Open Access Repository of Indian Theses

SUBRAMANIAN, S. S.

Abstract…




ABSTRACT

The field of nonlinear optics has attracted lot of interest in the past three decades not only because of the possible numerous applications in telecommunications, optical data storage etc., but also because of the fundamental research connected to issues like charge transfer, conjugation, polarization and crystallization into noncentrosymmetric lattices. In principle nonlinear optics (NLO) deals with the interaction of applied electromagnetic fields in various materials to generate new electromagnetic fields altered in frequency, phase or other physical properties.  A variety of systems including inorganic materials, organometallic materials, organic molecules and polymers have been studied for NLO activity. Owing to the combination of chemical tunability and choice of synthetic strategies, organic molecules in particular have received much attention. Second order nonlinear optical response in an organic chromophore is intrinsically based on electronic ‘push-pull’ mechanism operative in a molecule containing a donor linked to an acceptor moiety through a conjugated electron bridge. For a given set of donor and acceptor, enhancement of second order hyperpolarizability (a measure of non linear optical activity) is achieved by increasing the conjugation of the -electron bridge, which, on the other hand, causes undesired bathochromic shift of absorption and contributes to impart photo-instability to the system. In view of this, conceptual development of structural model based on alternate route to charge transfer (CT), other than  conjugation, is warranted.

In view of above drawbacks, novel strategy is proposed on the concept of charge transfer (CT) between -donor and -acceptors separated by -spacers. The CT in these molecules arises owing to the interaction of -orbitals of the donor and acceptor moiety through the -bond (( bond coupling).  The advantage in such CT is that the molecules have the absorption maxima, max, in region less than 300 nm thus being transparent in the region of interest for frequency doubling operations. The disadvantage is that these will have weaker oscillator strengths and hence smaller first hyperpolarizability, , values. An alternative to enhance the  value without losing the transparency in the visible region is 3-Dimensional molecules. Here three donor/acceptor molecules are attached to a central -acceptor/donor system by  bonds. Since there is a linear superposition of three CT molecules the  value should add up constructively. As -bond insertion between donor and acceptor may cause loss in thermal stability, hence conventional aromatics are replaced with heteroaromatics which are also possessing ease of polarization and synthetic feasibility. s-Triazine derivatives are found to be the right candidates to satisfy above requirements. Hence this thesis mainly focused on s-triazine derivatives. Various s-triazine derivatives have been designed using computational techniques and analyzed for their linear and nonlinear optical properties. Series of s-triazine derivatives have been synthesized and characterized based on various techniques like 1H-NMR, 13C-NMR, Mass, IR, and X-Ray crystallography. Linear optical properties (max) are recorded, compared with theoretical observations and analyzed. Nonlinear optical properties are measured with Hyper-Rayleigh Scattering (HRS) technique in solution () and Kurtz-Perry powder technique in solid-state (). 

This thesis entitled “Design and synthesis of novel s-triazine derivatives for second-order nonlinear optics: A combined experimental and theoretical investigation” and is divided into five chapters and two appendixes. 

Chapter 1 provides brief introduction about Nonlinear Optics, its origin, applications and various types of nlo effects. Detailed description is given for second-order effects particularly on second harmonic generation followed by brief review of organic materials studied so far. Emergence and necessity of octopolar systems over dipolar systems are discussed in detail. Theoretical models to determine and experimental techniques to estimate the magnitude of NLO activity are presented. Limitations of organic molecules to find applications and scope of the work followed by organization of the thesis are described at the end of the thesis.

Chapter 2 deals with nonlinear optical property evaluation of dipolar (D--A) molecules. In this chapter s-triazine is considered as an acceptor and ethylenic bond is spacer with conventional aromatic substituents as donors. Synthesis of two series of s-triazine derivatives and linear and nonlinear optical property evaluation are presented. Experimental results have been compared with theoretical observations.

Chapter 3 is composed of computational design and synthesis of 2,4,6-tris (benzylamino)-1,3,5-triazine derivatives along with nonlinear optical properties investigation in solution and solid-state. Conformational analysis is encountered in detailed manner and also tells the importance of conformationally flexible molecules.

In Chapter 4, -O-CH2- spacer is considered instead of –NH-CH2- in chapter 3. This chapter describes the synthesis and conformational analysis of 2,4,6-tris (benzyloxy)-1,3,5-triazine derivatives by means of computational tools and its consequences in nonlinear properties at molecular and macro level.

Chapter 5 describes synthesis of a series of s-triazine derivatives, characterization and nonlinear optical property evaluation using experimental techniques and computational methodologies.

In Appendix I, the detailed experimental set-up for the hyper-Rayleigh Scattering (HRS) measurements at 1064 nm are described. 

Appendix II explains synthesis and keto/amine-enol/imine tautomerism of bis (2,4-benzyloxy)-6-(5h)-one-1,3,5-triazine in the solid-state and gas phase and it involves a combined crystallographic and quantum-chemical investigation.












	
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English

STUDIES ON CITRACONASE, A NEW ENZYME AND ON CERTAIN ASPECTS OF BACTERIAL DEGRADATION OF C5-DICARBOXYLIC ACIDS

D.D., NANAVATI

MODIFICATION OF NIKEL CATALYST FOR THE HYDROGENATION OF VEGETABLE OILS AND APPLICATION OF K-BRUMOSUCCINIMIDE FOR THE CREATION OF CONJUGATION IN FATTY ACIDS AND OILS

N., THANKARAJAN

iii 
 
 
ABSTRACT 
 
 Fireworks mixtures are highly energetic compounds susceptible to 
explosive degradations on ignition, impact and friction. Several accidents 
during processing, storage and transportation have been reported in Indian 
fireworks manufacturing units. An analysis of accident data reported in the 
fireworks units during 1989-2013 in India has shown that the main cause of 
an accident is inadequate knowledge of the mechanical, thermal and 
electrostatic sensitivity of fireworks mixtures. Nevertheless, the sensitivity of 
a mixture to its explosion cannot be theoretically predicted as it depends on 
the reactive nature of the mixture components and the conditions employed 
during its preparation and handling. Though Material Safety Data Sheets 
(MSDS) of pure chemicals are readily available, no such data are available for 
mixtures. Additionally, the mixture’s composition varies from company to 
company for the same type of application.   
 There are no standard procedures or techniques to estimate 
performance related concepts applicable to mixing ratios. This leads to 
difficulties in providing a standard scale for assessing the quality of fireworks. 
Due to the non-availability of standard manufacturing equipment, tools, 
manufacturing procedures, and inadequate understanding of the chemistry of 
pyrotechnics and their explosive nature, accidents continue to take place in 
fireworks industry. The extensive review revealed that detailed studies were 
iv 
 
not available for ternary fireworks mixtures, currently employed in the 
fireworks industry. The only ternary mixture studied in detail was the flash 
mixtures containing potassium nitrate, sulphur and aluminium. To address the 
causes of frequent blast accidents, there is a need for a comprehensive study 
on thermal and mechanical sensitivity of ternary firework mixtures currently 
employed in fireworks industry. The consequences of fireworks accident are 
devastating, it is important to know the TNT equivalence of fireworks 
mixtures in order to determine the blast effects or scaled distance. The focus 
in this thesis is to assess the thermal and mechanical hazards of the firework 
mixtures, atom bomb cracker, Chinese cracker and palm leaf cracker and tip 
mixtures (flowerpot tip and ground spinner tip) which are involved in blast 
incidents. 
 The DSC heat flow responses for atom bomb cracker, Chinese 
cracker and palm leaf cracker mixtures show a sharp single exothermic 
transition in the temperature range 320-355°C. Of the three cracker 
compositions, atom bomb cracker has the earliest exothermic peak 
temperature. The behavior of this mixture can be attributed to the high 
concentration of potassium nitrate (oxidiser) in its composition. DSC plots 
with varying water content for ground spinner tip mixtures revealed that with 
increase in water content, heat release pattern also increased in the 
temperature range of 300-340°C. Kinetics treatment by Ozawa and 
Kissinger’s method revealed a fair agreement of activation energy of 179.38 
and 178.9kJ mol-1 respectively.   
v 
 
 The ARC data showed that the fireworks mixture decomposition 
process under adiabatic condition was vigorous and therefore dangerous. 
Although explosive potential is required for it to be a cracker, the same 
property could be dangerous if the event occurs during handling, 
manufacturing, storage or transport. The self heat rate plot shows a maximum 
heat release rate of 1088.1°C min-1 at 320°C. The observed large heat release 
rate confirms the vigor of exothermic explosive process of atom bomb cracker 
mixture. One gram of atom bomb cracker mixture can contribute to a peak 
pressure rise of 25.9bar at 342.3°C. Both Chinese and palm-leaf crackers 
show delayed onset temperatures of 295°C and 290°C respectively. The delay 
in initiation of the exothermic activity can be related to their composition, 
especially the quantity of the oxidiser KNO3. The sudden jump in self heat 
rate from 10 to 50°C min-1 and finally to 100°C min-1 (in a discontinuous 
manner) indicated the explosive nature of the process.  
 The ARC studies at different iso-ageing conditions are discussed in 
the following sections. The sample with 40% water (w/w) content recorded a 
maximum heat rate of 4.19°C min-1 at 147.65°C. The self heating nature of 
the ground spinner tip mixture and its reactive thermal hazards with water 
under iso-ageing conditions were at the lowest onset temperature of 40°C. 
This study (perhaps for the first time) could successfully correlate the 
accident occurring conditions. The exothermic onset of these samples at 
ambient conditions appeared to be responsible for triggering blast accidents. 
In fireworks industry, the unused water mixed ground spinner tip mixture at 
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the end of the day was left overnight for next day use. Explosive accidents 
were reported after shutting down the factory. In practice this temperature can 
be attained by thermal and mechanical stimulus.  Therefore, this mixture is to 
be handled carefully. It can be observed that for the crackers and tip mixtures 
the overpressure increases as the TNT equivalence increases. As the weight 
increases, TNT equivalence increases since overpressure and TNT 
equivalence have a direct correlation. It can be observed that the overpressure 
decreases as the distance increases for cracker samples and tip compositions. 
This is because of the inverse relation between the distance and the 
overpressure. The firework mixture, under certain conditions can be 
equivalent to an explosive and hence has to be handled carefully.  
 Impact sensitivity measurements for different firecrackers mixtures 
indicated that the impact energy varied when concentration of the component 
of the mixture was changed. This behavior showed that the impact sensitivity 
was directly related to the reactivity of each component. Higher concentration 
of sulphur is critical for increased impact sensitivity. All the cracker mixtures 
studied could be categorized as Class III explosives of the Andrejev and 
Beljajev classification. Frictional sensitivity of atom bomb cracker mixture 
shows that increase in sulphur concentration from 13-34% increased the 
friction sensitivity. Increase in potassium nitrate and aluminum concentration 
in the mixture showed a significant increase in friction sensitivity. 
  


STUDIES ON METAL CHELATES OF ?-DICARBONYL COMPOUNDS

PATIL, SHASHIKUMAR

V 
 
ABSTRACT 
 
The thesis entitled “Aggregation and Prototropic Behavior within Ionic Liquid based 
Media” is concerned with understanding the behavior of important prototropic probes and 
cyanine dyes within neat and aqueous ionic liquid (IL) systems. Specifically, prototropism of 
common prototropic probes and aggregation properties of various cyanine dyes that reveal 
important properties of neat and aqueous IL media are explored with detailed optical 
spectroscopic investigations to understand the unique role of these unusual substances. The long 
term aim is to establish ILs as important and preferred media as far as prototropism and dye 
aggregation are concerned. 
ILs have received increased attention from both academia and industrial research 
communities due to their unusual features. Many chemical processes demonstrate effects and 
outcomes that are very different within a milieu constituted of only ions as opposed to a 
molecular solvent. ILs also show increased applicability in a variety of analytical methodologies 
encompassing electroanalysis, separation, extraction, spectrometry, and sensing. The recent 
investigations on ILs are partly due to their potential environmentally-benign nature as well. Due 
to their important absorbance and luminescence properties, various probes are used routinely to 
determine the properties of solvent media. Solvent milieu, in turn, may affect the photophysical 
properties of such probes resulting in altered aggregation and prototropic behavior. The 
importance of prototropism is manifested through various applications, such as, in developing 
effective photoprotecting agents, in laser dyes, in photodynamic therapy, and in developing 
fluorescence probes for biological molecules, among other. Furthermore, molecular aggregates 
of dyes, specifically those of cyanines, are known to play important role in many technological 
applications, such as, in photography, as sensors, as photoconductors, in biology, in medicine, in 
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nanotechnology through the formation of self-assembled materials, in future light harvesting 
systems, to name a few. Recent studies by numerous researchers, have demonstrated the 
potential of ILs in altering the behavior of certain dyes and prototropic probes. The hypothesis 
that ILs and aqueous IL systems afford altered (and perhaps unusual) outcomes towards 
Prototropism and aggregation is thoroughly tested in this thesis.                         
 The Chapter titled “Prototropic Behavior of Naphthols within Neat and Aqueous Mixture 
of Ionic Liquids” deals with the study of the prototropic behavior of 1- and 2-naphthols in IL 
based media. The study has been divided into two parts. The first part of the Chapter highlights 
IL-controlled excited-state behavior of naphthols. Unusual excited-state properties of 1- and 2-
naphthols dissolved in various ILs that depend heavily on the molecular architecture of the IL 
cation are reported. Specifically, we found that the fluorescence from the anionic prototropic 
form of the photoacid is observed when the solubilizing IL contains pyrrolidinium and C2-
substituted imidazolium cations, whereas no fluorescence characteristic of naphtholate is seen 
when solubilizing IL is composed of imidazolium cation having acidic C2 proton. This is 
attributed to the formation of a ‘dark’ complex between imidazolium cation (Im+) and excited 
naphtholate involving C2-H of Im+. The second part describes the effect of addition of water-
miscible ILs, [bmim][BF4], [bmim][OTf], and [bmpyrr][OTf], on photophysical properties of 2-
naphthol at various pH. Electronic absorbance behavior of 2-naphthol dissolved in aqueous 
mixtures of ILs is observed to be similar to that found in water at different pH. The excited state 
properties, however, are changed dramatically in presence of ILs. Presence of imidazolium ILs 
results in significant quenching of the fluorescence emission from 2-naphtholate anion. On the 
contrary, pyrrolidinium IL does not quench the fluorescence from the anionic species. Aromatic 
imidazolium cation acts as an electron/charge acceptor during the quenching process where 
VII 
 
formation of a weakly fluorescent complex between imidazolium cation and excited 2-
naphtholate anion possibly involving the acidic C2 proton of imidazolium is proposed. Due to 
the absence of such acidic proton, nonaromatic pyrrolidinium cation is not able to form a 
complex with excited 2-naphtholate and cannot act as an electron/charge acceptor.  
The Chapter titled “Prototropism of Acridine within Neat and Aqueous Mixture of Ionic 
Liquids” deals with the study of prototropic behavior of acridine when dissolved in neat and 
aqueous mixture of ILs. The study has been divided into two parts.  The first part of the Chapter 
describes the role of the acidity of IL cation on the prototropic behavior of acridine. We observed 
that acidity of IL cation can tune the prototropism of acridine in the excited-state. More 
specifically, fluorescence emission from the protonated form of acridine  is observed when it is 
dissolved in imidazolium ILs having acidic protons at C2 position, whereas neutral acridine 
emission is observed in case of pyrrolidinium ILs due to the absence of such acidic proton. 
Interestingly, acidity of C2-methyl substituted imidazolium ILs is found to be temperature 
dependent and hence fluorescence from the protonated as well as neutral forms of acridine can 
be observed depending upon the temperature. In the second part, effect of addition of small 
amount of aqueous acid or base to acridine dissolved in ILs is studied. It is found that addition of 
small amount of such aqueous component can fine tune the properties of ILs and make them 
suitable medium so as to have desired prototropism of acridine. Protonation and deprotonation of 
acridine dissolved in ILs can be achieved irrespective of the identity of the IL, in ground as well 
as in excited state, by addition of small amount of  0.1 M aqueous HCl or 1 M aqueous NaOH, 
respectively. 
The Chapter titled “Aggregation Behavior of Cyanine Dyes in Neat and Aqueous Mixture 
of Ionic Liquids” is divided into two parts. The first part describes the aggregation behavior of 
VIII 
 
cyanine dyes TDBC, DMTC in neat and 1 M aqueous NaOH added ILs. It was found that TDBC 
and DMTC are triggered to form fluorescent H-aggregates by addition of only 2 wt% 1 M 
aqueous NaOH to ILs 1-alkyl-3-methylimidazolium tetrafluoroborate [Cnmim][BF4], whereas 
addition of 2 wt% 1 M aqueous NaOH to cyanine dye solutions in ILs with other anions results 
in J-aggregation. H-aggregation is unambiguously demonstrated to be specific to the [BF4]–-
based ILs. We propose the different hydrolytic properties of BF4– as compared to other anions in 
ILs to be the reason for this dramatic difference in IL-dependent cyanine dye aggregation. In the 
second part, effect of addition of ILs on the aggregation behavior of a cyanine dye, TDBC, was 
investigated. In basic aqueous buffer solutions (pH ≥ 10), TDBC preferably exists in its J-
aggregated form. Addition of > 5 wt% hydrophilic ILs is observed to disrupt the TDBC J-
aggregates converting them to monomeric form most likely because of the interaction between 
bulky IL cation and the J-aggregates in a time dependent manner. This is evidenced by the 
observed increase in monomer band absorbance at the expense of the absorbance band due to J-
aggregates over time. Inorganic salts at similar molar concentrations do not cause this 
phenomenon but instead induce TDBC precipitation. At low concentrations (< 5 wt%), the added 
IL acts similar to the inorganic salts reducing the overall absorbance of TDBC in the solution 
most likely due to cation exchange causing TDBC precipitation. The unique and dual behavior of 
ILs as additives toward affecting cyanine dye aggregation is demonstrated.      
Chapter titled “Conclusions and Future Perspectives” presents highlights and salient 
features of the work presented in the thesis. In brief, it is concluded that ILs are unique in 
affecting prototropism and aggregation due to their inherent architecture. Further, it is 
emphasized that the photophysical behavior of probes and dyes in IL based media can be 
different than that in conventional organic solvents. All-in-all, the findings of this work will help 
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in understanding the different types of molecular interactions within IL-based media and could 
be of potential use to both academia and industry. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


CONDUCTION THROUGH THIN FILMS OF SEMICONDUCTOR

RAGHAVAN, K.S.

i 
 
Preface 
The growing need for artificial biomaterials for the restoration of bone and 
dental defects has urged the search for optimally designed materials as a joint effort of 
physicians, engineers, chemists and physicists. Among the several materials available 
today, hydroxyapatite (HA) was recognized as one of the attractive materials for the 
restoration of bone and dental defects since it resembles the mineral component of 
bones and teeth. Synthetic HA exhibits specific properties of biomaterials such as 
biocompatibility, bioactivity, osteoconductivity, etc., hence it has been widely used as 
a bone defect filler, coating on metallic implants, scaffold for tissue engineering and 
carrier for drug delivery. The major drawbacks of HA are poor mechanical 
performance, less resorption and uncontrolled drug delivery that has limited its 
utilization for advanced functional applications. Hence tailoring the different 
properties of HA bioceramics for enhanced biomedical applications has got great 
attention and has been the topic of research interest for many years. 
The first chapter provide a comprehensive source of information on research 
and development of nano-HA biomaterial and various approaches to tailor the 
properties of nano-HA. Successive five chapters are on tailoring the properties of 
nano-HA by employing different approaches for biomedical applications. 
In chapter–2 we present a systematic facile strategy for the synthesis of 
nanocrystalline HA with enhanced sinterability and mechanical properties via 
precipitation method coupled with ultrasonication and freeze drying process. Role of 
ultrasonication and freeze drying on the characteristic of HA are explained based on 
observed results from the various characterizations. Thermal stability, crystallinity, 
microstructure and densification behavior of HA obtained from conventional and 
ultrasonication assisted freeze drying methods are discussed. Comparison of 
mechanical properties of HA (sintered at 1100 ºC) obtained from both methods also 
forms part of this chapter. 
ii 
 
In chapter–3, we demonstrate a novel method for in situ fabrication of          
HA - calcite nanocomposite using calcium nitrate, di-ammonium hydrogen phosphate 
and citric acid as the starting materials. The mechanism of formation of HA - calcite 
nanocomposite is discussed elaborately in this chapter. In addition, effect of citric acid 
concentration on the composition of HA - calcite nanocomposite is also discussed. 
Density, porosity, mechanical properties, in vitro bioactivity, in vitro dissolution and 
biocompatibility of the in situ formed HA - calcite nanocomposite are presented in this 
chapter.  
Chapter–4 is focused on the effect of Zn and carbonate co-substitution on the 
properties of nanocrystalline HA. Zinc and carbonate co-substituted nano-HA have 
been synthesized by precipitation method and characterized by various techniques. 
Effect of Zn and carbonate substitution on the thermal stability of HA has been 
investigated in detail. Subsequently in vitro studies such as apatite forming ability, 
dissolution behavior and cytocompatibility are reported. It is found that simultaneous 
substitution of Zn and carbonate in HA alters the crystallite size, crystallinity, lattice 
distortion, etc., which consequently affects the thermal stability, dissolution and 
bioactivity of nanocrystalline HA. 
Chapter–5 deals with the preparation of flower-like Mg containing carbonated 
HA nanostructure from eggshell biomineral using a simple and rapid microwave 
irradiation method with the help of EDTA as a complexing agent. Various 
characterization studies on prepared HA are described. The mechanism of the 
formation of flower-like Mg containing carbonated HA from eggshell biomineral is 
presented in detail. This chapter also provides apatite forming ability, 
biocompatibility, drug adsorption/desorption behavior, antibacterial activity and 
photoluminescence property of prepared flower-like HA nanostructure. 
Chapter–6 deals with the in situ preparation of ciprofloxacin loaded HA 
nanoparticles and its characterization by different techniques. The in situ formation of 
ciprofloxacin loaded HA nanoparticles and effect of concentration of ciprofloxacin on 
iii 
 
the crystallite size and particle size of HA are explained based on above studies. 
Finally, drug release behavior, antibacterial activities, in vitro apatite forming ability 
and cytocompatibility of ciprofloxacin loaded HA nanoparticles are discussed.  
The final chapter (chapter–7) presents an overall summary of all the chapters 
and suggestions for future work. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


THE INTERACTION OF METAL IONS WITH PEPTIDES AND SIMILAR MOLECULES

MALHOTRA, SHADI LAL











Abstract

	The thesis entitled “Multipotent action of Rhizophora mucronata (Red mangrove) as neuroprotective drug against Alzheimer’s disease – Evidence from preclinical studies” deals with the study of neuroprotective effect of Rhizophora mucronata against Alzheimer’s disease (AD) through its multipotent action in  in vitro (PC12 cells) and in vivo model systems (mice). The thesis is typically divided in six chapters.

	Chapter I: In vitro screening of Rhizophora mucronata for its multipotent neuroprotective effect - Antioxidant, Cholinesterase inhibitory, Metal chelating and Anti-amyloidogenic property.
	Chapter II: Nutritional evaluation of Rhizophora mucronata leaves.
	Chapter III: Safety and Toxicity evaluation of Rhizophora mucronata methanolic leaf extract: in vitro and in vivo study.
	Chapter IV: Neuroprotective effects of Rhizophora mucronata against beta-amyloid-induced cell death in PC12 cells via its antioxidant mechanism.
	Chapter V: Anti-amnestic and neuroprotective effect of methanolic leaf extract of Rhizophora mucronata against A (25-35) induced toxicity in Swiss Albino mice.
	Chapter VI: Bioassay-directed isolation of active compounds with neuroprotective effect from Rhizophora mucronata leaf extract.
	
	Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized clinically by insidious onset of memory and cognitive impairment, emergence of psychiatric symptoms and behavioral disorder, and impairment of activities of daily life. AD one of the fourth leading causative of death, affects about 26.6 million people worldwide, which might quadruple to 106.8 million by 2050 if no therapy intervenes. Multiple causative factors such as - amyloid (A) aggregation,-protein aggregation, oxidative stress and cholinergic hypothesis plays major role in the pathogenesis of AD, among which, Aβ peptide plays a pivotal role. Currently AChE inhibitors such as tacrine, donepezil, rivastigmine and galanthamine are used for the treatment of mild to moderate AD as approved by Food and Drug Administration. However these drugs possess severe gastrointestinal side effects and are effective only for mild to moderate forms of AD. Diverse pathogenic factors and safety limitation of currently used synthetic drugs necessitates exploration of multipotent drug from natural source to hit more than one target implicated in AD. 
	Rhizophora mucronata commonly termed as Asiatic mangrove have been valued for its timber and ethanomedicinal properties. In traditional medicine, leaves of R. mucronata have been used for the treatment of elephantitis, hematoma, hepatitis, ulcers, inflammation and febrifuge.  In addition the leaves have been used as alternative source of tea and as animal feed. The leaves have also been reported for its pharmacological properties such as anti-diabetic, anti-inflammatory, anti-viral activity and anti-diarrheal effect. Despite of its wide pharmacological properties, the application of R. mucronata in the treatment of neurological disorder is still at its infancy. Mangroves in general are halophyte which possess rich source of antioxidant system to overcome the oxidative stress induced by salt stress environment. Since A peptides leads to neuronal death and cognitive deficits through oxidative stress, it was attempted to screen the mangrove R. mucronata, which has rich antioxidant and anti-inflammatory property, for its neuroprotective effect. We have attempted to evaluate the neuroprotective activity of Rhizophora mucronata against AD through its multipotent action.
	The chapter I “In vitro screening of Rhizophora mucronata for its multipotent neuroprotective effect - Antioxidant, Cholinesterase inhibitory, Metal chelating and Anti-amyloidogenic property” deals with screening different mangroves for its potent antioxidant, cholinesterase inhibitory activity, metal chelating and antiaggregation property. Nine mangroves (Avicennia marina, Rhizophora mucronata, Rhizophora lamarkii Rhizophora apiculata, Rhizophora annamalayana, Ceriops decandra, Sweda maritima, Pruscaria celligrica and Lumnitzera racemosa) collected from Pichavaram, Vellar estuary, Tamil Nadu, India were used for the present study. Screening for cholinesterase and antioxidant activity of mangroves showed that of the nine mangroves screened, leaf extract of R. mucronata exhibited potent antioxidant and dual cholinesterase inhibitory activity. Further, successive solvent extracts from the leaves of R. mucronata were assessed for its multipotent neuroprotective effect (antioxidant, cholinesterase inhibitory and metal chelating activity).  Of the various solvents, methanolic leaf extract of R. mucronata (MERM) displayed remarked inhibition against both AChE and BuChE exhibiting dual cholinergic effect.  In vitro antioxidant assays showed that MERM showed excellent reducing power and free radical scavenging activity. Assessment of antiamyloidogenic property illustrates that MERM inhibits Aβ fibrillogenesis and also destabilizes the preformed fibrils. To the best of our knowledge, this is the ﬁrst study describing anticholinesterase and antioxidant activity using ferrous ion-chelating, nitric oxide, hydroxyl radical scavenging and ferric-reducing antioxidant power (FRAP) assays of the afore-mentioned mangrove species. Since MERM acts as multifunctional agent exhibiting cholinesterase inhibition, antioxidant, metal chelating property, antiaggregation and deaggregation of Aβ fibrils, it can act as a potent drug to combat the multiple targets of AD. The multipotent activity of MERM could be attributed to the presence of high amount of total phenol and ﬂavonoid contents.
 	The chapter 2 “Nutritional evaluation of Rhizophora mucronata leaves” deals with nutritional analysis of R. mucronata, its proximate composition, amino acid profiles and physicochemical properties in comparison with green leafy vegetables consumed in India.  Proximate composition analysis showed that R. mucronata act as a good source of dietary fiber, lipid and protein content suitable for human consumption. The study also detected the presence of seventeen amino acids with higher essential amino acid content which were comparable to FAO/WHO requirement. Presence of higher levels of Linoleic acid, alpha linolenic acid and low levels of saturated fatty acid signifies the leaves as nutritionally balanced diet. Leaves also act as rich source of ascorbic acid, thiamine, riboflavin, tocopherol, Na+, Fe2+ and Ca2+ which could help in overcoming micronutrient malnutrition at a negligible cost. Physicochemical properties of leaves had a high positive correlation with their total amount of fiber and protein content. Overall, the leaves of R. mucronata could be used as a potential health food in human diets and may be of use to the food industry as a source of ingredients with high nutritional value.
	Safe use as plant-based medicine, beside historical and experienced applications on humans and animals, requires toxicity evaluation of these medicinal herbs.  Hence Chapter III “Safety and Toxicity evaluation of Rhizophora mucronata methanolic leaf extract: in vitro and in vivo study” deals with assessment of toxic effect of MERM under in vitro (mutagenic, genotoxic, cytotoxic) and in vivo conditions (Acute and Sub-acute toxicity). Results of Salmonella/microsome assay shows that MERM produced neither frame shift mutation in TA98 and TA1538 nor base pair substitution in TA100 and TA1535.  Lack of mutagenic effect of MERM at concentrations up to 4000 µg/plate in all the tester strains provides reassurance on the safety of the extracts widely used in traditional medicine. In addition MERM exhibited antimutagenic effect against direct acting mutagens (Sodium azide) in both TA98 and TA 100 tester strains. Investigation of cytotoxic effect by trypan blue exclusion assay and MTT assay showed that MERM had no cytotoxic effect on PBMC up to 24 hrs. Evaluation of genotoxicity (comet assay) revealed that MERM (2000 µg/ml) showed no significant difference in comet tail moment and tail scores which concludes that MERM has no genotoxic effect. In vitro hemolytic activity showed that MERM showed no significant (p  0.05) hemolytic effect even at the maximum dose (2000 µg/ml), which indicates that leaf extract preserves the integrity of cell membranes. In acute toxicity studies oral administration of MERM (250-2000 mg/kg) in Wistar rats produced neither mortality nor any noticeable changes in behavior. Hematological and biochemical parameters showed no difference, except for a significant increase in ALT and AST at the highest dose. Histopathological findings revealed hepatotoxicity and neurotoxicity at highest dose of extract. In sub-acute toxicity studies administration of MERM (1000 mg/kg) for 28 consecutive days neither altered the body weight gain nor behavioral parameters. No significant change was observed in the hematological and biochemical parameters analyzed. Histopathological examination showed normal architecture suggesting no morphological disturbances. Collectively, these data demonstrate that consumption of MERM for various medicinal purposes is safe.
	Chapter IV “Neuroprotective effect of Rhizophora mucronata against beta-amyloid-induced cell death in PC12 cells via its antioxidant mechanism” deals with various mechanism by which MERM protects against A induced toxicity in PC12 cells. PC12 cells used for the current study are widely used for in vitro research on AD as these cells contain many membrane-bound and cytosolic molecules associated with neurons, and are electrically excitable and neurosecretory. In addition PC12 cells were found to be more sensitive to Aβ insult hence it has been used extensively to study Aβ induced cellular toxicity. A (25-35) treatment in PC12 cells (2  105 cells/ml) for 24 hrs at 20 M concentration reduced cell viability, altered the morphology,  induced ROS/RNS production, decreased mitochondrial membrane potential, deregulated intracellular calcium levels and caused macromolecular damage. MERM pretreatment prior to A (25-35) exposure significantly elevated cell viability, reduced A (25-35) induced apoptosis, decreased ROS/RNS production, stabilized mitochondrial membrane potential, attenuated caspase 3 activation, maintained intracellular calcium homeostasis and  inhibited protein and lipid oxidation.  MERM also restores the antioxidant status of the cell by maintaining the antioxidant enzymes (SOD, CAT). MERM maintains the intracellular Glutathione level by regulating glutathione metabolizing enzyme (GR, GPx and GST). MERM also inhibits both acetyl and butyryl cholinesterase activity. Our findings suggest that MERM, a multiple-target drug holds potential for neuroprotection and therefore, may be promising for the treatment of AD. This effect of MERM could be due to the active compounds present in it, which may increase the capacity of endogenous antioxidant defense and may modulate the cellular redox state.
	Animal models play a critical role in understanding the pathology and screening of therapeutics for AD.  Although no available model can meet all the full pathologic spectrum of AD disease, the injection of Aβ into the brain was shown to impair learning and memory, and elicit a degree of AD type neurodegeneration. Chapter V “Anti-amnesic and neuroprotective effect of methanolic leaf extract of Rhizophora mucronata against A (25-35) induced toxicity in Swiss Albino mice” deals with possible mechanism by which MERM combats A (25-35) induced toxicity under in vivo condition. Mice were fed orally with 200 and 400 mg/kg bw for 21 days followed by single intracerebroventricular (i.c.v.) injection of A (25-35) (10 g/mouse). Behavioral changes in the mice were evaluated using water maze, Y-maze and passive avoidance tests. The consumption of MERM significantly ameliorated the cognitive deficits caused by i.c.v. injection of A (25-35) by restoring spatial memory, improving short term memory (STM) and long term memory (LTM). MERM enhances cholinergic effect by inhibiting both AChE and BuChE, attenuating lipid peroxidation and protein oxidation, reducing ROS and RNS level by activating antioxidative defense system (SOD, CAT) and  restoring glutathione metabolizing enzyme systems (GPX, GR and GST). MERM inhibits BACE1 activity thereby reducing the formation of neuronal tangles as observed in histopathological analysis. MERM inhibits neuronal apoptosis by suppressing both caspase-3 and MAO-B activity. Taken together, the results conclude that exogenous administration of MERM provides neuroprotective effect against toxic effect of Aβ peptide in regions related to cognitive functions providing new insight for future therapeutic approaches of AD.
	Chapter VI “Bioassay-directed isolation of active compounds with neuroprotective effect from Rhizophora mucronata leaf extract” deals with isolation and identification of bioactive compound responsible for the neuroprotective effect. MERM was fractionated using silica column chromatography (60-120 mesh) with solvents from nonpolar to polar (benzene to water) in various ratios and the eluted fractions were freeze dried. The fractions were subjected to bioactive guided fractionation using in vitro antioxidant and cholinesterase inhibitory assays. Among the various fractions Ethyl acetate: Methanol (1:1) fraction (Fr 14) showed the highest antioxidant and cholinesterase inhibitory activity. TLC analysis of the active fraction using Vanillin-Sulphuric acid showed the presence of four pink and two yellow colored bands. Preliminary phytochemical analysis showed the presence of flavonoids, terpenoids and glycosides. TLC chromatogram sprayed with p-anisaldehyde sulphuric acid and 1% Alcoholic aluminium chloride showed purple colored bands in visible light and fluorescent yellow colored bands under UV 366 nm, confirming the presence of terpenoids and flavonoids. The active fraction 14 was further fractionated by HPLC using methanol : water by binary gradient method. Among the eight fractions eluted, fraction 3 showed the highest AChE and BuChE inhibitory activity with IC 50 value of 32.25  0.001 and 40  0.002 g/ml respectively. Enzyme kinetic studies showed that the active fraction exhibited competitive and mixed type of inhibition against AChE and BuChE. 
TLC chromatogram analysis of active fraction sprayed with alcoholic aluminum chloride showed single fluorescence band with Rf value of 0.6 which confirmed the presence of flavonoids. Structural elucidation using FTIR, UV scan, C13 and H1 NMR spectrum illustrated that the content present  in bioactive fraction 3 was (+) - catechin (C15H14O6) a flavones-3-ol with molecular weight of 290.27. The identity was further confirmed by comparison with authentic sample (Sigma Aldrich, Bangalore) using LC-MS. 
	To conclude, the present work represents a comprehensive study of multipotent neuroprotective role of Rhizophora mucronata and its constituent (+)-Catechin against AD. Overall results of in vitro and in vivo studies validated the hypothesis that exogenous administration of MERM exerts neuroprotective actions against toxic effects of Aβ peptides in regions related to cognitive functions providing new insight for future therapeutic approaches of AD. Results of safety evaluation illustrates that MERM used for the present study is devoid of toxic effect both under in vitro and in vivo conditions which assures the safe usage of traditional drug. Despite of its neuroprotective effect, the rich nutritive value indicates that leaves can be used as nutraceuticals.  Moreover this is the first report regarding the neuroprotective role of mangrove, hence the present work offers new insight for exploration of drugs from marine natural products for the treatment of AD. 























1





Studies in Co-ordination Polymerization

KHANCHANDANI, K.S

Microbiological transformations of terpenes

A., NARAYANAN

 SUMMARY 
 
III 
 
Wilson disease (WD, OMIM# 277900) is a rare inborn error of copper accumulation inherited 
in an autosomal recessive manner. It is caused by mutations in the copper transporting P-
type ATPase gene (ATP7B) (Thomas GR et al., 1995a). The disease is manifested at a 
median age of 12 to 23 years as hepatolenticular degeneration resulting from accumulation of 
copper primarily in the liver and brain. Patients mostly present with hepatic, neuropsychiatric 
or mixed symptoms (Das SK and Ray K, 2006). Varied clinical and biochemical 
manifestations often complicate diagnostic workup and diagnosis can be challenging 
especially for patients who are asymptomatic or present with liver disease. Failure to 
diagnose WD before its substantial progression is the leading cause of morbidity and 
mortality. But if patients can be diagnosed early enough, lifelong de-coppering with 
appropriate chelating agents can lead to gradual improvement and make them largely 
symptom free for the rest of their lives (Walshe JM, 2007). There is no single clinical or 
biochemical test to exclude WD. Genetic analysis still remains to be the confirmatory 
approach to detect the disease especially in sporadic cases with no family history and 
ambiguous clinical indications. 
When the work for this dissertation was initiated, several studies have revealed the 
ATP7B mutation spectrum from North Indian (Kumar S et al., 2005), South Indian (Santhosh 
S et al., 2006) and East Indian (Gupta A et al., 2005, 2007a) (our group) WD population. 
However, the WD mutation pattern from West India was yet to be elucidated. Hence, the 
genetic, clinical and functional studies leading to this dissertation have sequentially focussed 
on the following areas (i) further characterization of molecular defects present in ATP7B gene 
in East Indian WD patients and exploration of the ATP7B mutation spectrum from West India 
(ii) screening of the ATP7B regulatory region in patients with single detectable coding 
mutations or lack of any, and followed it up with functional characterization of variants in the 
promoter and 5'UTR, (iii) Investigating the genotype-phenotype relationships in WD by 
generating a matrix for exploration of the clinico-genetic interface, followed by evaluation of 
candidate modifier loci, and (iv) functional evaluation of variants detected in the ATP7B 
coding region by localization and trafficking studies. 
A total of 199 WD patients (174 East Indian and 25 West Indian) mostly with 
neurological problems from 178 unrelated families comprising of 687 individuals have been 
included in this study prospectively. This group is an extension of a previous study from our 
lab where 25 East Indian patients had been characterized with both ATP7B mutations and 
single mutations found in a few (Gupta et al., 2007a). ATP7B mutation screening has been 
undertaken in 174 patients (East Indian: 149, West Indian: 25). Finally, a comprehensive 
mutation profile of all 199 patients is described. In this study, we detected 29 reported and 22 
novel mutations after screening most of the ATP7B coding region (exons 2 to 21) and flanking 
intron-exon boundaries. Of the reported ones, 19 defects had been reported from other 
populations but detected for the first time in our patient cohort and 10 variants were previously 
described by our group (Gupta A et al., 2007a) in addition to finding those during the study 
related to my dissertation. Adding up all mutations, our patient cohort harbors a total of 10 
 
Summary 
 
IV 
 
common and 48 rare lesions in the coding region of ATP7B. Three mutations (p.G1061E, 
p.N1270S and p.A1049A-fs) are shared between East and West. The common mutations 
have been found to account for 74% of characterized mutant alleles with p.C271X (63/260) 
and p.G1101R (7/31) being the most prevalent in East and West Indian patient cohorts, 
respectively. Comparison of mutation pools across India suggested that the exons 2, 8, 9, 13, 
14, 15, 18 and Intron 4 harbor the prevalent mutations and should be screened with priority in 
the Indian WD population. In addition to the mutations, 28 innocuous variants could be 
detected during this study. Mutations in both the alleles could be characterized in 114 
patients, while in 63 patients only one mutation could be detected in the coding region. No 
coding mutations could be detected in 22 patients. Further studies on these patients with 
missing mutation(s) are described in relevant sections below. 
WD is one of the few genetic diseases, the progression of which can be controlled if 
diagnosed early enough. Knowledge of common mutations in a population would pave the 
way for rapid diagnosis, even in some suspect case without family information, before the 
manifestation of overt clinical symptoms. But mutation screening is a time consuming 
process. So, a parallel strategy was employed to identify the carriers and presymptomatic 
individuals in 41 East Indian and 19 West Indian WD affected families with multiple sibs to 
provide relevant information faster without waiting for determination of the underlying causal 
mutation. By studying the inheritance of linked polymorphic microsatellite markers D13S314 
and D13S316 in the affected pedigrees, we could detect 5 presymptomatic, 40 carrier and 36 
normal individuals. Mutation information, when available was utilized to resolve the 
ambiguous cases. 
Most genetic studies reported earlier focussed on studying the ATP7B coding region 
but the gene promoter, though characterized, remained largely unexplored. As already 
described, 85 patients (63 + 22) in our pool could not be characterized for either one or both 
ATP7B mutations even after screening much of the coding region. In order to identify the 
missing mutations, we screened the ATP7B promoter and both the 5'- and 3'-UTRs in these 
patients. We detected 5 novel and 6 reported variants in the promoter and 5'UTR. Promoter 
cloning, site-directed mutagenesis and dual luciferase assay was carried out for functional 
evaluation of these variants. Five variants and one haplotype in the 5' regulatory region could 
be implicated in WD pathogenesis based on significant down regulation of the promoter 
activity. We carried out segregation studies in pedigrees to dissect the phase of alleles with 
respect to promoter variants and downstream coding mutations. We further hypothesized that 
ATP7B promoter variations altering its activity might also be present in patients where both 
coding mutations have been detected and influence the expression of the mutated protein. 
The differences in promoter strengths could have the potential to add on to the observed 
clinical heterogeneity in addition to the influence of other confounding genetic and 
environmental factors. Thus, the role of promoter variants in the regulation of ATP7B merits 
further investigation. 
 
Summary 
 
V 
 
Till date, no nucleotide substitutions in the ATP7B 3'UTR have been associated with 
WD phenotype. This study reports 3 novel variations in this region in WD patients, however, 
further experiments are required to ascertain their status as mutations or rare innocuous 
variants. We reached a mutation detection frequency of 77% in our study. 
We devised a Neurological Involvement Score (NIS) to explore the spectrum of 
neurological association across cognition, behaviour and motor domains in WD patients with 
preferential neuropsychiatric manifestation. The scale was found to have high internal 
consistency (Cronbach’s α 0.802) and inter-rater reliability (ICC 0.888). Thus NIS was highly 
reproducible. Subsequently we undertook a novel unified approach of genotype-phenotype 
(G2P) correlation by utilizing NIS as a simple tool to explore the neuropsychiatric arena of WD 
patients in light of the mutations identified across different ATP7B protein domains. Using age 
at onset and neurological involvement score, we generated a G2P matrix. The scheme of 
G2P matrix provided opportunities to examine and compare the patient phenotypes with 
respect to same genotype and also across different protein domains. We observed that the 
truncation mutations could be equally deleterious irrespective of the site of termination being 
early or late in the polypeptide chain. Compound heterozygotes bearing combination of 
missense and truncation mutations indicated missense mutations to retain more functionality 
in the mutated protein as reported previously (Nicastro E et al., 2009). Specific mutations in 
the nucleotide binding domain were found to attribute hepatic predominance. 
Patients with same/similar mutation background would be expected to have similar 
presentation. The most interesting observation was made when we encountered a few 
patients who depicted stark difference in their clinical manifestation as compared to other 
patients in the same mutation cluster. We counted the patients with divergent phenotypes as 
“outliers” in the matrix. Possible roles of COMMD1 and ATOX1, two genes in copper 
homeostasis pathway, were also investigated for their potential to act as modifier loci in the 
outliers, though no variant could be implicated for the cause. Since the ‘basal level 
heterogeneity’ of presenting symptoms in WD is not exclusively explained by the ATP7B 
mutations, and cases of misdiagnosis and phenotypic overlap have been reported implicating 
additional genes such as NPC1, SLC33A1, and SLC30A10 (Connemann BJ et al., 2012; 
Huppke P et al., 2012a; Quadri M et al., 2012), we propose that these loci should be studied 
for their putative role in imparting WD features by some novel pathophysiological 
mechanisms. 
The diagnostic delay emerged to be a confounding factor for G2P analysis. The mean 
diagnostic delay in our study was 19.5 months (0.2 to 180 months) and there is opportunity 
for further improvement. 
Missense mutations might have a spectrum of consequences on the mutant protein. 
In this context, we made an attempt to explore the effect of missense mutations on cellular 
localization and anterograde trafficking of ATP7B. GFP fusion constructs for 9 different 
ATP7B mutants selected from different domains were investigated for their localization and 
 
Summary 
 
VI 
 
copper responsive redistribution. While 6 of 9 mutants showed normal behavior, 2 mutants 
(p.G1061E and p.G1101R) were found to be grossly mislocalized to endoplasmic reticulum 
while 1 mutant (p.S1362A) was found to be unresponsive to copper induction. Mutations 
might affect several properties of ATP7B which could alter its precise conformation, catalytic 
and kinase mediated phosphorylation, ATPase activity, copper binding and transport rate 
across membranes etc. Each of these factors might have deleterious consequences on 
normal copper distribution and may lead to WD. Thus, a detailed functional exploration of the 
effect of missense variants on other properties of the protein would provide deeper insight of 
their effect on the clinical phenotype of the patients. 
The studies leading to this dissertation have resulted in the following publication as 
noted below and additional manuscript describing unpublished work is under preparation: 
Mukherjee S, Dutta S, Majumdar S, Biswas T, Jaiswal P, Sengupta M, Bhattacharya A, 
Gangopadhyay PK, Bavdekar A, Das SK, Ray K. Genetic defects in Indian Wilson disease 
patients and genotype-phenotype correlation. Parkinsonism and Related Disorders 2013; (In 
press); DOI, 10.1016/j.parkreldis.2013.09.021 


STUDIES ON SOME ASPECTS OF THE AQUEOUS CHEMISTRY OF MOLYBDENUM (AND RHENIUM)

MADYASTHA, K.M.

190 
 
 SUMMARY 
_________________________________________________________________________ 
 
Puntius shalynius Yazdani and Talukdar, 1975 is a small indigenous ornamental fish of 
Meghalaya. Commonly called as Shalynai in Khasi languages, it is an important hill stream 
fish occurring in almost all the streams and rivers of Khasi and Jaintia Hills.  
In the present investigation, distribution study of P. shalynius in the water bodies of 
Meghalaya was carried out. A stream and a pond were selected for detailed ecobiological 
studies. It includes habitat ecology, morphometric measurements, meristic counts, 
biometric index, length-weight relationship, condition factor, relative condition factor, 
food and feeding habits, reproductive cycle and ultrastructural studies.     
The distribution study showed fragmented and sparse population of this endemic fish 
in the State. It may be attributed to loss of habitat and feeding ground due to anthropogenic 
activities. It is represented in water bodies of four of the seven districts of the State viz. 
East Khasi Hills, West Khasi Hills, Jaintia Hills and Ribhoi Districts. The fishes were 
found both in lentic and lotic water bodies situated between 1000 m and 1400m msl. The 
habitat of the fish was associated with forest, agriculture and inhabited land use. It was 
observed that the water was slightly acidic with pH ranging from 6.23-6.60.  The dissolved 
oxygen was relatively low and conductivity high in all the water bodies. The over-
exploitation of this fish, chiefly for food, and also for export as an ornamental species is a 
major concern. Of the late the population of this species has declined to an extent that 
presently they are categorized as a vulnerable species.  
P. shalynius belongs to the family of freshwater fishes Cyprinidae, the most species-
rich vertebrate family under the class Actinopterygii and order Cypriniformes. It has a 
short and compressed body. Head is short; mouth arched, anterior; eyes moderate, 
dorsolateral, not visible from below ventral surface; lips thin covering the jaws; jaws 
simple, covered by lips without any knob or tubercle at the symphysis. Barbell is absent; 
abdomen rounded. There are three undivided dorsal rays, the first almost indistinct and the 
third longest with serration on its posterior face. Dorsal fin short inserted nearly opposite 
pelvic fins and anal fin also short. Caudal fin forked. Scales fairly big, hexagonal with 
anterior margins distinctly wavy; two black spots on tail and a horizontal blue line on the 
body; lateral line incomplete, ending on or before 11
th
 scale. In some males, minute white 
tubercles present on head.       
 
191 
 
The abiotic and biotic parameters of stream and pond were investigated for a period 
of two years in stream and for one year in pond. Seasonal variations of 11
o
C (January) to 
25
o
C (July) observed in water temperature of stream and 12
o
C (December) to 25
o
C (July) 
in pond affirmed the subtropical nature of the two systems. The pH of water ranged from 
4.87 to 6.69 in stream and 5.11 to 6.42 in pond indicating the acidic nature of the water. 
The conductivity value ranged between 0.09 mS (December) and 0.13 mS (April) in 
stream and 0.09 mS (December) and 0.11 mS (April) in pond water highlighting „soft-
water‟ character with low electrolyte concentration. The dissolved oxygen content was 
moderately high with values of 9.7 mgl
-1
 (May) in stream and 13.1 mgl
-1
 (March) in pond. 
Free carbon dioxide was present throughout the study period with the maximum value in 
March (18.5 mgl
-1
) and minimum in December (4.6 mgl
-1
) in stream. In pond, the 
maximum value was observed in December (15.4 mgl
-1
) and minimum in October (6.0 
mgl
-1
). The alkalinity values were maximum during February (40.3 and 34 mgl
-1
) in stream 
and May in pond (42.3 mgl
-1
) while minimum in June (20.6 mgl
-1
) and December (22mgl
-
1
) in stream and February (16 mgl
-1
) in pond water. The value of total hardness was 
maximum in April (61.2 mgl
-1
) whereas minimum in August (14.6 mgl
-1
) in stream. In 
pond water, the total hardness value fluctuated between 20.6 mgl
-1
 (December) and 99.4 
mgl
-1
 (October). However, all the recorded parameters exhibited slight seasonal variations. 
Climate of the study area plays a significant role on the habitat and reproduction the fish 
species surviving therein.  Climatic data on air temperature, relative humidity and rainfall 
were from the Meteorological centre. The data have been correlated with the monthly 
changes in the physico-chemical parameters of the stream and pond water. 
Studies on biotic factors have shown that the phytoplankton dominanted over the 
zooplankton in both the systems. The phytoplankton was represented by six groups namely 
Bacillariophyceae, Chlorophyceae, Euglenophyceae, Myxophyceae, Xanthophyceae and 
Zygnematophyceae. A total of 34 genera were identified and out of these 13 belonged to 
Bacillariophyceae, 15 to Chlorophyceae, 3 to Euglenophyceae, 1 to Myxophyceae, 1 to 
Xanthophyceae and 1 to Zygnematophyceae in stream while 12 each from 
Bacillariophyceae and Chlorophyceae, 2 from Euglenophyceae, 1 to Myxophyceae, 1 from 
Xanthophyceae and 1 from Zygnematophyceae in pond. In regards to zooplankton, a total 
of 7 genera belonging to 4 groups were recorded from the stream and pond water namely 
Rotifera, Cladocera, Copepoda and Rhizopoda.   
192 
 
Morphological variations have been observed between the two populations i.e. from 
the stream and pond. Based on Johal (1994) classification, in stream population of P. 
shalynius sixteen out of the twenty morphological characters studied were found to be 
genetically controlled, one belonged to the intermediate controlled characters and three 
falls under environmentally controlled characters where as in pond population, all the 
twenty characters were seen to fall under the genetically controlled characters. The 
maximum growth rate („b‟ value) in relation to total length was recorded in forked length 
and minimum in head depth, while it was observed maximum in interorbital width and 
minimum in eye diameter in relation to head length, for stream population. In pond 
population, the „b‟ value of forked length was observed maximum and caudal fin length 
minimum in relation to total length, while it was maximum in post orbital length and 
minimum in eye diameter in relation to head length. High values of correlation coefficient 
(r>0.75) obtained indicated a high degree of positive correlation of the different 
morphometric characters with the total length and head length of P. shalynius in both the 
population. Meristic counts were similar in stream and pond populations except a slight 
variation in caudal fin rays. The growth of the standard length, forked length, pre-anal 
length and head depth in relation to total length was found to be isometric in both the 
systems.  
The value of length-weight regression coefficient „b‟ obtained  for male, female and 
combined in all the four seasons did not follow the ideal value of 3 indicating that the 
species exhibit allometric pattern of growth in both the system. No differences were found 
between sexes and seasons in both the population. Condition factor (K) value showed 
monthly changes which is apparently related to the reproductive period with a peak in 
April before the spawning season and declined in September/October after the spawning in 
both the sexes and systems. From this study, we can conclude that P. shalynius reproduce 
in between May to September/October since the lowest K value was recorded during this 
period. K value for males and females were calculated for different length groups. K values 
increased almost gradually as they grow from juvenile to adult stage. But, the K values 
decreased with the increasing length in female fishes. This result indicated that their gonad 
developed gradually until the maturation stage, when their body length was between 5.0- 
5.5cm as observed in both the systems. The best fish condition was recorded for those 
individuals with total length of 6.0-6.5cm in stream and 5.0-5.5cm in pond for males. It 
was the stage when the gonads of most of the individual male fish were fully grown, thus 
193 
 
contributing to the higher K values. Relative condition factor (Kn) value was ≥1 both in the 
stream and pond populations. It suggests the health of the fish was good in both pond and 
stream populations.  
Food and feeding habit was analyzed in P. shalynius for stream and pond population. 
The relative gut length value indicates an omnivorous nature of feeding habits of the fish 
as the RLG value is generally <1 in carnivorous fish, between 1 to 3 in omnivorous fish 
and >3 in herbivorous fish. The feeding intensity or Gastrosomatic index (GaSI) was 
observed high in the month of December (4.541±0.386) for male and April (5.281±0.499) 
for female while the lower GaSI value was recorded in the month of June (1.687±0.202 for 
male and 1.645±0.127 for female) for both the sexes in stream population. In pond 
population, the high feeding intensity was recorded in the month of February 
(4.802±0.218) for male and during November (5.150±0.749) for females. The low value 
was observed in July (2.152±0.209 for male and 2.032±0.281 for female) for both the 
sexes. Feeding intensity in different length group suggested that feeding intensity was high 
in the smaller length group while the larger length group showed a lower value of feeding 
intensity. GaSI was calculated for different maturity stages. The lowest value was recorded 
in spent stage (1.892±0.164) and highest in immature stage (3.502±0.286) for males. Again 
in females, the lowest value was found in spent stage (1.897±0.154) and highest in 
maturing stage (2.689±0.219 for stream individuals. For pond individuals, the low value 
was observed in spent stage for both the sexes (1.942±0.257 for male and 1.871±0.241 for 
female) and highest value was seen in the immature stage for both the sexes (4.394±0.378 
for male and 4.182±0.310 for female).  
Gut content analysis revealed that the food items of P. shalynius can be divided into 
the following components (i) Detritus (ii) Phytoplankton (iii) Nematodes (iv) Zooplankton 
(v) Insects (vi) Plant matter and (vii) Unidentified algae based on the frequency of 
occurrence and volumetric method. Detritus dominated the P. shalynius diet in both the 
sites studied irrespective of natural lotic or artificial lentic habitat.  Detritus constitute of 
49.23% (stream) and 61.17% (pond) of the diet in both the sites studied irrespective of 
natural lotic and artificial lentic habitat and therefore can be regarded as the basic food of 
the fish. This is an indication that the species is bottom grazer. Qualitatively as well as 
quantitatively, the diet composition of P. shalynius was dominated by the detritus and 
phytoplankton in all months. The food composition of the fish in variation of respective 
length groups revealed that food habits changed with the increase in size as the fish grew. 
194 
 
Detritus were the dominant food item in all the size class. Smaller individuals feed mainly 
on the detritus, phytoplankton, less amount of zooplankton, nematodes and insects whereas 
larger individuals preferred detritus, phytoplankton, zooplankton, nematodes, insects and 
plant matter. On the basis of index of preponderance, the preference for detritus was 
evident in both the systems followed by the phytoplankton, insects, plant matter and 
zooplankton in their diet. This indicates the stenophagic feeding habit of the fish. The 
study revealed that the P. shalynius is omnivorous and bottom feeder in nature mainly 
feeding on detritus and that the environment plays an important role in governing the food 
and feeding habits of the species. 
Sex ratio in different months showed that there exists a variation between number of 
males and females in both the population. The overall sex ratio was 1:1.16 (male: female) 
in stream population whereas in pond population, 1: 0.91(male: female). Sex ratio was in 
favor of females in stream and males in pond population. In stream population, the mean 
values of Gonadosomatic index (GSI) of male attained peak in the month of April and for 
female in June. The GSI of male and female fishes gradually increased from January 
onwards and attained the peak in April for male and June for female. Subsequently, there 
was a decreased from the month of May onwards in male and July in female. The lowest 
value was observed in December in both the sexes. Moreover, in pond population, both the 
sexes were seen to attain maximum value in the month of July. It increased from the month 
of March attained peak in July and then decreased to lowest in November for males. In 
females, the value increased from the month of January attained peak in July and decreased 
to lowest in December. It can be concluded that the fish spawns once in a year with single 
spawning peak. The Dobriyal index (DI) showed the same trends as GSI which confirms 
the above observation. 
Based on the macroscopic and microscopic study, five different maturity stages of 
gonads had been described. These stages were: I= Immature or Virgin, II= Maturing, III= 
Mature, IV= Ripe or Spawning, V= Spent or Regressing. Maturity stages in different 
months indicate that the ripe species were found during April to October in male and May 
to September in female both in stream and pond population. 
Fecundity study revealed that P. shalynius is a low fecund fish. The absolute 
fecundity and relative fecundity varied from 47 to 1240 eggs and 45 to 633 eggs per 
individual in stream population while in pond population, absolute fecundity and relative 
fecundity varied from 67 to 531 eggs and 42 to 515 eggs per individual. The correlation 
195 
 
coefficient (r) for the relationship between fecundity and body length (0.500 for stream and 
0.268 for pond), fecundity and body weight (0.673 for stream and 0.182 for pond) and 
fecundity and gonad weight (0.717 for stream and 0.718 for pond) indicates that the gonad 
weight has a better correlation with reproductive capacity than the body weight. Further, 
the correlation coefficient between fecundity and ovary weight was found to be quite high 
both in stream and pond population. The analysis of the ova diameter revealed that it varies 
between 0.375 mm (March) to 0.745 mm (July) along with the progression of the maturity 
stages (Fig 6). The ripe ova found in the month of July (0.745 mm) which indicated that 
the fish spawns once in a year with single spawning peak. It has been observed that 50% 
individuals (M50) of P. shalynius mature at 5.6-6.0 cm (males) and 5.1-5.5 cm (females) 
length group in stream population. In pond population, it was 5.1-5.5 cm (males) and 4.6-
5.0 cm (females).  This suggests that females reach their first sexual maturity before males. 
Scanning electron microscope (SEM) study in the scale of P. shalynius shows the 
general architectural pattern of a cycloid cyprinid scale having focus, circuli and radii.  No 
annuli were observed indicating that age of fish was less than one year. The morphological 
defect due to exposure in low pH of water was observed in the partial or complete damage 
to the structure of lepidonts. This suggests that the declining population of this fish can be 
correlated to the pollution occurring in water bodies. SEM of each gill was found to consist 
of gill arch, gill rakers and gill filaments. From the gill arch arise a number of gill 
filaments which are equidistant from each other giving a leaf like structure. The gill rakers 
are located on the internal side of the gill arches. Gill rakers in fish act as a selective screen 
preventing the entrance of large and undesirable organisms, besides protecting gill 
filaments from damage. SEM studies of oral cavity in P. shalynius show a narrow anterior 
opening transversely placed, bordered by the upper and lower jaw considered to be a 
slightly protractile mouth. The narrow mouth opening may assist the fish in sucking or 
picking up small food particles. The outer surface of the upper jaw exhibit cap-shaped 
mucous cells arranged parallelly.  The lower jaw was distinguished by a tubercle in the 
middle at the union of its two halves. In P. shalynius, the jaws were edentulous. In this 
fish, which feeds on small food items, the presence of teeth on jaws, required to hold or 
grasp and to tear the food in the oral cavity may not be of much significance. The 
morphological features of P. shalynius such as narrow mouth opening, absence of teeth in 
jaws, blunt gill rakers, well developed stomach, high RLG value (>1) and high percentage 
of detritus and plant diet confirmed its omnivorous feeding habit.  
196 
 
P. shalynius do not possess a true stomach and have been divided into anterior and 
posterior intestine based on the depth of mucosal fold. Both the anterior and posterior 
intestine consists of four layers viz., serosa, muscularis, submucosa and mucosa. Mucus 
secreting goblet cells were found in mucosal layer. The anterior and the posterior intestine 
shared similar anatomical and histological structures but anterior intestine showed 
prominent longitudinal mucosal folds and thick muscularis. The longitudinal fold probably 
helps in facilitating conduction of food.  
Transmission electron microscope study on intestine revealed that the columnar 
epithelial cells (enterocytes) were elongated with apical part bearing numerous microvilli. 
Cytoplasm of the epithelial cells contained mumerous mitochondria, a prominent nucleus 
was along with lipid vesicles. The enterocytes were joined at the apical surface by typical 
junctional complexes including the desmosomes which provide the cells with adhesion to 
one another. Microvilli help in absorption of nutrients from digested food increasing the 
cell surface area.  
The present findings from two different habitats will add to the knowledge on 
ecobiology of P. shalynius in water bodies of Meghalaya. P. shalynius is one of the 
promising species which can be taken up for aquaculture not only as food fish but more so 
as ornamental. Cultivation of P. shalynius which is vulnerable in IUCN status due to 
habitat destruction will add to conserve the species and enrich the icthyodiversity of the 
state.  
 
 
 
 
 
 
 
 
 
 


MICRONIOLOGICAL TRANSFORMATIONS OF TERPENES

SAKORE, TUKARAM

Synopsis | 1 
 
The thesis entitled “Culture independent detection and characterization of 
Streptococcus pyogenes and its transcriptional gene regulation upon subinhibitory 
exposure to antibiotics” deals with the use of culture independent approaches for the 
detection of pharyngitis causative bacteria directly from the throat swabs; molecular 
characterization and comparison of S. pyogenes isolated from different infection cases 
and the in vitro and in vivo analysis of S. pyogenes challenged with subinhibitory 
concentrations of antibiotics. The work elements have been divided into five major parts 
viz., 
1. Culture independent analysis of throat swabs from pharyngitis patients and 
healthy subjects by Denaturing Gradient Gel Electrophoresis.  
2. Culture independent analysis of throat swabs from pharyngitis patients and 
healthy subjects by Terminal Restriction Fragment Length Polymorphism. 
3. Development of multiplex PCR assay for the detection of pharyngitis causing 
bacteria directly from the throat swabs of pharyngitis patients. 
4. Comparative molecular analysis of Streptococcus pyogenes isolates from ocular 
infections, pharyngitis and asymptomatic children in south India. 
5. Effect of subinhibitory concentrations of fluoroquinolones on biofilm production 
by clinical isolates of Streptococcus pyogenes. 
6. In vitro and in vivo analysis of genes involved in virulence, biofilm formation and 
antibiotic resistance in Streptococcus pyogenes exposed to subinhibitory 
concentrations of antibiotics. 
 
Streptococcus pyogenes is one of the most common cause of bacterial pharyngitis 
in human, which upon left untreated may result in deadly post-infection sequelae such as 
scarlet fever, rheumatic heart disease and necrotizing fasciitis. Apart from S. pyogenes, a 
number of other bacteria are also involved in the pharyngitis. A greater understanding of 
the bacteria involved is needed to improve our knowledge as well as prescribing right 
method to eliminate the causative pathogen. Increasing knowledge in the field of 
molecular diagnostics opens up new strategies to explore the changes in bacterial 
community structure between the throats of pharyngitis and healthy subjects. In addition, 
PCR based methods allow rapid, accurate and simultaneous detection of more than one 
causative bacterium directly from pharyngitis throat swabs by means of multiplex PCR. It 
also allows molecular comparison of S. pyogenes strains isolated from different infection 
Synopsis | 2 
 
cases. Apart from diagnostics and molecular characterization, the present study is focused 
on the in vitro and in vivo effects of subinhibitory concentrations of various antibiotics 
against S. pyogenes isolates. 
      The section “Culture independent analysis of throat swabs from pharyngitis 
patients and healthy subjects by Denaturing Gradient Gel Electrophoresis” deals 
with the comparative analysis of throat metagenomes of pharyngitis patients and healthy 
subjects using nucleic acid based culture independent method Denaturing Gradient Gel 
Electrophoresis (DGGE). In this study, 40 throat metagenomes each from healthy and 
pharyngitis patients were taken. The 16S rRNA gene of the metagenomes was amplified 
through two rounds of PCR. In the first round PCR, ~1500 bp 16S rRNA gene was 
amplified using universal 16S primers. The second round PCR is a nested PCR in which 
first round PCR amplicons were used as templates and V3 region of 16S rDNA (~250 bp) 
was amplified using GC-clamped primers. DGGE reference marker for inter-gel 
comparison of DGGE gels was prepared using Streptococcus pyogenes, Staphylococcus 
aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Haemophilus influenzae. 
DGGE banding patterns generated directly from the throat swabs showed considerable 
difference between healthy and pharyngitis samples. The number of separate bands in the 
studied samples ranged from 3 to 26 in healthy subjects and 2 to 29 in the case of 
pharyngitis samples. Comparative band analysis showed the presence of more pathogens 
in pharyngitis samples than the healthy subjects. S. pyogenes in 8 (20%); S. aureus in 9 
(22.5%); P. aeruginosa in 4 (10%); H. influenzae and K. pneumoniae in 7 (17.5%) in 
healthy subjects’ throat swabs and S. pyogenes in 20 (50%); S. aureus in 16 (40%); P. 
aeruginosa in 9 (22.5%); H. influenzae in 13 (32.5%) and K. pneumoniae in 19 (47.5%) 
in pharyngitis samples. Sequencing of dominant bands revealed the presence of 
uncultured Capnocytophaga sp., Enterobcter sp., Serratia sp. and Aeromonas sp. in the 
throat metagenomes.  
The section “Culture independent analysis of throat swabs from pharyngitis 
patients and healthy subjects by Terminal Restriction Fragment Length 
Polymorphism” deals with the comparative analysis of throat metagenomes of 
pharyngitis patients and healthy subjects using terminal restriction fragment length 
polymorphism (T-RFLP). For T-RFLP analysis, metagenomic DNA was extracted from 
10 throat swabs each from healthy and pharyngitis patients. The 16S rRNA genes of 
Synopsis | 3 
 
bacteria present in throat metagenomes were amplified by PCR with 6-carboxy-
fluorescein (Fluorophore) - labeled universal forward primer (6-FAM - 27F) and a 
universal reverse primer (1513R). The 16S rDNAs were digested with restriction 
enzymes with 4bp recognition sites such as MspI or RsaI and analyzed by using an 
automated DNA sequencer. T-RFLP patterns were numerically analyzed using computer 
programs. From analysis of the throat bacterial community, patterns derived from MspI 
and RsaI digested samples of healthy subjects and pharyngitis patients were grouped into 
different clusters, though RsaI digested samples showed some uncertainty. Pharyngitis 
throats generated an average species richness of 9 (2.1 [SD]) and 10 (2.9) for MspI and 
RsaI digests respectively, whereas healthy throats generated 6.3 (1.2) and 6.1 (1.5) in 
MspI and RsaI digests, respectively. These results suggest that samples from pharyngitis 
patients contain an unexpected diversity of causative bacteria.  The pharyngitis throats 
were colonized with a rich diversity of bacterial species than that of healthy throats. In T-
RFLP analysis, the reference strain, Streptococcus pyogenes SF370 was used for T-RF 
comparison. The T-RF patterns of the reference strain coincides with the Streptococcus 
pyogenes T-RFs generated from the metagenomes.  
 The section “Development of multiplex PCR assay for the detection of 
pharyngitis causing bacteria directly from the throat swabs of pharyngitis patients” 
deals with the development of a pentaplex PCR assay for rapid and simultaneous 
detection of five oral pathogens namely S. pyogenes, S. aureus, P. aeruginosa, N. 
gonorrhoeae and K. pneumoniae directly from the throat swabs of pharyngitis patients. 
The designed species-specific primers detected the five targeted pathogens without any 
cross reaction. Multiplex PCR amplification of the five species specific genes was 
achieved by optimizing the PCR conditions. 16S rRNA gene was taken as internal 
control. Annealing temperature of 58°C for 1 min was found to be the optimum 
temperature for the pentaplex PCR amplification. The sensitivity checking using serially 
diluted DNA (20ng to 1.25ng) showed unambiguous amplification up to 2.5ng of DNA 
and slight amplification at 1.25ng. Metagenomic DNA (n=39) isolated from throat swabs 
of pharyngitis patients were used to evaluate the developed assay. The multiplex PCR 
assay revealed the presence of P. aeruginosa, S. aureus and S. pyogenes in 2 throat swab 
samples each. None of the metagenomes showed the presence of N. gonorrhoeae and K. 
pneumoniae or the combination of P. aeruginosa, S. aureus and S. pyogenes. This study 
Synopsis | 4 
 
once again proves that multiplex PCR based pathogen detection is an effective tool in the 
field of clinical diagnostics. 
 The section “Comparative molecular analysis of Streptococcus pyogenes 
isolates from ocular infections, pharyngitis and asymptomatic children in south 
India” deals with the molecular characterization of S. pyogenes isolates from ocular, 
pharyngitis and asymptomatic carrier cases. In the present study, emm type surveillance 
as well as its association with toxin gene profiles was analyzed. S. pyogenes was detected 
in 20 isolates of the ocular infected cases such as lacrimal abscess, corneal ulcers and 
mucocoele. For noninvasive isolates, 370 pharyngitis cases and 400 asymptomatic school 
children were screened and recovered 33 pharyngitis and 14 carrier isolates respectively.  
Fourteen emm type distributions were observed in ocular isolates whereas 11 emm types 
each were noticed in pharyngitis and asymptomatic carrier isolates. The two dominant 
emm types, emm49 and emm63 were accounted for 33% of the total S. pyogenes isolates. 
Among ocular isolates, slo, smeZ, speB and speG were found in >50% of isolates and in 
pharyngitis smeZ (48%), speB (45%) and speG (42%) genes were found to be prevalent. 
Alarmingly, carrier isolates showed more prevalence to virulence genes than the ocular 
and pharyngitis isolates with speF (79%), speB, speG (64%), slo and sil (64%). Among 
the three groups, pharyngitis isolates harbored more prtF1 (33%) and prtF2 (94%) genes 
than the asymptomatic carriers (28% and 71%) and the ocular isolates (45% and 40%). 
450 bp size band in prtF1 and 350 bp size band in prtF2 showed dominance. Among the 
three groups tested, the distribution of ermB and mefA was high in pharyngitis isolates 
(30%) where 10 isolates showed the presence of both genes. None of the isolates showed 
the presence of ermA and tetO genes. Dendrogram generated based on the virulence and 
antibiotic resistance gene profiles revealed that the isolates are intermingled (except few 
clusters), irrespective of their emm types and isolation source. 
 The section “Effect of subinhibitory concentrations of fluoroquinolones on 
biofilm production by clinical isolates of Streptococcus pyogenes” deals with revealing 
the effects of sub-MICs of fluoroquinolones (FQs) on S.  pyogenes biofilm formation. In 
this study, biofilm forming six M serotypes M56, st38, M89, M65, M100 and M74 of S. 
pyogenes strains were challenged against four FQs namely, ciprofloxacin, ofloxacin, 
levofloxacin and norfloxacin. The antibiofilm potential of these FQs were analyzed at 
their subinhibitory concentrations (1/2-1/64 MIC) using biofilm assay, XTT reduction 
Synopsis | 5 
 
assay, Scanning Electron Microscopy and Confocal Laser Scanning Microscopy. From 
the four FQs tested, ofloxacin and levofloxacin at 1/2 MIC showed the maximum 
inhibition (92%) of biofilm formation against M56 and M74 serotypes.  FQs effectively 
interfered in the microcolony formation of S. pyogenes strains at 1/2-1/8 sub-MICs. 
Interestingly, inhibition of biofilm formation was greatly reduced beyond 1/16 MICs and 
allowed biofilm formation. XTT reduction assay revealed the increase in metabolic 
activity of S. pyogenes biofilm against the decrease in FQs concentration. In addition, 
SEM and CLSM validated the potential of sub-MICs of FQs against the six S. pyogenes. 
These results confirm that all the four FQs are concentration dependent in inhibiting S. 
pyogenes biofilm formation. FQs at proper dosage can be effective against S. pyogenes 
and lower dosage concentrations may allow the bacteria to form barriers against the 
antibiotic in the form of biofilm.  
Finally, the section “In vitro and in vivo analysis of genes involved in virulence, 
biofilm formation and antibiotic resistance in Streptococcus pyogenes exposed to 
subinhibitory concentrations of antibiotics” deals with the in vivo and in vitro effects 
of sub-MICs of antibiotics on biofilm and non-biofilm forming S. pyogenes strains. MIC 
determination by broth microdilution method showed the highest MIC value of 1 µg/ml 
for SP7 and SF370 isolates against ofloxacin. SF370 showed the lowest MIC value of 
0.03 µg/ml against erythromycin. Sub-MICs of 1/2 to 1/64 MICs were utilized to 
challenge S. pyogenes strains. Sub-MICs treated S. pyogenes were assessed for 
autoaggregation ability and found that it varies between the strains.  In biofilm forming 
stains SP7and SF370, the average autoaggregation was observed to be 0.25 and 0.7nm. 
Whereas, the non-biofilm former SP24 showed an average of 0.03nm of autoaggregation. 
mRNA isolated from the sub-MIC treated bacterial cells were utilized for analyzing the  
virulence, biofilm and antibiotic resistance associated genes. Real time PCR analysis 
showed gene regulation in concentration dependent manner. The analysis showed 
increase in expression at the higher and lowest sub-MICs and showed a decrease in gene 
expression at the middle sub-MICs. These results have correlation with the 
autoaggregation assay results where, most of the antibiotics showed autoaggregation in 
concentration dependent manner. In vivo analyses using C. elegans through nematode 
liquid killing assay shows biofilm forming strains SP7 and SF370 extending its survival 
up to six days, whereas complete mortality occurred within four days in the case of non 
Synopsis | 6 
 
biofilm former SP24. In addition, lower sub-MICs such as 1/8 to 1/64 MICs showed 
lethal effects within 48h whereas the control showed less lethal effect. The results of the 
present study substantiate that the lower sub-MICs of antibiotics enhance the virulence of 
S. pyogenes. Hence, from the present study, it is evident that usage of antibiotics at 
proper concentration is very crucial to protect the mankind from life threatening 
infections.   


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