Open Access Repository of Indian Theses

MANDAL , DEENDAYAL

Abstract
 
Chapter 1
Human being, with a very short life span, has expanded his conception of the duration
of time, in order to study his own planet earth and the various processes that are
continually operating on it through geologic times. The coda of this geologic time,
represented by the Quaternary period, is accorded with the highest interest and
significance due to its proximity to the present age and most importantly, it is
characterized by the emergence and development of the hominids.
The Quaternary period has witnessed series of frequent and rapid climatic changes
along with epeirogenic movements, which in turn had continually modified the
surface expressions of the continents. This second period of Cenozoic Era represents a
total span of about 1.8 Ma and has been sub-divided into Lower Pleistocene (1.8 –
2
0.75 Ma), Middle Pleistocene (750 – 125 Ka), Upper Pleistocene (125 – 10 Ka) and
Holocene (10 – 0 Ka) comprising thick accumulation of sedimentary deposits.
The south Gujarat alluvial plains, occupying a significant position along the western
margin of Indian sub-continent, comprise considerable accumulation of these
Quaternary sedimentary deposits. Owing to its uniqueness from the point of view of
geomorphic diversity and climatic significance, these alluvial plains represent one of
the most important and interesting segments of the west coast of India. Climatically,
the south Gujarat terrain is represented by an overall humid tropical type, with a good
seasonal rainfall and the presence of perennial river systems, characterized by a
westward flow. These river systems, which form one of the most important geologic
agents on the earth, had continually sculptured the landforms of south Gujarat terrain.
The northern portion of the south Gujarat alluvial plains is demarcated by the lower
Tapi river basin (LTRB) i.e., the study area, which is characterized by the presence of
Quaternary deposits. The LTRB has its own distinct identity in terms of sedimentation
patterns vis-à-vis active tectonism and climatic vicissitudes. It is characterized by
variety of landscape as well as tectonic features along with a well-developed drainage
network. The LTRB also comprises an interesting assemblage of well-preserved
palaeo-geomorphic landforms and tectonically accentuated features. However, a
glance through the available literature has revealed that the LTRB has received less
attention from the earlier workers and not much work is reported, except meager
information merely highlighting the geomorphic setup of the south Gujarat as a whole.
Hence an attempt has been made through the present study, to comprehend the role of
various geological processes operating in the LTRB and also to envisage the overall
sedimentation history vis-à-vis the role of neotectonism during the Quaternary times.
Although these geological processes seem to be infinitely slow today, but over a
3
period of several thousands of years, have resulted in the present-day landscape
configuration.
The LTRB is located on the threshold of semi-arid and humid climatic domains and
its northern and sout hern boundary is distinctly demarcated by the presence of Kim
and Purna rivers respectively; whereas the trappean highlands and the Arabian sea
delimits its eastern and western boundary respectively. The LTRB constitutes a thick
pile of Quaternary sedimentary deposits resting unconformably over the Tertiary and
Trappean basement. Tectonically, the LTRB forms a part of the two major pericontinental
rift basins i.e., the Cambay and the Narmada rift basins, which seems to
have governed its tectonic setup. It is also evident that the episodic reactivation along
these tectonic elements coupled with the climatic perturbations, have controlled the
sedimentation pattern and geomorphic evolution of the landforms in the LTRB.
Owing to this, a multi-disciplinary approach has been adopted in the present study to
understand the intricacies related to the Quaternary sedimentation in the LTRB. The
combined evidences from the extensive field observations, sub-surface bore-hole data,
precise laboratory investigations and various other persuasive studies, have helped in
the reconstruction of the basin configuration of the LTRB, its sedimentation history
and the governance of tectonism over it, during the Quaterna ry times.

Chapter 2
The study area forms an integral part of the three major tectonic elements bordering
the western margin of India such as the Cambay, Narmada and Tapi, comprising
variety of geological formations, ranging in age from Proterozoic to Recent. Although
majority of the geological formations are reported in the subsurface data, however the
exposed outcrops belong to Deccan Traps, overlain by Tertiary and Quaternary
deposits.
The geological aspects of the lower Tapi river basin ha ve received less attention from
the earlier workers. Blanford (1867) gave the earliest account on the geological
attributes in his memoir on the “Geology of the Taptee and lower Nerbudda valleys
and some adjoining districts”, followed by Foote (1898), who had described the vast
10
alluvial stretch between Kim and Tapi rivers. Various other contributions related to
the geological aspects, directly or indirectly pertaining to the LTRB, have been
provided by several workers associated with institutes such as, Geological Survey of
India, Directorate of Geology and Mining, Government of Gujarat, Oil and Natural
Gas Corporation Ltd., National Geophysical Research Institute, Hyderabad and the M.
S. University of Baroda.

Chapter 3

This chapter is mainly concerned with the systematic description and interpretation of
the present day as well as the past geomorphic features in the study area, taking into
account the field observations aided with remote sensing techniques, and ground
checks. According to Davis (1909), the geomorphic processes and the prevailing
litho-tectonic framework along with the climate, plays a significant role in controlling
the successive sculpturing of the landscape configuration of that region, with
increasing time.
The south Gujarat area in general and the study area in particular aptly represents an
assemblage of diverse geomorphological features exhibiting a complex interplay of
various erosional as well as tectonic processes (Scheidegger & Padale, 1982; Vyas,
33
1984). Earlier workers (Radhakrishna, 1965; Scheidegger & Padale, 1982; Ollier &
Powar, 1985; Alavi, 1990) have attempted to describe the various geomorphic
attributes of south Gujarat terrain; however, no specific studies have been made so far
as the study area is concerned. The present study is aimed to bring out systematic
analyses of these features and the factors responsible for their genesis with the help of
field observations and satellite data (IRS-1C LISS III, 1998) (Fig. 3.1).

Chapter 4
“Neotectonism” has been one of the most familiar terms amongst the geoscientists,
engaged in unraveling the Neogene and Quaternary history, throughout the globe.
This term was first introduced in the literature by Obruchev (1948), to describe the
study of the young and recent movements taking place at the end of the Tertiary and
the first half of the Quaternary periods. Stewart and Hancock (1987) have defined
“Neotectonism as a branch of tectonics concerned with the understanding of earth
movements that occurred in past and are continuing in the present day”. Mukherjee
(1980) has defined “Neotectonics as the study of structures of the earth’s crust created
by the latest movements, having a distinct influence on the formation of present day
surface relief”. According to Slemmons (1991), “Neotectonics can be broadly
68
described as tectonic events and processes that have occurred in post-Miocene
times…”. Although neotectonism has been defined in various ways by several
workers, however Morner (1990) is of the opinion that the neotectonic phase starts at
different times in different places, depending on the tectonic regime.
The south Gujarat in general and the study area in particular, have not remained an
exception as far as neotectonic activities are concerned. The studies carried out in
these areas, have clearly demonstrated the influence of these activities, persisted
throughout the Quaternary times. Owing to the potential nature of the study area and
its surroundings in terms of the neotectonic activities, the present study is aimed to
understand the role of these activities and their signatures, well imprinted on its
geological and geomorphological expressions, mainly based on the field observations
and sub-surface data. The following paragraphs furnish a detail account of the
neotectonic signatures observed within the study area and their vital role in the
Quaternary history of the terrain.

Chapter 5
The contents of this chapter forms a part of field investigation, dealing with the study of the
Quaternary sediments that occupy the various cliff-sections exposed all along the major river
channels such as the Kim, Tapi and Mindhola as well as their tributaries, in the study area, in
order to bring out a systematic record of the stratigraphy, spatial distribution pattern and
sedimentary characteristics. The sedimentological studies include the identification of various
lithological units, their vertical and lateral relations hips with the adjacent units and their
lateral mapping, in order to understand their continuity and extent. Along with the mapping
of the lithological units, due emphasis has been given on the identification of primary as well
as secondary structures wherever observed. The representative samples of each of the
lithological units have been collected by means of trenching of the entire cliff-sections, for
further laboratory investigations.
88
Although, the sedimentological studies have been attempted for the lithological units exposed
along the cliff-sections, however, an attempt has been made to incorporate the bore-hole
information so as to understand the sub-surface details of these sediments and to bring about
the overall Quaternary basin configuration.
The succeeding pages of this chapter, provides the location-specific sedimentological deta

Chapter 6
The contents of this chapter reflects the observations pertaining to the detail laboratory
investigations carried out for the representative samples of the various lithological units
exposed within the Quaternary successions along the Kim, Tapi and Mindhola river valleys.
The various laboratory techniques adopted in the present study includes the hand specimen
observations under binocular microscope, granulometric analyses of arenaceous sediments,
X-ray diffraction studies for mineralogical details, scanning electron microscope studies for
surface morphological attributes, geochemical investigations for trace element concentrations
and micro-palaeontological analyses. The results obtained from the laboratory investigations
have been interpreted and integrated to understand the influence of various processes during
129
the deposition of Quaternary sediments within the study area. The succeeding pages of this
chapter provide the detailed account of each of the laboratory techniques employed in the
present work and their results so obtained.

Chapter 7
The preceding chapters of the present study have provided an elaborate information of
the observations made on the nature of exposed Quaternary sediments of Lower Tapi
River Basin (LTRB), their lateral as well as vertical distribution, field characteristics,
geomorphic and tectonic vicissitudes and laboratory details. The contents of this
chapter mainly focuses on the integration and interpretation of the results to bring out
a comprehensive picture of the sedimentation history during Quaternary – Holocene
times, encompassing pre-Quaternary basin configuration, stratigraphy, depositional
environments of the sediments vis-à-vis the role of tectonism, sea level fluctuations,
climatic changes and geomorphic processes.





English

Fungal Mediated Biotransformations Of Organic (Carbonyl Group) And Inorganic Compounds.

NASARE, KANCHAN SHASHIKANT

Abstract



ABSTRACT

Natural products are gaining enormous importance and attention from chemists and pharmacologist to discover biologically lead molecules. In Natural Product Chemistry, marine organisms have proven to be a rich source of unusual secondary metabolites. Marine environment is a huge potential resource for Natural Products. In Ocean, the circumstances are quite different from those on the land. Ocean environment is a closed system with high salinity, high pressure and relatively constant temperature. Therefore marine organisms are expected to produce quite different compounds as compared to those produced by terrestrial organisms.
       Marine Natural Products chemistry originated in 1970's and developed rapidly in 1980's and matured in the last decade. During last three decades a great number of new natural products are isolated from various organisms.     
The aim of research projects concerned with Marine Natural Products may be
1.	To find out the novel compounds those are useful as leads for Drug Development.
2.	To provide good tools for basic research of life sciences.
3.	To study the roles and biological functions of secondary metabolites in the life of marine organisms.    
Inspired by the trends in the modern marine chemical investigation, the author has made a modest effort to investigate on marine organisms. In this thesis the results of chemical investigation of a sponge Dendrilla cactos, a coelenterates Zoanthus sp. and unidentified soft coral have been discussed. Chemical investigation of above marine organisms has lead to the isolation of some new and known compounds and synthesis of natural product analogues. The biological activities of some of compounds have been presented. The development of some new synthetic methodologies during the present studies has been described. To discuss all the investigations systematically, the thesis has been divided into three chapters. 

CHAPTER-I
Chemical investigation of the sponge Dendrilla cactos:
As part of our ongoing investigation on bioactive compounds from marine organisms we describe the isolation of bastadins alkaloids from the sponge Dendrilla cactos. The freshly collected organism was made into small pieces and preserved in excess MeOH at the collection site. Later the initial aqueous MeOH extract was decanted, the organism (1 Kg wet wt.) was reextracted with fresh CH2Cl2: MeOH (1:1, 3 x 1.5 L). The combined extract was concentrated under reduced pressure to a predominantly aqueous extract, which was extracted into ethyl acetate. Concentration of ethyl acetate extract yielded dark brown gummy mass (4gm). The water extract was lyophilized and dissolved in the methanol and filtered off and concentrated of methanol extract under reduced pressure to yielded 2.5 gm of crude extract. Both extracts were mixed and subjected for gel filtration chromatography (Sephadex LH-20, 1:1 CH2Cl2-MeOH, 47 mm x 820 mm) followed by silica gel column chromatography eluting with chloroform to methanol and followed by preparative (2.1cm x 25 cm) reversed phase HPLC column (C-18) using MeOH : H2O  (80:20) as eluents to yield nine pure compounds DC-1 to DC-9. DC-1 and DC-2 are new bastadin alkaloids and DC-3 to DC-8 are known bastadin alkaloids. DC-9 is a common sterol. Many of these bastadin alkaloids are bioactive. A literature survey revealed that the genus Dendrilla cactos has yielded several diterpenes, lamellarin alkaloids, and amino acid derivatives. Further, so far bastadins are reported from the sponges Ianthella sp. and Psammaplysilla purpurea. This is the third species to be reported for the presence of bastadins in the sponge Dendrilla cactos. The structure of compounds were established by interpretation of their NMR (1H, 1H-1H COSY, NOESY, ROSEY, HMQC and HMBC) and mass spectral data. 




CHAPTER-II

Chemical investigation on Zoanthus sp.
During our search for biologically active secondary metabolites from marine organisms we have investigated a zoanthid Zoanthus sp. collected from the Mandapam coast during November 2003. A literature survey reveled that the genus Zoanthus yielded previously various types of secondary metabolites, which include zoanthamine nor-zoanthamine alkaloids and ecdysones. 
The freshly collected zoanthus sp. specimens (1/2 kg wet weight) were soaked in MeOH at the site of collection until workup. The initial methanol extract was decanted and the material extracted with fresh 1:1 CH2Cl2 : MeOH (3x1L) at room temperature. The combined extract including initial methanol extract was concentrated under reduced pressure to give a predominantly aqueous extract and extracted into ethyl acetate. The ethyl acetate extract was concentrated to give a dark brown gummy mass (4gm). The water extract was freeze-dried and added methanol. The methanol soluble portion concentrated under reduced pressure to give brown crude extract (2g). Both the methanol and ethyl acetate extracts were combined and subjected to gel filtration chromatography (Sephadex LH-20, 1:1 CH2Cl2-MeOH, 47 mm x 820 mm) followed by silica gel column chromatography eluting with chloroform to methanol to yielded seven pure compounds ZA-1, ZA-2, ZA-3, ZA-4, ZA-5, ZA-6 and ZA-7. In these three are ecdysones ZA-1, ZA-2 and ZA-3 (Compound ZA-1 is first report from the marine source), ZA-4 ergo sterol, ZA-5 thymidine, ZA-6 uracil and ZA-7 ergost-4, 24 (28) - dien-3-one.





	


Effect of ecdysteroids on growth and metamorphosis of II instar larvae of Aedes aegypti.

S.No	Activity	Control*		20- Hydroxyecdysone		24, (28) dehydromakisterone A
				(ZA-2)		(ZA-1)
				Conc. in ppm		Conc. in ppm
				0.01	0.025	0.05	0.1		0.01	0.05	0.1	0.25
1	Percent mortality after 24 hours	0		4	19	25	36		7	21	27	38
Pupal formation from the survival of larvae
2	Total no. of survived larvae	100		96	81	75	64		93	79	73	62
	Pupation	% Normal	1000		63.54(61)	38.27(31)	17.33(13)	7.81(5)		55.91(52)	39.24(31)	23.29(17)	3.23(2)
		% Abnormal	00		34.38(33)	54.32(44)	68.00(51)	67.18(43)		39.78(37)	51.89(41)	64.38(47)	69.35(43)
		% Mortality	00		2.08(2)	7.41(6)	14.67(11)	25.00(16)		4.30(4)	8.86(7)	12.32(9)	27.41(17)
Adult emergence based on the number of survived pupae
3	Total no. of pupae (formed)	100		94	75	64	48		92	74	66	47
	Adult Emergence	% Normal	1000		59.57(56)	49.33(37)	14.06(9)	6.25(3)		66.30(61)	41.89(31)	19.70(13)	4.26(2)
		% Abnormal	00		18.09(17)	21.33(16)	32.81(21)	14.58(7)		21.74(20)	25.68(19)	18.18(12)	6.38(3)
		% Non- emergence	00		22.34(21)	29.33(22)	53.13(34)	79.17(38)		11.96(11)	32.43(24)	62.12(41)	89.36(42)

*With out Toxicant
The initial sample size of each concentration is 100 numbers.
Number in parenthesis indicate the sample size 

Chemical investigation of an unidentified soft coral (IIC-556):

The soft coral (IIC-556) was collected at Gulf of Mannar, Tamilnadu and was preserved in methanol at the site of collection. The initial inspection of 1H NMR spectrum of 1:1 dichloromethane : methanol extract revealed that the soft coral is rich of cembranoid and africanene derivatives. The silica gel column chromatography of 1:1 DCM: MeOH extract afforded five known compounds US-1, US-2, US-3, US-4 US-5 and US-6. The studies were confirmed by interpretation of spectral data.





Chemical transformation of ∆9 (15)-africanene and evaluation of antibacterial activities:

∆9 (15) Africanene, a medicinally important natural sesquiterpenoid has been subjected to chemical transformations. Various aromatic, hetero aromatic and aliphatic esters were prepared. Their structures were established by means of IR, NMR and mass spectral data. The derivatives were evaluates for their anti bacterial activities comparing with standard antibacterial agents like Penicillin and Streptomycin.




Table-1 Minimum Inhibitory concentration (MIC) of hydroxy monoesters of   ∆9 (15) africanene

 														
	9a	9b	9c	9d	9e	9f	9g	9h	9i	9j	7	8	Penicillin	Streptomycin
Bacillus subtilis	6.25	12.5	50	50	12.5	50	12.5	12.5	25	25	50	12.5	1.562	-
Bacillus sphaericus	6.25	12.5	25	25	12.5	25	12.5	12.5	25	12.5	25	12.5	3.125	-
Staphylococcus aureus	12.5	6.25	25	25	25	12.5	25	12.5	12.5	12.5	25	12.5	1.562	-
Pseudomonas aeruginosa	-	-	-	-	-	-	-	-	-	-	-	-	-	3.125
Klebsiella aerogenes	25	12.5	25	25	12.5	50	25	50	50	25	25	50	-	1.562
Chromobacterium violaceum	6.25	12.5	25	25	6.25	25	25	50	12.5	25	12.5	50	-	3.125
Values are indicated in g/ml.

CHAPTER-III

Development of new synthetic methodologies:  

	Development of new synthetic methodologies is an important subject in organic chemistry. Synthesis involves expensive reagents and catalysts, which are not easily available. To replace all such reagents and catalysts different improved processes have now been discovered to carry out the reactions efficiently and conveniently with readily available inexpensive materials. Modern methodologies are also concerned with the yield and selectivity of the products.     

1) Bismuth triflate catalyzed conjugate addition of indoles to , -enones

Reaction of indoles with electron deficient olefins under the influence of
bismuth triflate has been studied at ambient temperature and affords the
corresponding 3-alkylated indoles in excellent yields.
Tetrahedron Lett. 2003, 44, 6260



2). Synthesis of Dihydropyrimidinones: An Improved Conditions for the Biginelli
     Reaction.
      	A mild and selective method for the synthesis of dihydropyrimidinones   using CBr4 in methanol in the Biginelli three component cyclocondensation reactions has been developed. Yields are significantly higher than utilizing classical Biginelli reaction conditions.
      Heterocyclic Communications 2002, 8(5), 459


3)    HY-Zeolite Catalyzed Synthesis of Bis-Indolyl methane's: 
        HY-Zeolite is found to be a versatile catalyst for the electrophilic substitution  
      reaction of indole with carbonyl compounds to afford the bis indolyl methenes in   
      good yields. Indole and its derivatives are important intermediates in organic  
      synthesis in view of their pharmacological activities. During the past few years a  
      large number of natural products containing bis indolyl methane's, bis indolyl  
      ethane's have been isolated from marine sources. Some of these have been found  
      to  have interesting biological activity. 
       Synthetic Communications 2003, 33, 3681



4) Amberlyst-15-Catalyzed Efficient Synthesis of 1,1-Diacetates from Aldehydes

Amberlyst-15 was found to be an efficient catalyst for acylation reaction of  
    aldehydes with acetic anhydride at room temperature in dichloromethane.
    Synthetic Communications 2003, 33, 1531 






























































































































































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i





Morphological Characterization and DNA fingerprinting of a Prasinophyte flagellate isolated from Kerala coast

NITURE, SURYAKANT K

Abstract

The thesis entitled “Studies on the Synthesis of Selected Isobenzofuranone and Angucycline Natural Products” describes the total synthesis of two naturally occurring 3-(2,6-dihydroxyphenyl)isobenzofuranones, namely isopestacin (10) and cryphonectric acid (11), and synthetic endeavors towards A and/or B-ring non-aromatic angucyclinones.
DBU-catalyzed aldol type reaction of 1,3-diketones (e.g., 2) with phthalaldehydic acid (1) has been developed for the synthesis of 3-substituted isobenzofuranones (e.g., 3).

These condensed products have been aromatized by treatment with Hg(OAc)2-NaOAc system to yield 3-(2,6-dihydroxyphenyl)isobenzofuranones. The methodology has been applied to the total synthesis of isopestacin (10) and cryphonectric acid (11).

A carbonyl-ene reaction of 2-methallylated anthraquinone aldehyde 16 has been proposed for the synthesis of angucyclinones. But attempted synthesis of 16 via allylation of anthraquinone 12 provided a tetracyclic compound 13. In another attempt, oxidation of 14 produced an unusual product 15. Synthesis of 16, attempted by a variety of routes was elusive.


A new method has been developed for the preparation of ortho-allylated phenols (e.g., 18) at room temperature. It involves an unusual reaction of quinol ethers (e.g., 17) with allylindium reagents.

Application of the Diels-Alder reaction of an in-situ generated o-quinone dimethide, prepared by the pyrolysis of sufone 22, with a dienophile was envisaged for the regioselective synthesis of highly substituted non-aromatic A-ring of angucyclinones. To this end, preparation of compound 22 via naphthoquinone monoketal 20 could not be achieved. Phenyliodonium diacetate (PhI(OAc)2) oxidation of phenol 19 failed to give any product. On the other hand, oxidation of 23 furnished a thiophene fused compound 24 instead of sulfide analog of 20.

The attempts on trapping the intermediate 27 of Hauser annulation, obtained by condensation between 3-phenylsufonylphthalide 25 and 4-fluorocyclohexa-2,5-dienone 26 are described. Though the intermediate could not be isolated, the results established that fluoro derivative 26 more readily participates in Hauser annulation in comparison to the corresponding 4-methoxy analog. The method has been shown to be useful for the synthesis of anthraquinone-steroid hybrids, fused indoloquinones and fluoroanalogs of angucyclinones.


A DBU promoted aldol cyclocondensation of hydroxyisobenzofuranone with cyclohexane-1,3-dione, followed by aromatization has resulted in the first short synthesis of isopestacin

Quinol ethers and quinone monoketals are shown to undergo formal anti-Michael addition reactions with allylindium reagents at room temperature to give only ortho allylated phenols in good yields.

4-Fluorocyclohexa-2,5-dienones are introduced as new acceptors for the Hauser annulation. In cases where the corresponding methoxy analogs fail to undergo annulation, the former smoothly do so with phenylsulfonylphthalides to furnish anthraquinones in good yields. Steric effects are implicated to explain the inertness of the methoxy analogs

A review on the recent advances on Hauser annulation, an anionic [4+2] cycloaddition

DBU catalyzed condensation of phthalaldehydic acids and 1,3-diketones have been developed to be a general method for the synthesis of 3-substituted phthalides. This method, in combination with mercuric acetate mediated oxidative aromatization has been utilized for the regiospecific synthesis of isopestacin and cryphonectric acid



BIOCHEMICAL ECOLOGY OF FUSARIUM MONILIFORME ISOLATED FROM AN ESTUARINE ENVIRONMENT

SETH, ROHIT

Abstract
Communication between the brain cells is fundamental to the mechanism by which neurons generate brain function. This communication generally occurs by the release of chemical substances called neurotransmitters. Discovery of short chain of amino acids that are active in nervous system and later termed as 'neuropeptide', further revolutionized the study of neural compartment. In some cases they transmit signals between nerve cell and in some cases also serve as hormone. 
Arginine vasotocin (AVT) and mesotocin (MT) are avian neurohypophysial peptides that belong to 16 membered family of neurohormones found among vertebrates. These two neurohormones are analogous to mammalian Arginine vasopressin (AVP) and Oxytocin (OT) respectively. Vasopressin and oxytocin turn out, to be an example of a family of peptides whose structure is well conserved across a wide range of species. Unlike mammals where both these hormones perform individual function of water balance and parturition, in birds, AVT performs dual function of antidiuretic and oxytocic hormone, while role of MT remains unclear. AVT is synthesized in the hypothalamic paraventricular (PVN) and supra optic nucleus (SON), transported to posterior pituitary through hypothalamo-hypophysial tract and from there these neuropeptides are secreted in the peripheral circulation in response to appropriate physiological stimuli. 
Availability of AVT mRNA sequence of chicken has revolutionized the study of involvement of AVT in regulation of various physiological events at the genetic level. Recent cloning of two vasotocin receptors designated as VT1 receptor; expressed in brain and shell gland and VT1a receptor expressed in the pituitary has further facilitated the study of AVT and its physiology. Experimental study described in the present thesis was undertaken to investigate physiological role of AVT hormone and the expression of AVT gene in hypothalamus of two poultry species chicken and Japanese quail in relation to osmotic stress and oviposition. This research work for the first time will investigate expression of AVT and AVT receptor genes in the hypothalamus as well as in shell gland (uterus), in response to egg laying. Effect of in ovo administration of sex steroids on development of vasotocinergic system in chicken has also been studied in detail. This project will provide an insight in understanding of these biological phenomenon at the gene level. 
Specific objectives of present thesis are as follows: 1. To investigate age, sex and species dependent effect of osmotic stimulation (water deprivation) on hypothalamic AVT gene expression. 2. To analyse the effect of sex steroid on dehydration induced expression of hypothalamic AVT gene. 3. To study and establish temporal relationship of expression of AVT and its receptor gene in hypothalamus and shell gland in relation to oviposition. 4. To determine the response of vasotocinergic neurons of different hypothalamic nuclei following in ovo administration of sex steroids in response to various developmental stages. 
The thesis work has been divided into four chapters preceeded by review of literature. Methodology used for present investigation are northern blot analysis, in situ hybridization, reverse transcriptase - polymerase chain reaction (RT-PCR), immunohistochemistry, osmometry and radioimmunoassay. 
Chapter 1: Age and sex dependent variation of Arginine vasotocin gene in response to water deprivation in chicken and Japanese quail. 
To investigate and compare age and sex dependent central and peripheral effects of osmotic stress, sexually immature (young) and mature (adult) chicken (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix japonica) of both the sexes were water deprived for four days. Both young and adult quail of the two sexes exhibited significant loss in body weight and increase in plasma osmolality compared to their hydrated controls. More or less similar effect was noted in chicken although degree of difference varied between two age groups and sexes when compared to that of quail. Northern blot analysis indicated 1.24 & 1.94 fold increase in AVT transcripts of water deprived adult male and female and 1.45 & 1.42 fold increase in young male and female quail respectively. On the other hand, 1.27 & 1.6 and 1.44 & 1.65 fold increase was observed in water deprived adult and young male and female chicken respectively compared to controls. 
Present study indicates difference in the response of hypothalamic AVT system of sexually immature and mature birds following osmotic stimulation. Since young ones are more adversely affected in terms of peripheral effects (body weight loss) it is quite reasonable to suggest a possible role of sex steroids in body homeostasis. Smaller species with less surface area appear to be affected more than those with larger surface area. When comparing two sexes, females are affected more adversely than their male counterparts which may be attributed to increased requirement of AVT to perform dual function of water balance as well as oviposition. At the peripheral level decrease in body weight after water deprivation was more in young birds compared to adult, suggesting that sensitivity of AVT system to osmotic stress is higher in adults, hence they are relatively less affected compared to young ones which are more adversely affected. It is concluded that osmotic stress induced by water deprivation is a potent stimulator for hypothalamic AVT gene expression in chicken and Japanese quail and response of AVT system varies with the reproductive age and sex of the birds. 
Chapter 2: Expression of hypothalamic arginine vasotocin gene in response to dehydration and sex steroid administration in female Japanese quail. 
To study the effect of sex steroid administration coupled with dehydration on hypothalamic AVT gene expression of sexually immature Japanese quail, three weeks old female Japanese quail maintained under short days (8L:16D), were divided into 4 groups [Control (C), Water deprived (WD) for 2 days, Testosterone propionate (TP) treated (1mg/day for 16 days), Testosterone propionate treated + Water deprived for 2 days (TP + WD)]. Aim of the study was to observe the effect of water deprivation and testosterone treatment independently, as well as simultaneously on hypothalamic AVT system. 
A significant increase in plasma osmolality, sodium ion concentration and AVT was observed following water deprivation, but administration of sex steroids has no significant influence on these parameters. However, number as well as size of ir - AVT neurons and hybridization signals (in the form of silver grains representing AVT mRNA in corresponding neurons of paraventricular nuclei) along with hypothalamic AVT transcript (northern analysis) increased significantly in all the treated groups compared to control. These findings indicate that although testosterone administration has no effect on plasma osmolality and AVT concentration unlike water deprivation, but it may affect AVT neurons of PVN, supporting the possibility of sex steroid receptors on these neurons. It is concluded that in quail, osmotic stress not only up regulates the expression of AVT gene but also causes AVT release by increasing the rate of its synthesis in PVN neurons, while sex steroid appears to have an effect in these neurons only at the level of transcription and hence influences / modulates hypothalamic AVT gene expression in response to osmotic stress. This study also indicates the influence of breeding status on hypothalamo- neurohypophyseal axis (AVT system) of quail and further suggests the interrelationship of AVT and reproduction in birds. Chapter 3: Expression of AVT and its receptor gene in relation to egg laying in chicken and Japanese quail. 
Number of reports indicate increased plasma AVT concentration in relation to oviposition supporting its oxytocic role in avian physiology. But no information is available on the expression of AVT gene in both hypothalamus and shell gland in relation to oviposition and its co-relatation with vasotocin receptor gene expression in shell gland. In view of above following two sets of studies were performed. 
(A). Expression of hypothalamic AVT gene in response to egg laying in chicken and Japanese quail. For this experiment actively laying quail were constantly monitored for 2-3 weeks to check the egg laying pattern and exact time of oviposition. One set of quail were sacrificed just before egg laying and another set immediately after egg laying. Hypothalamus of these quail were processed for total RNA extraction followed by northern blot analysis to detect AVT transcript. Following normalization with house keeping gene GAPDH, results indicated a remarkable increase in amount of AVT transcript prior to oviposition which falls significantly after the egg is laid. A similar experiment was also performed in chicken but unlike quail, the difference in AVT transcript before and after egg laying was not obvious. Hence another experiment was performed with modified protocol. White leg horn hens having a regular laying cycle of 6-7 days were used in this experiment. Expression of hypothalamic AVT gene was studied 2 hours before (-2 hr), immediately after (0 hr) and 2 hours after egg laying (+2 hr). 
Our results indicate a significant increase in plasma AVT concentration at the time of egg laying (78.7 ? 12.4), which remain in basal condition 2 hr before (13.3 ? 1.6) and 2 hr after egg laying (14.1 ? 2.9). Plasma osmolality and other ionic concentration did not vary in response to egg laying in chicken. Northern blot analysis showed an increase in hypothalamic AVT transcript at the time of egg laying which remained low 2 hour before and 2 hour after egg laying. Low levels of AVT mRNA (in situ hybridization) in the magnocellular neurons of PVN, observed 2 hr before egg laying increased remarkably at the time of egg laying, followed by a decrease 2 hr after egg is laid. The number of ir- AVT neurons and immunostaining also increased significantly in PVN at the time of oviposition compared to that of 2 hr before and 2 hr after egg laying. 
(B). Expression of shell gland AVT and its receptor gene in response to egg laying in chicken Changes in expression of AVT and its receptor gene in shell gland of chicken was also studied 2 hours before (-2 hr), immediately after (0 hr) and 2 hours after egg laying (+2 hr). In view of relatively few copies of AVT / AVT receptor (VT1) transcript in the shell gland, RT-PCR was performed. To detect very low signals of immunofluoroscent - AVT, sections of the shell gland were studied using confocal laser microscope. Our results indicate increased expression of AVT gene in shell gland 2 hr prior to oviposition which declines sharply immediately after egg laying and remained low until 2 hr after egg laying. Contrary to this, AVT receptor gene expression is up regulated immediately after egg laying which remains low 2 hours before and 2 hours after egg laying. 
Findings of the above two studies indicate physiological and temporal relation of AVT gene expression at the central (hypothalamic) and peripheral (shell gland) level. While endocrine function of hypothalamic AVT via its release from neurohypophysis is well documented, we suggest a paracrine role of shell gland AVT in causing contraction of uterine smooth muscles via some factors like prostaglandins and also by influencing number / sensitivity of shell gland receptors of AVT (VT1) directly or indirectly. This study indicates a physiological interrelationship of AVT gene expression at the central (hypothalamus) and peripheral (shell gland) level suggesting the co-ordination of endocrine and paracrine release of AVT with that of its receptors for expulsion of egg. 
Chapter 4: In ovo administration of sex steroid alter vasotocin synthesis in hypothalamic magnocellular and parvocellular neurons of chicken. 
Present study was performed to check the effect of in ovo administration of sex steroids on expression of AVT gene in magnocellular (SON and PVN) and parvocellular neurons of sexually dimorphic nuclei, the bed nucleus of stria terminalis (BnST) in chicken. Sex steroids (estradiol benzoate / testosterone propionate; 20 ?g / egg) were administered in chicken egg at day 6 of incubation (E6), and effect was monitored on the expression of AVT gene both transcriptionally (in situ hybridization) and translationally (immunohistochemistry) during embryonic development (E18) and after hatching at day 7, 21 and 40. 
It has already been reported by other workers that from embryonic day 14 (E14) onwards, BnST starts exhibiting sexual dimorphism and ir - AVT can not be located in this region of the hypothalamus of female chicken. However, AVT transcript can be detected in this nuclei up to D35 post hatch. But so far no report is available indicating role of in ovo administration of sex steroids in sexual dimorphism of AVT system in developing brain. Present study for the first time indicates that in ovo administration of sex steroid can alter the expression of AVT gene in both magnocellular as well as parvocellular group of neurons. Following in ovo administration of sex steroid, the ir - AVT which was not observed after E14 in control females, was detectable even up to D40. Further, estradiol was more potent in increasing the amount of AVT transcript in BnST during different stages of development except at D40 when testosterone appeared more effective. 
Altered expression of AVT gene in BnST following in ovo administration of sex steroids, may be due to its sexually dimorphic nature and supports the role of BnST in reproductive behaviour. Further, a single injection (in ovo) of sex hormone during early embryonic stage (s) modulates activity of vasotocinergic neurons by altering the expression of AVT gene as well as its secretion in the developing brain of chicken. These findings also support the possibility of presence of sex steroid receptors on AVT neurons. Moreover, administration of sex steroids appears to have an irreversible and long lasting effect on AVT neurons of SON, PVN and sexually dimorphic BnST nuclei. It is concluded that vasotocinergic system of developing brain is sensitive to sex steroids which appear to have a long lasting effect on the activity of hypothalamic AVT neurons and thus supports possible role of AVT in reproduction in addition to its established role in water balance. 
In Summary, increased synthesis and secretion of AVT hormone from magnocellular neurons of hypothalamus following osmotic stimulus, which varies with the age and sex of the species, may be altered by sex steroids / breeding condition of the bird. Taking in account oxytocic function of AVT it is further stated that both endocrine and paracrine release of AVT from hypothalalmus and shell gland respectively, controls oviposition in co-ordination with expression of its receptor gene in shell gland. Further, administration of sex steroids at early embryonic / developmental stage(s) alters AVT transcript / synthesis in both parvocellular sexually dimorphic (BnST) and magnocellular neurons (SON, PVN). It is concluded that in addition to its classic / established role as a hormone in water balance, paracrine release of AVT also influences / regulates reproductive behaviour including oviposition. Further, both endocrine (hypothalamic) and paracrine (BnST, shell gland) synthesis / release of AVT may be modulated by sex steroids indicating role of AVT in reproduction. 




Arginine vasotocin gene expression in Chicken (Gallus gallus domesticus) and Japanese Quail (Coturnix coturnix japonica) in relation to dehydration and oviposition

VELURI, RAVIKANTH

ABSTRACT:The changes in the concentration of an ion in soil or in plant tissue are affected not in a singular manner but are accompanied by secondary changes in the soil system or in the tissue content of other dissimilar ions. Continuous application of plant nutrient may result in the increased concentration and buildup in the soil, which may interact with other nutrient elements and alter their availability and uptake. Micronutrient nutrition of plants not only depends on the ability of soil to supply these nutrients but it also depends on several other factors. Interaction of a micronutrient with other micro or macronutrient is an important factor. Such interaction may take place in soil and/or in plant. The interaction may lead to the increased availability of other plant nutrients (synergistic) or it may adversely affect the availability of other nutrients (antagonistic). Since S and B play a vital role in the oilseeds’ growth and development, a study was conducted to assess the “Interaction between boron and sulphur as measured by crop response and soil analysis” on the soils deficient in sulphur and boron.The release of sulphur in presence of boron and vice-versa at various levels was quantified based on a incubation study conducted at two temperatures (i.e., 25° and 40°C). The effect of boron on sulphur release and vice-versa was found to be non-significant. The interaction between sulphur and incubation was found to be significant at both the temperatures under study, i.e., 25° and 40°C. All other interactions were found to be non-significant. The temperature significantly influenced the sulphur and boron mineralization, which increased rapidly up to 28 days of incubation. Further, the adsorption and desorption study of boron in presence of sulphur and vice-versa was described more satisfactorily with the help of a Freundlich adsorption equation as compared to Langmuir and Temkin equations.  Results of greenhouse experiments with various levels of sulphur (0, 20, 40 and 60 mg kg-1) and boron (0, 1, 2 and 3 mg kg-1) with mustard (var Pusa Bold) and sunflower (var Morden) as test crops indicate that the interaction effect between boron and sulphur significantly and synergistically influenced the dry matter and seed yields of both the crops, which was observed highest at 60 mg  kg-1 of S in conjunction with 2 mg kg-1 of boron. 
The contents and uptake of sulphur and boron were found to increase due to the application of both the nutrients in combination. The oil and protein contents of sunflower and mustard seeds were significantly and synergistically improved by the application of both boron and sulphur.Fractionation of boron and sulphur into different forms after the harvest of mustard and sunflower revealed that the readily soluble, specifically adsorbed, oxide bound and organically bound forms of boron were found to increase with increasing levels of boron. Excluding the oxide bound boron, all other forms had positive relationships with the plant parameters of mustard and sunflower.  Water soluble S, sulphate S and heat soluble S forms increased with increasing levels of S. At same level of sulphur with increasing levels of boron, the water soluble sulphur and sulphate sulphur forms were found to decrease, but the heat soluble fraction did not show any significant change. Water soluble S and sulphate S forms were found to be positively and significantly correlated with all the plant parameters of mustard and sunflower. Finally, it was established that the boron was not influenced by sulphur and vice-versa, because both have different adsorbing sites and are non-competing in nature.




Phytochemical Examination of Some Folklore Medicinal Plants and Their Pharmacological Evaluation

H., YAMINI SHRIVASTAVA

Organic molecules play vital role in various biological processes of living systems.  The specific chemical properties of organic molecules are the result of presence of particular functional groups that are clusters of atoms with characteristic structure and functions.  The biomolecules and macromolecules (proteins, DNA, carbohydrates etc.) are constructed by small organic molecules as monomers, for example, amino acids, sugars, nucleobases etc., which are very essential for life.  Apart from these, most of the drugs are small organic molecules, and they become effective by interacting with specific molecules in the body, especially with proteins, by modulating their functions.  From toxicity angle organic and inorganic molecules can be broadly divided into toxic and non-toxic chemicals. ‘Toxic chemical’ means any chemical, which through its chemical action on life processes can cause death, temporary incapacitation, or permanent harm to humans or animals.  Toxic chemicals causes illness to all living bodies including plants.   Various forms of herbicides (​http:​/​​/​www.infoplease.com​/​ce6​/​sci​/​A0823452.html​) and defoliants are also known to destroy crops and vegetation. Plants, leaves and flowers change colour if they are exposed to toxic chemicals. Some of the nitrogen, sulphur containing organophosphorus compounds are generally classified as chemical toxins. These chemicals are often used as pesticides and sometimes as warfare agents. Chemical toxins are two types, one is lethal and the other is non-lethal. Lethal chemicals directly attack the skin, blood, nervous, or respiratory system and require hospitalisation of the victim, whereas the non-lethal chemicals cause temporary physical disability.  The potential misuse of chemical toxins has increased with improved methods of dissemination, such as artillery shells, grenades, missiles, aircraft and submarine spray guns. 
Chlorine and mustard gas (​http:​/​​/​www.infoplease.com​/​ce6​/​sci​/​A0834601.html​)es were first used during World War I by the Germans against the Allies. Efforts to control chemical and biological weapons began in the late 19th century. In 1993 an international treaty banning the production, stockpiling, use of chemical weapons and calling for the establishment of an independent organization to verify compliance was adopted. The agreement, which became effective in 1997, has been signed and ratified by 160 nations. The treaty is enforced by the Organization for the Prohibition of Chemical Weapons (OPCW), The Hague, The Netherlands. The analysis of Chemical Warfare Agents (CWAs) and their precursors and degradation products, in samples collected from suspected production/ storage facilities or from environment in case of alleged use, is an important activity under the Chemical Weapons Convention (CWC) for taking further action. The chemical warfare agents are classified into three groups by CWC, namely schedule 1, 2 and 3. 
Schedule 1 contains chemicals that are deemed to pose a high risk to the purposes of the CWC, but have very limited, if any, commercial applications. This schedule includes nerve agents (e.g. Sarin) and blister agents (e.g. mustard gas), these chemicals are listed below.
A) Toxic chemicals:
O-Alkyl (R2: <C10, including cycloalkyl) alkyl (R1: Me, Et, n-Pr or iso-Pr) phosphonofluoridate:


O-Alkyl (R3: <C10, including cycloalkyl) N,N-dialkyl (R1, R2: Me, Et, n-Pr. iso-Pr) phophoramidocyanidate:

O-Alkyl (R4: <C10, including cycloalkyl) S-2-dialkyl (R2, R3: Me, Et, n-Pr. iso-Pr)-aminoethyl alkyl (R1: Me, Et, n-Pr or iso-Pr) phosphonothiolates and corresponding alkylated or protonated salts (R5: alkyl or H; X: anion):




2-Chloroethylchloromethylsulfide:		

Mustard gas: Bis(2-chloroethyl)sulfide:


n = 1: 1,2-Bis(2-chloroethylthio)ethane
n = 2: 1,3-Bis(2-chloroethylthio)-n-propane
n = 3: 1,4-Bis(2-chloroethylthio)-n-butane
n = 4: 1,5-Bis(2-chloroethylthio)-n-pentane

Bis(2-chloroethylthiomethyl)ether:

Bis(2-chloroethylthioethyl)ether:

2-chlorovinyldichloroarsine:


Bis(2-chlorovinyl)chloroarsine:


Tris(2-chlorovinyl)arsine:


Bis(2-chloroethyl)methylamine:                        


Bis(2-chloroehtyl)ethylamine:


Tris(2-chloroethylamine):

B) Precursors:
Alkyl (R1:Me, Et, n-Pr or iso-Pr)phosphonyldifluoridates:

O-Alkyl(R2: H or <C10, including cycloalkyl) O-2-dialkyl (R3, R4: Me, Et, n-Pr or iso-Pr)-aminoethyl alkyl (R1: Me, Et, n-Pr or iso-Pr) phosphonites and corresponding alkylated or protonated salts (R5: alkyl or H; X:anion):




O-isopropyl methylphosphonochloridate:


O-Pinacolyl methylphosphonochloridate:

Schedule 2 contains chemicals that are deemed to pose a significant risk to the purposes of the CWC and includes toxic chemicals that could be used as chemical warfare agents and key purposes to the chemicals in schedule 1. Some of these chemicals have limited commercial applications and are listed below.
A) Toxic chemicals: 
Amiton: O,O-Diethyl S-[2-(diethylamino) ethyl phosphorothiolate and corresponding or protonated salts (R1: alkyl or H; X:anion):




 
1, 1, 3, 3, 3-pentafluoro-2-trifluoromethyl)-1-propene:


3-Quinuclidinyl benzilate (BZ):


B: Precursors:
N,N-dialkyl (R1, R2 = Me, Et, n-Pr or iso-Pr) phosphoramidic dihalides (X, Y:Halogen):



O,O-dialkyl (R1, R2 = Me, Et, n-Pr or iso-Pr) N,N-dialkyl (R1, R2 = Me, Et, n-Pr or iso-Pr) -phosphoramidates:




Arsenic trichloride:    AsCl3

2, 2-diphenyl-2-hydroxyacetic acid:


Quinuclidin-3-ol:

N,N-dialkyl (R1, R2 = Me, Et, n-Pr or iso-Pr) Aminoethyl-2-chlorides and corresponding protonated salts (X: anion):



N,N-dialkyl (R1, R2 = Me, Et, n-Pr or iso-Pr) Aminoethyl-2-ols and corresponding protonated salts (X: anion):



N,N-dialkyl (R1, R2 = Me, Et, n-Pr or iso-Pr) Aminoethyl-2- thiols and corresponding protonated salts (X: anion):



Thiodiglycol: Bis(2-hydroxyethyl)sulfide:  S(CH2CH2OH)2

Pinacolyl alcohol:

Schedule 3 contains other chemicals that are considered to pose a risk to the purpose of the CWC, including 'dual purpose' chemicals such as phosgene (used as a chemical weapon in world war I. but with legitimate commercial uses) and selected precursors for chemical warfare agents and are presented below.
A: Toxic chemicals:
Carbonyl dichloride:		COCl2                        
Cyanogen chloride:		ClCN
Hydrogen cyanide:	            HCN	
Trichloronitromethane:	Cl3CNO2

B: Precursors:
Phosphorus oxychloride:	POCl3
Phosphorus trichloride:	PCL3
Phosphorus pentachloride:	PCl5
Trimethyl phosphite:		P(OCH3)3
Triethyl phosphite:		P(OC2H5)3
Dimethylphosphite:		(CH3O)2POH
Sulfur monochloride:		SCl
Sulfur dichloride:		SCl2
Thionyl chloride:		SOCl2
Ethyldiethanolamine:		CH3CH2N(CH2CH2OH)2
Methyldiethanolamine:	CH3N(CH2CH2OH)2
Triethanolamine:		N(CH2CH2OH)3

All the CWAs can enter into the body through orally or from respiratory system or diffusion from skin. Death usually occurs within 15 minutes after absorption of these chemicals. The main property that allows the classification of a substance, as warfare agent is its toxicity and applicability on the battlefield. Chemical toxins are divided into different groups according to their effect on the organism and are discussed below.

The chapter reviews the methods for the analysis of chemical toxins, their precursors and degradation products that are present in different environmental and biological sample matrices using chromatographic methods such as gas chromatography and liquid chromatography with different types of detectors. GC is the popular option for the identification of volatile and non-polar compounds with elemental specific detectors like FPD, PFPD, NPD, AED, FID and AED and also for the pre-monitoring of these compounds in different sample matrices. Derivatization is needed for polar compounds prior to GC analysis.  However, unambiguous identification of these chemicals is done by GC- coupled with mass spectrometer under EI and CI conditions. EI mass spectrum is a fingerprint of the compound to deduce its structure and the molecular weight. CI is complementary technique to EI to confirm the molecular weight of an unknown compound. Non-volatile and polar compounds which are present in trace quantities in water with other interfering solutes are directly analysed by liquid chromatography coupled with MS under TSP and API conditions and structure of the compounds are identified by MS/MS analysis.  Hence, mass spectrometry is a powerful tool compared to other analytical techniques for the unambiguous identification of chemical toxins.








Studies On the Interaction Of Chromium(III) Complexes With Proteins

JOSHI, MADHUMITA V.

PAGE  





Abstract



ABSTRACT
This thesis entitled “Development of some important synthetic methodologies and their applications in the synthesis of biologically important compounds” is divided in to three chapters.
Chapter-I: This chapter deals with the “Stereoselective synthesis of (-)- pestalotin”.
Chapter-II: This chapter deals with the “TEMPO-oxidation of alcohols to aldehydes on solid-phase”.
Chapter-III: This chapter divided in two sections.
Section-A: This section deals with the “A versatile and efficient synthesis of (2S)-2-(hydroxylmethyl)-N- Boc-2, 3-dihydro-4-pyridone”.
Section-B: This section deals with the “Studies directed towards the synthesis of (+)-5-epi-tashiromine”.

CHAPTER-I: Stereoselective synthesis of (-)-pestalotin.
	Pestalotin 1, a gibberellin synergist, was first discovered by Kimura et al from a culture broth of Pestalotia cryptomeriaecola Sawada, which is a fungus pathogenic for the Japanese cedar, Cryptomeria japonica. Later Ellestad et al., also isolated 1 as a minor metabolite from the fermentation culture P880, an unidentified penicillium species and gave the code LLP-880α. The 6-substituted 5,6-dihydro-2-pyrone skeleton in pestalotin 1 which also occurs in many natural products show diverse biological activity. Especially those with oxygen function on the pyrone ring and C6-side chain are known to exhibit plant growth inhibitory, antifungal, and antitumor activities.  




	
	The presence of two contiguous stereogenic centers in (-)-pestalotin 1 has made it an attractive synthetic target since its discovery. Several synthesis of racemic and optically active forms of pestalotin have been reported. In most of these syntheses the two chiral centers were built stepwise and also in some cases low stereoselectivity was observed. 		 
	In connection with our interest in utilizing substituted aromatic system as masked, 1,3-dione or 1,3-diol and 1,5-dione in the synthesis of natural products, we report here a facile synthesis of (-)-pestalotin where the m-methoxy substituted benzene was utilized as masked -ketoester in turn to obtain pyranone skeleton of compound 1. Also in our approach the asymmetric dihydroxylation (AD) developed by Sharpless was used to get the two chiral centers present in 1.
Our synthesis commenced with the Wittig homologation of 1-pentanal 2 with (carbethoxy ethylidene) triphenyl phosphorane to afford the --unsaturated ester [diastereomeric ratio (trans: cis = 91.37:6.7) ] as a colourless liquid in 74%(trans) isolated yield .

Scheme-I

Sharpless asymmetric dihydroxylation reaction of (E)-ethyl-2-heptenoate 3 using (DHQ)2PHAL as the chiral ligand, catalytic OsO4, and methanesulfonamide at 0 C for 16 h provided the corresponding ethyl- 2, 3- dihydroxy-(2R, 3S)-heptanoate 5 in 71% yield, ( Scheme-II) [97.35% ee, determined by chiral HPLC analysis].



Scheme-II

	Compound 5 when exposed to 2, 2-dimethoxy propane in the presence of catalytic PTSA gave 6. The ester 6 was smoothly reduced by LiBH4, generated in situ, to afford the alcohol 7. The sulfonate ester 8 was readily prepared from 7 using p-toluene sulfonyl chloride, triethylamine and DMAP (catalytic). Deprotection of the isopropylidene moiety of 8 in acid medium followed by basification with K2CO3 in MeOH/H2O afforded the epoxy alcohol 9. When compound 9 was subjected to nucleophilic epoxide ring opening with m-methoxyphenyl magnesium bromide in the presence of catalytic CuI, gave 10 (Scheme III). 

Scheme-III

	The crucial intermediate, β-keto ester 12 was unmasked from 10 via Birch reduction-ozonolysis sequence. Accordingly, when 10 was treated with Li/liq.NH3, EtOH the dihydroanisole intermediate 11 was produced. Ozonolytic cleavage was performed on the unpurified Birch product to give the β-keto ester 12, which was carried to the next step without any purification.  The δ- lactone 13 was obtained from 12 by treatment with 1N aqueous NaOH solution which was subsequently methylated using dimethyl sulphate to furnish, the (-)-pestalotin 1 as a white solid after recrystallization [hexane:benzene (1:1)]. The NMR (1H, and 13C) spectral data of the synthetic sample were consonant with those of the reported product.
	In summary, we have accomplished the synthesis of (-)-pestalotin 1 using asymmetric dihydroxylation and Birch reduction-ozonolysis sequence of m-substituted anisyl ring as latent  β-keto ester synthons for generating the 6-substituted 5,6-dihydro-2-pyrone unit. Use of m-anisole unit as a masked form of -ketoester has an advantage, it can be introduced easily in to the skeleton and can be converted to -keto ester at an appropriate stage in two step sequence.

CHAPTER-II: TEMPO-oxidation of alcohols to aldehydes on solid-phase.
The solid-phase organic synthesis (SPOS) has been developed for the synthesis of chemical libraries in solid-phase using an excess of reagents, which can be removed by simple filtration and washings without the need for a chromatographic work up. Several functional group transformation have been developed on solid phase to conduct SPOS. One of the important transformations is oxidation of alcohol to aldehyde. Although several reagents are known to oxidize alcohol to aldehyde in solution phase chemistry, but there are several limitations to adopt these reagents on to solid phase.
	So far only three approaches are known on solid-phase for oxidation of alcohol to aldehyde. (i) TPAP (tetra-n-propyl ammonium perruthenate)/NMO in DMF (ii) Pyr.SO3. DMSO, NEt3 (iii) Dess-Martin periodinane 
	Herein we report a convenient method of oxidizing alcohol to aldehyde using TEMPO conditions. Though there are many reports where TEMPO-free radical was used for oxidation reactions (solution phase) the one particularly attracted us is the work of Jacques Einhorn et al., where they used NCS (1-3eq), TEMPO (0.1eq), TBACl (0.1eq) and aqueous solution of NaHCO3 (0.5M) and K2CO3 (0.05M) in DCM at r.t for oxidizing primary alcohol to aldehyde. Under these conditions alcohol gives good yields of product and over oxidation product was not obtained even with slight excess NCS. Therefore it was thought to adopt these conditions on solid-phase for oxidation of alcohol to aldehyde. Though water is used in slight amounts in the reaction, but it is required only for maintaining PH, mostly oxidation is taking place in CH2Cl2 medium.

Scheme-IV



As a first example Wang resin was taken in CH2Cl2 and is subjected to TEMPO-oxidation conditions, after filtration and drying the resin, Magic angle NMR of the solid showed clean formation of aldehyde. The resin aldehyde was cleaved under TFA/CH2Cl2 condition to give crude p-hydroxy benzaldehyde whose 1HNMR showed clear aldehyde signal. Generally aldehydes may have unstability under cleavage condition therefore product formation was conformed by 1HNMR of magic angle of the aldehyde on the resin.  Several examples (see the table-I) have been studied under the above conditions the 1HNMR (magic angle) clearly showed aldehyde formation (δ 9.5- 10.0).
Thus simple general and useful method for the conversion of alcohol to aldehyde on solid-phase was developed using TEMPO-oxidation condition.

TEMPO-oxidation of alcohols to aldehydes on solid support


Table –I
Entry	Substrate	Resin	Time	Product
1		Merrifield	24h	
2		Chlorowang	24h	
3		Chlorowang	24h	
4		Merrifield	24h	
5		Merrifield	24h	

CHAPTER-III:  Section-A: A versatile and efficient synthesis of (2S)-2-(hydroxymethyl)-N-Boc-2, 3-dihydro-4-pyridone. 
2, 3-Dihydro-4-pyridones and their N-acyl derivatives are interesting building blocks for a large variety of nitrogen-containing heterocycle syntheses. The amino-enone moiety can be used in various reactions leading to key intermediates and is also particularly useful in the synthesis of biologically active compounds and alkaloids. In particular, the chiral dihydropyridones of type-28 (Fig.II) have been utilized for the synthesis of some chiral piperidine skeletal natural products e.g. (+)-dienomycin C, (+)-deoxoprosopinine, and 1-deoxynojirimycin.  

                                                     
Herein we report a novel and simple method for the synthesis of 28 starting from 3-aryl-2-aminopropanol 25 (obtained from aryl amino acid) in three steps, using Birch reduction and ozonolysis.
      	Compound 25 is commercially available and can also be easily prepared from L-(-)-phenylalanine in three steps. L-(-)-phenylalanine 22 was converted to N-Boc-methyl ester 24 in the presence of acetyl chloride and methanol followed by treatment with  (Boc)2O. The resultant ester 24 was reduced to alcohol 25 using LiBH4.

Scheme-V

	
Birch reduction of 25 gave the corresponding dihydro derivative 26 which on ozonolysis followed by quenching the resultant ozonide with H2/Pd(OH)2 yielded the -keto aldehyde 27. Compound 27 without isolation was subjected to acid catalysed cyclisation to give the dihydro-4-pyridinone 28.                                               



Scheme-VI

This method is novel and general in nature to obtain the skeleton of a 2,3-dihydro-4-piperidinone, by using an appropriate amino-aryl precurser.                

Section-B: Studies directed towards the synthesis of (+)-5-epi-tashiromine.  
	(+)-5-epi-tashiromine is an indolizidine alkaloid isolated from subtropical asian deciduous shrub, “maackia tashiroi”. It is interesting from the perspective of both chemotaxonamy and biosynthesis maackia species. Indolizidine alkaloids have been isolated from poison dart frogs and may have neurological properties.




Our synthesis of 33 and 36 commenced with the mono benzyl protection of the commercially available butane- 1, 4- diol 31 and pentane- 1, 5- diol 34  (Scheme-VII). One of the hydroxyl of 31 and 34 was protected as benzyl ether by using BnBr, NaH, in DMF at 0 0C to room temperature for 12 h to afford compound 32 and 35 in 66% and 67% yield. TEMPO oxidation of the free hydroxyl in 32 provided the required aldehydes 33 in 82% yield, which without further characterization was taken forward for the next reaction.
Scheme- VII

The compound 35 under goes TEMPO oxidation of free hydroxy provided the required carboxylic acid 36 in 70% yield, which was taken forward to couple with oxazolidinone 39 to get 40. The two chiral centers in (+)- 5- epi- tashiromine were planned to generate by an Evan’s aldol reaction between the aldehyde 33 and the chiral oxazolidinone 40, which in turn can be prepared from L- phenyl alanine 37, using a known procedure. Accordingly, L- phenylalanine 37 was reduced to phenylalaninol 38 using sodium borohydride and iodine in THF (Scheme- VIII).
The amino alcohol was protected as a carbamate 39 using diethyl carbonate and catalytic amount potassium carbonate at 135 0C. The analytical data of compound 39 matched with the reported value.

Scheme-VIII




In presence of 1.6 eq. of LiCl and 2.0 eq. of Et3N in dry THF, acid 36 (1.1eq.) reacted with the amide nitrogen of 39 to give 40 in 62% yield. Dibutyl boryl trifluromethane sulphonate mediated Evans aldol reaction between chiral oxazolidinone 40 and the aldehyde 33, gave the syn aldol product 41 in 73% yield.
Scheme-IX


Our next objective was the non-destructive removal of the chiral oxazolidinone without recemization to get enantiomercally pure primary alcohol 42 (Scheme- XI). To realise this we initially treated compound 41 with NaBH4 in THF- H2O at 0 0C to r.t for  3 h. The -position to the carbonyl carbon in 41 under went recemization when subjected to reduction with NaBH4 furnishing 43. To circumvent the possibility of recemization at -position to the carbonylcarbon 41 was treated with LiBH4/Et2O in the presence of cat.amount of water at 0 0C to furnish 42 in 72% yield and the recovery of oxazolidinone 39. Selective protection of primary hydroxyl group of compound 42 was then subjected to TBDMS protection, provided 44 in 87% yield.

Scheme-X





The hydroxy group in 44 was converted to its azide via mesylation. The reduction of azide functionality of 46 treated with TPP/THF-H2O and reflux at 60 0C for 19 h gave the amine, which was subsequently Boc protected using (Boc)2O to give 47. Carbonate 47 was debenzylated under the Li/ liq NH3 conditions in THF at -78 0C to afford the compound 48. 

Scheme-XI


The diol 48 was then convert to corresponding ditosylate 49 which after subsequent deprotection of the N- Boc followed by nutralization should afford the cyclized product. Accordingly the ditosyl was subjected to N-Boc deprotection protocol using TFA/CH2Cl2. Treatment of 51 with Et3N in CH2Cl2 at r.t did not realise the cyclization, then the reaction mixture was subjected to reflux condition, however, we failed to obtain the cyclized product. We then thought of changing the leaving group ability. Thus we converted the diol 48 into the corresponding di-mesylate 50 using MsCl, Et3N in CH2Cl2 and DMAP(catalytic) at 0 0C for 3h. The compound 50 was subjected to amine deprotection with TFA/CH2Cl2 to give salt 52. The expected salt failed to under go cyclisation with Et3N in CH2Cl2 at r.t., even after changing the solvent system to CH3CN and DMF did not afford the cyclization product 30.
The steric hinderence executed by the hydroxyl methyl group in 51/52 during cyclization could be a reason for the unsuccessful cascade cyclization to form the indolizidine skeleton.










Fig. III

Fig. II


Fig. I



PAGE  



I





Studies on In Vitro Regeneration and Genetic Transformation in Peanut (Arachis hypogaea L.)

A, ANITHA

Abstract
India's cultural and genetic diversity is of remarkable interest due to the peculiarity of its social structure and breeding patterns in the different populations. It has a population of one billion plus, with 631 cultural, ecological and economic traits, which are assigned to 4635 communities. Kerala, the southern most state of India, is known for its largest biodiversity reserve, maximum cultural heterogeneity and high rate of literacy. It has approximately 35 ethnic communities scattered in various regions of the state; they form about 5% of the total population. These endogamous tribes are some of the oldest known and are evolutionarily conserved. It would therefore be very interesting to study the genetic structure and its inheritance in these primitive tribes. By making a genetic database of the population, one will be able to answer various questions pertaining to the demography and migration of different populations in Kerala. In addition, it can also find the root cause for population decline and other such health related problems here. The present study is the first extensive study regarding the genetic diversity aspects of Dravidian language groups in Kerala. We analyzed the genetic diversity of ethnic populations in Kerala using a series of autosomal dinucleotide microsatellite markers. Microsatellite variation was analyzed in eight ethnic communities (7 Scheduled Tribes and 1 Scheduled Caste) belonging to different geographical regions of Kerala. Samples were collected only from those communities, which had remained isolated from the mainland for several generations. The study was aimed at the molecular characterization of tribal populations in Kerala based on variations in the functional and non-functional regions of chromosomes. The following markers were used in our study: (i) The non-functional microsatellites in the autosomes and the Y chromosome (ii) The SNPs in the functional NAT2 gene Two types of microsatellites were used in the autosomal analysis. The first type included five unlinked STRs from chromosomes 9, 10, 13 and 22. Since the results from this analysis were not informative and could not be used for interpreting the anthropological history of study populations, we further analyzed these populations using 33 unlinked STRs belonging to chromosomes 12 and 13. The average linkage distance between the markers was 8.3 cM. These STRs had an average heterozygosity value of 0.81; the average genetic differentiation of the markers based on allelic variance was found to be 0.09 across the various populations. This variation is within the limits of human species. This indicates that these STRs are highly informative in population diversity analyses. The heterozygosity values indicate that the Kurichiyas are the most genetically diverse group. This observation is also significant from the point of view of anthropological history, which says that the Kurichiyas are the most advanced among the tribal populations of Kerala. The study shows that the Malapandaram is the least diverse group. The UPGMA tree showing the genetic relationships between populations calculated based on Nei's unbiased minimum distance method (1978), places the Malapandaram tribe in a separate group. Here, an important aspect to be noted is that, Malapandaram is a nomadic tribe, whereas the other tribes have a more or less permanent settlement; hence, the observation is significant. The Kani tribe in the southernmost part of Kerala is placed in a cluster along with other tribes in the northern part of Kerala. In addition, the dendrogram indicates that the Kani and Kattunaikkar tribes are genetically very close. Thus, it is clear that geographical distance is not linked to the genetic distance of the populations. The AMOVA analysis indicates that the there is greater diversity within populations than between populations. The lesser degree of diversity between populations suggests that these ethnic populations might have had a common origin, and might have been separated out only recently. Of the 264 locus-population combination for HWE, 53 significant deviations were observed. Significant deviations were observed in Adiya, Kani and Paniya tribes. Markers with higher homozygote frequency showed deviations from HWE. These markers showed higher inbreeding coefficient as well. Linkage disequilibrium was observed for 40 locus pairs in chromosome 12, and 36 locus-pairs in chromosome 13. Maximum deviations from the locus-pair linkage disequilibrium were observed in Adiya and Kani for chromosome 12 and in Adiya and Kattunaikkar for chromosome 13. In Adiya and Kani communities, the deviations were observed in the same locus-pair combinations in chromosomes 12 and 13. Markers having smaller recombination distance were found to show significant deviations. It is interesting to note that the linkage disequilibrium between locus-pairs remained inconsistent with populations, with the exception of Adiya, Kani and Kattunaikkar. This suggests that these populations might be closely linked and they might have had a common origin. We found strong linkage association among markers placed up to a distance of 18 cM in chromosome 13 and up to a distance of 22 cM in chromosome 12. Some neighborhood markers did not show significant association in comparison to distantly placed markers. This shows that these markers are differently placed in our population in comparison with CEPH families. Linkage disequilibrium between microsatellite markers could reflect on the functional genetic profile of an individual or a population. Therefore, it would be interesting to study the role of functional genes located between these markers. This might help us to identify the ethnic predisposition factors, and to understand the impact of inbreeding in these populations. Y chromosome analysis, which is an indication of male migration and gene flow, gave some interesting results. Only minimal haplotype sharing was observed between the populations. The Adiya and Kani tribes were found to be the most genetically diverse groups, whereas the Kuruma exhibited very low heterozygosity. Haplotype sharing was observed between Kani and Kuruma tribes, though only to a limited extent. The UPGMA tree showed two clusters, one cluster comprising mainly of populations speaking Kannada dialect of the Dravidian language, and the other cluster comprising mainly of populations speaking Malayalam dialect of Dravidian language. The AMOVA analysis indicated that greater diversity was observed within populations than between populations. This also supports the idea of a common origin for these tribes and / or a gene flow between them. Network analysis on these markers suggests that there is a unidirectional pattern of mutation, which indicates that the evolution might also be unidirectional in these populations. SNP analysis in the functional NAT2 gene indicated that majority of the people in the study populations are slow acetylators. The only exception is the Kuruma tribe, where there was a slight predominance of rapid acetylators. The results from this study are concordant with the studies in other Asian populations, where ~60% of the people are genotyped as slow acetylators (Cascorbi et al. 1995). However, the populations are distinctly different from the Japanese and Chinese populations, which are predominantly rapid acetylators. The allele 5B, which is supposed to have an European origin, was present in very high frequency in these ethnic populations. An overall predominance of rapid acetylators was observed in males compared to females. A new deletion mutation was observed at 859 site (BamHI site), in Malapandaram tribe; it was found to be linked to the mutation at 481 site. Consequently, two new alleles were observed: (i) 282, 341, 481 and 803 mutations (GenBank Accession no. AF348074) and (ii) 341, 481, 803 and 859 mutations (GenBank Accession no. AF348075). The new mutation and the new alleles were observed only in the Malapandaram tribe. Thus, the study shows the impact of ethnicity in pharmacogenetic profiles. The UPGMA tree based on Nei's unbiased minimum distance method (1978), places the Malapandaram in a separate group. The remaining seven tribes are placed together in a separate cluster. This observation is significant because the Malapandaram is a nomadic tribe, unlike the other tribes. The phylogenetic relationships were found to be basically similar in LMS microsatellite analysis and NAT2 SNP analysis. This shows that the mode of differentiation of study populations with respect to the NAT2 gene is related to the mode of genetic differentiation in these populations. This indicates that the variation in pharmacogenetic traits with respect to NAT2 gene has a genetic background as well, in addition to the possible environmental and physiological factors. This study is a preliminary step towards establishing a genetic database of ethnic populations in Kerala. The observations made during the study can be utilized in preparing individual gene cards. The study might open up several avenues in characterizing inbreeding and resultant genetic diseases. The implications of our observations might find their application in a variety of fields such as medical genetics, forensic science, anthropology and pharmacogenetics. 
  	 




Molecular Signatures of Tribal Populations in Kerala Based on DNA Fingerprinting

K., Venkata Subba Rao

Syntheses of heterocyclic compounds by cyclization and oxidation reactions on semiconductor photocatalysts supported over zeolites

JHA, KULA NAND

Abstract
Fertilization is a unique event in the life of an organism. It results from the reciprocal interaction of the sperm and the oocyte. Spermatozoon or the male gamete is the end product of a complex and highly ordered chain of events occurring in the seminiferous tubules of the testis. The series of events leading to the transformation of undifferentiated germ cell into highly differentiated spermatozoa are termed spermatogenesis. However, the testicular spermatozoa lack the ability to recognize the oocyte and hence it is unable to fertilize. Testicular spermatozoa undergo further maturational processes both in the epididymis as well as in the female reproductive tract in order to acquire the fertilization-competence. Post-testicular sperm maturation during the passage through the epididymis confers progressive forward motility to spermatozoa which is essential for them to reach the exact site of fertilization in the famale. However, even after epididymal maturation the spermatozoa remains fertilization-incompetent. But the spermatozoa on reaching the female reproductive tract undergo yet another maturational event which makes them capable enough to fertilize the oocyte. This maturational event associated with spermatozoa while they transit through the female reproductive tract is termed 'capacitation'. Capacitation culminates in acrosome reaction followed by fertilization and thus, it is absolutely required for successful fertilization. The physiological site of capacitation is either the uterus or the oviduct and it requires many factors present in the female reproductive tract secretion. However, it can be achieved in in vitro in a defined medium containing certain essential components such as, energy sources (glucose, lactate, pyruvate), a protein source (usually BSA) and certain electrolytes (Ca2+, HCO3-). During the process of capacitation the spermatozoa undergo a number of physiological and biochemical changes such as changes in intracellular ion concentrations, plasma membrane fluidity, metabolism and a distinct transformation in motility termed hyperactivation. Hyperactivated spermatozoa are more vigorous and less progressive in motility than the non-hyperactivated spermatozoa. Although, these changes are known to accompany the process of capacitation, it is still not clear if they are absolutely required for capacitation. Furthermore, the molecular basis of capacitation and the events concomitant with this are very poorly understood. But, considering that the spermatozoon is a terminally differentiated cell, it is possible that post-translational modifications such as phosphorylation, glycosylation etc play an important role in capacitation. The present study was undertaken to elucidate the molecular basis of capacitation in golden hamster (Mesocricetus auratus) spermatozoa and the specific objectives were:1. Capacitation-associated changes in hyperactivation, protein phosphorylation and acrosome reaction.2. The involvement of protein kinases and phosphatases in capacitation and,3. The protein(s) which undergo phosphorylation during capacitation.Chapter 1 is a review outlining the changes associated with spermatozoa during capacitation viz. hyperactivation and acrosome reaction. It describes the motility changes occurring during capacitation as studied by the computer-aided sperm analysis in a variety of species, the physiological significance of hyperactivation, its linkage to capacitation and the possible mechanism of regulation of hyperactivation. It also describes the mechanism and the signaling events known to be operative in acrosome reaction. Further, the physiological regulators of capacitation and the existing model for the signaling cascade during capacitation are described at length. The changes in protein phosphorylation during capacitation and the involvement of protein kinases and protein phosphatases associated with them are discussed. It also describes the phosphoproteins which undergo phosphorylation during capacitation. Chapter 2 deals with the materials and methods employed, the source of the various chemicals used and the details of the animal system used. The basic definitions of the parameters set for computer-aided sperm analysis and the set parameters conditions are also described. In Chapter 3 a detailed account of the experimental results is given. Section 3.1 up to 3.3 includes the capacitation-associated changes in hyperactivation and acrosome reaction and the effects of BSA, Ca2+ and HCO3- on these two events. The results indicate that hamster spermatozoa undergo a distinct changes in motility pattern and motility parameters during capacitation and thus capacitated spermatozoa can be distinguished from the non-capacitated ones by computer-aided sperm analysis. Further, among the media components BSA or PVA and Ca2+ are essential for hyperactivation whereas in the absence of HCO3- hyperactivation is delayed. It also indicates that PVA can substitute for BSA. Hamster spermatozoa also undergo a time-dependent increase in spontaneous acrosome reaction during capacitation. BSA or PVA and Ca2+ are absolutely required for acrosome reaction and HCO3- does not seem to be important if the medium pH is maintained by other buffering agents such as HEPES. Section 3.4 up to 3.6 deals with the changes in protein tyrosine phosphorylation during capacitation and the effects of the media components on protein tyrosine phosphorylation. The results indicated that a set of proteins in the range of 50-150 kDa get tyrosine phosphorylated during capacitation in a time-dependent fashion and two proteins of mol. wt. 83 and 97 kDa were the most prominent tyrosine phosphorylated proteins. When the effects of the media components were studied on protein tyrosine phosphorylation it was observed that Ca2+, HCO3- and BSA or PVA were required for the protein tyrosine phosphorylation. Furthermore, the inhibitory effect on protein tyrosine phosphorylation due the absence of both Ca2+ and HCO3- was significantly greater than when either of the two was not added. Thus, protein tyrosine phosphorylation appears to be a very important regulatory event during capacitation. The results from 3.1 up to 3.6 also indicate that capacitation is linked with hyperactivation and/or acrosome reaction in hamster. If protein tyrosine phosphorylation is involved in capacitation, then protein tyrosine kinase (PTK) and phosphatase (PTP) must be involved in the process. The results described in section 3.7 up to 3.9 provide evidences that protein tyrosine kinase(s) and protein tyrosine phosphatase(s) are probably involved in capacitation. Specific inhibitors for PTK and PTP were used to study the effects on motility, hyperactivation and protein tyrosine phosphorylation. Among the three inhibitors used for PTK piceatannol inhibited the motility and tyrosine phosphorylation very significantly and hyperactivation was totally inhibited. Sodium orthovanadate, a PTP inhibitor also inhibited the motility and no hyperactivation was observed in its presence. Further, it increased the protein tyrosine phosphorylation as expected. Once the involvement of PTK and PTP was established we were interested in characterizing the proteins which undergo protein tyrosine phosphorylation during capacitation (Section 3.10 to Section 3.16). For this purpose the N-terminal sequence of the most prominently phosphorylated protein (83 kDa) was analyzed and it showed high homology with a testis specific protein called AKAP82 (a kinase anchoring protein) from mouse. It's homologue from bovine, human and rat were also already reported. However, it was not known if this protein was involved in motility only and/or capacitation. Therefore, to get more insight into its possible role in hamster sperm capacitation further experiments were carried out using degenerate primers from the N-terminal sequence of the 83kDa protein and the database the cDNA was made and amplified by RT-PCR. The cDNA sequence showed high homology to its homologue from mouse, human, rat and bovine. It had the conserved proteolytic cleavage site and the RII binding site characteristic feature of the testis AKAP82 homologues. By RT-PCR approach the expression of AKAP83 was investigated and it was found to be testis specific in hamster. In order to know its localization which would also help in knowing its possible role, polyclonal antibodies against AKAP83 were generated. Immunoblot studies with anti-AKAP83 antibody indicated that AKAP83 is synthesized as a precursor protein (mol. wt. 97 kDa), it is tyrosine phosphorylated only in capacitated and not in non-capacitated spermatozoa, and it is associated with the fibrous sheath as the signal was observed only in the principal piece of the tail. Taken together these results indicate that AKAP83 is important for capacitation. This is the first protein characterized from hamster spermatozoa which undergoes differential phosphorylation during capacitation. We were also interested to know if the spermatozoa also show differential serine/threonine phosphorylation during capacitation. Section 3.17 up to 3.21 deals with the results obtained from the experiments designed to investigate the role of serine/threonine phosphorylation in capacitation, hyperactivation, and acrosome reaction. Hamster spermatozoa showed changes in serine/threonine phosphorylation in a time-dependent fashion when incubated in a medium supporting capacitation. Protein serine phosphorylation did not show any change with respect to the intensity of the bands during the incubation of the spermatozoa in a medium supporting capacitation until 7-8 h, except a 100 kDa protein which showed a time-dependent decrease. However, two proteins of mol. wt. 49 and 63 kDa showed time-dependent increase in threonine phosphorylation during capacitation. Unlike protein tyrosine phosphorylation which was observed to begin in 1-1.5 h these two proteins started getting phosphorylated at threonine by 3 h and reached the saturation in 7 h much later than the former which was saturated by 5 h. Media components did not have any affect on the differential phosphorylation seen in these proteins. The results indicated that probably these proteins were more crucial for acrosome reaction. Furthermore, by using specific inhibitors the involvement of serine/threonine kinase and phosphatase was demonstrated in hyperactivation and acrosome reaction. The chapter ends with the localization changes with respect to ser/ thr phosphorylated proteins during capacitation.Chapter 4 includes the discussion of all the above results in light of the data already available at present on this subject. The chapter concludes with a model depicting the signaling cascade involved in hamster capacitation hypothesized on the basis of present results and the results reported in literature so far.



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