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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 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 theophyllineItem 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 A Study of Antibacterial and Anti-Inflammatory Activity of Zinc Oxide Nanoparticles (ZnONPs) on Multiple Antibiotic-Resistant Staphylococcus Aureus Induced Inflammatory Rat ModelSikdar, MausumiConventional treatment strategies involving antibiotics are gaining accelerated challenges as a result of increasing antibiotic resistance by pathogenic microorganisms. A significant quantity of therapeutic research had been focused on implementing green synthesized nanoparticles. In this study, the green synthesis method has been used to prepare ZnONPs from leaf extract of coriander. The green synthesized and chemically synthesized commercially available ZnONPs showed almost no variation concerning their size and morphology when characterized by methods like XRD, DLS, etc. Comparison of antibacterial potential among both types of ZnONPs, used against strains of (Control and Clinical) reveals, lower MIC and MBC of green ZnONPs were required to generate the same quality of bactericidal effect as achieved by using the commercial one. This signifies that, apart from easy synthesis procedure, lower doses are enough to generate the desired anti-bacterial efficacy. After inducing septic arthritis by clinical strain (KC-02) of S. aureus, both ZnONPs (chemical and green) were capable of lowering the increased serum level of inflammatory markers, without producing the measurable amount of serum oxidative stress. The combination of ZnONPs (both chemical and green) and antibiotics shows excellent antibacterial and anti-inflammatory potential. The part of this study involving the in-vivo model indicates that both the ZnONPs when used in the mentioned concentration alone and also in combination with an antibiotic, are incapable of precipitating histological changes in selected organs like the liver, kidney, spleen, and adrenal gland. Therefore, the use of ZnONPs, a preferably green one for treating inflammatory diseases like septic arthritis, could be a better choice over conventional antibiotic therapy.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 Studies on hormonal regulation of oocyte maturation, steroidogenesis and mercury-induced reproductive impairments in banded gourami, Trichogaster fasciataPramanick, KousikThe endocrine regulation of gonadal development and annual variation of key sex steroids is the basic knowledge to understand the reproductive cycle of teleost fish. Present study was aimed to investigate the levels of gonadotropins in relation to the follicular development and plasma steroids during the reproductive cycle of female Trichogaster fasciata. Furthermore, Mercury is one of the key pollutants responsible for the degradation of natural aquatic ecosystems. Pollutant may exert their action on organisms or populations by affecting their normal endocrine function as well as reproduction. Thus, the present study also tried to understand the effect of mercuric chloride (HgCl2) on reproductive function and to decipher the molecular mechanism of Hginduced reproductive impairments of female T. fasciata. In female fish, relatively higher level of FSH was observed till the ovary reaches in late vitellogenic stage confirms that FSH regulates the early folliculogenesis of the ovary, whereas LH peak was observed in the postvitellogenic stage, which indicates that maturation and ovulation were controlled by LH. Seasonal steroid profiles show that both T and E2 reach its maximum level prior to the 17,20β-P which attain itspeak value in the month of August. Moreover, this study analyzes the other key factors of ovarian function such as cyp19a1a gene expression, aromatase activity and SF-1 localization throughout the year. cyp19a1a gene expression and the aromatase activity were highest in vitellogenic stages indicate that relatively higher E2 production in this stage is regulated by FSH. Immunohistochemical localizations of aromatase and SF-1 in the cellular layer of oocytes demonstrated that aromatase is FSH-dependent and SF-1 could be regulated by both FSH and LH. Both in vivo and in vitro experiments were performed by using ecologically relevant doses of HgCl2 and the resulting effects on follicular development, steroidogenic potentiality, aromatase activity, aromatase gene expression and steroidogenic factor-1 (SF-1) expression pattern were analyzed. In vivo experiments confirm the inhibitory role of HgCl2 on follicular development, steroid biosynthesis and SF-1 activity. In vitro experiments revealed that aromatase activity, steroidogenesis, aromatase and SF-1 expression were blocked by HgCl2. The results obtained from this study contribute to understand the molecular mechanism of HgCl2- induced reproductive impairment of T. fasciata.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 Selection of high yielding elite chemotypes of paclitaxel producing Himalayan yew (Taxus wallichiana) from different geographical regions of the HimalayasDey, AbhijitTaxus wallichiana Zucc. (synonym Taxus baccata subsp. wallichiana (Zucc.) Pilg.) or Himalayan yew, has drawn a lot of interest globally. This is mostly attributed to its remarkable abundance of paclitaxel, an exceptionally potent anti-cancer drug, as well as various other significant secondary metabolites. Paclitaxel, commercially known as Taxol®, has demonstrated efficacy in treating several types of cancers including breast, cervical, head, lung, neck, ovarian, urothelial carcinoma. All taxa under the genus Taxus, including T. wallichiana, have slow growth rate over annually. Consequently, the extraction and isolation of paclitaxel from Himalayan yew for industrial applications pose significant challenges due to its limited presence in the bark, stem, and needles. The cost of paclitaxel has increased due to a combination of factors including its limited availability and growing demand in cancer therapy. The major goal of this research is to quantify the marker component, paclitaxel, which is extracted from Himalayan yew samples obtained from different locations. The goal is to find the most effective chemotypes of Himalayan yew based on different altitude in the Himalayan region. Furthermore, the current study aims to find other phytochemical compounds. The aim of this research is to establish a standardized and verified HPTLC (high performance thin layer chromatography) procedure for the quantitative measurement of marker anticancer compound paclitaxel. Furthermore, the study also encompasses a comprehensive analysis of antioxidant and toxicological study of various plant extracts. This investigation conducted in the selected region will significantly contribute to the scientific application and exploitation of these findings.Item Screening of some phytochemicals and bioactive properties of selected naturally grown and micropropagated medicinal plants of the Apocynaceae familyRay, PujaBackground of the research: Medicinal plants, their identification, preservation and phytochemical profiling have become extremely important due to excessive demand of plant derived bioactive secondary metabolites as natural products, which have potentiated present day herbal drug, pharmaceuticals and nutraceuticals based industries in an unprecedented manner. Medicinal plants are marketed for their therapeutic effect but those particular plants, where root part is more valuable than the other parts, have higher threat of eradication from the wild due to overexploitation. Under in-vitro culture system application of elicitor molecule reportedly increases total root biomass and content of secondary metabolites. In West Bengal, there is a gap in medicinal plant research especially in the southern part. So, after mindful consideration, three high-value, ethno-medicinally important medicinal plants have been selected from the Apocynaceae family, namely, Hemidesmus indicus, Tylophora indica and Cryptolepis buchanani, which, i. are marketed for the root part; ii. are found mainly in the southern plain of West Bengal; iv. have significant therapeutic efficacy. Application of micropropagation, must be accompanied with phytochemical screening (crucial for quality control and selection of elite chemotype) as well as pharmacological studies. Aim of the study: Establishment of fast, effective and innovative micro-propagation protocol, for selected medicinal plants, which can aid in their conservation, sustainable usage and could be useful for higher accumulation of plant biomass. Qualitative and quantitative assay of phytochemicals and marker compounds of selected medicinal plants as well as the comparative assessment, between wild and invitro counterparts, for the selection of elite chemotype. Screening of selected pharmacological activities with solvent extract/powder/fraction obtained from mother plant and regenerated plantlets to estimate therapeutic and commercial value of in-vitro culture of selected medicinal plants. Materials and methods: PGR plus elicitor fortified, MS media based micropropagation protocol of these medicinal plants was established where the regenerated plantlets were exposed to in-planta elicitation. The goal was to check its role on biomass generation and marker compound accumulation. Assessment of clonal fidelity was done; evaluation of various parameters and establishment of experimental model to estimate the role of different variables under in-vitro culture was performed. The mother plant (wild) and regenerated plantlets were tested cum compared for phytochemical content (TPC, TFC, TTC); photosynthetic pigment content; anti-oxidant efficacy (DPPH, FRAP, SOD, CAT, APX assay); anti-bacterial property and pancreatic lipase inhibitory activity. Moreover, quantification of marker compounds done via simple, robust and standardized HPTLC technique. Results: As root is the main part for the accumulation of marker compounds in all these three plants, the impact of different variables on the root length has been analyzed. MS- BAP (1.5 mg/l) combo has produced the highest (6.7) ‘number of shoots / explant’ and the ‘average shoot length’ is 11.7 cm in case of H. indicus. The values of these two parameters are 4.1 and 9.2 cm, respectively as observed in T. indica. In case of C. buchanani, Kin (1mg/l) has generated maximum shoot length (6.8 cm) despite of a trend of slow growth. In all these plants, IBA 1.5 mg/l have promoted rooting phenomenon more than the other combinations of auxin. Profuse base callus formation and increase in fresh weight of nodular meristemoids have been noted in T. indica which may be connected with lower multiplication rate in this plant. Methyl jasmonate (50 μM) based elicitation of healthy microshoots has yielded promising result in T. indica and H. indicus but this phenomenon is absent in C. buchanani. ISSR marker based genetic fidelity assessment has been done in support of the monomorphic plant production trend in tissue culture. Phytochemical screening has revealed the TPC, TFC, TTC, PPC content in mother plants and when the values were compared with the in-vitro ones, significant content wise compatibility has been observed in other two plants except C. buchanani. The activities of antioxidant enzymes (SOD, CAT, APX), DPPH, FRAP assays plus pancreatic lipase inhibitory activity of all these plants have confirmed the assumption that in-vitro raised plants can be a sustainable option against the wild plants in terms of basic bioactivity and phytochemical content. This idea was further supported by the rapid, validated, and sensitive densitometric HPTLC based quantification of marker compounds. In H. indicus and T. indica the values of the markers (MBAld and tylophorine respectively) are noteworthy in elicitor treated root samples and similar with naturally grown root sample. In C. buchanani, HPTLC was applied to assay the content of cryptolepine in leaf tissues of wild and micropropagated ones. The naturally harvested samples of C. buchanani have yielded better result in all phytochemical and pharmacological screening. In case of the anti-bacterial activity, the marker compounds have demonstrated significant result. Conclusions: The plants which have been selected for the present study; have traditional acceptance and commercial application. Considering their pharmaco-therapeutic importance plus overexploitation scenario the present work has recommended a fast and effective micropropagation technique, Multifaceted screening of all three medicinal plants; which have been collected from the southern part of West Bengal, is reported in the present work for the very first time. The results have also highlighted the prospective application of in-vitro micropropagation technique as a sustainable cum potential remedy against overexploitation of valuable medicinal plants.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 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 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 Studies on the diversity and ecology of zooplankton population in Sundarban Estuarine System (SES), West Bengal, IndiaMandal, SumitZooplankton play pivotal role in estuarine ecosystem. The relative importance of different environmental parameters in determining the diversity and ecology of zooplankton have been investigated from Sundarban Estuarine System (SES), India. The initial observation was designed to decipher the effect of monsoon on zooplankton species diversity, abundance along with physiochemical factors at river Saptamukhi of SES. Significant intra-monsoonal changes of zooplankton population correlated with several environmental parameters were documented. Additionally, the spatial-temporal distribution of zooplankton were recorded based on different multivariate statistics and indicator value (IndVal) analysis from the river Matla in the eastern part of SES. Seasonal occurrence of dominant taxa with high IndVal index was noticeably observed and it can be used as potential bioindicator for particular season and environmental condition in this estuarine system. Exclusive occurrence of warm water species indicated that the area is under threat of climate change mediated temperature rise. Altogether, 56 zooplankton taxa were identified with copepods forming the predominant population. The zooplankton density was highest in winter, followed by monsoon, summer and spring. Different time series observations were also carried out to unravel the specific diel and tidal variations of economically important shellfish larvae. It has been established that, their early developmental stages utilize the tidal currents for ingress and egress mechanisms in order to migrate horizontal distances and/or remain in their preferred habitats. Moreover, Metapenaeus dobsoni was also selected as a proxy to understand the diel, tidal and ontogenetic changes in their abundance within SES during two different tidal phases. Finally, the impact of climate change mediated elevated temperature on the physiology of a meroplankton species has been assessed through mesocosm experiment by using Penaeus monodon post larvae as a model organism. The growth, survival percentages, ingestion and absorption rate were decreased; howbeit respiration and ammonia excretion rate had been increased in elevated temperature resulted a negative Scope for Growth in stressful environment. The present work provides a benchmark for Sundarban zooplankton species diversity and distribution as well as their diel and tidal variations with respect to different environmental parameters for planning suitable conservation strategies and sustainable development in future.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 Structural and Biochemical characterization of a class III sirtuin, OsCobB in Oryza sativa indica and its association to stress response in plantsDey, SanghamitraSirtuins have attained recognition in the sphere of epigenetic regulation, owing to its affiliation with numerous biological processes including metabolism and genomic stability. Many post translational modifications (PTMs) are controlled by these sirtuins, playing critical roles in escaping abiotic and biotic stresses, although their pattern in plants have been poorly understood. In our investigation, a novel class III sirtuin has been discovered in Oryza sativa var indica with a high sequence identity with bacterial CobB. No class III sirtuins have been reported in plants till this study. Correlating molecular docking analysis with in-vitro and in-vivo deacylation assays helped in the understanding of various aspects of enzymatic mechanisms of OsCobB. We identified the important motifs for substrate and NAD+ binding like GAGISA, FGE, YXXR and TQNID, which were unique to class III sirtuin family. Although all modifications could be accommodated at the catalytic site, its selectivity also depended on the orientation of the peptide backbone and its interaction (bonded and non-bonded) with the sirtuin. OsCobB had a preference for the negatively charged lysine modifications like malonyl, succinyl and glutaryl through H-bonds with conserved YXXR motif. OsCobB can also accommodate the longer acyl modifications like myristoyl by a possible shift of its α4 helix. It was also established that an initial attachment of its co-substrate, NAD+ was mandatory to facilitate a conformational change for the correct binding of the peptide. In contrast to some class III members, OsCobB didnot exhibit any ADP-ribosyl transferase activity. We have identified this sirtuin to be majorly localized in the mitochondria with trace amounts in the nucleus. However, the preferential localization of this protein in the nucleus in response to low temperature and dehydration conditions could be related to its functioning. Histones (H3 and H4) as well as ACS were identified as the nuclear targets for this enzyme. Though OsCobB was incapable of efficiently deacetylating histone H4, it could deacetylate H3 at K9 and K18 residues. To cope with dehydration, OsCobB was capable of regulating the metabolism by removing different acyl groups like succinyl in H4 and butyryl, HIB and BHB in H3. Arsenic toxicity in the soil was also linked to OsCobB catalyzed H3 demalonylation and H4 desuccinylation. This enzyme was also capable of modulating mitochondrial ACS and IDH2 activity, favouring its deglutarylation and desuccinylation. This is the first mitochondrial CobB targeting important plant machinery under pathogen attack. NAD+ could be replaced by NADP+ as a cosubstrate in deacetylation reaction. OsCobB activity is insensitive to its product, NAM while showing sensitivity towards Fe2+ and Mn2+ ions. All-inclusive, this study discovered the catalytic abilities of rice class III sirtuin in conjunction with their substrates. A correlation between OsCobB overexpression and its preferential removal of lysine modifications on target substrates may hint at its regulation under stress.Item A multibiomarker study to delineate the effects of environmental stressors on aquatic shellfishes of West BengalMandal, SumitOver the past decade a great attention has been paid to decipher the consequences of impending climate change scenario on the physiological and biochemical functioning of aquatic organisms. Anthropogenic activities primarily combustion of fossil fuel is the prime cause behind the increased concentration of CO2 into the atmosphere. As a consequence, marine environments are anticipated to experience more shift towards lower pH (ocean acidification) and elevated temperatures (ocean warming). Global climate change induced ocean warming and acidification have complex reverberation on physiological functioning of marine ectotherms. Moreover, since the industrial revolution the growing demand for petroleum-based products has been mounting up worldwide leading to severe oil pollution. Widespread use of pesticides in modern agricultural system has a significant contribution in polluting aquatic habitats across the world; and enclosed freshwater ecosystem being the ultimate receptacle is at great risk. In recent years, application of nanotechnology also has become more widespread and nanomaterials are constantly being released into aquatic environments and posing a potential risk to various organisms and ecosystems. Research works merging climate change stressors and incipient chemical pollutants are imperative to better comprehend and interpret biological effects in altering aquatic habitat. Environmental factors and pollution generally do not act in isolation, but usually perform collectively. Several researches have recognized the discrete effects of these drivers on species and ecosystems separately but studies regarding the interactive effects of multiple drivers are scarce. Animal physiologists have now unambiguously recognized the fact that anticipated climatic vicissitudes will impose noteworthy consequences on physiological functioning of most species. Ectotherms are of specific interest as their physiology principally relies on the external environment. The purpose of present study was to compare and decipher the impacts of multiple stressors and specifically whether global environmental drivers aggravate the effects of a local driver (pollution) on physiological energetics and antioxidant responses of two economically significant shellfishes from West Bengal. Two species from two different ecosystems- freshwater and estuarine were studied to compare and decode the ecological functioning of these realms distinctly. Multiple biomarkers from different levels of biological strata (physiological energetics, energy budget, thermal performance, antioxidant and detoxification defence mechanisms, lipid peroxidation levels, DNA damage) of the species were evaluated to understand responses. Stress levels were amalgamated from individual biomarker responses using an “Integrated Biomarker Response (IBR)” approach. The present doctoral work can provide an outline for more integrated management of commercially exploited shellfish populations, and shed light into coastal fisheries and aquaculture prospects in the impending future.Item Investigation of stress induced Tbx20 function in autophagy in heart with comparative analyses of extracellular matrix remodeling with multiple stress inductions in cultured cardiac cell lineChakraborty, SantanuTbx20, a T-box transcription factor, is known to be crucial for cardio genesis and murine models with Tbx20 knockout fail to survive beyond E10.5. Tbx20 mutations correspond with severe congenital heart defects including valvulogenesis and septal defects. 1–3 All T-box proteins have a DNA binding domain. The T-box domain of Tbx20 consists of 180 amino acid residues and Tbx20 has a binding affinity to T/2 site [(5’-…AGGTGTGA…-3’ over the consensus T-site 5’-…TCACACCT…-3’. 25 Tbx20 promotes cardiomyocyte proliferation via the Bmp2/pSmad1/5/8 and PI3K/AKT/GSK3β/β-catenin signaling pathways.4 Tbx20 has also been found to act as a cardio-protectant against oxidative stress and downregulation of Tbx20 has been linked to increased apoptosis in cultured rat cardiomyocytes.5 Further, the cardio-protective role of Tbx20 under ROS and hypoxic conditions in the H9c2 cell line was reported. 6 Tbx20 is known to interact with and induce other transcription factors like Nkx2.5 and Gata4 7,8 which are also important for cardiogenesis, maintaining cardiac homeostasis and promoting the expression of Troponin-I and myosin heavy chain protein. Autophagy is an evolutionarily conserved catabolic phenomenon that recycles cellular components to provide for bioenergetics and fuel for cellular survival under stress conditions. At the basal level, the goal of the autophagic machinery is to maintain cellular and organ homeostasis. This is achieved by providing catabolites like fatty acids and amino acids which in turn serve as substrates for many metabolic processes. The cargo (can be organelles, protein aggregates, lipids, cellular proteins) to be degraded is sent to lysosomes via autophagosomes whereby fusion of lysosomes with the latter forms autolysosomes and the cargo is thereafter degraded by lysosomal hydrolases. Autophagy can also be activated under stress conditions like nutrient scarcity/ caloric restriction, ROS (Reactive Oxygen Species) accumulation, ER (Endoplasmic Reticulum) stress, and mitochondrial damage where autophagy serves as a substrate recycling machinery remove protein aggregates and provides much-needed ATP for the survival of cells. 9–11Autophagy has been known to be a critical factor in the survival of neonates postpartum wherein it was found that Atg5 knockout mice models failed to survive after birth. Furthermore, in mice that survived the brief period of nutrient deprivation postpartum (which is the usual scenario), massive upregulation of cardiac autophagy was observed giving away the importance of autophagy in pro-survival while also bringing cardiac autophagy to the centre.12 Impaired and altered autophagy has been an underlying cause of cardiac diseases like AMI (acute myocardial infarction), Ischemic heart disease (IHD) and cardiomyopathy. A similar role of autophagy has been found in acute myocardial infarction wherein inhibition of autophagy enlarges the infarct zone and decreases the ATP content of cardiomyocytes and there have been theories that augmenting autophagy would be a therapeutic approach in restoring the cellular integrity and cardiac functioning in these case13–17 Aging is perhaps one of the greatest risk factors responsible for failing hearts. In fact, aged individuals with no underlying cardiac condition show poor cardiac functionality, diastolic function and left ventricular dilatation. 18–20Accumulation of protein aggregates, misfolded proteins, a poor balance between ROS and anti-oxidants, mitochondrial derangements, attenuated expression of Sirtuins (a class of NAD+ dependent deacetylating enzymes) especially Sirtuin 1, 3 (Sirt 1,3), GSk-3 contribute to cardiac aging.21–23 As such, impaired and poor levels of autophagy are prevalent in aging hearts while apoptotic levels are on a surge. Augmenting autophagy by either calorific restriction or Rapamycin (Rap) administration has shown improvement in cardiac functioning and improved life longevity.24–26 Here in this study for the first time, we demonstrate the role of Tbx20 as a potential candidate to induce anti-senescence-like characteristics in the aging mice population. Autophagy induced expression of Tbx20 activates GSK-3 and transcription factors Nkx2.5, Gata4 and Sirt1 after subjection to starvation (Strv) and rapamycin (Rap) treatment in both in-vivo (BALB/c mice) and in-vitro (H9c2 cell line) model systems. The upregulation of Nkx2.5 and Gata4 following autophagy induction is indicative of progression towards progenitor like cardiomyocyte characteristics, while activation of Sirt1 and GSK3 suggests an anti-aging/ senescence since Sirtuin1 is closely linked with aging, is known to be a mediator of caloric restriction and Sirt1 transgenic mice prevent early mortality. With pre-existing knowledge of the expression of Sirt1 and GSK-3 under autophagy conditions along with the cardioprotective roles they play, these two were chosen as possible candidates that could interact with Tbx20. Further, Tbx20 loss of function (LOF) assay in the H9c2 cell line validated Tbx20-dependent expression of Sirt1, GSK-3, Nkx2.5 and Gata4. On the other hand, ECM remodeling in heart or cardiac remodeling remains an important factor in the pathophysiology of cardiovascular diseases.27,28 Collagen-I forms the major component of the matrix interstitium of the myocardium in addition to Collagen-III, fibronectin, proteoglycans, tissue inhibitors of matrix metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs). The three stages of cardiac remodeling following cardiac injury are inflammatory, proliferative and maturation phases leading to a mature scar formation.29 The preliminary stages of ECM remodeling are necessary as it prevents rupture of the ventricular wall, however, exacerbated ECM remodeling leads to progressive fibrosis in the heart and cardiac malfunctioning.30,31 The MMPs (zinc-dependent proteases) are involved in the turnover of matrix proteins like Collagen.32 Adamts4, a member of Adamts family is an important MMP. Adamts4, also is a disintegrin with thrombospondin like motifs.33,34 The mode of action of Adamts4 is by binding to ECM proteins and thereafter cleaving ECM proteoglycans like aagrecan, versican, brevican in addition to regulating Collagen turnover.35,36 Adamts4 modulates the pathophysiology of osteoarthritis through degradation of matrix proteoglycans and eventually lead to cartilage degradation which manifests as degenerative osteoarthritis.37,38 Besides osteoarthritis, Adamts4, has also been linked with cancer and angiogenesis where its role remains controversial. Some studies report it to be an indicator of early-stage cancer like in cases of colorectal cancer, others findings suggest that its mutated and truncated fragments may suppress tumour growth through inhibition of angiogenesis. 39,40 However, the involvement of Adamts4 in cardiac remodeling is relatively less known. Only a few studies have shown the involvement of Adamts4 in atherosclerotic plaque development.41 and recent studies have shown elevated expression of both Adamts4 and Adamts1 in patients with acute aortic dissection and coronary artery disease.42,43 To decipher the molecular cascade of Adamts4 induction and associated signaling pathway, cultured H9c2 cells were used for in vitro experiments. Adamts4 expression was induced in H9c2 cells following hypoxia (Hyp) and ROS and Hyperglycaemic stress inductions. Additionally, Adamts4 expression was manipulated by siRNA-mediated loss of function and TGF- inhibitor studies with SB431542/ALKI treatment in-vitro to evaluate the hierarchy and dependency on TGF- signaling. TGF- is a known marker for inflammatory and fibrotic responses following pathological stress like Myocardial Infarction, ischemia and reperfusion (I/R) injury.44–48 Ultimately, ADAMTS4 expression was also assessed in patients with cardiac diseases namely Dilated Cardiomyopathy (DCM) and MI. Overall, this study focusses on the role of Tbx20 under stress conditions and ECM remodeling.Item Understanding the Role of Epstein Barr virus essential Nuclear antigen EBNA3C in regulating unfolding protein response UPR Autophagy networkSaha, AbhikIn rapidly proliferating cancer cells, endoplasmic reticulum (ER) tends to be overloaded with unfolded and misfolded proteins due to high metabolic demand. With the limited protein folding capacity of ER, cancer cells suffer from Unfolded Protein Response (UPR) stress and subsequently elevate autophagy, in which the protein-aggregates are degraded and provide free energy in form of amino acids allowing cancer cells for an uninterrupted proliferation. Consequently, several anticancer molecules targeting UPR-autophagy network are currently under clinical trials. Epstein-Barr virus (EBV) is associated with a number of B-cell lymphomas. EBV can readily transform quiescent B-lymphocytes into continuously proliferating lymphoblastoid cell lines (LCLs), providing an excellent model system for studying EBV associated B-cell lymphomagenesis. EBNA3C, one of the essential viral oncoproteins for B-cell transformation, manipulates multiple cell pathways – most strikingly cell-cycle/apoptosis and ubiquitin-targeted protein-degradation machineries. Interestingly, several components of these pathways were previously shown to be intricately connected with the UPR-autophagy network in the development of human malignancies. In our study, we have shown that EBNA3C transcriptionally upregulates several autophagy genes (ATG3, ATG5, and ATG7), particularly involved in autophagosome formation, under nutrient deprived conditions. EBNA3C recruits several histone activation epigenetic marks to regulate autophagy gene transcription. Moreover, EBNA3C specifically elevates PERK-eIF2α-ATF4 UPR signaling cascade in normal growth conditions, whereas in the presence of UPR-inducers it blocks further UPR activation, indicating EBNA3C promotes an adaptive condition for cell survival. Overall, our study provides a new role of an essential EBV oncoprotein in regulating autophagy-UPR cascade and offers novel targets for potential therapeutic expansion against multiple EBV induced B-cell lymphomas.Item Understanding the Regulation of Mitochondrial Homeostasis by SARMI and its role in NeurodegenerationMukherjee, PiyaliWith the increase in life expectancy neurodegenerative diseases like Alzheimer s disease (AD) and Parkinson s disease (PD) are on the rise. While neurodegeneration has been studied in the context of various pathological conditions, not much is known on the molecular mechanisms that cause/regulate axonal retraction during aging. Identification of key targets that delay the process of neuronal death before they reach the point of no return may enhance their longevity and prevent subsequent disease pathology. SARM1 (Sterile alpha and TIR motif-containing 1 protein) is a key molecule that plays a pivotal role in axonal death. To study the role of endogenous SARM 1 we established a cellular model of neurodegeneration in SH-SY5Y cells by treatment with the mitochondrial complex I inhibitor rotenone. We showed that rotenone induced neuronal death through SARM1 activation that was accompanied by increased inflammation, deregulation of electron transport chain (ETC) complex genes and defective autophagy. To study age-associated neurodegeneration, we established a drosophila model of aging for the study of age-dependent vulnerability to rotenone, a pesticide that has been implicated in sporadic cases of PD. Our results showed that age plays a major role in the increased susceptibility to rotenone that is accompanied by decreased lifespan, severe locomotor deficits, and loss of dopaminergic neurons. Rotenone exposure results in the SARM1 induction that is accompanied by an increased inflammatory response and independent of ROS generation. Thus, this study aims to provide a detailed mechanistic insight into the regulation of neuronal homeostasis by SARM1 and its implication in age-associated neurodegeneration.