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Item Apoptosis regulators as targets for induction of apoptosis of Imatinib resistant chronic myeloid leukemia cellsBiswas, NabenduApoptosis is a regulated cell death characterized by cell shrinkage, nuclear condensation, DNA fragmentation, membrane blebbing. There are several apoptosis regulators eg: BCL2, Bcl-xL, BAX, Survivin, FLIP, XIAP, cIAP etc. Targeting these apoptosis regulators may be a viable strategy for the treatment of cancer because one of the hallmarks of cancer is the deregulation of apoptosis. In our first study we tried to induce cell death in Imatinib-resistant chronic myelogenous Leukemia cell line K562, by using TRAIL, a well-studied anti-cancer agent. Normally these cells are also resistant to TRAIL. We used Hydroxychavicol, a Piper betel leaf derived polyphenol, to make this Imatinib- resistant K562 cells sensitive to TRAIL. In imatinib resistant K562 cells, XIAP, FLIP have emerged as targets by hydroxychavicol to sensitize the cells to TRAIL mediated apoptosis. Reactive Oxygen Species, particularly H2O2 has been shown to be a key player for this TRAIL sensitization by Hydroxychavicol. We found that ROS decreased XIAP, FLIP in imatinib sensitive K562(S) and imatinib resistant K562(R) both. These XIAP and cFLIP downregulation abrogated their inhibitory effect on Caspase activation and removed its break from extrinsic apoptotic pathway activation by TRAIL and this leads to apoptosis of Imatinib-resistant K562 cells and imatinib sensitive K562 cells. In Imatinibresistant K562 cells, FLIP and XIAP were differentially regulated by JNK and ERK respectively. Akt phosphorylation was decreased by ROS-activated ERK. Dephosphorylation of Akt inhibited its binding to XIAP and that lead to the destabilization of XIAP. On the other hand, ROS-activated JNK increased the expression of an ubiquitin ligase ITCH which degraded FLIP by binding to it and ubiquitination. However, interestingly, when we checked these finding on K562(S) cells, some of the findings were different. In K562(S) cells, ROS degrades XIAP, FLIP by lysosomal degradation pathway. Moreover, JNK alone instead of JNK and ERK played important role in this XIAP and FLIP downregulation. Thus, our findings suggest anti-apoptotic proteins XIAP and FLIP as a viable therapeutic target for Imatinib-resistant CML. We have also identified a novel ROS mediated regulatory pathway of these two proteins which may be further explored for therapeutic targets.Item Assessment of fluoride induced male reproductive disorders in adult Wistar rats and its amelioration by supplementation of vitamin C and EMukhopadhyay, Prabir KrFluoride is necessary for tooth and bone development, but excessive exposure causes different system disorders including male reprotoxic consequences. The purpose of this study was to investigate the extent of fluoride toxicities in the male reproductive system and its possible management by vitamin C and E. The study was undertaken to find out the optimum dose of fluoride responsible for causing considerable reprotoxic effects in adult male Wistar rats. The rats were divided into four groups; control (group I) rats received vehicle only and treated rats (group II, III, and IV) were administered sodium fluoride (NaF) orally at 10, 15, and 20 mg/kg/day doses, respectively, for 30 consecutive days. Assessments were done on the following parameters: histoarchitecture of testis and epididymis; spermatozoal potential and DNA integrity; testicular oxidative status, activities of functional marker enzymes and DNA integrity. All these parameters were altered in a dose-dependent manner and based on these; 15 mg/kg/day was selected as the minimum dose responsible for considerable alterations. Further experiments were carried out with this dose. The protective role of VC&VE was assessed on different animal models. Rats were divided into four groups. Group I was the control; Group II received NaF at 15 mg/kg/day dose; Group III was provided with VC (200 mg/kg/day) and VE (400 mg/kg/day) along with NaF; Group IV received only VC&VE for 30 consecutive days. The following studies were undertaken: structure-function integrity of testis, epididymis and spermatozoa; oxidative status of testis and epididymis; testicular functional and steroidogenic status; apoptotic pathway of testis and epididymis; testicular inflammatory status along with spermatozoal apoptotic state. All these parameters were altered due to the fluoride threat. Supplementation with VC&VE restored all the anomalies either to normal or near normal levels. The group IV rats showed a similar pattern of the results to the group I rats.Item Bioactivity, Phytochemical Screening and Synthesis of Potent Nanoparticle from some Mangrove and Weed Species of South West Bengal against UropathogensSikdar (née Bhakta), MausumiPlants are generating an ever increasing interest due to their effectiveness, low cost and minimum side effects associated with drugs derived from them.Nowadays infection from common pathogens are mostly resistant to several antibiotics which undermine their ability to control the infections. Sufferings from Urinary Tract Infection (UTI) across the globe is alarming. Four mangrove species found in Sundarban areas of West Bengal, namely-Avicennia alba Blume.,Avicennia marina(Forssk.) Vierh., Brugeiracylindrica(Linnaeus) Blume and SonneratiaapetalaBuch.-Ham and two weed species found in Southern part of West Bengal, namely,- LippianodifloraL., Chlerodendruminfortunatum(Linn)are selected to investigate their antibacterial activity against two epidemiologically most prevalent uropathogens of Eastern India Escherichia coli and Klebsiellapneumoniae.Antioxidant content and LC-MS analysis of the plant leaves were studied. As they synthesize a large amount of bioactive compoundsthe selected plant leaves are employed to reduce silver nitrate to silver nanoparticles. Their efficacy as antimicrobials and their characterization were investigated in this study. The characterization of the AgNPwere done by UV-vis spectrophotometer, Fourier Transformed Infra Red Spectroscopy, Zeta Potential and X-ray Diffraction, Transmission Electron Microscope and Scanning Electron microscope. The study of synergistic effect of AgNP with different antibiotics depicted enhanced antibacterial activity of the antibiotics. The cytotoxic level of the synthesized nanoparticles against human red blood cells and human kidney cells were also reported. This study forms the foundation for development of alternate drugs to treat resistant human pathogens. Further investigations of the plant extracts to identify the bioactive compounds will be beneficial in alternate drug formulations as well as drug modifications.Item Characterization of Aluminum Stress Tolerance in Mung Bean for Crop ImprovementGanesan, M.Under acidic soil and aluminum stress conditions, the crop plants are facing several growth defects. Particularly significant decrease in root growth, nutrient uptake and low yield are the very common problems. Besides, several organic acid transporters are involved in Al sensing, transport and detoxification mechanisms. In this study, Al stress tolerance of mungbean plants was studied under different Al stress conditions. The results showed that the mungbean plants are severely affected by Al stress and released significant amount of malate in hydroponics media when compared with control plants. Based on this study and expression analysis of Al stress responsive genes, further, we developed ALMT1 overexpressors and ALMT1-RNAi transgenic plants of mungbean to analyze the acid soil tolerance. Later, these transgenic mungbean plants were challenged with different Al concentrations to check their stress responses. The ALMT1-RNAi lines showed almost no root growth variations in hydroponics media when compared to WT plants under normal growth conditions but displayed significant decrease in root length on exposure to Aluminum. Further, when AtALMT1 was overexpressed, even in absence of Aluminum stress, there was approximately 12% increase in length of primary roots when compared with WT. Malate efflux was almost doubled in the AtALMT1 overexpressed plant lines whereas KD lines showed around 36% drop in malate efflux under Al stress conditions. Therefore, based on these observations, we concluded that VrALMT1 has a significant role on Aluminum stress tolerance responses in mungbean plants.Item Characterization of Indigenous Sewage Microbial Communities of Kolkata and their Bioremediation Potentiality in Heavy Metal Contaminated Soil(Biswas) Sarkar, ShampaCulturable bacterial profile, bacterial chromium tolerance, chromium removal efficiency, their role in plant well-being and environmental cleaning were investigated in this study. The literature survey revealed that the microbes growing in heavy metal polluted soil can furnish an impressive mode of environmental heavy metal degeneration. In order to understand the activity of microbial heavy metal tolerance, rhizospheric microbes were isolated from the soil of three hot and humid, heavy metal depository sewage reservoirs in the south (Circular Canal, Tolly Nullah), east (East Kolkata Wetland), and north (Kestopur Khal) of Kolkata. Different morphological, physiological, and biochemical characteristics and antibiotic resistance patterns were analyzed with these isolated microbes. In order to compare the composition of the bacterial community, the selected microbes were subjected to molecular characterization after fulfilled the criteria of polyphasic classification accordingly. The heavy metal resistance patterns and possible biochemical mode of action behind chromium reduction of selected microbes and their impact in reducing the chromium concentration were subsequently investigated. The microbe inhabited at anoxic to hypoxic chromium polluted sewage environment, adapted itself by such a manner that, it can utilize the redox potential of chromium reduction to fulfil its own physiological needs in the absence of oxygen. The microbe produced extracellular organic acids, chelating compounds siderophore and EPS helped in microbial chromium absorption by inducing soil chromium mobility. Soil chromium mobility also influenced the reduction of plant chromium absorption and facilitated plant growth in chromium polluted environment by means of microbial chromium affinity. The Plant Growth Promoting Rhizobacteria (PGPR)-like traits of the isolated soil microbes were investigated and their influence on rhizospheric heavy metal absorption by the plant roots was studied. Three neutrophilic mesophilic chromium resistant microbial strains (Microbacterium radiodurans K12016, Bacillus xiamenensis 1E0018 and Bacillus xiamenensis 26K018) were chosen for their selective antibiotic sensitivity and effective chromium absorption and reduction capability, which are characteristics of a good bioremediating candidate. They may help to protect humans from heavy metal toxicity by reducing soil metal accumulation and also reduce heavy metal absorbance of edible plants.Item Characterization of the Bambusa tulda Roxb inflorescences and flowers at the morphological anatomical and genetic levels to understand floral organ developments and differentiation in bambooDas, MalayBamboos belong to the monocotyledonous plant family Poaceae and subfamily Bambusoideae. Bamboos represent an extreme example of perennialism, where the reproductive phase begins after completion of an extended vegetative phase, which may vary across species. Other than delayed flowering time, existence of two different types of inflorescences, genetics of floral development and nature of pollen compatibility make bamboo unique in comparison to other grasses. The main aim of this study was to understand the floral organ development and pollination mechanism in Bambusa tulda. In this study, the relative abundance of two different types of inflorescence in an entire flowering cycle were analysed and rate of seed setting was compared. One potential limitation to perform gene expression analyses on Bambusa members was the absence of suitable reference genes to normalize expression data. In this study, reference genes were identified, which were stable across species, organs and developmental stages. In order to understand the molecular mechanism that regulates flower development, the floral meristem identity genes MADS14, MADS15 and MADS18 were sequenced from B. tulda and their expression pattern was analyzed across different vegetative and reproductive tissues. In order to understand the genetics of pollen compatibility in bamboo, in vivo pollination experiments were performed. The finding indicated that B. tulda is primarily a cross pollinated species. In order to further understand the molecular mechanism regulating genetic compatibility in bamboo, selected members of RNase T2 and Fbox gene families were studied. Overall, the findings of this study will be useful to understand the morphological, ecological and molecular basis of flower development and genetic compatibility in bamboos.Item Comparative Analysis of Eukaryotic Homo sapiens and Oryza sativa indica Class IV SirtuinsDey, SanghamitraSirtuins are a class of proteins biologically acting as epigenetic modulators. They are generally found to be involved in regulating the essential cellular processes like metabolism, aging and genome stability. In our studies we have investigated the class IV sirtuins to explore the mechanistic and regulatory aspect of this protein family. To understand the role of class IV sirtuins better we took representatives from both human and plant. Human class IV sirtuins HsSIRT6 and HsSIRT7 are nuclear and nucleolar proteins, respectively whereas OsSRT1 is a nuclear protein. Using HsSIRT6 as a control/model, we investigated the biochemical and epigenetic activities of HsSIRT7 and OsSRT1. From our studies, we found that N and C terminal region of these proteins have important role in their enzymatic activities and substrate recognition. N-terminal region in HsSIRT7 is associated with protein-protein interactions and C-terminus is often associated with subcellular localization. In case of OsSRT1, the extended C-terminal region plays important role in substrate recognition and catalysis. Sirtuins in general have a unique substrate specificity. There were available reports of H3K9Ac deacetylation by OsSRT1 and H3K18Ac deacetylation by HsSIRT7. Our studies shed light on its dual enzyme capability with preference for mono ADP ribosylation (mono ADPr) over deacetylation. Just like its plant counterpart, a comparison of these dual activities suggests HsSIRT7's preference for the mono ADPr transfer over its deacetylation of H3K18Ac. Several new targets for both the deacetylation and ADP ribosylation were detected. Class IV sirtuins can specifically transfer the single ADP ribose group on its substrates in an enzymatic manner. So, this study is the first one to report the ADP ribosylation activity in HsSIRT7 and OsSRT1, which were previously reported as only deacetylases. This mono ADPr effect is not well known in plants, more so for deacetylases. The products of this reaction (NAM and ADP ribose) have immense negative effect on these enzymes suggesting a tighter regulation. Under various ranges of abiotic stress conditions in rice plants, we could link this ADPr activity to the DNA repair pathway by activating the enzyme PARP1. The histone specific deacetylation is also related with OsSRT1 upregulation under these stress situations. Metal toxicity in plants also influences these enzyme activities. Mono ADP ribosylation in cells is often linked to different metabolic disease conditions. This kind of modification of transcription factors, p53 and ELK4 by HsSIRT7 may play a key role in maintaining the tumor phenotype. Thus, HsSIRT7 becomes an important therapeutic hotspot for drug designing to counter several disease conditions. Screening of several chemical compounds with HsSIRT7 active site has been carried out in this study.Item Comprehensive morphological and molecular analyses of flowering events in Bambusa tuldaDas, MalayFlowering is one of the most important adaptations in the history of plant evolution. Our current understanding of flowering and the genes involved in the regulation of flowering time are based on the studies conducted on the model plants Arabidopsis thaliana and Oryza sativa. Since both these plants are annual and herbaceous, it is unlikely that the information obtained from these studies can directly be translated to explain the unusually extended flowering time in bamboo. The ecological impacts caused due to bamboo flowering are known for decades, but our knowledge about flower development and also the genes controlling such unique flowering behaviour is very inadequate in bamboo. a major objective of this study was to understand the detailed reproductive features of B. tulda Roxb. by studying the morphological (inflorescence, flower and pollen), histological (inflorescence) and biochemical (pollen viability) analyses. This study observed that protrandry, limited pollen liberation, low pollen viability and low seed setting are prevalent in B. tulda. Another major objective of this study was to characterize important flower induction and pathway integrator genes in order to understand their role in flower induction in tropical bamboos. Four copies of FLOWERING LOCUS T (BtFT1, BtFT2, BtFT3 and BtFT4) and two copies of FD (BtFD1, BtFD2) genes were identified and their tissue-specific as well as diurnal gene expression analyses revealed possible functional divergence among the closely related homologs. Taken together, this study indicates that copy number expansion and expression divergence may lead to new regulation of flowering gene functions in bamboo.Item Crosstalk between Proteasomal and Lysosomal Protein Degradation Pathway in EBV-Induced B-cell LymphomasSaha, AbhikEpstein-Barr virus (EBV) nuclear oncoprotein EBNA3C plays an important role during B-cell transformation and subsequent development of several B-cell lymphomas specifically in those who are from immuno-compromised background. EBNA3C manipulates several important cellular pathways including ubiquitin-proteasome machinery and deregulate multiple cellular oncoproteins and tumor suppressor proteins. Studies have revealed that EBNA3C is ubiquitinated at its N-terminal domain and can interact with 20S proteasome machinery. However, In vitro, the viral protein is extremely stable in EBV transformed growing B-lymphocytes. EBNA3C can also bypass autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Moreover, recently, our group demonstrated that in response to metabolic stress, EBNA3C elevates the basal level of autophagy through epigenetic alternation and transcriptional activation of several autophagy related genes (ATGs). This also serves as a prerequisite for B-cell survival under growth limiting conditions. The crosstalk between EBNA3C mediated proteasomal and autophagy-lysosomal machinery further prompted us to investigate the underlying proteolytic mechanism governing EBNA3C’s turn-over. We demonstrate that proteasomal inhibition accelerates EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, in presence of proteasomal inhibitors, two EBNA3 family oncoproteins–EBNA3A and EBNA3C were degraded, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated and colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, thus facilitating autophagy-mediated degradation. We further describe that the degradation signal is located at the first 50 residues of the N-terminal domain of EBNA3C. The colony formation ability of this important viral oncoprotein is also reduced when proteasome is blocked. In addition, proteasomal inhibition induces apoptotic cell death and accelerates transcriptional activation of both latent and lytic gene expression which further induces viral reactivation from EBV transformed B-lymphocytes. Overall, this study provides rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas.Item Deciphering micro/nanoplastic induced phytotoxicity with special emphasis on the cytogenotoxic effects on Allium cepa L.Pramanick, KousikConsidering the present plastic pollution crisis, this thesis work reported the phytotoxicity of polystyrene micro/nanoplastics (M/NPs) in onion with effects on seed germination, root growth, and cytotogenotoxicity. The detection of negative impacts in environmentally relevant concentrations of polystyrenes (PS) clearly showed the harmful effects of polystyrene particles on the plants health, indicating its possible negative impacts on biodiversity. The root growth rather than seed germination was negatively affected by the increasing concentrations of polystyrene particles. The tested diameter of polystyrene particles caused an imbalance in redox homeostasis through the elevated production of ROS, which in high concentrations of polystyrenes subdues the antioxidant defence and ensues in different harmful effects. Different chromosomal alterations and DNA damage potential of micro/nanopolystyrenes (M/NPS), as recorded in this work, may indicate the potential threat of micro/nanoplastics to other organisms because, the basic genetic makeup is same for all eukaryotes. The combined effects of nanoplastics and heavy metals such as Cd and Hg were also evaluated showing an increasing level of toxicity of Cd and a decreasing toxicity of Hg in the presence of polystyrene particles. This observation signifies a differential effect of plastics in modulating the toxicities of heavy metals depending on metal species. Another interesting finding of this work was the positive impact of high temperature or temperature stress (TS), but not the salinity stress (SS), in aggravating the toxicity of micro/nanoplastics. The temperature stress improves the root accumulation of polystyrene particles with notable deleterious effects on oxidative stress, microtubules stability, and organization. In this study, we have used the term micropolystyrene and nanopolystyrene interchangeably for the same diameter (100 nm or 0.1 µm) of polystyrene particles, because the selected diameter remains at the transition between micro and nano particles and there is no accepted distinction on nanoplastics size unlike metal nanoparticles. Some authors have used the term micro for this diameter, while others have used nano for the same.Item Evaluation of physiological stress due to Schoolbag carriage among children in West Bengal and some suggestive remedial measures an ergonomic studySen, DevashishSchoolbag carriage among children is an intriguing cause of concern since it generates multiple manifestation of stress. Several studies have reported escalation in subjective parameters like pain occurrence and severity. Augmentations in ground reaction force, trunk inclination angle, muscle activity, heart rate, oxygen consumption and energy expenditure were experimentally observed. The study embodied exploration of the effect of schoolbag carriage among male and female children belonging to urban and rural habitat of high and low socioeconomic status (SES). The primordial task of collating baseline data regarding schoolbag weight and duration of carriage among children of West Bengal was conducted. The objective of the work was to suffice the prerequisite of devising interventions aiming to ameliorate the resultant stress experienced by school children. Majority of the children carried schoolbag load exceeding the recommended limit of 10% load of body weight (BW). Duration of carriage among the children belonging to low SES was prolonged than their counterpart from high SES. The influence of schoolbag weight and duration of carriage on reported pain was observed among children. The fallacy of posture analysis tools like rapid entire body assessment and ovako working posture assessment system involving consideration of absolute weight for generating scores of risk assessment was probed into with reference to children representing an array of SES. Occurrences of self-reported pain were identified to be predominant in shoulder, neck and back among children. Variation in reported pain was found to exist with SES of the children. Combined effect of schoolbag weight expressed as the percentage of BW and duration of carriage was found to be associated with pain reported by the children of different groups. Children from different SES were exposed to cardiopulmonary stress even due to schoolbag carriage of 8% load of BW. No significant difference was observed in means of heart rate, oxygen consumption, energy expenditure when assigned with 8%, 12% and 16% loads of BW between children of high and low SES (p>0.05). This study corroborates ground to re-consider the established load limit for children and suggests recommendation to accommodate changes in perspective.Item Exploring the possibilities of enhancing enzymatic activities of phytopathogenic fungi for their utility in biological control of waterhyacinthRay, PujaGlobal infestation of invasive hydrophyte waterhyacinth [Eichhornia crassipes (Mart.) Solms-Laub. (Pontederiaceae)], has led to an intensive investigation of several methods for its control. Among the most eco-friendly methods implemented, several phytopathogenic fungi are often found to cause damage to their host-plants tissue, through production of phytotoxic metabolites that induce disease symptoms (such as chlorosis, necrosis, etc.), eventually leading to the death of the plants and in the case of weeds, their control. Newline During this study many fungal cultures were isolated from waterhyacinth. 152 fungal isolates, belonging to more than 25 genera were identified by morphological studies. Several potential isolates were subjected to molecular characterization by Sanger sequencing using 18S rDNA. They were evaluated for their biocontrol potential and host-range studies. Among the potential fungal pathogens of waterhyacinth, few have shown to produce an array of lytic, cell-wall-degrading enzymes (CWDEs) that can degrade the polysaccharides of plant cell walls and break the structural moiety of the plants. On the basis of the damage intensity incurred by the primary metabolites on the host weed, the present study highlights on one of the first reports, where xylanases (in its optimised condition), with a molecular weight of 24 kDa, produced by Fusarium oxysporum sp. lycopersici 4287, have shown potency against its targeted host, waterhyacinth (a noxious aquatic weed) by aiming the xylans, which are a major hemicellulosic component of the cell walls. Evolutionary diversion of the responsible enzyme-producing genes (XYL2, XYL3, XYL5) among related species of the selected fungi, also gives an idea of perception of the involvement of these genes in cell-wall degradation. The knowledge of the mode of action with their evolutionary significance eventually helps to predict the breaking of the structural rigidity and moiety of the plant and leading to control its invasiveness, via close relatedness.Item Functional role of Interleukin-6 and Interleukin-10 in the reproductive processes of a teleost fish, Anabas testudineusPramanick, KousikIn mammals, interleukin-6 (IL-6) and interleukin-10 (IL-10) has an important function during the reproductive processes, however, the functions of IL-6 and IL-10 in fish have not been elucidated. In the present study, there was quantification of de novo synthesis of ovarian IL-6, IL-10 and tumor necrosis factor-alpha (TNFα) in control and hCG treated fish and results were compared with those from an in vitro study where there was evaluation of the regulatory functions of gonadotropins and TNFα of IL-6 and Il-10 secretions. Relatively greater concentrations of ovarian IL-6 at the post-GVBD (post-germinal vesicle breakdown) stage and higher concentration of ovarian IL-10 in post vitellogenic (PV) and post-GVBD stages indicates IL-6 and IL-10 can modulates reproductive processes. The hCG-induced increase in relative abundance of IL-6 and IL-10 (in vitro) mRNA transcript and secretion from the ovary were attenuated when there was administration of the inhibitor of TNF-α secreting enzyme, TAPI-I, which indicates TNF-α modulates IL-6 secretion and facilitate the production of IL-10. Treatments with IL-6 induced a marked increase in ovulation rate but there was no direct involvement of IL-10 during the time of maturation and ovulation. In vitro established that Il-6 induced ovulation by activating matrix metalloproteinase (MMP). Furthermore, treatment with IL-6 resulted in production of prostaglandin as indicated by the IL-6 induced increase in the abundance of ptgs2 mRNA transcript in the ovary of Anabas testudineus. IL-10 during this process mostly play the regulatory role of the cytokine environment in the oocyte the inhibition of IL-6 when treated with IL-10 in PV follicle establish the fact. Furthermore, results indicate the source of IL-6 and IL-10 in the ovary, is the granulosa cells for IL-6 and both theca and granulosa cells for IL-10 with secretion of IL-6 and IL-10 being induced by the additions of hCG and TNFα in the medium. There was also an IL-6-induced increase in abundance of receptors (IL-6 Rα and gp130) to which it binds indicating IL-6 autoregulates this population of receptors. There was also marked increase in IL-10 receptor IL-10R1 and IL-10R2 suggesting their involvement during the reproductive processes. Stat-3 activation ensured the involvement of IL-6 during the time of ovulation. Results from this study, for the first time, elucidate the reproductive functions of IL-6 in a teleost fish.Item Identification and molecular characterization of important flowering genes in Bambusa tulda Roxb with major emphasis on photoperiodic pathway and integrator genesDas, MalayBamboos belong to the subfamily Bambusoideae, family Poaceae and are phylogenetically close to the reference monocot rice (O. sativa). They display extensive variations with respect to flowering time, which may extend upto 120 years. Besides, another ecological feature is mass death of all the flowering culms, known as semelparity. Therefore, it has direct impact on forest dynamics and indirect impact on the sustainability of crops. Flower induction is a very important developmental events in plants' life. It takes place in a favorable environment as a result of regulatory crosstalk at the molecular level. Signals from multiple external and internal sources ger synchronized to decide the timing of flowering. The two major environmental stimuli that influence flowering are light and temperature. In particular, duration and nature of light plays very important role, based on which plants can be characterized as long day, short day and day neutral plants. The main focus of this study is to understand the role of light mediated flowering pathway on bamboo flowering. Therefore, in this study, four circadian clock genes (LHY, TOCI, ZTL, GI) and two clock integrators (CO A. B) of photoperiodic pathway were identified from Bambusa tulda and sequence characterized in comparison to homologous genes. Detailed studies on expression patterns in different tissues, seasons and diurnal conditions provide important clues regarding their possible involvement in flowering. Finally, in planta overexpression analyses of BFD1 and BFD2 genes in Arabidopsis combined with bioinformatics analyses demonstrated that the two genes perform diverse developmental roles in bamboo.Item Identification of candidate tumor suppressor genes’ loci on chromosome 9 associated with the development of arsenic induced urinary bladder cancer in West Bengal, IndiaGhosh, AmlanThe etiological link of arsenic with bladder cancer (BC) is well established across the globe. The molecular pathogenesis of arsenic induced BC should be explored to identify potential markers of clinical importance for better disease management. Arsenic toxicity is a burning health issue along Gangetic belt of West Bengal and higher BC incidence was recorded in affected areas of the state than safe areas. On this background, present hospital-based study aimed to analyse the association of arsenic with development/progression/prognosis of BC in West Bengal and, explore molecular pathogenesis of arsenic-induced BC targeting candidate tumor suppressor genes (TSGs) of chromosome-9 due to previous reports of alteration of the chromosome in BC. In this hospital-based study, majority of BC patients were documented from arsenic affected areas of the state and in these patients, concordantly high tumor arsenic level (AsH, >100 ppb) was detected. High tumor arsenic level was found to be associated with higher proliferation potential (assessed by immunohistochemical analysis of ki67) and pathological stages of tumor and poor patient survival. Thus, in exposed individuals, arsenic accumulates in bladder tissue to influence tumorigenesis and favour acquisition of aggressive tumor phenotypes that affect disease outcome. To explore molecular pathogenesis, 9p22-21 and 9q22.3 were selected based on previous report and candidate TSGs deleted in these regions in AsH tumors were detected by analysing whole genome CGH+SNP array data of our previous study. In 9p22 region, SH3GL2 (SH3 domain containing GRB2 like 2) showed molecular alteration (deletion & promoter methylation) preferably in AsH tumors similar to array based data and, concordant reduced expression. SH3GL2 inactivation seemed to affect receptor mediated endocytosis of active EGFR as evident from overexpression of the later without any amplification of gene in same sample set. SH3GL2 inactivation and associated dysregulation in “negative regulation of ERBB signalling pathway” was seen to have prognostic significance. In 9q22.3 region, deletion, promoter methylation, and concordant reduced expression of PHF2 was seen preferentially in AsH tumors. PHF2 (PHD finger protein 2) involves in “histone lysine (H3K9me2) demethylation pathway” essential for p53 functioning. In present study, PHF2 inactivation might affect p53 mediated transcription as evident from reduced p21 expression in same set of tumors over- expressing p53 protein. CDKN2A and CDKN2B loci from 9p21 region, FANCC and PTCH1 loci from 9q22.3 region were also analysed in this study. As evident from comparable and high frequency of molecular alteration in AsH and AsL (arsenic low; ≤100 ppb) tumors, the gene loci did not seem to have important contribution in pathogenesis of arsenic induced BC. The information on association of arsenic with disease progression, prognosis and related molecular pathogenesis will have implementation in designing preventive and management strategies for arsenic induced bladder cancer in future.Item Identification of Molecular Markers associated with Development and clinical outcome of Gestational Diabetes Mellitus in West Bengal, IndiaGhosh, AmlanGestational diabetes mellitus (GDM) is considered as health hazard with respect to short and long term adverse effect on maternal and neonatal health. On the background of fragmented knowledge, present study aimed to analyze prevalence, population specific risk factor, pathogenesis and adverse disease outcome of GDM on case control basis in a Kolkata based population of West Bengal, India. Prevalence of GDM in Kolkata was 17.2%. Older maternal age, presence of acanthosis nigricans and diabetic family history was found as major risk factors. Association of oxidative stress and related inflammation with GDM indicated incidence of insulin resistance in disease pathogenesis. Genetic predisposition seemed to play major role in development of GDM of our studied population; this was evident from significant association of genetic variants of type-2 diabetes from insulin synthesis/ processing (rs7903146, rs4132670 and rs12255372 of TCF7L2 and rs2269023 of PCSK2) and secretion pathways (rs5219 of KCNJ11, rs757110 of ABCC8, rs10830963 of MTNR1B) with disease condition. As disease specific adverse neonatal outcome, macrosomia (newborn with excess birth weight) and neonatal hypoglycaemia (abnormally low blood glucose level of newborn) were highlighted in studied population. In GDM pregnancy, phenomenon of intrauterine hypoxia was evident from higher incidence of placental histological anomalies like villous edema, increased syncytial knots, chorangiosis, and thickening of basement membrane than normal one. Over-expression of GLUT1 and GLUT3 in GDM placenta was found than normal one, indicating enhanced glucose transport to fetus and was predicted as consequence of hypoxic condition. In logistic regression, villous edema, chorangiosis and placental GLUT1 were established as independent predictors for both macrosomia and neonatal hypoglycaemia and increased syncytial knots for neonatal hypoglycaemia; this indicated importance of intrauterine hypoxia and enhanced fetal glucose transport in pathogenesis of GDM associated neonatal health.Item In vitro propagation and phytochemical screening of three important ethno-medico-botanicals used by the ethnic communities of Purulia district, West Bengal, IndiaGiri, KalyanBackground: In order to explore the traditional medicine practised by the ethnic communities residing in the Purulia district of West Bengal, India, a quantitative ethnobiological approach was adopted. Three medicinal plants viz. Bacopa monnieri, Aristolochia indica and Rauvolfia serpentina were selected, micropropagted, assessed for clonal fidelity and phytochemical markers. Materials and methods: Ethnobiological surveys were conducted by interviewing a number of informants or traditional healers with the help of a semi-structured questionnaire. The survey included questions on botanical ingredients and additives, vernacular names of the plants and animals, methods of preparation and administration and restrictions during medications. Additional quantitative indices such as use value, informant’s consensus factor and fidelity level were used for data analysis. On the basis of use value three plants viz. B. monnieri, A. indica and R. sepentina were selected and micropropagted in presence of various plant growth regulators (PGRs) and/or polyamines (PAs). Clonal fidelity of the three micropropagated plants were assessed by RAPD and ISSR based markers. Further, the plants were analyzed by validated HPTLC methods for the presence of marker compounds such as bacoside A, aristolochic acid I, reserpine, ajmalicine and stigmasterol. Results: A number of folkloric use of botanicals were reported by the traditional healers. High frequency in-vitro multiplication and regeneration in the three selected plants were achieved via use of PGRs and/or PAs. Clonal fidelity assessment of micropropagated plants using two markers (RAPD and ISSR) systems revealed high genetic homogeneity and low level of polymorphism. Comparative HPTLC analysis of bacoside A, aristolochic acid I and reserpine, ajmalicine and stigmasterol content in natural and in-vitro grown B. monnieri, A. indica and R. serpentina respectively indicated higher concentrations of bioactive phytochemicals in tissue-culture raised plants. Conclusion: Possible synergistic interactions among phytochemicals and additives were indicated to explain enhanced therapeutic efficacy of mixed herbal formulations. Moreover, in-vitro high frequency multiplication and regeneration from various explants may serve as exciting culture material for propagation and conservation of this valuable medicinal plant. HPTLC studies clearly indicated that in-vitro raised plant samples can be used as excellent resource for qualitative and quantitative estimation of high value pharmaceuticals.Item Investigating the Molecular Details of Hepatitis C Virus Entry EventsMukhopadhyay, AparnaThe RNA genome containing Hepatitis C Virus (HCV), remains an enigma due to the presence of many genotypes and quasispecies. To understand genotypic differences of HCV glycoprotein E2, 29 nucleotide sequences of genotypes 1 and 3 were analysed by in silico studies. Nucleotide analysis revealed a preference for Cytosine which was attributed to a bias for Cytosine rich codon in genotype 1 and a similar but lower preference in genotype 3. Comparison of amino acids revealed a relatively conserved N-terminal region with majority of the inter-genotypic changes concentrated at the C-terminal portion. Modelling followed by docking analysis between de novo models of E2 glycoproteins and receptor protein CD81 revealed unique interacting residues for both proteins with genotype 3a exhibiting the strongest interaction with the receptor. In our quest to understand the early events of HCV entry, fluorescent pseudoparticles (HCVpp) were generated and used to study HCV trafficking. We observed internalization and recycling of fluorescence within 15 minutes of HCVpp entry and maximum colocalisation with plasma membrane marker FM 4-64 also at 15 minutes. We predict that fusion of viral and cellular membranes occurred within this time. To understand the role of regulatory GTPases Rab1a, similar experiments were repeated in a Rab1a knockdown cell lines. In these cells, we observed impaired trafficking indicating an important role of Rab1a in HCV entry. To identify entry inhibitors of HCVpp from medicinal plants, methanolic leaf extracts were prepared from Psidium guajava L., Plumeria alba L., Syzygium cumini L. and Tamarindus indica L., plants with antiviral and hepatoprotective property. We observed that Tamarindus extract exhibited an inhibitory role by both qRT-PCR and microscopic experiments. In conclusion, we have established stark differences in structure and binding activities of the glycoprotein E2 across genotypes 1 and 3. We have established a trafficking assay using fluorescent HCVpp and predict a probable time line of HCV entry. We have also reported a role of Rab1a in HCV trafficking. To establish a potent natural HCV therapeutics, we found Tamarind leaf extract to act as a potent entry inhibitor for the virus. However further mechanistic details still need to be investigated.Item Investigating the relationship between Pro Apoptotic Anti Apoptotic and Proliferation pathways mediated by TNF Related Apoptosis Inducing Ligand TRAILPal, RanjanaTriple Negative Breast Cancer (TNBC) being devoid of the hormonal receptors manifests a difficult challenge for different therapeutic approaches. As TRAIL (TNF Related Apoptosis Inducing Ligand) is known for its ability to cause damage specifically to the tumor cells. Whole transcriptome analysis of MDA-MB-231 cell line treated with rhTRAIL showed upregulation of more than 1.5fold in nine genes from the complement pathway. Kaplan-Meiers plotter showed Complement component 4B (CFB) to be positively corelated with relapse free survival in breast cancer patients. CFB was also observed to be co-related and co-expressed with TRAIL in TNBC patients. Because of the heterogenicity of breast tumor population, some cells are inherently resistant to TRAIL induced apoptosis. Via microarray analysis of the selected resistant cells, we observed CDH1 to be downregulated in the resistant cell population in comparison to the sensitive cells. Upregulation of CDH1 sensitized the resistant cells towards rhTRAIL mediated cell death. Another commonly used approach to increase the effectiveness of the TRAIL treatment is through combination therapy. Theophylline has a cytotoxic effect on the MDA-MB-231 cell line. It increased ROS production causing DNA damage and lipid peroxidation. We further observed theophylline to increase the level of TNFR1, thereby activating the caspase mediated apoptotic pathway. Moreover, rhTRAIL in combination with theophylline caused increased cell death in comparison to rhTRAIL or theophylline alone. Combination treatment increased the levels of DR5 in the rhTRAIL and theophylline treated cells. Therefore, we can conclude that CFB and CDH1 can be used as biomarkers for TRAIL therapy in breast cancer. Furthermore, rhTRAIL in combination with theophylline could be a possible alternative for breast cancer therapy. Keywords: rhTRAIL, CFB, CDH1, DR4, DR5 and theophylline