Open Access Repository of Indian Theses

SHAIKH, ABBAS-ALLI GHUDUBHAI


Molecular cloning and characterization of cDNAs encoding Glyceraldehyde - 3 - phosphate dehydrogenase and NC1 domain of Collagen XVIII from Indian shrub frog, Polypedates maculates


Increased blood vessel density, abnormal vasculature and a milieu analogous to tumors necessitated an evaluation of the angiogenic imbalance in keloids. The angiogenic phenotype is regulated by a complex mechanism involving the interactions between pro - angiogenic and anti - angiogenic factors. The status quo of angiogenic balance was studied with reference to the circulatory and tissue - specific profiles of VEGF (a pro -angiogenic factor) and Endostatin (an anti - angiogenic factor) by Enzyme linked immunosorbent assay (ELISA), semi - quantitative RT - PCR, and Western blot analysis. Endostatin was found to be downregulated in sera and skin tissue of keloid patients in comparison to normal controls. Anti - angiogenic therapeutics in combination with current curative strategies hence, provide a new scope for effective treatment of keloids.

Endostatin of mouse and human origin have been extensively investigated due to their suppressive activities on endothelial cells and inhibition of tumor growth. However, they are known to exhibit differential effects on endothelial cells in spite of remarkable similarity in their amino acid sequences. 

Investigations on anti - angiogenic cues from amphibian sources are scanty, although the latter are known for their intriguing facets of scarless wound healing and tissue regeneration. Tadpoles have been increasingly used as models to unravel the genetic basis of angiogenesis so as to accelerate discovery of genes and drugs related to it. Anti - microbial peptides identified in amphibians have mammalian counterparts that perform different roles inspite of either sequence or structural similarities. Thus, based on our knowledge that genes are highly conserved between species, the prospect of anti - angiogenic principles from amphibians presenting effective scarless wound healing capabilities in humans would be significant. Amphibian endostatin has not been structurally and functionally characterized, although Collagen XVIII has been identified in the Xenopus toads. 

Therefore, the thesis presents investigations on molecular cloning and characterization of cDNAs encoding NC1 domain of Collagen XVIII and Glyceraldehyde - 3 - phosphate dehydrogenase (GAPDH) from Indian shrub frog, Polypedates maculatus of the Rhacophoridae family of amphibians. Identification of amphibian endostatin would augment appreciation of its distinguishing characteristics and probably unearth a more functionally potent angiogenesis inhibitor.




English

SYNTHESIS AND CHARACTERIZATION OF POLY(ARYL CARBONATE)S AND POLY(ARYLESTER-CARBONATE)S VIA CARBONATE INTERCHANGE REACTIONS WITH DIALKYLCARBONATE

SHAIKH, ALAMGIR ABDULLA

CHARACTERIZATION OF PHOTOSYSTEM II 
HETEROGENIETY 
IN RESPONSE TO SALINITY STRESS 
 
 
 
 
 
SYNOPSIS FOR 
Ph.D. 
REGISTRATION 
 
 
 
 
 
 
SUBMITTED BY 
POOJA MEHTA 
SCHOOL OF LIFE SCIENCE 
    DEVI  AHILYA  VISHWAVIDYALAYA 
KHANDWA  ROAD 
INDORE 
 
 
 
 
 
SUPERVISOR:                                                                         CO-SUPERVISOR: 
Dr. Anjana Jajoo                        Prof. Sudhakar Bharti 
SCHOOL OF LIFE SCIENCE    SCHOOL OF LIFE SCIENCE 
DEVI  AHILYA  VISHWAVIDYALAYA   DEVI  AHILYA  VISHWAVIDYALAYA 
INDORE    INDORE 
 
      
INTRODUCTION 
 
Life on Earth ultimately depends on the energy derived from the Sun. Photosynthesis is 
the only process of biological importance that can harvest solar energy. In 
photosynthesis, the plants use solar energy to oxidize water, thereby releasing oxygen and 
reduce carbon dioxide forming large carbon compounds primarily sugars. The 
Photosynthetic complex comprises of two photosystems: Photosystem II and I. 
Photosystem II (PSII) is a multisubunit pigment protein complex embedded in the 
thylakoid membrane of the chloroplast. It utilizes light for photochemical energy 
conversion and is also involved in the regulation of energy flow. PS II consist of at least 
25 different types of protein subunits many of which are bound to the thylakoid 
membrane. Some subunits are involved in the capturing of solar energy and the 
regulation of energy flow; while others are directly or indirectly responsible for 
photochemistry, including the oxidation of water to molecular oxygen. The central part of 
complete PS II unit is formed by the so called core complex, a well defined structure of 
which the reaction centre protein D1, D2 and core antenna protein CP 47 and CP 43 are 
largest and functionally the most important components (Boekema et al. 1999) and The 
PS II complex also consist of three extrinsic  proteins, a cluster of four manganese atom 
and two specific tyrosine residue, one on D1 called TyrZ (Yz) and other on D2 called Tyr 
D  (YD) (Bacon Ke 2000) 
 PS II complex is not homogenous in nature and differs in its structure and 
function both. This diverse nature of PS II is known as heterogeneity. Heterogeneity is 
mainly classified in to two classes i. e. static heterogeneity and dynamic heterogeneity. 
Static heterogeneity is correlated with the grana or stroma structure and heterogeneity of 
antenna size heterogeneity with respect to the ability of sustaining electron transfer. 
Dynamic heterogeneity is implied when the functional changes of the photosynthetic 
apparatus occur under different circumstances (Lavergne and Braintais 1996). Extent and 
nature of PS II heterogeneity may vary under different physiological states i.e. salinity 
stress, temperature stress etc. Photosynthetic performance is limited due to the biotic and 
abiotic stress factors.  
 REVIEW OF LITERATURE 
 
Photosystem II has been found to be more heterogeneous than other components such as 
PS I and Cyt b6f in various aspects. Two major fractions i.e. PS II α and PS II β   were 
distinguished with respect to PS II antenna heterogeneity and heterogeneity related with 
energy connectivity. Determination of antenna heterogeneity of PS II from fluorescence 
rise curve method, measured with 3-(3’ 4’dichlorophenyl)-1, 1 dimethyl urea (DCMU) 
for the first time was introduced by (Melis and Homann 1975, 1976,  Strasser 1981). By 
using this method three types of heterogeneities were determined i.e. PS II, PS IIand 
PS II (Sinclair and Spence 1988 , Hsu and Lee 1991). PS II α is mainly located in grana 
region of thylakoid membrane and is characterized by a large light harvesting antenna 
while PS II β is mainly located in the stroma region of the thylakoid membrane and is 
characterized by a small light harvesting antenna When only PS II α and PS II β were 
considered, analyses of the DCMU fluorescence rise curve revealed about 65-80% PS II 
α and rest PS II β centres (Lazar et.al. 2001). PS II β differ from PS II α among other 
thing, in the fact that the former has an exponential fluorescence rise in presence of 
DCMU in comparison with PS II α which has sigmoidal one. It is generally accepted that 
the exponential increase in the fluorescence intensity reflects energetic separation of PS 
II (Melis 1979, Melis and Anderson 1983). The determination of PS II antenna 
heterogeneity was also done without DCMU. In the flash fluorescence induction 
measurement, a very strong 100 μs flash is applied which causes transient closure of PS 
II (Nedbal et al. 1999). On the basis of biochemical and fluorescence method it was 
found that about 20 -40% PS II can not reduce Q B and PQ pool. These PS II centres were 
called the Q B non-reducing centres or inactive PS II centres   as different from the Q B   
reducing or active PS II centres    that can reduce   Q B and PQ pool. It was suggested that 
Q B reducing type PS II centres can be of   α type (Ghirardi and Melis 1988). Another 
heterogeneity related to PS II is in the formation of domains with different rates of the PQ 
pool reduction; 50-70% of PQ molecules from the PQ pool are reduced fast and 
suggested that fast reducing PQ pool represents the pool from grana region of thylakoid 
membrane. (Joliot et al.1992, Kirchoff et al. 2000) 
 Increasing salinity in soil affects the plants. Effects of NaCl stress are mainly of 
two types. Primary stress i.e. direct membrane damage leading to metabolic alteration 
and secondary stress i.e. osmotic stress. The increase in NaCl concentration above 100 
mM inhibited the photoreduction of ferricyanide in uncoupled isolated spinach thylakoids 
and consequently decrease the rate of the primary electron flow to ferricyanide. Exposing 
leaf cell to high salinity can causes ionic imbalance and toxicity effect as well as drought 
effect in plants (Robert and Nott 1982). The high salinity of surrounding solution causes 
changes in water and ionic status in cell. High salt stress in wheat leaves causes 
retardation of chlorophyll accumulation, partly by reducing the rate of porphyrin 
formation (Abdelkader et al. 2007) High salinity markedly reduced oxygen evolution in 
isolated thylakoids from NaCl treated plants. Salt stress inhibited the apparent quantum 
efficiency of photosynthesis and PS II actively while stimulating PS I activity and dark 
respiration significantly. Salt stress also resulted in decrease in overall electron transport 
rates which could not be restored by DPC. Salt stress resulted in decrease in growth and 
relative water content as well as net photosynthesis in maize. The effect of salinity on in 
vitro growth morphogenesis, photosynthetic rate, PS II efficiency (Fv/Fm) and 
chlorophyll content were investigated in Centaurium erythraea (Siler et al. 2007). 
Chlorophyll a, b and total chlorophyll content had a decreasing trend with increasing 
supply of NaCl in Growth medium. The accumulations of intracellular sodium ion during 
salt stress change the ratio of K: Na, which probably affects the bioenergetic process of 
photosynthesis.  
 
RATIONALE OF THE WORK 
 
Increase in salinity in soil and an effect on plants has become a vast problem for 
agriculture. Increase in salt concentration in soil reduces the productivity of the plants, so 
it necessary to know the cause of reduction of productivity. 
In this project we explore the alteration of photosystem II heterogeneity under salinity 
stress. The result obtained from this study may help in designing plant resistant or 
tolerant to salt stress in future. This is a basic study to establish if stress affects the 
structural and functional heterogeneity of PS II. The effects on PS II heterogeneity may 
affect photosynthesis and eventually crop yield.  Assortment of measurements using both 
preparative biochemistry and biophysical techniques are likely to provide the markers for 
monitoring the PS II heterogeneity during early stages of salinity stress as well as for 
screening the tolerance in varieties and transgenics. 
 
OBJECTIVES 
 
1. To study the effect of salt treatment on the rate of electron transport mediated by 
PS II and PS I. 
 
2. To measure heterogeneity in antenna size by measuring PS II α and PS II β 
centres as a function of salt stress. 
 
3. To determine functional heterogeneity by monitoring Q B reducing and Q B non-
reducing centres as a function of salt stress. 
 
4. To study heterogeneities in the arrangement of PS II in granal and Stromal 
lamellae in response to salt stress. 
 
METHODOLOGY 
 
HYDROPONICS: For the salinity stress we adopt the hydroponics and grow the plant in soil 
less culture. The term hydroponics originally meant nutrient solution culture. 
Hydroponics or soil less culture is a technology for growing the plant in nutrient solution 
that supply all nutrient elements needed for optimum growth with and without use of 
inert medium such as gravel, vermiculite, rock wool, saw dust, coir dust etc. to provide 
mechanical support. Hydroponics or soil less culture reduces the possibilities of soil born 
disease and pests. 
 
PREPARATION OF THYLAKOID MEMBRANES: The present work involves working with 
young plants, intact leaves and isolated thylakoid membranes under salt treatment. The 
thylakoid membrane fragments prepared by following the method described in Kuwabara 
and Murata (1982).  
Various biophysical and biochemical tools will be used to analyze PS II heterogeneity as 
a function of salt stress. 
 
ELECTRON TRANSFER MEASUREMENT:  Different acceptor and donor will be used to 
monitor the partial electron transport. 
(i). THE HILL REACTION: Water to DCPIP reaction will be used for studying electron       
transfer of PS II. In the Hill reaction measures PS II rates by measuring amount of dye 
reduced mg chl
-1
 h
-1.
  
(ii). OXYGEN EVOLUTION: To study the effect of salinity stress on oxygen evolving 
complex, oxygen evolution rates will be measured by using oxygraph system. 
(iii). OXYGEN CONSUMPTION: The electron transfer rates of PS I (DCPIP to MV or 
DAD/TMPD to MV) will be studied by measuring oxygen consumption rates on the YSI 
oxygen monitor. 
 
STEADY STATE FLUORESCENCE: The fluorescence measurement will be done in order to 
understand the change in energy spill over due to the effect of salinity stress. Change in 
fluorescence intensity with salinity treatment in PS II is monitored at room temperature. 
 
LOW TEMPERATURE FLUORESCENCE: Any change in the energy distribution between two 
photosystem by salinity stress monitored at 77K by low temperature fluorescence 
emission spectra. 
 
FLUORESCENCE INDUCTION KINETICS: In photosynthetic system the processes by which 
the energy is dissipated are either photochemical or non photochemical. Photochemical 
processes utilize absorbed energy for photochemistry during which electron donation 
from pigment to an acceptor molecule occurs and such processes direct energy for the 
chemical work involved in photosynthesis. Non-photochemical processes dissipate 
energy from the photosynthetic apparatus in a manner that does not result in 
photochemistry. Energy is usually reemitted from the sample in form of heat and red far-
red radiation, which is known as chl a fluorescence. The fluorescence induction curves 
use to measure quantum yield of chl a fluorescence in leaves as well as in isolated 
thylakoid membranes. Fluorescence induction curves are useful for the differentiation of 
QB reducing and QB non reducing PS II centres as well as PS IIα and PS IIβ centres. The 
fluorescence induction curves will be measured by photosynthesis efficiency analyzer 
(PEA Hansatech instruments).  
 
GEL ELECTROPHPRESIS: Protein profile will be monitored during different steps of the 
experiments by SDS- PAGE electrophoresis. 
 
EPR STUDIES: Experiments will be done to explore measurement of PS II heterogeneity 
using EPR 
 
 
BIBLIOGRAPHY 
Abdelkader, AF, Aronsson, H and Sundqvist, C (2007). High salt stress in wheat leaves 
causes retardation of chlorophyll accumulation due to limited rate of protochlorophyllide 
formation.Physiologia Plantarum 130 , 157-166. 
 
Anderson, JM and Melis, A (1983). Localization of different photosystems in separate 
region of chloroplast membrane. Proc. Natl. Acad.Sci.USA 80, 745-749. 
 
Bacon Ke, (2000).Photosystem II In: Advances in Photosynthesis, Photochemistry and 
Photophysics Vol.10 pp 199-214, Dordrechet : Kluwer Acedemic Publishers. 
 
Boekema, E.I, Henny Van Roon, Calkoen,F,Bassi,R, Dekker, P(1999). Multiple type of 
association of Photosystem II and Light harvesting antenna in partially solubilized 
Photosyste II membrane. Biochemistry 38(8), 2233-2239. 
 
Gamborg, OL, Wetter, LR (1975). Plant tissue culture method. Saskatoon: Natl. Res. 
Council of Canada.  
 Ghirardi, ML and Melis, A (1988). Chlorophyll b deficiency in Soyabean 
mutants.I.Effect on photosystem stoichometry and chlorophyll antenna size. 
Bioche.Biophys.Acta 932, 130-137. 
 
Hsu, BD and Lee, YS (1991). A study on the fluorescence induction curve from DCMU 
poisoned chloroplast. Bioche.Biophys.Acta 1056, 285-292. 
 
 Joliot, P, Lavergne, j and Beal, D (1992).  Plastoquinone compartmentation in 
chloroplast Evidence for domain with different rates of photoreduction. 
Bioche.Biophys.Acta 1101, 1-12 
 
Kirchoff, H, .Horetmann, S and Weis, E (2000).  Control of the photosynthetic electron 
transport by PQ diffusion microdomain in thylakoids of higher plants. 
Bioche.Biophys.Acta 1459, 148-168. 
 
Kuwabara T, Murata N  (1982). Inactivation of photosynthetic oxygen evolution and 
concomitant release of three polypeptides from photosystem II particles of spinach 
chloroplasts. Plant Cell Physiol 23,533–539 
 
Lavergne, J and Braintais J.M.(1996). Photosystem II Heterogeneity, In: Oxygenic 
Photosynthesis: The Light Reaction (Ort, D.R. and Yoccum,C.F,eds) pp265-287 
.Dordrecht:Kluwer Acedemic Publishers. 
 
Lazar, D, Tomet, P, Ilik, P and Naus, L(2001). Determination of antenna heterogeneity of 
photosystem II by direct simultaneous fitting of several fluorescence rise curve measured 
with DCMU at different light intensities. Photosynth.Res. 68,  247-257. 
 
Melis,A and  Anderson, JM (1983). Structural and functional organization of 
photosystem in spinach chloroplast. Antenna size, relative electron transport capacity and 
chlorophyll composition. . Biochem.Biophys.Act 724, 473-484. 
 Melis, A and Duysen, LMN (1979). Biphasic energy conversion kinetics and absorbance 
difference spectra of photosystem II of chloroplasts. Evidence for two different 
photosystem II reaction centres . Photochem.Photobiol.29, 373-382. 
 
Melis, A and Homann, PH (1976). Heterogeneity of the photochemical centres in System 
II of chloroplast. Photochem.Photobiol.23, 343-350. 
 
Melis, A and Homann, PH  (1975) . Kinetic analysis of the fluorescence induction in 3-
(3,4 dichlorophenyl)-1, 1 dimethyl urea poisoned chloroplast. Photochem.Photobiol.21, 
431-437. 
 
Nedbal, L, Tritilek, M and Kaftan, D (1999). Flash fluorescence induction: a novel 
method to study regulation of Photosystem II. J.Photochem.Photobiol.B:Biol.48, 157-
157. 
Robert, M, Simillie and Robyn Nott (1982). Salt tolerance in crop plants monitored by 
chlorophyll fluorescence in vivo. Plant Physiology 70, 1049-1054  
 
Siler, B , Misic, D, Filipovic, B, Popovic, Z,Cvetic,T, Mijovic, A (2007). Effect of 
salinity on in vitro growth and Photosynthesis of common Centuary (Centauium 
erythrae). Arch. Biol. Sci., Belgrade 59(2), 129-134. 
 
Sinclair, J and Spence, SM (1988). The analysis of fluorescence induction transients from 
dichlorodiphenyldimethylurea- poisoned chloroplast. Biochem.Biophys.Act  935, 184-194   
 
Strasser, RJ (1981). The grouping model of plant photosynthesis: heterogeneity of 
photosynthetic units in thylakoids. In: Photosynthesis III: structure and molecular 
oganisation of the photosynthetic apparatus (Akoyunoglu.G, ed.) pp.727-
737.Philadelphia.Balban International Science Service. 
 
 
   
 
 
 
 
 
(Pooja Mehta) 
Candidate 
 
 
 
 
 
 
 
(Dr. Anjana Jajoo)     (Prof. Sudhakar Bharti) 
Supervisor      Co-supervisor  
School of Life Science     School of Life Science 
Devi Ahilaya Vishwavidyalaya   Devi Ahilaya Vishwavidyalaya 
Indore       Indore 
 
 
 
 
Head 
School of Life Science 
Devi Ahilaya Vishwavidyalaya 
Indore 
 
 
 
  
 
 
 
 
 
 
 
 
 
 


SYNTHESIS AND CHARACTERIZATION OF MORDENITE ZEOLITE

REDDY, SRINIVASA

Circadian heart rate and blood pressure variability in apparently healthy subjects using ABPM.

Objectives: 
1.	To study circadian variability in blood pressure (BP) and heart rate (HR), using ABPM, in a population of apparently healthy human subjects belonging to the South-Eastern India as function of age, gender, type of job, and dipping pattern.
2.	To examine the circadian variability and nocturnal dipping patterns in blood pressure (BP) and heart rate (HR) in hypertensive subjects with and without medications, using ABPM. 
3.	To evaluate circadian rhythm characteristics of blood pressure (BP) and heart rate (HR) in young healthy women as function of phases of their menstrual cycles. 
Significant Results
Chapter I: Circadian rhythm in blood pressure and heart rate in apparently healthy subjects as function of age, gender and type of job
A statistically significant circadian rhythm, gauged by a null amplitude hypothesis test, was documented for most of the studied variables, namely SBP, DBP, MAP, DP and HR, irrespective of age, gender, work type and dipping pattern. However, the rhythm detection ratio in all studied variables was lower in older subjects and non-dippers. Further, gender-linked variability was observed for circadian Mesors (24-h averages) of most of the studied variables. Values of SBP, DBP and MAP were significantly higher in males than that of females, however, vice versa was noticed for HR. Results of one-way ANOVA demonstrate that the factor dipping pattern produced significant effect on amplitudes of SBP, DBP, MAP, and DP. Amplitudes of all these variables were significantly higher in extreme dippers (ED) than those of the dippers (D) and non-dippers (ND). Further, the prevalence of non-dippers in this study was about 32%. We further validated that the characteristic of day-night variability of blood pressure and heart rate are influenced by the factors, such as age, gender, dipping pattern, body surface area, and body mass index.
On the basis of present findings we can conclude that variability in BP may be associated with factor gender, whereas nocturnal dipping in BP is independent of gender. Interestingly in the present study, about 32% subjects were non-dippers that may be an indication of higher risk of cardiovascular diseases among individuals belonging to younger generation of this region. Information on day-night variation in BP, reported in the present study, using ABPM in human subjects may be paramount in clinical diagnosis.
Chapter II: Circadian variability in blood pressure and heart rate in hypertensive subjects
Results demonstrate that the rhythm detection ratio of all studied variables, except DBP, was high in hypertensive subjects with medication as compared to subjects without medication. In both groups the ratio was low in DBP. Further, the ratio was low in SBP, DBP, and MAP in non-dippers hypertensive. Results further indicate a statistically significant higher Mesors of all studied variables, except HR, in subjects without medication than that of subjects with medication. Results of one-way ANOVA demonstrate that factor dipping pattern produced significant effect on amplitudes of SBP, DBP, MAP, and DP. Amplitudes were significantly higher in ED than those of D and ND. In this study the prevalence of non-dippers was higher among the hypertensive subjects without medications (45.45%) as compared to their counterparts (33.33%) who were on medication. Two-way ANOVA was employed to examine the effects of factors medication (with vs. without) and time (daytime vs. nighttime) on SBP, DBP, MAP, and HR. Both the factors produced a statistically significant effect on all these variables.
We concluded that the prevalence of non-dippers is remarkably higher in hypertensive subjects without medication than that of subjects with medication. However, the limitation of our study is the smaller sample size, particularly in the group of subjects without medication. Therefore, studies including larger human population of India should be carried out to strengthen the above findings. 
Chapter III: Blood pressure and heart rate variability as function of ovarian cycle in young women
Results of the present study demonstrated a statistically significant circadian rhythm for most of the studied variables, irrespective of phases of menstrual cycle. However, the rhythm detection ratio of all studied variables was low during follicular phase as compared to other phases. The factor phase did not produce any significant effect on Mesors of SBP, DBP, MAP, DP, and HR. Nonetheless, decrement in amplitudes of all rhythms was observed during follicular phase as compared to other phases. Further, day/night ratio of SBP declined significantly during follicular phase than luteal phase.
In conclusion, we report low rhythm detection ratio, reduced day/night ratio and decrement in amplitude during follicular phase. These observations may have a bearing during the diagnosis of hypertension especially in young human females. However, the above conjecture needs further validation.
Recommendations
In recent times there has been an alarming increase in the incidence of hypertension and cardiovascular diseases (CVD) in India. It is presumed that the phenomenon could be attributed to concomitant prevalence of abnormality in the blood pressure variability (BPV). Surprisingly, studies on BPV from chronobiological point of view have not yet been started in the country. Therefore, we recommend that a nation-wide program be initiated to study BPV in Indian population consisting of both healthy and hypertensive subjects.
We aspire that the results obtained and documented in this thesis will provide the scientific community with the novel data on BPV in human population of India that has never been studied earlier. In addition, a comparative account of BPV among healthy and hypertensive subjects may contribute significantly to the existing knowledge on the discipline. The outcome of the study might help in: (a) establishing the standard values of blood pressure and heart rate with special reference to gender and age, (b) assessing the relative role of predictors of hypertension, and (c) delineating plans to curb various cardiovascular diseases in India.



A STUDY OF METHYLALUMINOXANE CATALYZED HOMOGENEOUS ZIEGLER-NATTA POLYMERIZATION OF ETHYLENE AND PHOTOPOLYMERIZATION OF ACRYLIC MONOMERS

VARMA, VIVEK

Preparation of anticorrosive thin films for steel materials
Metals and alloys, depending on their mechanical, chemical and physical properties, are chosen for variety of applications.  They are used not only in the industry but also in our daily life as utensils.  These materials are used in one or the other form from complicated equipments such as engines, automobiles etc to simple toys, cooking tools etc [1-2].  
The steel is an engineering material which covers very high range of applications pertaining to different fields.    As they are exposed to aggressive environments during their usage the corrosion/surface deterioration is expected.  The phenomenon of corrosion by definition is the deterioration of material due to its environmental effect or interaction.  The cause of corrosion of these key materials has become fundamental, academic and industrial interest.  The process of corrosion has many serious consequences including economic, health, safety, technological and cultural aspects.  As far as economic loss is concerned, the costs associated with corrosion losses can be estimated with reasonable accuracy.  The typical estimations were US $275 billion dollars in 1998 and US $364 billion dollars in 2005, draining about 3.1% of the GDP from the US economy.  In India, the corrosion problem is serious than cold countries because of its tropical climate.  The direct loss due to corrosion is estimated to be approximately `250 crores per year.  The money spent on its prevention is about `50-70 crores per year.  These statistics alone justify much greater need for development of newer corrosion protection techniques.
Various protection methods are conventionally applied to prevent the materials from the corrosion.  The most common method is fabrication of metal or alloy thin films onto the surface of the material that is to be protected.  A thin film is defined as a low-dimensional material created by condensing one-by-one, atomic/molecular/ionic species of matter. The thickness is typically less than several microns. Formation of a thin film takes place via nucleation and growth process.  However, any thin film growth process involves three main steps.
1.	Production of the appropriate atomic, molecular or ionic species
2.	Transport of these species to the substrate through a medium
3.	Condensation on the substrate, either directly or via a chemical and/or electrochemical reaction; to form a solid deposit.
Thin film techniques include chemical vapor deposition (CVD), physical vapor deposition (CVD), sputter deposition, electroless / electrodeposition etc [2].  Electroless deposition and electrochemical deposition are most frequently used techniques.  The film developed on the surface can protect the material sacrificially or by just being a physical barrier depending on the nature of metal coated.  Electroless deposition is of autocatalytic kind wherein metal ions are reduced by a suitable reducing agent like sodium hypophosphite.  Electroless nickel is very frequently employed coating via this method.  The nickel alloys such as Ni-W-P, Ni-Cu-P, Ni-Co-P etc. are practiced due to their enhanced surface properties including corrosion resistance [3-6].  With respect to the process of electrodeposition, zinc and its alloy films are employed for sacrificial protection for steel materials [7-10].  
With the interest of increasing the service life of these films various addition agents are used.  However, the inclusion of the nano materials is of very high growing interest because of various exciting properties possessed by these substances.  The nano materials frequently studied are TiO2, Al2O3, WO3, CeO2, BN, WS2, MoS2, graphite, PTFE, CNTs and many more.  These incorporated secondary materials modify the characteristics of the host material which is the metal matrix and gives new functional features.      Recent advances in deposition and processing techniques of thin films have lead to the design and manipulate the materials with unique properties that are often unachievable in bulk materials. Improvements in physical and mechanical properties have been achieved by incorporating nano structured materials to the metal/alloy thin films deposited via electroless and electrodeposition methods [11-21].  

The present thesis comprises of seven chapters.
Chapter -1 contains the general and basic aspects on electroless and electrodeposition of thin films and corresponding composite coatings. Also contains models, methods, factors affecting, process conditions, applications of the composite films with literature survey.
Chapter-2 deals with the aim and scope of the work and also highlights the importance of the present investigations carried out.
Chapter- 3 gives the following methods and materials adopted for fabrication of the thin films and electrochemical studies.
	Pre-plating, plating and post-plating processes and preparation of the plating bath solutions.

	Preparation of plating baths for electroless nickel and its different alloys and electrodeposition of zinc and Zn-Ni alloy in the presence of different nanomaterials.

	Deposition of pure metal/alloy and composite coatings and measurement of thicknesses by weight gain method

	Determination of composition of coating by Energy dispersive X-ray analysis (EDX) method

	Analysis of the surface morphology using scanning electron microscopy (SEM)
	Determination of average grain size and crystallographic orientation by X-ray diffraction studies   

	Corrosion resistance studies by open circuit potential (OCP), polarization, impedance spectroscopy and voltametry

	Microhardness measurements
	
	Measurements of Friction coefficients
	Spectral studies- FTIR and UV-Vis
Chapter-4 deals with the results pertain to the preparation of electroless Ni-P composite coatings and the alloys as well, containing different nanomaterials.   There are four subchapters
	 Chapter-4.1 focuses on fabrication of electroless Ni-P and Ni-P-Halloysite nanotubes composites. The corrosion resistance of the composite coating has been established over plain Ni-P coating.  Apart from the electrochemical studies, the microhardness and friction coefficients are also studied.
	Chapter-4.2 describes the preparation and electrochemical and mechanical properties of electroless Ni-Zn-P-TiO2 composite thin films.  The performance of electroless Ni-Zn-P-TiO2 composite coating is studied in comparison with the plain Ni-P and Ni-Zn-P film and found to be better.  Also, the effect of heat treatment on the performance of the film is studied.  
	Chapter-4.3 related with the preparation and properties of electroless Ni-Cu-P-ZrO2 nanocomposite coatings.  The corrosion performance is examined by varying the different nickel-copper alloys and composites with different compositions.
	Chapter-4.4 is divided into two subchapters.
		Chapter-4.4.1 deals with the preparation of electroless Ni-W-P-WS2 composite thin films and their corrosion and mechanical performance.
		Chapter-4.4.2 deals with the fabrication of electroless Ni-W-P alloy coatings with MoS2 particles and their electrochemical and mechanical behavior.
Chapter-5 presents design, studies of zinc films by adopting electrodeposition technique and their composites with different nano particles.   
	Chapter-5.1 deals with the electrodeposition of zinc composite coatings with CeO2 nano particles.  Also the effect of cationic surfactant Cetyltriammonium bromide (CTAB) is studied.
            Chapter-5.2 focuses on the preparation of Zn-HNTs coatings.  The electrochemical studies are made for these composite films and compared with plain zinc.
Chapter-6 presents design, studies of zinc-nickel alloy films and their carbon nanotubes composites, by adopting electrodeposition techniques. 
            Chapter-6.1 focuses on electrodeposition of Zn-Ni alloy films.  The various alloys are deposited with different composition and corresponding multi walled carbon nanotubes (MWCNT) composites are prepared.  The corrosion studies of the resulting composites are made.
	Chapter-6.2 deals with the electrodeposition of Zn-Ni alloy films with highly dispersed MWCNTs.  The dispersion of the tubes is increased using different surfactants.  The UV-Vis spectroscopy is utilized to study the dispersion of CNTs.  Corrosion studies of the films are studied with well dispersed CNTs.
           Chapter-7 summarizes the main conclusions of the presented work.




SOME MOLECULAR STUDIES ON PROLAMIN AND GLUTELIN SEED STORAGE PROTEIN GENES IN BASMATI-370 (ORYZA SATIVA L.)

KHARAT, DINKAR KISAN RAO

237 
 
SUMMARY 
 
 ISOLATION AND DIVERSITY OF HALOALKALIPHILIC 
BACTERIA 
 Thirty four different haloalkaliphilic bacteria were isolated from salt-enriched 
soil collected from salt panes located in Okha (Latitude 22.20 N, Longitude 
70.05 E), Gujarat, India. Isolates were initially diversified based on their 
enrichment conditions followed by the studies on colony characteristics, gram 
and metabolic reactions, cell morphology, antibiotic profiles, enzyme 
secretion and protease amplification profile.  
 Based on 16S rRNA gene homology, three potential isolates, O.M.A18, 
O.M.E12, O.M.C28 were related to their nearest homologus and the sequences 
were deposited in NCBI as Oceanobacillus iheyensis O.M.A18, 
Haloalkaliphilic bacterium O.M.E12 and Oceanobacillus oncorhynchi 
O.M.C28. The two potential isolates, O.M.A18 and O.M.E12 were also 
analyzed by FAME confirming their status as genus Bacillus. 
 Along with classical and molecular parameters, proteolytic activity was 
considered as one of the parameter for judging the diversity of haloalkaliphilic 
bacteria. Isolates secreted alkaline proteases in the broad range of NaCl (0-
20%, w/v) and pH (8-11) and were further diversified on the basis of their 
optimum pH and salt. Alkaline protease genes were amplified by using sets of 
primers. Diversity was evident in terms of product size and concentration of 
amplicons. Size of the protease gene products varied form 0.5 to 1.2 kb. 
 The antibiogram profile was quite useful in generating the microbial diversity 
profile among the isolates. Overall, isolates were more resistant against 
Tobramycine and highly sensitive towards Oleandomycin, Cephaloridine, 
Norfloxacin, Cephradin, Erythomycin, and Cefuroxime. The biochemical and 
metabolic reactions revealed the production of catalase and oxidase by all 
isolates. Isolates were diversified on the basis of sugar utilization and none 
238 
 
produced gas in Durham’s tube. Only few isolates from O.M.6.2 and O.M.6.5 
utilized all the sugars.  
 PURIFICATION AND CHARACTERIZATION OF 
ALKALINE PROTEASES  
 The enzyme characteristics reflected that although O.M.A18 and O.M.E12 
strains were from the same site they reflected quite distinct traits. 
 The enzymes were purified to their homogeneity by single step on phenyl 
sepharose 6 FF affinity column. The purification was achieved with specific 
activities of 16945 and 24600 U/mg for O.M.A18 and O.M.E12 proteases. The 
apparent molecular mass of the proteases were estimated 35 and 29 kDa for 
O.M.A18 and O.M.E12, respectively. The effect of NaCl was assessed on 
enzyme activity and thermostability. NaCl enhanced the enzyme activity of 
purified enzyme, while activity declined in crude and partial purified stage of 
the enzyme. For both; O.M.A18 and O.M.E12 strains, there was shift in 
temperature profile with NaCl. Enzyme preparations maintained their 
activities and stability in wide range of pH; 8-11, with maximum activity at 
10. 
 One of the foremost points of the study was related to the effect of temperature 
on enzyme catalysis. The O.M.A18 enzyme had broader range of temperature 
for catalysis (37-90
o
C), while that for O.M.E12 was over a relatively narrower 
range (37-70
o
C). The high activity at 90°C for O.M.A18, would be among the 
few reports where mesophilic organisms catalyze reaction at elevated 
temperature. This feature holds novelty from diversity view point as well; 
since there may be only limited reports where such a unique and contrasting 
characteristic features are observed from the same site of isolation. 
 The partially purified and purified proteases from O.M.A18 and O.M.E12 were 
subjected to urea denaturation. Partially purified enzyme from O.M.A18 was 
exceptionally resistant against urea denaturation at 90
o
C after 48 hours with 
8M urea. The O.M.E12 enzyme, however, was relatively less resistant to urea. 
The effect of NaCl was assessed on urea denaturation; the findings revealed 
239 
 
that there was no significant change in the denaturation profile of crude, 
partially purified and dialyzed enzymes. Crude and partially purified enzymes 
were quite stable in various commercial detergents and oxidizing and reducing 
agents. 
  On the basis of phylogenetic prediction, the next most homologus sequences 
for all the proteases sequences were found with the reported extracellular 
protease sequences. The characteristic features; i.e. G+C content, PI, aliphatic 
index, hydrophobicity resembled to known haloalkaline proteases. 
 The significance of the work relates to the fact that while alkaline proteases 
are extensively studied, only few haloalkaliphilic bacteria have been explored 
towards this end. Despite the fact that the saline habitat in the study possessed 
significant bacterial diversity, it remains unexplored in terms of it 
characterization, biocatalytic potential, enzymatic characteristics, enzyme 
structure-function analysis and phylogenentic status.  
 Further, some of the novel features of the enzymes, such as stability over the 
wide range of pH and salt, catalysis and thermostability of enzyme at higher 
temperatures make them attractive candidates for future studies. The results 
are also important from the diversity viewpoint as although, both the strains 
are from the same site, they displayed distinct features of growth, protease 
secretion and enzymatic characteristics, highlighting their ecological 
significance. 
 CLONING, SEQUENCING, OVER EXPRESSION AND 
CHARACTERIZATION OF SERINE ALKALINE 
PROTEASES  
 Cloning of alkaline protease genes from the genomic DNA of O.M.A18 and 
O.M.E12 was carried out in an over-expression vector, pET21a
+ 
(Novagen). 
Vector construct of O.M.A18 and O.M.E12 were transformed in Escherichia 
coli strain BL21. The positive clones exhibiting resistance against ampicillin 
(30µg/ml) were selected for plasmid isolation. 
240 
 
 With respect to over-expression of protease genes from both isolates, the 
optimum production of recombinant proteases was at 27
o
C and 1mM IPTG. 
 The molecular weights of both proteases were estimated ˜30 kDa, which were 
quite comparable to the enzymes produced in native haloalkaliphilic 
organisms. 
 Over-expressed proteases were purified to the homogeneity by single step 
purification using nickel column exploiting His-tag property of pET21a
+
. High 
level of purified enzymes obtained as evident from the specific activity and 
yield.  
 Enzymes were characterized for physico-chemical properties, as for their 
native counterparts. In general, the recombinant enzymes were more sensitive 
towards NaCl, temperature and chemical denaturant compared to the native 
O.M.A18 and O.M.E12 proteases. 
 Molecular characterization of recombinant enzymes; distribution of amino 
acids, 3D structure analysis, protein secondary structure analysis and 
hydropathy analysis were carried out. The presence of serine residues in active 
site confirmed the serine nature of the enzyme. Further, stability of enzyme 
structure was confirmed by in-silico analysis.   
 METAGENOMICS: ISOLATION OF ENVIRONMENTAL 
DNA AND CAPTURING ALKALINE PROTEASES GENES 
 Several protocols were attempted and optimized for the isolation of 
metagenomics DNA from O.M.6.2 and O.M.6.5 the saline sites used for the 
isolation and enrichment of bacterial isolates. DNA extraction methods were 
evaluated in terms of DNA purity, yield and humic acid content. Diversity 
based analysis and amenability for molecular biology work was assessed by 
16S rRNA amplicons. Followed by amplification profile, metagenomics 
nature of amplicons was elucidated from DGGE-molecular fingerprinting 
technique. Beside, the source also provided a huge and comprehensive 
platform for capturing novel alkaline protease gene sequences. Successful 
241 
 
cloning and expression of alkaline protease from metagenomic derived 
amplicons revealed interesting features. 
 Physico-chemical and molecular characterization of metagenomic protease 
confirmed that some characteristic properties of un-cultivable clone were quite 
similar to recombinant clones earlier discussed. However, the enzyme was 
found to be more sensitive. These findings were further enriched by protein 
structure and function predictions. 
 ORGANIC SOLVENTS TOLERANCE OF NATIVE, 
RECOMBINANT AND METAGENOMIC DERIVED 
PROTEASES  
 Native alkaline proteases exhibited resistance towards organic solvents as 
compared to recombinant enzymes. While, metagenomics derived enzyme 
displayed minimum resistance against solvents.  
 Similar trends were also reflected with reference to enzyme stability. The 
results on organic solvents holds novelty as similar results exhibiting 
comparative view on varied enzyme preparations are not studied extensively.  
 Catalysis in the presence of solvents was further studied in combination of 
salt, pH and temperatures.  
 To get more insight into phylogenentic relatedness, the protease sequences of 
O.M.A18, O.M.E12 and metagenome clone O.M.6.2 were aligned with known 
protease sequences available in the database. The enzyme sequences were 
quite diverse in its sequence similarity; they were more closely related to 
enzyme sequences of other halophilic organisms as compared to enzymes 
studied in the present study. 
 The present study highlighted the properties and capabilities of the 
haloalkaliphilic bacteria and their enzymes. The properties of the O.M.A18 
and O.M.E12 native enzymes and its recombinant counterparts displayed 
unique features with its ability to function under extreme conditions. Besides, 
the biotechnological potentials, extensive studies on diversity, physiology and 
242 
 
metabolic reactions would further be supportive to understand the 
bioenergetics and adaptation strategies of these organisms as they are 
subjected to different environmental stresses. Moreover, the wide occurrence 
of the alkaline protease and their properties among cultivable isolates as well 
by soil derived clone clearly indicated that they could be used as marker for 
studying bacterial heterogeneity and population dynamics of unexplored 
habitats. The result on catalytic efficiency in the presence of organic solvents 
significantly addressed that they can be employed in non-aqueous 
enzymology. 
 
 


SOLID STATE (CHEMICAL) SYNTHESIS AND ELECTRICAL CHARACTERIZATION OF ZINC OXIDE (ZnO) BASED ELECTRONIC CERAMIC SENSORS

UBALE, RAJMOHAN

Abstract/Synopsis 
Mixed valency in a ligand bridged symmetric polynuclear set up arises when the metal 
centers exist in their different oxidation states. The intermetallic electron and charge-
transfer aspects in such molecular frameworks have significance in understanding the 
electron transfer kinetics in biological processes involving redox-active metalloproteins 
as well as in developing molecular electronics and optical devices. The extent of 
intermetallic electronic coupling in the mixed valent state(s) of polynuclear complexes 
varies primarily depending on the electronic nature of bridging and ancillary ligands as 
well as on the specific oxidation states of the metal ions. On the other hand, the use of 
redox active (redox non-innocent) bridging ligands in the complex frameworks creates 
ambiguities towards the assignment of valence and spin configurations of such 
complexes as both the metal and ligand centers can take part in electron transfer 
processes because of closeness of their frontier orbitals. In consequence precise 
determination of valence and spin situations in transition metal complexes comprising of 
redox non-innocent ligands is considered to be a formidable challenge. However, further 
complicacy develops when asymmetry is imposed on such systems through systematic 
synthetic strategies. In this context, the research work in the thesis has primarily been 
focused on the synthesis of symmetrical and asymmetrical diruthenium complexes 
encompassing redox non-innocent bridging ligands. To understand the valence and spin 
distribution processes in symmetrical dinuclear system, the following complexes 
[(acac)2Ru(μ-BL1)Ru(acac)2]n (acac: 2,4-pentanedionato; BL1: 1,2,4,5-tetraimino-3,6-
diketocyclohexane) and [(acac)2Ru(μ-BL2)Ru(acac)2]n (BL2: 2,5-pyrazine-dicarboxylato) 
have been synthesized. The extent of bridge mediated metal-metal coupling at the mixed 
valent {RuII(-BL)RuIII} state has been established by electrochemistry, 
spectroelectrochemistry (UV-vis-NIR-IR and EPR) and finally by theoretical calculations 
(DFT). The specific effect of σ-donating ancillary ligand, acac on the electronic structural 
aspects of the molecules has been scrutinized. On the other hand, to understand the 
electronic structural aspects of asymmetrical diruthenium complexes, the following 
molecular frameworks have been synthesized: [(acac)2Ru(μ-BL3)Ru(bpy)2]n and 
[(acac)2Ru(μ-BL3)Ru(Cym)Cl]n (BL3: 2,2/-azobispyridine, bpy: 2,2/-bipyridine or Cym: 
p-cymene). The valence and spin situations of the complexes in the accessible redox 
states have been authenticated by various experimental techniques and theoretical 
calculations. Finally, redox non-innocent behavior of the modified β-diketonato system 
(9-oxidophenalenone = L–) has been established in the molecular frameworks of [Ru(L–
)(bpy)2]k, [Ru(L–)2(bpy)]m, and [Ru(L–)3]n via detailed experimental and theoretical 
calculation. 
 
 
 
 
 
 
 
 
 
 


STUDIES IN HOMOGENEOUS CATALYSIS: CARBONYLATION OF ALCOHOLS AND ESTERS

Sathaye, Sangeeta S.

Abstract/Synopsis

Transition Metal Derived Catalysts for Selective Oxidation and Hydrogenation Processes

The thesis primarily focused on the development and their potential application of various homogeneous and heterogeneous catalytic systems. The introductory chapter highlights various important aspects around the transition metal catalyzed selective oxidation and hydrogenation processes which essentially develops the necessary platform of the work presented in the subsequent six chapters. In this regard, generalized catalytic protocol for the direct functionalization of terminal alkenes to corresponding dimethyl acetals and terminal aldehydes have been developed with absolute anti-Markovnikov regioselectivity by using Fe(BF4)2.6H2O as catalyst and pyridine-2,6-dicarboxylic acid as ligand. Moreover, the effect of isomeric structural features of the molecular composition of [RuII(trpy)(L)Cl] (trpy = 2,2/:6/,2//-terpyridine, L = deprotonated form of quinaldic acid, HL) on the catalytic epoxidation process have also been evaluated under mild and environmentally benign reaction condition. Isomeric identities have been established by their single-crystal X-ray structures which reveal that under the meridional configuration of trpy, O and N donors of the unsymmetrical L are in trans, cis and cis, trans configurations, respectively, with respect to the Ru-Cl bond. The origin of the differences in catalytic behavior of both the isomers has been investigated experimentally and by DFT calculations. The preferential radical mechanism for the said epoxidation process has been established via various controlled reactions in presence of radical scavengers which has also been well integrated by DFT calculations. Similar type of ruthenium complex of the molecular composition of [RuII(trpy)(L)H2O]PF6 (trpy = 2,2/:6/,2//-terpyridine, HL: Quinolinecarboxylic acid) has been synthesized and characterized properly to behave as an efficient water oxidation catalyst. It exhibits pH dependent proton coupled electron-transfer (PCET) processes which indeed facilitates the in situ formation of {RuIII-OH} and {RuIV/V=O} species as has been revealed by the Pourbaix diagram. The detailed mechanistic aspects of the said water oxidation process have also been established by experimental and DFT calculations which reveal the involvement of water nucleophilic attack (WNA). The newly designed palladium complex, [Pd(L)Cl2] where L: N,N/-bis(diphenylphosphino)-2-(diphenylphosphino)ethanamine, has been found to be effective in generating selective phenol derivatives from a wide variety of arylboronic acids in solvents with low dielectric constant using molecular oxygen through an ‘oxidase catalytic pathway’. However, in solvents with high dielectric constant values, homo-coupled product, biaryl is also formed along with phenol. The controlled experiments have revealed that the reaction is initiated by in situ formation of palladium-peroxo species and the phenol formation takes place through the reductive elimination step. Finally, catalytic activity and selectivity of [Pt15(CO)30]2– derived nano-catalysts have been evaluated and compared with the reported [Pt15(CO)30]2– derived MCM-41 and a water soluble polymer supported analogues catalysts.



Tissue Culture Studies on Peanut ( Arachis hypogaea)

G., SATYANARAYANA

Abstract
Characterization of a DEAD-box RNA helicase mutant ΔcrhR in Synechocystis sp. PCC 6803

We investigated the role of a cold-inducible and redox-regulated RNAhelicase, CrhR, in the energy redistribution and adjustment of stoichiometery between photosystem I (PSI) and photosystem II (PSII), at low temperature in synechocystis sp. PCC6803. The results suggest that during low temperature incubation, i.e., when cells are shifted from 34oC to 24oC, wild type cells exhibited light-induced state transitions, whereas the mutant deficient in CrhR failed to perform the same. At low temperature, wild type cells maintained the plastoquinone (PQ) pool in the reduced state due to enhanced respiratory electron flow to the PQ pool, whereas in  crhR mutant cells the PQ pool was in the oxidized state. Wild type cells were in state 2 and crhR cells were locked in state 1 at low temperature. In both wild type and crhR cells, a fraction of PSI trimers were changed to PSI monomers. However, in crhR cells, the PSI trimer content was significantly decreased. Expression of photosystem I genes, especially the psaA and psaB, was strongly down-regulated due to oxidation of downstream components of PQ in crhR cells at low temperature. We demonstrated that changes in the low temperature-induced energy redistribution and regulation of photosystem stoichiometry are acclimatization responses exerted by Synechocystis cells, essentially regulated by the RNAhelicase, CrhR, at low temperature.
 



NOVEL MAGNESIUM - TITANIUM CATALYST SYSTEMS FOR THE POLYMERIZATION OF UNSATURATED MONOMERS

SARAWADE, BHIMRAO DHONDIBA

SYNTHESIS AND CHARACTERIZATION OF POLYMERS CONTAINING s-TREACLE RINGS IN THE MAIN CHAIN

RAO, P. RAJA HARI PRASAD

Structural studies of silicalite and metallosilicate molecular sieves of MFI type using non-ambient powder X-ray diffraction technique

The thesis describes the hydrothermal synthesis of silicalite-1 and metallosilicate molecular sieves, various physicochemical characterization and in particular, powder XRD studies at non-ambient conditions. The thermal stability of the metallosilicate molecular sieves is checked in-situ using HTXRD technique. Studies on the negative thermal expansion behavior of silicalite-1 and metallosilicate molecular sieves are investigated by in-situ HTXRD analysis. Kinetics of template decomposition from silicalite-1 framework is also studied with in-situ HTXRD technique and the results are correlated with results obtained from thermogravimetric analysis. Orthorhombic to monoclinic phase transition studies in metallosilicate molecular sieves are carried out using low temperature XRD technique and attempts have been made to locate the Fe and Ti atoms in respective silicalite samples. This chapter present a brief summary of the work described in previous chapters and general conclusions arrived from the work. 

Chapter 1 provides a general introduction to the zeolites and MFI type molecular sieves. A brief discussion about the powder X-ray diffraction technique with different geometries and its application for various property studies such as determination of crystallinity, crystallite size, phase analysis qualitatively as well as quantitatively, determination of unit cell parameters and structure determination is given in this chapter. Rietveld refinement technique is outlined in this chapter with its principle and various parameters that can be monitored during the Rietveld analysis and information about GSAS (General Structure Analysis System) software used for Rietveld refinement is described briefly. This chapter also describes the applications of powder X-ray diffraction technique for the characterization of zeolite molecular sieves and summarizes the available literature on it. Different applications viz., isomorphous substitution of ‘T’ atoms by heteroatoms, location of ‘T’ atoms in the framework of zeolites and orientation of organic molecules inside the zeolite channels are outlined. The in-situ powder X-ray diffraction technique used at high temperature and low temperature conditions is discussed. The applications of high temperature powder X-ray diffraction in kinetics of solid-state phase transitions, determination of thermal expansion coefficients and reaction kinetics are described briefly. The applications of LTXRD for the study of materials, which are liquids or gases at room temperature, charge density analyses, phase transitions and thermal expansion coefficients, are outlined. It also elucidates the physical principles of other analytical techniques used for the characterization of metallosilicate molecular sieves such as UV-vis and MAS-NMR spectroscopy. This Chapter is mainly a review of the literature on the above said topics. 

Chapter 2 describes the procedure for the hydrothermal synthesis of metallosilicate molecular sieves with different Si/M (M=Fe, Ti and Zr) ratio in synthesis gel. The characterization of the metallosilicate molecular sieves by different techniques viz., X-ray diffraction, TG-DTA, FTIR, SEM-EDAX, DRUV-vis spectroscopy, 29Si MAS NMR etc. is described in this chapter. Theory and experimental procedure employed for each technique is discussed in this section. This chapter also describes the Rietveld refinement technique employed to extract the unit cell parameters from the powder XRD patterns collected at room temperature. The expansion in unit cell parameters as a result of isomorphous substitution of Si by Fe, Ti and Zr is discussed in this chapter. Determination of Si/M ratio in the framework, with the experimental and theoretical values of unit cell volume using powder XRD data is also discussed in this chapter. The data from the XRD supports the isomorphous substitutions of Si by Fe, Ti and Zr but some of the portion remain as extraframework or other than tetrahedral coordinated atoms, which is concluded by the results of other spectroscopic techniques.

Chapter 3 describes in-situ high temperature X-ray diffraction (HTXRD) studies on silicalite-1 and metallosilicate molecular sieves (FeS-1, TS-1 and ZrS-1) with MFI structure (Si/M = 50) to study the thermal stability and thermal expansion behavior as a function of temperature in the temperature range 298–1623 K. The structure of silicalite-1 and FeS-1 (monoclinic and orthorhombic phase of silicalite-1 and FeS-1) collapsed at 1173 and 1323 K and transformed to α-cristobalite phase. This transformation is irreversible. The structure of TS-1 and ZrS-1 are stable even upto 1623 K. The thermal behavior of trivalent and tetravalent cations in the MFI framework depends on the role of counter cation present in the former. The sharp negative thermal expansion seen in all the samples is anisotropic, with relative strength of contraction in ‘a’ axis is being greater than ‘b’ and ‘c’ axes in TS-1 and ZrS-1 while contraction is less along ‘a’ axis in FeS-1. Lattice thermal expansion coefficients (av) in the temperature range 298–1023 K are -6.75x 10-6 K-1 for silicalite-1, -12.91x 10-6 K-1 for FeS-1, -16.02x 10-6 K-1 for TS-1 and -17.92x 10-6 K-1 for ZrS-1. The highest lattice thermal expansion coefficients (av) obtained are -11.53 x 10-6 K-1 for FeS-1 in the temperature range 298–1173 K, -20.86 x 10-6 K-1 for TS-1 and -25.54 x 10-6 K-1 for ZrS-1, respectively, in the temperature range 298–1623 K. The strength of contraction increases with increase in framework density in the three metallosilicate molecular sieves studied. Tetravalent cation substituted metallosilicate molecular sieves show very high thermal stability, while trivalent cation substituted samples are structurally/thermally less stable. The results of the thermal stabilities are discussed and compared with the theoretical studies in literature. 
Detailed study of the negative thermal expansion behavior of these materials is carried out using Rietveld refinement of HTXRD patterns collected in the temperature range 373-673 K for silicalite-1 sample. The bond angles and bond distances are determined from the Rietveld refinement data to look for any structural changes taking place as a function of temperature. The Si-O bond distances, Si-O-Si bond angles and Si-Si non-bonding distances are calculated from the Rietveld refinement results and proposed the intrinsic NTE mechanism. The Si-O bond distances seem to play no role in the NTE, as there is no change in their distances as a function of temperature. The transverse vibrations of the Si-O-Si bridging oxygen atoms decreases the Si-Si non-bonding distances which causes the contraction in the material as a function of temperature. The observed decrease in Si-Si non-bonding distances with the constituent Si-O bond distances remaining constant, supports the correlation that the transverse vibrations of the bridging oxygen atoms in the structure are responsible for the NTE in this material. The chapter also describes the HTXRD studies carried out on the metallosilicate molecular sieves TS-1, FeS-1 and ZrS-1 of different Si/M ratio. All the samples exhibit negative thermal expansion on heating in the temperature range 373-773 K and the strength of NTE increases with decreasing Si/M ratio

Chapter 4 describes the non-isothermal reaction kinetics of the template decomposition from the as synthesized silicalite-1 framework which has been carried out using thermogravimetric analysis (TGA) and high temperature powder X-ray diffraction (HTXRD) techniques and are compared for the first time in literature. Template burning (or calcinations) of the as-synthesized material is a key step in the activation of a zeolitic material for its application as a catalyst or as a membrane. HTXRD experiments are carried out at different heating rates such as 1, 3 and 5 C/min, while TG were measured at 10, 15 and 20 C/min. The kinetic analysis is carried out using the plots of conversion factors (α) versus temperature. Conversion factor for template decomposition is calculated using two methods viz., % weight loss from the thermogravimetric analysis and changes in the intensities of the Bragg reflections 101/011 and 200/020 in the HTXRD patterns scanned at different temperatures (range 298-823 K). A kinetic analysis based on the % weight loss from the TG data and the intensity change of the low angle peaks in the HTXRD patterns is performed using Kissinger and Flynn–Wall–Ozawa methods. According to the Kissinger method, the slope of the plot of (ln(β/Tp2) versus 1/Tp is proportional to Ea, according to the following equation: β is the heating rate, R is the molar gas constant and k is the reaction rate constant. The calculated apparent activation energy for template decomposition in air is 129 kJ mol-1 (TG) and 125 kJ mol-1 (HTXRD) using the Kissinger method. The alternative method by Flynn-Wall-Ozawa (where the slope of the plot of lnβ vs. 1/T is proportional to Ea) is also used to calculate the activation energy using the equation. Apparent activation energy for template decomposition in air is 123 kJ mol-1 (TG) and 124 kJ mol-1 (HTXRD) by Flynn–Wall–Ozawa approach. An advantage of this method is that any changes in the mechanism of the reaction are immediately apparent as variations in the slope for different α. The reaction order is determined by Avarami equation using the method of Kennedy and Clark. Avarami equation is successfully used to fit the kinetic data. The value for reaction order is nearly equal to two for all the heating rates. The reaction order determined using the method of Kennedy and Clark, is close to 2 for both the experimental data (TG and HTXRD), which rule out the diffusion limitation. The apparent activation energy Ea is determined to be 125 kJ mol-1, which is in good agreement with the results reported in the literature. The second order of template decomposition can be attributed to the fact that TPA+ is positioned in two different orientations inside the silicalite-1 framework. The second order of template decomposition can be attributed to the fact that TPA+ is positioned in two different orientations inside the silicalite-1 framework; the so called TPA1 and TPA2 having an occupancy of 0.6 and 0.4, respectively. Part of the TPA+ ion in the sinusoidal channel is in the folded conformation, while extended conformation is present in the straight channel. Hence, there may be different mechanism present for template decomposition in these two physically different environments of the template molecule.
Thermogravimetric analysis is used extensively in the past to study the decomposition of the template cation, TPA+. The weight loss of a sample as a function of temperature is recorded using thermogravimetric analysis. Over the temperature range from 573 to 823 K, the decomposition of template molecules takes place. The loss in weight corresponds exactly to the TPA+ content of the zeolite, for e.g. four template molecules per unit cell (12% weight loss). 
Rietveld analysis has been carried out to look in to the structural changes taking place in the framework as a function of temperature. Template removal occurs with a contraction in unit cell dimensions. There is a large contraction in the unit cell volume as the template is removed from the framework and it is attributed to the transverse vibrations of Si-O-Si bridging oxygens as a function of temperature. In addition, the unit cell dimensions approach the same values regardless of heating rate.

Chapter 5 describes the investigation of the phase transition temperature for various TS-1 and FeS-1 samples (concentration of Ti and Fe vary) to determine the effect of Si/M ratio on the phase transition temperature. These metallosilicate molecular sieves show orthorhombic phase in Pnma space group at room temperature whereas at low temperature the structure is transformed to monoclinic symmetry in space group P21/n. The powder X-ray diffraction patterns were recorded at various temperatures from 298 K to 80 K at regular intervals. The scans in the 2 range 22 to 26° were monitored for the phase transition from the splitting of 1 3 3 reflection of the orthorhombic symmetry into the –3 1 3 and 3 1 3 reflections of the monoclinic symmetry. Attempts have been made to locate the heteroatoms in the silicalite framework for TS-1 and FeS-1 samples at low temperature (below the room temperature and above the temperature of phase transition) by using the Rietveld refinement technique. The experiment to locate the T atoms in the metallosilicate molecular sieves are extremely challenging due to the low concentration of T atoms. Contribution from the thermal atomic displacement parameters should be necessarily lower and hence XRD patterns at low temperature are collected to locate the ‘T’ atoms. The results of the location of heteroatoms are also discussed and compared with the results in literature.




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