop3 which may be 
responsible for altered pore size or permeability. 
Mutations both within and outside the transpeptidase domain (AA340-570) of penA gene 
encoding for penicillin binding protein are responsible for providing resistance for 
penicillin and other beta lactams. An insertion of aspartic acid at 346th position in 
transpeptidase domain is shown to be associated with the altered structure and acylation 
of β lactam antibiotics. All the isolates showed Asp 346 insertion and mutations F504K, 
A510V and A516G. Mutant P551L was found to be one of the most frequent mutations 
while only two isolates had H541N mutation. In our study, we also found two novel 
mutations G550R, N562L and S567. We had also observed new substitution at I566N 
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along with the previously reported mutations, thus giving rise to new mutation patterns 
(Table 6). Mosaic pattern IX (11/26; 42.3%) and II (10/26; 38.46%) were most prevalent 
patterns observed in isolates from Delhi hospitals. Isolates which carried triad of 
mutations but were PPNG negative also showed either reduced susceptibility or were 
sensitive for penicillin supporting the previous contention that single mutations at F504K, 
A510V and A516G are not responsible for development of resistance against penicillin 
and ceftraixone. It has been previously shown that substitutions at F504L and P551S lead 
to decreased penicillin acylation rates but mechanism of G542S has not been elucidated. 
Thus, in present study we had tried to elucidate mechanism using homology modeling 
and docking methods. Using crystal structure of PBP2 and modeling techniques, the 
mutant protein was generated and active sites were compared. It was observed that unlike 
wild type, serine 542 formed hydrogen bond with tyrosine 544 whose side chain points 
into the active site. This may alter the spatial arrangement of the tyrosine with respect to 
active site and thus is responsible for higher affinity of the mutant for penicillin. 
Mutations in the structural genes of Mtr operon encoding for MtrCDE efflux pump or 
its regulatory protein (MtrR) as well as in promoter region could result in increased 
efflux of drug and thereby make the cell resistant to the said drug. MtrE encodes for 
outer membrane part of the efflux pump. K181E, K191R and I429S are mutations 
found in various clinical isolates. K181 resides in non conserved region of MtrE and 
is found in resistant as well as susceptible isolates suggesting that this mutation has no 
direct contribution in imparting antimicrobial resistance. K191R and I429S mutations 
reside in conserved region of MtrE. The above mutation resides in the lining of 
intraprotomer groove of the pump which binds with the MtrC and plays role in the 
channel formation and thus the function of the pump. Comparing the resistance 
pattern of isolates, it appears that K191R substitution may be crucial in imparting high 
resistance towards penicillin and tetracycline. We also compared the MIC values for 
tetracycline in PPNG positive isolates and found that isolates with K181E and K191R 
double mutants were less susceptible in comparison to K181E and K181E and I429S 
double mutant indicating some role of K191R in resistance. 
The most common mutation observed in MtrR gene was H105Y present in 11 out of 
25 clinical isolates and was mostly coupled with A/T deletion in promoter region 
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(10/11 isolates). This mutation resides in C Terminal of the protein possessing 
dimerization domain as well as ligand binding cavity. The G45D and A39T mutations 
in DNA binding domain of MtrR protein are known to provide high resistance against 
penicillin and tetracycline. Only G45D mutation was observed in clinical isolates in 
present study. In present work L33V, a novel mutation is also observed in two 
tetracycline resistant clinical isolates along with G45D mutation. To decipher the role 
of L33V mutation, we compared the MIC values of the isolates which possess similar 
mutation pattern for rest of the genes studied (penA, mtrE, porB and mtrR promoter 
region). Thus comparing the MIC value for tetracycline we found that single mutant 
had higher MIC value of 12 µg/ml as compared to 8 µg/ml possessed by the double 
mutants. This has led us to hypothesize that L33V is not advantageous to Neisseria 
with respect to antibiotic resistance. Although mechanism of action of G45D mutant 
MtrR is well recognized (i.e abrogation of binding of the protein), no direct evidence 
elucidating the mechanism of action H105Y mutation is known. We hypothesize two 
plausible mechanisms for the action of this mutation. 
 MtrR is known to bind as homodimer to its promoter.  Mutation H105Y may 
affects the dimer formation and thus its binding with DNA. 
 H105Y may alter the binding of antibiotics in ligand binding cavity. The binding 
of antibiotics like penicillin may induces conformational change and thus its 
binding with the DNA. 
With a view to understand the functional implications of mutant MtrR, we decided to 
check whether mutations in MtrR are responsible for any conformational change in 
the protein and if so, does it affect its interaction with DNA and small ligands like 
penicillin. Therefore, to identify a plethora of processes in which MtrR has some role 
to play, we selected the following objectives:  
 Bioinformatics approach to study the change in the conformation of MtrR and 
its interaction with penicillin and promoter DNA  
 Comparative analysis of the DNA binding activity of wild type and mutant 
MtrR protein using purified protein. 
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To achieve these objectives, in-silico modelling of wild type and mutant MtrR 
proteins was carried out using swiss PDB server and Discovery studio. 
Conformational change was studied by superimposing mutant protein over wild type. 
Change in conformation majorly in Helix 1 (N terminal) and helix 4 (C terminal) was 
observed. Size of the ligand binding cavity decreased from 356 Å3 of wild type to 256 
Å3 in H105Y mutant. This change is in contrast to G45D mutant wherein a minor 
increase is observed in the volume of the cavity (364 Å3). We also observed that 
presence of penicillin has an effect on MtrR conformation and/or DNA binding as 
suggested by fluorescence spectrophotometeric results. The assays were performed 
using purified wild type and H105Y mutant MtrR titrated with increasing 
concentration of penicillin which showed shift in lambda max is more pronounced in 
case of mutant. Appearance of second peak is observed around 420 nm in case of 
H105Y mutant MtrR. Both of these results suggested altered conformation of the 
mutant. We then docked penicillin in wild type as well as H105Y mutant MtrR to 
check if penicillin affects MtrR conformation by directly binding to it.  Change in 
pose and thus interacting partners was observed in wild type and Mutant MtrR. On 
comparing the various parameters, H105Y mutant appears to possess higher affinity 
for penicillin probably due to hydrogen bond formed by substituted Tyrosine 105 thus 
stabilizing the protein penicillin complex.  
Next we checked the effect of the mutation on DNA binding of MtrR using EMSA, 
fluorescence spectrophotometeric assays and in silico analysis. Electrophoretic 
mobility gel shift assay performed using crude lysate of over expressed wild type, 
mutant G45D and mutant H105Y showed that higher concentration of mutant H105Y 
protein is required to bind with its promoter DNA to cause visible gel shift. 
Competition assays suggested that far less DNA is required to compete with mutant 
protein as compared to wild type suggesting H105Y has either low DNA binding 
affinity or increased rate of dissociation. Fluorescence spectrophotometry experiments 
clearly indicated that unlike wild type MtrR, H105Y mutant MtrR failed to bind 
effectively to its binding site on the promoter. The binding affinity of the mutated 
MtrR with promoter was about 1.13 folds lower than that of wild type. Although 
change is small, it could be possible that two mechanisms act synergistically and 
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binding is effectively altered in the presence of penicillin. Further to decipher the 
change in hydrogen bond profile of protein DNA complex assembly, MtrR was 
docked with the promoter DNA. Since homodimer binds to the promoter, we first 
docked monomers of wild type and the mutants to form homodimers. Homodimer of 
H015Y mutant was most stable energetically, but increased centre to centre distance 
between helix1 and helix 1’ of two monomers suggested altered alignment of the two 
monomers on the pseudo-repeat binding site on the DNA which may reduce its 
binding with DNA as compared to that of wild type MtrR. To check this hypothesis, 
protein DNA docking was performed using refined monomer form from the dimer 
complex. Best pose was screened using structural similarity with QacR (complexed 
with its promoter), another member of TetR family. No optimal binding pose was 
obtained for G45D mutant as expected as this amino acid is critical for DNA binding. 
For H105Y mutant, two best poses which imperfectly resembled that of wild type and 
QacR DNA complex were selected. This may be explained in the light that due to 
increased centre to centre distance, residues of helix3 are not able to reside close to 
DNA residues in second half site of pseudodirect repeat of the promoter. Hydrogen 
bond analysis shows formation of different hydrogen bond between wild type DNA 
complex and H105Y mutant DNA complex.  Based on these results, we envisage that 
although different hydrogen bonds are formed between H105Ymutant MtrR and DNA 
complex, they may be sufficient to provide only partial stability to the complex which 
may account for the observed protein-DNA binding using EMSA and fluorescence 
spectrophotometry. It remains to see whether mutation in H105Y affects the rate of 
dissociation from DNA which will indirectly result in increased expression of the operon. 
Thus even a single amino acid change outside the DNA binding domain causes a 
conformational change in the binding domain of MtrR both in the presence and 
absence of penicillin which may be responsible for altered DNA-protein interaction.  


English

STUDIES ON PLANT TISSUE CULTURE (ANNONA SPP.)

WADGAONKAR, PRAKASH

Abstract
The present thesis describes : 
1) Telechelic diene polymers : a review, 2) Synthesis of azobisnitriles containing functional groups. 3) Decomposition rate studies of azobisnitriles in solution 4) Hydroxyi-terminated polybutadienes, 5) Isocyanate-terminated polydienes, and 6) Silicon-containing polyirnides. 
Chapter -1: Telechelic Diene Polymers:A Review 
This chapter embodies a brief and comprehensive review of literature on preparation, characterization, curing, and application of telechelic (functionally terminated) diene polymers. 
Chapter - 2 : Synthesis of Azobisnitriles Containing 
functional Groups It was realized in 1950's that functionally terminated polymers could be produced from monomers which terminated by combination by polymerizing in the presence of initiators containing the functional groups. From that time, a considerable number of azo initiators containing functional groups have beer synthesized with a view to use them for the syntheses of functionally terminated polymers1-4. Bifunctional azobisnitril, have also been used to prepare macro-azonitriles, which are useful in the preparation of block copolymers5. This chapter describes synthesis of the following azobisnitrilas containiig functional groups: (1) 4,4l-Azobis(4-cyanopontanol), (ABCP) (2) 5,5'-Azobis(5~cyano~2-hexanol), (A3CH) (3) 4,4'-Azobis(-4-cyanopentanoic acid), (ABAC) (4) 4,4'-Azobis(4-cyanope.ntanoyl azide), (ABAZ) (5) 4,4'-Azobis (4-cyano-2-methylpentanoic acid), (AMAC) The azo compounds (ABCH), (ABAZ), and (AMAC) have been synthesized for the first time. All these azobisnitriles were characterized by spectral data, and elemental analysis. 
Chapter 3 : Decomposition Rate Studies of Azobis nitriles in solution The present investigation reports the results of studies on thermal decomposition in solution of the following azobisnitriles : (l) 4-4'-Azobis(4-cyanopentanoic acid), (2) 4,4'-Azobis(4-cyanopentanol), (3) 4,4'-Azobis (4-cyano-2-methylpentanoic acid) , (4) 5,5'-Azobis(5-cyano-2-hexanol). The rates of decomposition of these azo compounds were determined in dioxane solution at four different temperatures in the range of 65-80 C, by measuring nitrogen evolution as a function of time . All the azo compounds exhibited first-order kinetics. The activation energies and half-life time values for all the azo initiators were determined. The effect on structure of azo compound on the rate of decomposition has been discussed. 
Chapter 4: Hydro -Terminated Polybutadienes Liquid polybutadiene with reactive end groups is among the most promising liquid polymers7. Polybutadienes carrying term hydroxyl groups are particularly desirable materials on account of the multifold quantitative derivatization possibilities they offer. One of the most promising materials that can be prepare from hydroxyl-terminated polymers are polyurethanes. Hydroxyl-terminated polybutadianes are mainly used as binders in advanced solid rocket propellants, where polymers carrying secondary hydroxyl end groups are preferred8 because the cure reaction of such polymers with diisocyanate can be better controlled with respect to pot-life and casting.Hydroxyl-terminated polybutadienes are generally prepared 3,4,7 by either an anionic polymerization or free radical polymerization method. In the anionic polymerization method, the type of hydroxyl groups can be fixed by selection of appropriate chain-terminating reagent. Polydienes carryin all types of hydroxyl end groups - allylic primary, primary1 and secondary hydroxyl-have been prepared. In the freeradical polymerization method, the most commonly used initiate:are hydrogen peroxide, and 4,4'-azobis(4-cyanopentanol) Hydrogen peroxide produces polybutadienes carrying predominantly terminal primary hydroxyl groups of the allylic type, 4,4'-Azobis(4-cyanopentanol) produces polymers carrying primary hydroxyl end. groups. Surprisingly, synthesis of polybutadienes carrying secondary hydroxyl end groups by the free radical polymerization method was not attempted so far. This chapter describes the synthesis of polybutadienes possessing secondary hydroxyl end groups by free radical polymerization method, employing 5,5'-azobis(5-cyano-2-hexanol as an initiator. Primary hydroxyl-ended polymers were also prepared by using 4,4'-azobis(4~cyanopentanol) as an initiator The effects of reaction parameters such as initiator concentration, polymerization time, and monomer concentration on yields and properties of polymers were studied. The polybutadienes obtained were characterized by number average molecular weight, hydroxyl content, functionality, IR and NMR spectra, and microstructure. Preliminary curing experiments of hydroxyl terminated polymers with diisocyanate were also studied. 
Chapter 5 :Isocyanate-Terminated Polydienes In the poly?rethane elastomer industry, polydiene-based polyurethanes have been developed as a new field. Polydic based polyurethanes are of interest because they offer the advantage of low moisture permeability and hence they have been. the subject of several studies aimed at exploiting this property for adhesive and electrical potting compounds, Polydiene-based polyurethanes are prepared from hydroxylterminated polybutadienes by either one-shot or two-step technique. 'Whilst there is a vast literature concern,with the preparation of hydroxyl-terminated polydienes, there is a lack of information on the free radical synthesis of isocyanate-terminated polymers. In the present research, free radical polymerization method, for the direct synthesis of isocyanate-terminated polymers has been developed for the first time. The initiator employed was azobis(4-cyanopentanoyl azide). Liquid polyisoprenes and polybutadienes carrying aliphatic isocyanate end groups were prepared and characterized by number average molecular weight, isocyanate content, functionality, IR spectra and NMR spectra. The isocyanate end groups of low molecular weight polybutadienes were subjected to chemical modifications to introduce other interesting functional groups such as tertiary-amine, and peroxycarbamate. 
Chapter 6:Silicon -containing Polyimides Recent advances in space technology have a growing need thermally stable polymers. Polyimidos have excellent mechanie properties even at high temperatures and find uses in a varies of ways such as high-temperature adhesives, coatings, sealantetc. This chapter describes the synthesis of two new aromatic diisocyanates having a silicon-containing tricyclic fused ring system. Further, phenoxasilin rings containing polyimides were synthesized by polycondensation of these diisocyanates with aromatic dianhydrides, viz., (l) pyromellitic dianhydride, and (2) benzophenone tetracarboxylic acid dianhydride. Polyirnides exhibited good heat resistance but were insoluble in organic solvents. 
  	 
	




STUDIES IN (I) TELECHELIC DIENE POLYMERS, (II) SILICON-CONTAINING POLYIMIDES

MALVANKAR, RAMNATH B.

Abstract
CULTURED DIVERSITY OF PURPLE ANOXYGENIC PHOTOTROPHIC RHIZOBACTERIA OF PADDY AND THEIR PLANT GROWTH PROMOTING ATTRIBUTES

The “Anoxygenic phototrophic bacteria (APB)” encompasses an extremely heterogeneous group of prokaryotes that are capable of converting radiant energy into chemical energy under anaerobic conditions but release no oxygen. The heterogeneity of APB is demonstrated by great variations they show in their habitats, morphology, physiology, radiation capturing pigments and phylogeny. In contrast to oxygenic cyanobacteria and plants, they posses only one photosystem (PSI or PSII). Instead of water, they use electron donors which are more reduced than water such as reduced sulfur compounds (sulfide, thiosulfate, sulfite, tetrathionate), hydrogen, a number of small organic compounds [acetate, pyruvate; (Imhoff, 1995)], or ferrous iron (Fe2+) (Heising et al., 1999; Heising & Schink, 1998).
     APB are widely distributed in various aquatic and terrestrial habitats, which include rice fields (Arunasri, 2004; Harada et al., 2001; Elbadry et al., 1999; Habte & Alexander 1980; Matsuguchi & Tangcham, 1974; Kobayashi et al., 1967). The rice fields which are characterized by the diurnal light irradiance, eutrophic environment and oxic-anoxic interface (Liesack et al., 2000), provide purple phototrophic bacteria (a major physiological group of APB) with an ideal niche for their growth (Feng et al., 2009). The purple bacteria were found to be repeatedly enriched and found in high numbers at different stages of the rice plant (Archana, 2002; Madigan, 2001; Elbadry et al., 1999; Ormerod et al., 1996). Among them, the purple non sulfur bacteria are the most numerous to occur in paddy soils and their numbers are reported to be comparable to those of other N2 fixing bacteria (Elbadry et al., 1999; Okuda et al., 1975; Haque et al; 1969; Kobayashi et al., 1967). However, the previous studies available on the distribution and enumeration of purple bacteria from rice fields have been restricted to countries like Japan, Egypt, Phillipines, Thailand, Taiwan and Malaya with only sporadic reports from rice fields of India (Archana, 2002; Sasikala et al., 2002). The present thesis focuses on the distribution, characterization and identification of purple bacteria from the rice field habitats of India. Furthermore, the role played by these bacteria in rice rhizosphere soils in terms of exhibiting plant growth promoting attributes was studied.



STUDIES IN ANALYTICAL METHODS IN ORGANIC CHEMISTRY

MESHRAM, NAMDEO RAMJI

I 
 
 
 
 
 
 Synopsis 
 
 
 
 
 
 
 
Synthesis of New Somatostatin Receptor Binding Radio-labelled 
peptides: Physico-chemical and Biological Evaluation and 
Scintigraphic Studies 
 
 
II 
 
Peptides are important regulators of growth and cellular functions not only in normal tissue 
but also in tumors. Peptides are becoming of increasing interest in nuclear oncology for 
targeted tumor diagnosis and therapy. 111In-diethylenetriaminepentaacetic acid (111In-DTPA) 
labelled Octreotide (Octreoscan; Mallinckrodt / Inc.) was the first routinely used tracer in 
nuclear medicine for the localization and staging of somatostatin receptor positive tumors. 
Stable somatostatin derivatives such as Octreotide / Lantrotide are the most frequently used 
radiopharmaceuticals acting through specific binding to somatostatin receptors; however, 
they do not bind with high affinity to all five receptor subtypes. 111In-DTPA-Octreotide based 
imaging served as a sensitive and specific technique for somatostatin receptor scintigraphy 
that can detect in-vivo somatostatin receptors in various tumors mainly of endocrine origin. 
111In-DTPA-Octreotide is commercially available for imaging; however the use of 111In as a 
radioisotope entails high cost, limited availability, suboptimal image quality and a high 
radiation burden to the patient. These disadvantages were overcome by the use of 99mTc 
labelled somatostatin analogs. Thus newly developed somatostatin analogs labelled with 
99mTc were intensively studied in the proposed research work. 
New somatostatin analogs (octreotide derivatives) like HYNIC-Tyr3-Octreotide 
(TOC), HYNIC-His3-Octreotate and HYNIC-Met3-Octreotate (TATE) were synthesized by 
standard Fmoc Solid Phase Peptide Synthesis (SPPS) on O-t-butylthreoninol-2-chloro trityl 
resin for TOC derivatives and H-Thr(tBu)-2-Cl trityl resin for TATE derivatives on a semi 
automated peptide synthesizer according  to our laboratory protocol. Coupling of prochelator 
to peptide was performed in the presence of excess of HYNIC-BOC in solid phase. BOC 
protected HYNIC was also synthesized and characterized in our laboratory. Then the peptide 
chains were cyclized by formation of cys-cys disulfide bond through Iodine oxidation 
method. These peptides were deblocked and deprotected from resin and lateral protecting 
groups by using trifluoro acetic acid cocktail. Then purification was achieved by semi-
III 
 
preparative HPLC. The structures of the peptides were then elucidated by 1D NMR (1H 
NMR, 13C NMR, DEPT 90, 135), 2D NMR (HSQC, HMBC, COSY), MALDI mass and FT-
IR spectroscopy. The peptides were again quantitated by amino acid analysis of a 24 h acid 
hydrolysate using Waters Accu-Tag chemistry together with a Waters HPLC system. 
Conformational analysis of the peptides was performed by circular dichorism spectroscopy in 
pure water and trifluroethanol-water (1:1). 
Labelling of peptides was performed with 99mTc using Tricine and EDDA as 
coligands by SnCl2 method to get products with excellent radiochemical purity ˃98%. 
Complexation efficiency and radiochemical purity of radiolabelled peptides were validated 
by ITLC and RP-HPLC.  In-vitro stability study on rat serum and PBS buffer pH 7.4 was 
performed. The percentages of radiolabelled complex and free pertechnetate were assessed by 
ITLC method. The stability of radiolabelled peptides was validated by RP-HPLC. Metabolic 
stabilities in rat kidney and urine indicate that the radio peptides were stable to enzymatic 
changes. Metabolic stability analysis did not show any evidence of breaking of the labelled 
compounds and formation of free 99mTc.  
C6 glioma cell (NCCS, Pune, India) was grown confluent in DMEM high glucose 
nutrient mixture supplemented by 10-20% (v/v) fetal bovine serum, 1 mM glutamine, 100 
U/ml penicillin and 100 μg/ml streptomycin in humidified air containing 5% CO2 at 37 °C. 
Internalization studies were done and IC50 values were determined in somatostatin receptor-
expressing C6 glioma cell line and rat brain cortex membrane, and the results compared 
against HYNIC-TOC as standard. Radioligand internalization, in-vitro receptor binding 
assays, blood clearance, scintigraphy, and biodistribution studies were performed to evaluate 
their biological characteristics. The results of scintigraphy imaging studies and renogram 
study also showed the highest uptake in the kidney which gradually decreased as time passed. 
Blood clearance was faster with rapid excretion through kidneys. Biodistribution and image  
IV 
 
 


TOLUENE DISPROPORTIONATION AND TRANSALKYLATION WITH C9 AROMATICS OVER ZSM-5 ZEOLITES

PATASKAR, SURYAKANT

STUDIES ON THERMAL DECOMPOSITION OF SOLIDS IN CATALYST PREPARATION

NADGAUDA, RAJANI SATISH

Abstract

Selectivity in some new methodologies for synthetic elaboration at α-C-H centres of tertiary amines

The research work describes investigations in new synthetic methodologies for α-C-H functionalization of tertiary amines, especially with respect to regioselectivity and stereoselectivity.  

Metal catalyzed regioselective CDC reactions of tertiary amines (N-methyl tetrahydroisoquinolines, N-methyl piperidine, N-methyl pyrrolidine, indolizidine) with alkynes have been studied. Applications of this procedure for synthesis of phenethylisoquinolines (biologically active alkaloids) are also evaluated. A metal free protocol for oxidative coupling of tertiary amines with different nucleophiles is also described. The mechanisms of these reactions, and the possible factors for the origin of regioselectivity are discussed in the context of earlier known examples.

 Stereoselective lithiation-substitution reaction sequence of tertiary amines was investigated. The α-lithiation-substitution of Lewis acid complexed bicyclic tertiary amine systems (pyrrolizidine and quinolizidine) and its enantioselective version is discussed. An alternative approach to induce enantioselectivity at the α-C-H position in N-methyl tetrahydroisoquinolines using chiral Lewis acid has also been evaluated.



BIOCHEMICAL STUDIES ON PLANT TISSUE CULTURES

SHARMA, G. V. MADHAVA


Abstract/Synopsis Development and characterization of Pigeonpea [Cajanus cajan (L.) Millisp.] EST-SSR markers and analysis of allelic variation across related species and legumes. Pigeonpea [Cajanus cajan (L.) Millspaugh] is one of the most important food legumes of semi-arid tropical and subtropical regions, has limited genomic resources, particularly expressed sequence based (genic) markers. Expressed sequence tags (ESTs) are fragments of mRNA derived through single sequencing reactions performed on randomly selected clones from cDNA libraries. In this study, ~1.7 million ESTs were generated from leaf, root, stem, pod, bud and immature seed of two pigeonpea varieties, Asha and UPAS-120 by Sanger and 454-next generation sequencing. Out of this, 43,324 unigene contigs derived from 454 GS-FLX pyrosequencing were used to generate 3,771 genic-SSR loci, excluding homopolymeric and compound repeats, were identified; of which 2,877 PCR primer pairs were designed for marker development. Dinucleotide was the most common repeat motif with a frequency of 60.41%, followed by tri- (34.52%), hexa- (2.62%), tetra- (1.67%) and pentanucleotide (0.76%) repeat motifs. Primers were synthesized and tested for 772 of these loci with repeat lengths of ≥18 bp. Finally we developed 550 validated genic-SSR markers in pigeonpea. From these, 20 highly polymorphic markers were used to evaluate the genetic relationship among 22 pigeonpea varieties and eight wild species. The number of alleles at these loci ranged from 4-10 and the polymorphism information content values ranged from 0.46 to 0.72. Neighbor-joining dendrogram showed distinct separation of the different groups of pigeonpea cultivars and wild species. These markers also proved very useful in intra and inter specific transferability showing high cross-species transferability ranging from 79.5 to 87.0% across the nine cajanus species and from 16.0 to 50.0% across the seven legume genera. We evaluated 200 pigeonpea genic-SSR markers and observed a clear correlation between transferability rate of markers and phylogenetic distance between the species. Sequence analysis indicated that the same size of PCR product was not always associated with conserved SSR sequence and different size PCR products were not necessarily due SSR size differences. Dendrogram constructed on the basis of these loci not only separated all the pigeonpea cultivars from their wild species and validated the proximity of C. cajan to C. platycarpus but also elucidated the phylogenetic relationships among the species of four major clades of subfamily Papilionoideae. These cross-transferable markers can be used for genetic mapping within and across species and will provide powerful tool for molecular breeding in different crop legume species. This study provides an insight into the distribution of SSRs in the pigeonpea transcribed regions and also demonstrates the development and utilization of genic-SSRs. In addition to proving their suitability for genetic diversity analysis, their high rates of transferability also proved their potential for comparative genomic studies and for following gene introgressions and evolution in wild species, which constitute the valuable gene pool in pigeonpea. 




MACROLIDES

MULLA, S. T.

 69 
 
Summary and Conclusion 
1. A non-functional BADH2 form was detected in P. amaryllifolius.  In scented rice 
and other 2AP synthesizing systems, such non-functional form of BADH2 is 
reported that blocks the GABA synthesis and leads in the accumulation of 
Δ1pyrroline, an immediate precursor of 2AP. The present finding of non-
functional form of BADH2 suggests similar mechanism of biosynthesis of 2AP in 
P. amaryllifolius. This is the first report of its kind in P. amaryllifolius. 
 
2. In addition to non-functional BADH2 form, functional BADH2 was also detected 
in P. amaryllifolius. Thus the study showed the co-existence of functional and 
nonfunctional BADH2 gene isoforms in P. amaryllifolius. This is also a first 
report of its kind in P. amaryllifolius. 
 
3. The higher relative expression of BADH2 along with 2AP synthesis supports the 
co-existence of two forms of BADH2 gene in P. amaryllifolius. 
 
4. The higher in vivo BADH2 enzyme activity also supports the existence of 
functional BADH2 gene in P. amaryllifolius. 
 
5. The in vivo enzyme assay confirms γ-amino butyraldehyde as the preferential 
substrate for BADH2 in P. amaryllifolius. 
 
6. The recombinant PaBADH2 protein activity also confirmed the substrate 
specificity with γ-amino butyraldehyde in P. amaryllifolius. 
 
7. The 9-10 pH optima of PaBADH2 suggests that the protein might be targeted in 
peroxisomes. 
 
8. The docking studies of PaBADH2 confirmed that it belongs to the ALDH_SF 
superfamily with a specific hit with the ALDH_F10_BADH, i.e. betaine aldehyde 
dehydrogenase family. 
 
9. The docking studies also indicated that the key regions for the substrate binding 
pocket, cofactor binding domain and oligomerization domain are conserved in P. 
amaryllifolius. 
 
10. The Ramchandran plot analysis showed that the PaBADH2 protein model had 
good stereochemical quality with 91.3 % of amino acid residues present in most 
favored regions. 
11. The partial and hybrid structures of PaBADH2 showed the same interaction 
energies with the substrates, suggesting that the partial PaBADH2 is active in P. 
amaryllifolius. 
 
 70 
 
Summary and Conclusion 
12. The histochemical studies explained the site specific expression of two forms of 
BADH2. The functional form is active in the mesophyll tissue while the non-
functional form is present in the epidermal tissue where 2AP is stored. 
 
13. The highest 2AP synthesis in P. amaryllifolius can be correlated with higher 
contents of its two precursors - proline and methylglyoxal than in scented rice. 
 
14. Thus, overall the present study explains the molecular mechanism of 2AP 
expression in P. amaryllifolius that seems to be similar as found in scented rice. 
 
  


SPECTROSCOPIC STUDIES OF HYDROGEN BONDING IN AMINO ALCOHOLS

SHROTRI, VIDYADHAR

Annexure I 
 
Abstract/Synopsis 
 Reptiles are ectothermic amniotes, providing the key link between 
anamniotic ectotherms, fishes and amphibians, and amniotic endotherms, birds and 
mammals. A greater understanding of reptilian immunity will provide important insights 
into the evolutionary history of vertebrate immunity. Hence, this study was undertaken to 
examine the effect of melatonin and testosterone hormones and photoperiodic modulation 
on immune responses of splenic macrophages. Also, seasonal as well as circadian 
variation in immune responses of splenic macrophages in the fresh-water snake was 
studied. Following immune parameters were taken into consideration: 
 Spleen mass and cellularity 
 Splenic macrophage Phagocytosis 
 Quantitative NBT reduction   
 Nitrite release  
 Splenic lymphocyte proliferation 
 
 To study the effect of melatonin hormone, melatonin injections in saline vehicle 
(dose: 5 µg /g body weight and 10 µg /g body weight) were given during evening hours, 
and alteration in lymphoid organ mass, splenic macrophage phagocytosis, Nitric oxide 
(NO) production (Nitrite assay), superoxide production (NBT reduction assay) and 
lymphoproliferation (MTT assay) were studied on the day following 10
th
 and 20
th
 
injection. No consistent and significant change was observed in phagocytic response of 
splenic macrophages harvested from snakes treated with either dose of melatonin for 
different duration. Nitrite release and superoxide production by splenic macrophages 
were significantly (p<0.05) higher in snakes receiving melatonin injections. Exogenous 
melatonin enhanced the mitogen-induced splenic lymphocyte proliferation only at the 
dose 10 µg/ g body weight, but not at that of 5 µg one.  In vitro melatonin also invariably 
has enhanced the mitogen-induced splenic lymphocyte proliferation, and vigorous 
response has been observed in splenocytes harvested from snakes receiving 10 µg /g 
body weight melatonin injection for 20 days, when splenocytes were induced by 
mitogens: 10 µg mL
-1
 of Con A and PHA and by 20 µg mL
-1
 of LPS. Taken together, the 
results of the present study demonstrate that in vivo melatonin treatment enhances innate 
immunity, in general except phagocytosis, in the fresh-water snake, N. piscator.  
 
To study seasonal variation in immune functions, the requisite number of animals 
were sacrificed under mild anaesthesia during mid of each month.  In all assay, sample 
size was 5 to 6. Analysis of variance reveals that there was no significant variation in 
splenic mass of the fresh-water snake during different months of study period, though 
spleen size has a trend to be high in autumn and winter months and low in spring and 
summer. Spleen cellularity was recorded high in winter months and again in September; 
while it remained low during rest of the year. No definite pattern was observed in 
phagocytosis by splenic macrophages. The percent phagocytosis varied between 42 to 60 
%, being highest in month of February. Analysis of variance revealed that free radical 
production and lymphoproliferation varied significantly during different months. Super 
oxide production, as judged by NBT reduction, was found to be high during spring 
season. Nitrite release by splenic macrophages was high in months of January, May and 
September - October. The splenic lymphocyte proliferation, both basal as well as Con A 
induced, was highest in month of May, followed by a little low in January and October: it 
remained low during rest of the year. 
 
 In the study of circadian variation, Cosinor analysis of the data revealed that no 
significant circadian rhythm was validated in fluctuation of splenosomatic index and 
spleen cellularity in all the three seasons. The percentage phagocytosis had significant 
rhythm of 24 h in summer and spring seasons, but not in winter; and no significant 
circadian rhythm was validated in phagocytic index. NBT reduction and nitrite release by  
splenic macrophages of fresh-water snake also varied significantly in circadian manner 
during winter season. But, significant circadian rhythm was absent in NBT reduction and 
nitrite release during summer season. A significant circadian rhythm was observed in 
splenocyte proliferation (Basal) and splenocyte proliferation (Con A10 stimulated) in all 
three seasons: summer, winter and spring. A significant phase shift in splenocyte 
proliferation (Con A10 stimulated) was obtained in all three seasons with a trend of 
delayed phase shift from winter to spring and spring to summer.  
  Most formal studies on seasonality have focused on day lengths i.e., photoperiod, 
as the environmental cue used by animals to coordinate intrinsic rhythms with extrinsic 
seasonal environmental changes. To study the effect of photoperiodic manipulation on 
immune parameters, snakes were maintained in 24h dark (DD), 24h light (LL) and at 
normal day length (10L:14D). Spleen mass was increased, but insignificantly, in the 
animals subjected to DD. No effect was observed in the animals kept under continuous 
light, i.e. LL, when compared with control ones. On other hand, spleen cellularity was 
also increased, but insignificantly, in the animals of both groups kept in DD as well as 
LL. No significant (p<0.05) change was observed in percentage phagocytosis and 
phagocytic index in animals kept under DD or LL, as compared with animals kept in 
10L: 14D. Super oxide production, as measured by NBT reduction assay, was reduced 
significantly (p<0.05) in the animals kept either in 24D or 24L. Production of nitric oxide 
by snakes’ splenic macrophages, as measured by nitrite assay, was significantly (p<0.05) 
decreased in animals kept under LL; while no change in nitrite release was observed in 
animals kept in DD, when compared with animals kept in 10L:4D. There was significant 
(p<0.05) enhancement of mitogen induced splenocytes proliferation in cultures obtained 
from animals kept under DD. To study whether melatonin could induce the splenocytes 
proliferation, splenocytes cultures obtained from all group animals were treated with in 
vitro melatonin (500 pg ml
−1
, final concentration). In vitro melatonin significantly 
(p<0.05) enhanced the splenocyte proliferation of the cultures obtained from animals kept 
in LL, as compared with cultures from 10L:14D animals, but not in those of DD animals.  
    
To study the effect of in vitro testosterone, splenic macrophages monolayer 
prepared on slides was exposed to different concentrations male sex hormone, (0.1, 1, 10, 
50 and 100 ng ml
–1
). After 4 hour of incubation, the monolayers were washed three times 
to remove testosterone, and phagocytosis was allowed to proceed. Effect of different 
concentrations of in vitro testosterone was also studied on NBT reduction, nitrite release 
and splenic lymphocyte proliferation (basal as well as mitogen induced). It was observed 
that percentage phagocytosis decreased significantly in correlation with the in vitro 
testosterone concentration. Maximum reduction of percentage phagocytosis was obtained 
at 100 ng ml
–1
 concentration of testosterone. Effect of in vitro testosterone on NBT 
reduction was found to be differential: as NBT reduction was decreased at 10 ng ml
–1
 
concentrations, but no decrease at 100ng ml
–1
, and again decreased at 1000 ng ml
–1
. The 
decrease was maximum at 1000 ng ml
−1
 testosterone concentration. Nitrite release was 
also significantly (p<0.05) reduced by in vitro testosterone treatment in a concentration 
dependent manner. When the macrophages were pre-incubated with testosterone receptor 
antagonist, Cyproterone Acetate (CPA), the decrease in Nitrite release was alleviated, as 
Nitrite release was comparable to that of control splenocytes incubated in medium alone. 
There was no change in basal proliferation of splenic lymphocytes in relation to in vitro 
testosterone treatment; while mitogen induced proliferative response reduced 
significantly (p<0.05) by in vitro testosterone. Present finding reveals testosterone as 
inhibitory to splenocytes immune responses in the fresh-water snake, Natrix piscator.     
 
Based on the results in the present work, it may be suggested that immune function in 
reptiles varies in different seasons. Pineal gland, by diurnal rhythm of synthesis and 
release of its principal hormone, melatonin, transmits signals to the immune system, and 
thus, regulates the immune responses. Melatonin injection enhanced the innate immune 
functions, and thus may be considered as an immunoregulatory factor in the development 
and maturation of immune system and in the progression of the immune response in our 
animal model, Natrix piscator. Our study also suggests there is circadian variation in 
innate immune parameters, some of which also show a clear phase shift according to the 
season. Further, in vitro testosterone reduces immune response in a manner dependent to 
testosterone concentration used. Differential effect of photoperiodic regimens, in immune 
parameters studied, is also established in our study. It is concluded that relationship exists 
between external environmental factors, melatonin, sex steroids and immune system 
which might have led to the adaptations to best fit the animal in the challenging 
conditions present. 
 
 
 
 


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