PhD Theses (Biosciences and Bioengineering)

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    To gain insights into the function of yeast Dnm1 in mitochondrial dynamics
    (2024) Banerjee, Riddhi
    Mitochondria, vital hubs of cellular metabolism, continuously modulate their shape and number through fission and fusion. The central mediator of mitochondrial fission is the GTPase Dnm1 in yeast, and its homolog DRP1 in humans. Dnm1 comprises four domains - an N-terminal GTP-binding domain, a middle domain, a variable B-insert domain, and a C-terminal GTPase effector domain. Dnm1 undergoes assembly/disassembly cycles driven by GTP binding and hydrolysis to facilitate fission. While substantial progress has been made in understanding the domain architecture, function, and interacting partners of Dnm1, critical aspects, including the regulation of oligomeric forms, their spatio-temporal distribution, and the impact of post-translational modifications (PTMs), remain unclear. Moreover, disruptions in the delicate balance between mitochondrial function and dynamics are associated with various human diseases, with specific mutations in DRP1 linked to pathological conditions. Despite previous studies on the effects of these mutations on mitochondrial morphology, their impact on protein localization, distribution, function, and structure remains unexplored. This study aimed to investigate specific residues in Dnm1 that may undergo modifications or are mutated in disease conditions, examining their impact on the protein's structure, localization, and function. To achieve this, functional FL-Dnm1-GFP and Dnm1-HisHA fusion proteins were constructed, for in vivo and in vitro assessment, respectively. Five putative Dnm1 phosphorylation sites were selected for mutagenesis based on stringent conservation criteria. Interestingly, mutating S624, analogous to the reported regulatory DRP1 S616 site, did not affect mitochondrial morphology in yeast. However, mutating T62 and S277 in G2 and G5 motifs of the GTPase domain yielded non-functional proteins despite differences in their localization and dynamics. Structurally, T62A/D formed atypical large puncta, while S277A/D resembled WT Dnm1. Further computational analyses and molecular dynamics simulations provided insights into conformational changes and altered atomistic motion, particularly highlighting the dominant-negative impact of S277 mutation without altering protein localization. Furthermore, the study extended the investigation to mimic four disease-causing DRP1 mutations in Dnm1, uncovering diverse functional outcomes. For instance, the A430D mutation led to a complete loss of Dnm1 function, disrupting typical punctate phenotypes and presence of diffused cytosolic fluorescence, indicating defective oligomerization. Simulations revealed the mutation induced major conformational and dynamics changes in a helix region. In contrast, the G397D mutation resulted in fewer, larger, and less dynamic puncta, likely due to change in orientation of a loop surrounding the mutated residue. Thus, investigating each mutation in detail is crucial for gaining insights into their respective roles in disease-associated alterations of mitochondrial dynamics. In conclusion, this research provides novel insights into the molecular basis of Dnm1 function and regulation, contributing to a deeper understanding of conserved mitochondrial fission processes. Additionally, it paves the way for the development of targeted therapies for several neurological diseases where mitochondrial fission-fusion regulation is impaired.
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    Cell functions and molecular mechanisms of zinc transporters in Neurospora crassa
    (2024) Ngiimei, Serena D
    "Thesis Title: Cell functions and molecular mechanisms of zinc transporters in Neurospora crassa In this thesis work, I studied the cellular functions and molecular mechanisms of zinc resistance-conferring 1 (ZRC-1), meiotic sister chromatid 2 (MSC-2), and zinc-regulated gene 17 (ZRG-17) that are members of the cation diffusion facilitator (CDF) family of zinc transporters in Neurospora crassa. The Δzrc-1 mutant was unable to grow under high zinc conditions (≥ 0.5 mM). However, the expression of zrc-1 was elevated ~3-fold under low zinc conditions in comparison to normal and high zinc concentrations. The Δmsc-2 mutant showed colony growth and aerial hyphae similar to wild type and the expression of msc-2 was independent of zinc. Furthermore, the double mutant Δzrc-1; Δmsc-2 and Δzrc-1; Δzrg-17 showed additive phenotypes of both the parental single mutants. However, the phenotypic defects such as slow growth rate, defective in asexual sporulation, and inability to degrade cellulose of the Δzrg-17 single mutant were restored in the Δmsc-2; Δzrg-17 double mutant, which showed phenotypes similar to the wild type. The double mutant Δzrc-1; Δzrg-17 showed severe growth defects, stunted aerial hyphae, short septa, and defects in conidiation. In addition, the Δzrc-1; Δmsc-2 and Δzrc-1; Δzrg-17 double mutants showed sensitivity to DTT-induced ER stress and were unable to grow in the medium containing cellulose. Furthermore, zinc-responsive activator protein 1 (ZAP-1) was also studied to understand the molecular mechanism and the interaction of the CDF zinc transporters with the transcription factor. The zap-1 of N. crassa was found to be crucial for survival under low zinc conditions and ZAP-1 was localized in nucleus under all zinc conditions tested. The double mutants Δzap-1; Δzrc-1, Δzap-1; Δmsc-2, and Δzap-1; Δzrg-17 showed slow growth under low zinc like Δzap-1, indicating that ZAP-1 might be functioning upstream of zrc-1, msc-2, and zrg-17. Furthermore, expression analysis of the CDF family of zinc transporter, zrc-1, msc-2, zrg-17, and zrt-3 in Δzap-1 mutant showed very low-level expressions compared to expression in wild type, indicating that the ZAP-1 transcription factor regulates the CDF zinc transporters under low zinc conditions."
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    New insight into the Adsorptive Removal of Organic and Inorganic Pollutants from Aqueous set up with tailored cellulosic and polymer-based biomaterials: Modeling and Ecotoxicological assessment
    (2022) Shahnaz, Tasrin
    The significance of water as a resource for the survival of humanity is incomparable. With the rapid advancement in various technologies, the demand for water usage has been augmented exponentially. From the industry domain to fulfilling the basic necessity for the people, water conservation with proper qualitative allocation has been at the forefront of all activities. While the swift developments in the technological industries have been a boon for appeasing the hefty consumer demand in the market, it leaves a deterrent impact on the environment. To eradicate the grave danger to the aquatic environment, the treatment of wastewater is crucial and imperative. The first study of the thesis work examines the efficiency of the removal of hexavalent chromium using physicochemically activated lignocellulosic biomass from Acacia auriculiformis (Fabaceae family). The treatment involved sulphuric acid activation followed by pyrolysis resulting in chemically modified activated carbon. Further, it was complexed with a chelating agent i.e. EDTA. Acid treatment and complexation with chelating agents improvised the biosorbent’s sorption capacity towards chromium species. Adsorption parameters like pH, adsorbent dosage, temperature, and initial metal concentration were optimised in the batch study.
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    Exploring the Potentials of FIKK Kinase(s) into Development of Antimalarial and Diagnostics
    (2022) Kumar, Anil D
    Kinome of Plasmodium sp. underwent unprecedented evolutionary divergence from other eukaryotic kinases. Some of these kinases do not come under the known kinase family due to their extreme divergence from their ancestors. All these kinases are clustered as orphan kinases, FIKK kinases are one among them. Plasmodium falciparum FIKK’s are the only kinase family among eukaryotic kinases that diverged extraordinarily and expanded from their ancestor. These are trafficked throughout the parasite-infected erythrocytes and pursue important tasks for the survival of plasmodium. The study explored detailed structural characteristics of FIKK9.1 and recommends possible interacting partners in host RBC. FIKK9.1 is essential for parasite survival, but its structural and biochemical characterization will enable us to understand its role in the parasite life cycle. The recombinant FIKK9.1 kinase is monomeric with a native molecular weight of 60 ± 1.6 kDa. Structural characterization of FIKK9.1 kinase reveals that it consists of N-terminal FHA like domain and C-terminal kinase domain. The C-terminal domain has a well-defined pocket, but it displayed an RMSD deviation of 1.38 - 3.2 Å from host kinases. ITC analysis indicates that ATP binds to the protein with a Kd of 45.6±2.4 μM. Co-localization studies revealed FIKK9.1 in the parasite cytosol with a component trafficked to the apicoplast and IRBC. FIKK9.1 has 23 pockets to serve as potential docking sites for substrates. Correlation analysis of peptides from the combinatorial library concluded that peptide P277 (MFDFHYTLGPMWGTL) fitted nicely into the binding pocket. The peptide P277 picked up candidates from parasites and key players from the RBC cytoskeleton. Interestingly, FIKK9.1 is phosphorylating spectrin, CD44, and band-3 from the RBC cytoskeleton. After that, our study explored potential antimalarial from synthetic molecules and natural sources. The rapid emergence of drug-resistant malaria parasites to all frontline antimalarial drugs urges a continuous search for new antimalarial drugs that are beneficial for chemotherapy and prophylaxis. Virtual screening of diverse organic structural scaffolds from the chemical library has identified seven molecules, which could arrest the growth of parasites by inhibiting FIKK9.1 kinase. Evaluation of top hit compounds in antimalarial activity assay indicates that the highly substituted 1,3-selenazolidin-2-imine 1 and thiophene 2 inhibit parasite growth. The functionalized heterocyclic compounds 1 and 2 are killing the malaria parasite with an IC50 of 2.68 ± 0.02 μg/ml and 3.08 ± 0.14 μg/ml, respectively. Isothermal titration calorimetry analysis indicates heterocyclic scaffolds 1 and 2 abolish the binding of ATP into the FIKK9.1 binding pocket. They in-turn reduces the ability of FIKK9.1 kinase to phosphorylate its substrate, and both compounds are potent inhibitor of FIKK9.1 kinase. Inhibition of FIKK9.1 kinase is disturbing the parasite life cycle and resulting in the parasite's death. Further as a natural source, we exploit potentials of Triphala and shukramatrika as antimalarial and efforts to find its mechanism and mode of action as antimalarial. The water extract of Triphala shows promising effects with schizonticidal and parasiticidal in-vitro plasmodium falciparum 3d7 cultures. The antimalarial activity reveals that inhibition of parasites follows parasiticidal nature. The cytotoxicity on HEK293 and hemolysis analysis suggests Triphala and Shukramatrika is safe to use as antimalarial. The underlying mechanism of parasitic death is evaluated through apoptotic biomarkers, including ROS generation, mitochondrial dysfunction, and in situ DNA fragmentation in Triphala, and Shukramatrika treated and untreated parasites. Our results showed that Triphala and Shukramatrika induce oxidative stress by increasing ROS levels, destabilizing the mitochondrial membrane, and increasing the population of fragmented DNA parasites. Certainly, all those major factors, such as the importance of FIKK’s in parasite survival, stable expression in all stages of malaria lifecycle, stable gene transfer, and regions specific to P. falciparum paves a new insight through the field of antimalarial drug discovery and development of a new diagnostic marker for malaria. Each FIKK has its unique N-terminal sequences with non-homologs to eukaryotic kinases and kinases in Plasmodium sp. These exclusive regions are highly potent to be antigenic which helps in producing specific antibodies against the FIKK kinase(s). Therefore we raised antibodies against FIKK9.1 kinase and identified the anti-FIKK9.1 antibody present in serum specifically detects purified FIKK9.1 as well as pfFIKK9.1 present in parasite lysate. These are found to have no cross-reactivity to proteins present in RBC lysate. The antibodies from serum are purified and analyzed for their sensitivity and selectivity. The sensitivity of purified anti-FIKK9.1 polyclonal antibody is validated by estimating the limit of detection using rFIKK9.1 protein. The purified anti-FIKK9.1 detects antigens even at 3 nmole. Further, the cross-reactivity is analyzed using human serum and complete media devoid of parasites. Both the samples show no cross-reactivity with anti-FIKK9.1. The anti-FIKK9.1 antibody detects FIKK9.1 antigens at least to 0.1% parasitemia grown under in-vitro conditions. Surprisingly, purified antibody detects FIKK9.1 in parasite culture media, and further, the detection increases with an increased level of parasitemia. The data suggests FIKK9.1 may export outside the infected parasite. The Semi-quantitative measurements of Parasite load in the infected RBC is studied by performing concentration curve analysis for both rFIKK9.1 and parasite lysate. Further, the anti-FIKK9.1 antibody is validated by mimicking the patient sample and mocking coinfection with other infectious agents. In conclusion, our study highlights the structural and biochemical features of FIKK9.1 to exploit it as a drug target. It provides new insight into the target FIKK9.1 kinase in malaria parasites to develop potent antimalarials. The study finds Triphala and Shukramatrika kill malaria parasites irreversibly through the apoptotic pathway. Further, the research finds that FIKK9.1 is a better biomarker for malaria diagnosis.
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    Structural and Functional Studies of a Phospholipid Transporter Involved in the Maintenance of the Outer Membrane Asymmetry in Gram-negative Bacteria
    (2023) Dutta, Angshu
    Gram-negative bacteria are more resilient than Gram-positive bacteria due to the presence of an outer membrane (OM). Unlike the inner membrane (IM), the OM is asymmetric in nature because of the presence of lipopolysaccharide (LPS) and phospholipid (PL) in the outer and inner leaflets, respectively. This asymmetric organization shields the bacteria from antibiotics, toxins, etc. However, the PLs have the tendency to flip back and accumulate in the OM, leading to the formation of patches and disruption of this barrier function. In order to restore the OM permeability, bacteria utilize the highly conserved inter-membrane ATP-binding cassette (ABC) transporter system, viz. maintenance of lipid asymmetry (Mla). The Mla system consists of three sub-complexes- OM-associated MlaA-Osmoporin F/C complex, periplasmic MlaC and IM-associated MlaFEDB complex. The components of the Mla system are involved in the movement of PLs between the membranes, thereby maintaining OM asymmetry. Owing to this, the system has been suggested to be an excellent drug target although in-depth studies highlighting the mechanism of action are still lacking. Thus, in this study, all the Mla proteins from Escherichia coli were computationally and structurally characterized. Computational studies of the Mla components reveal their unique features which are not observable in typical ABC transporters. These include the identification of conserved motifs, distinct evolutionary relationships, interaction profiles and interfacial residues. Structure elucidation of MlaC and MlaD (EcMlaC and EcMlaD) through X-ray crystallography reveals that both these proteins do not possess the conserved architecture of N-terminal and C-terminal domains present in substrate-binding proteins (SBPs). Instead, EcMlaC comprises two different domains that are arranged in a discontinuous fashion. On the other hand, EcMlaD forms a homo-hexameric ring with a central hydrophobic channel which is continuous but has varying dimensions. Owing to these structural peculiarities, these two proteins have been classified as non-canonical SBPs. Extensive structural analyses of these proteins have led to the proposition of two novel mechanisms of ligand binding that have not been reported in case of any SBP to date. Furthermore, through this study, global and local motions in EcMlaC have been identified that are critical for ligand binding. Altogether, the findings provide significant mechanistic insights into the functioning of the Mla system in E. coli and other Gram-negative bacteria.
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    Investigating the spectroscopic properties of Mammeigin from Mesua ferrea in micro-heterogenous systems
    (2022) Sanjana, S
    Mesua ferrea L. (Calophyllaceae), a medicinal plant abundant in Northeast (NE) India is a vast reservoir of phytochemicals. Mammeigin (MMG) is a neoflavonoid isolated from M. ferrea seed oil with high yield and purity. MMG is non-polar like several bioactive compounds, and its aqueous solubility was enhanced in surfactant micelles and Hen Egg-White lysozyme (HEWL) aggregates. The role of charges on the interaction with MMG was investigated in anionic Sodium dodecyl sulphate (SDS), cationic Cetyltrimethylammonium bromide (CTAB), and neutral Tween 20 (T20) using UV-Visible spectroscopy. The stability of MMG was enhanced at low ionic surfactant concentrations in the presence of sodium chloride at its physiological concentration. The interaction of MMG with HEWL aggregates was found to exist at pH 2 and pH 9, but not pH 5. This study provides insights for developing formulations and better molecular carriers. Furthermore, the application of MMG as acid-base pH indicator was explored in the surfactant systems. At alkaline pH, a stable and bright yellow color was developed by MMG in CTAB micelles. Similarly, the effect of specific ions was investigated in the surfactant systems and the sensitivity of MMG to fluoride ions in CTAB was observed in the range of 2.4-200 mM NaF.
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    De novo Design of Bioinspired Peptide based Molecular Constructs
    (2022) Prakash, Vivek
    Molecular self-assembly is a powerful tool for the generation of functional nanostructures in a bottom-up fabrication. Peptides have drawn significant attention to be used as basic building blocks for such hierarchical assemblies. Diversification of chain stereochemistry offers tremendous increase in the peptide and protein design space. The use of D-amino acids in the peptide sequence can possibly help in accessing the otherwise “forbidden” region of the Ramachandran map for the generation of novel functional peptide sequences composed of L and D amino acids. In this thesis, we have de novo designed different sets of peptide molecules for four different application fronts. In the first project, we have designed a 30 amino acid long artificial blue fluorescent protein by impregnating an unnatural amino acid in the hydrophobic core of an altogether novel fold, which gives blue fluorescence. In the second project, we have designed syndiotactic hexamer peptide (Ff)3-OH, which folds into a gramicidin helical architecture, facilitating an extended phenylalanine network forming quantum confinement. In the third project, we have synthesized Fmoc conjugated ultra-short peptide hydrogels, with interesting self-healing property, which make them potential candidates for tissue engineering and drug delivery. The antimicrobial property of the synthesized peptide hydrogel has been verified against Gram-positive and Gram-negative bacteria.
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    Exploring Potential of Silk 3D Matrices for Bioartificial Liver and Regenerative Applications
    (2022) Janani, G
    Advanced liver cirrhotic patients are limited with liver transplants as the only therapeutic option; indeed, the practical applications of liver transplantation possess several drawbacks. The criteria for engineering 3D hepatic constructs for liver regenerative medicine are (i) cell-cell and cell-matrix interactions assisting long-term cell viability, (ii) heterotypic culture of liver cells maintaining cell functionality, (iii) long-term liver-specific synthetic, metabolic, and detoxification functions, and (iv) facilitating the regeneration of damaged tissue. A suitable 3D matrix with appropriate biocompatibility, hemocompatibility, topography, and physicochemical attributes facilitates hepatocyte aggregation, polarity, differentiation, and proliferation. Herein, mulberry Bombyx mori (BM) silk fibroin, non-mulberry Antheraea assamensis (AA) silk fibroin, and decellularized liver extracellular matrix (ECM) are explored in the domain of liver tissue engineering owing to their biochemical composition, mechanical stiffness, biocompatibility, and biodegradability. The presence of intrinsic RGD (arginine-glycine-aspartic acid) motifs and the high mechanical strength of AA silk fibroin has made it a potential biomaterial as it enhances cellular attachment and cell-matrix interactions. The prominent role of LECM hydrogel in liver tissue engineering has been emerging, owing to the presence of growth factors, cytokines, and cell-secreted exosomes.
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    Evaluation of Synthetic Ligands as Staphylococcal Nuclease Inhibitors for Potential Anti-MRSA Therapy
    (2023) Konwar, Barlina
    Mitigation of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is challenging as the pathogen is resistant to a large number of therapeutic antibiotics. Hence, there is an impending need to develop alternate and effective therapeutic approaches in order to counter life-threatening MRSA infections. The current study is an endeavor to address this important and contemporary issue and highlights the prospect of deploying rationally designed small synthetic ligands and target the staphylococcal nuclease enzyme or MNase, which is a key virulence factor present in the pathogen. The present study demonstrates that an anthraquinone-based ligand (C1) could render a non-competitive inhibition, decrease the turnover number as well as catalytic efficiency of MNase, with an IC50 value of 323 nM. A potentially therapeutic C1-loaded HSA nanocarrier (C1-HNC) was developed, which rendered a sustained and protease-triggered release of the payload in presence of the cell-free extract of a clinical MRSA strain. Interestingly, the eluates from the payload nanocarrier could significantly inhibit MNase-catalyzed DNA cleavage. In another study, it was demonstrated that a benzimidazole-based ligand C2 could significantly inhibit MNase, preserve the integrity of the DNA scaffold and promote higher sequestration of MRSA by DNA, which in turn, enhanced the uptake of the DNA-entrapped-pathogen by activated macrophage-like cells. In continuation of the efforts to identify synthetic MNase inhibitors, model in vitro experiments revealed that a napthalimide-based ligand C1 could inhibit MNase, enhance MRSA entrapment in DNA and mediate enhanced pathogen uptake by activated macrophage-like cells. A pluronic F-127 nano-micellar carrier loaded with C1 was developed, wherein the anti-adhesion activity of both the carrier as well as the payload was leveraged in tandem, resulting in significant inhibition of MRSA adhesion onto collagen. Interestingly, the biocompatible C1-loaded nano-micellar arsenal could also hinder MRSA biofilm formation on orthopaedic titanium wire. The present study contributes to the continuous endeavor of discovering anti-MRSA therapeutics and demonstrates the potential of synthetic MNase inhibitors that can not only curb the virulence of MRSA but also empower the host innate immune cells for effective mitigation of infections caused by the pathogen.
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    Understanding the Molecular Mechanisms for Alleviating Boron Deficiency in Indian Mustard
    (2022) Muthuvel, J
    "Boron (B) is an essential micronutrient required for the optimal growth and development of vascular plants. Globally B deficiency is the second most important micronutrient deficiency that causes significant yield reductions in crop plants. Often, seed yield and quality are compromised in plants grown under limited soil B availability without any apparent visual symptoms. B forms borate diester crosslinking with a pectic polysaccharide, rhamnogalacturonan II, during the cell wall formation and therefore, B deficiency primary affects meristem growth, vitality of the pollen grains, flower development and seed set. Brassica juncea is an important oilseed crop in India and other parts of the world and is extremely sensitive to B deficiency. Although the application of foliar B fertilizer improves the yield significantly in B. juncea, excessive application of B can be toxic due to the narrow window between its deficiency and toxicity. Molecular mechanisms of B transport have been initiated by the discovery of B transporters in Arabidopsis thaliana. Several aquaporins (AQP’s) and borate efflux transporter (BORs) family genes have been reported to be involved in the efficient uptake and translocation of B to maintain optimal plant growth under low soil B condition and B exclusion under high soil B conditions. Soil B content in major mustard producing states of India is potentially low and hence it is important to study the B transport mechanisms in B. juncea for the optimal yield.
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    In silico prediction of precursor microRNA in insects
    (2023) Nath, Adhiraj
    Pre-MicroRNAs are the hairpin loops from which microRNAs are produced that have been found to negatively regulate gene expression in several organisms. In insects, microRNAs participate in several biological processes including metamorphosis, reproduction, immune response, etc. Numerous tools have been designed in recent years to predict novel pre-microRNA using binary machine learning classifiers where prediction models are trained with true and pseudo pre-microRNA hairpin loops. Currently, there are no existing tool that is exclusively designed for insect pre-microRNA detection.
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    Functional elucidation of CRISPR-Cas I-B interference machinery of Leptospira interrogans
    (2023) Hussain, Md Saddam
    Leptospira interrogans is the causative agent of leptospirosis, a zoonotic disease accounting for approximately 60,000 human deaths every year globally. The leptospires show incompetence to conventional genetic manipulation tools, and therefore, the molecular mechanism of its pathogenesis remains poorly comprehended using the reverse genetics approach. One possible reason for its incompetence in genetic manipulation is the presence of the CRISPR-Cas system in its genome. A CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) is an RNA-directed inheritable adaptive immunity in prokaryotes against invasive mobile genetic elements (MGEs), including bacteriophages and plasmids. The system encodes an effector complex (Cascade) that utilizes small crRNA (CRISPR RNA) to sense and interfere with the invasive MGEs having the crRNA-complementary sequence next to a protospacer adjacent motif (PAM). The predominant CRISPR-Cas type I-B system, marked in several pathogenic Leptospira genomes, presents a promising alternative to be explored as an endogenous genome editing tool. Moreover, to exploit Leptospira’s CRISPR-Cas type I-B (Lin_I-B) system for targeted genetic manipulation, characterization of its interference machinery and associated PAMs is a prerequisite. To this end, the present study reports the molecular and functional characterization of interference machinery of a Lin_I-B system, recently identified in L. interrogans serovar Copenhageni strain Fiocruz L1-130. The comprehensive biochemical analysis of the LinCas7, a major subunit of the Cascade, revealed it to be a Mg2+ ion-dependent non-specific endoDNase, unlike other Cas7 family proteins. Moreover, LinCas7 exhibits distinct binding to crRNA in the presence of Mg2+ ions, testifying its canonical role in Lin_I-B defense response. The molecular characterization of LinCas8b disclosed it as a genetic fusion of large (LinCas8b) and small subunit (LinCas11b) proteins of the Cascade (LinCascade). In this study, LinCas11b is demonstrated to co-translate from an in-frame internal translation start codon encoded within the lincas8b gene. LinCas11b displays structural and functional analogy with the well-studied Cas11 family proteins. In addition, the interference machinery of the Lin_I-B, when expressed in a surrogate host E. coli, was able to annihilate the target DNA with the predicted PAM. In sum, the current study presents the molecular and functional insight of the Leptospira subtype I-B interference machinery that, soon, may pave the way for scientists to harness the system as a programmable endogenous tool for genetic manipulation.
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    Lutein Production by Chlorella Vulgaris Using Poultry Litter Anaerobic Digestate and its Potential Applications
    (2022) Ramasamy, Surjith
    Lutein is an alpha carotenoid, and it is used as an antioxidant, colouring agent, and for treating age related macular degeneration. Lutein is currently produced from petals of marigold grown on agricultural land. Production and harvest of marigold flowers involves huge amount of land, man power and fresh water. Besides, utilization of fertilizers, and pesticides for its cultivation are drawbacks of lutein production from marigold flower petals. This study aimed to produce lutein from marine microalgae using anaerobic digestate and explore its potential applications. Four different halophilic microalgae strains, namely Chlorella vulgaris 92001, Chlorella vulgaris 52091, Chlorella vulgaris 10241 and Tetraselmis indica, and three different synthetic anaerobic digestate (municipal, dairy litter and poultry litter) were evaluated for lutein production. Lutein production was high using diluted poultry digestate for Chlorella vulgaris 92001 as compared to the other microalgal strains. Parameters involved in lutein production where screened and optimized using Plackett-Burman screening design and response surface methodology. Maximum amount of lutein obtained was 3.412 mg/L in the optimised condition, i.e. 22.12 dilution factor, 27 g/L, NaCl, 4.5 g/L NaHCO3 and 4.82 × 109 cells/L inoculum size. Kinetics of substrate utilization (NaHCO3 and CH3COONa), biomass growth and lutein production by C. vulgaris was studied and evaluated using different bio kinetic models. Lutein production by C. vulgaris 92001 was carried out in indigenously designed and fabricated split column and continuously stirred tank photobioreactor. In the batch mode operation, maximum biomass growth of 0.96 g/L and lutein production of 4.379 mg/L was attained at 336 h for split column photobioreactor. The lutein production in the continuously stirred tank photobioreactor was however lower as compared to that using the split column photobioreactor. Downstream processing strategies, namely electrocoagulation flocculation (ECF), sonication and nanofiltration were employed for cell separation, cell disruption and lutein purification, respectively. Cytotoxicity and insecticidal activity of the lutein was established against SF9 cell line and armyworm. Lutein enhancement in egg yolk was finally demonstrated by supplementing lutein, lutein ester and microalgae at different concentrations (75 to 450 mg/kg) in the basal diet of hen. Lutein content of the eggs increased significantly (p<0.001) with the increase in lutein concentration with lutein, lutein ester and microalgae containing basal diet. Increase in egg yolk colour was observed in all the diets for different lutein concentrations.
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    Plant tissue culture and chemo-profiling of Tinospora cordifolia and Stevia rebaudiana for therapeutic metabolite content analysis
    (2022) Srivastava, Vartika
    The present thesis was centralized on defining strategies for efficient germplasm conservation of two highly valued medicinal plants, Tinospora cordifolia and Stevia rebaudiana and providing alternative methods for their sustainable utilization. Their conventional conservation and propagation methodologies are unable to fulfil the demands of the soaring population. Plant tissue culture techniques are considered as best alternative strategy for large-scale propagation of the plant. It eliminates the seasonal and regional variability, provides yearly output, generates true-to-type elite lines and provides ease of physiological, metabolic and structural studies. An improved micropropagation methodology was developed for both the plant species. In the growth cycle of 6 weeks, rate of shoot multiplication was observed to be 10-fold in T. cordifolia, while multiplication rate in case of S. rebaudiana was noticed to be 54.0-fold and 52.1-fold in Sr1 and Sr2 experimental plant lines. Genetic integrity of elite lines was apprised using flow cytometry and inter simple sequence repeats (ISSR) molecular markers. The later technique revealed ≥95% monomorphism stating the true-to-type nature of the in vitro plants. With respect to T. cordifolia, in vitro callus cell line was also developed using leaf explants, for estimation of protoberberine alkaloids, jatrorrhizine and palmatine. Acid-base alkaloid extraction was employed for enhanced productivity of jatrorrhizine (11.0-fold) and palmatine (143.0-fold) with reduced phenolic contamination, from in vitro callus cultures in contrast to mother plant. Besides, leaves of S. rebaudiana was chosen for analyses of steviol glycosides, rebaudioside A and stevioside, being the reservoir for the same. In vitro propagated plants generate a vividly better ratio of rebaudioside A : stevioside as 2.1 : 1.7, as compared to 1.5 : 1.2 ratio in the mother plants. The target bioactive secondary metabolites have been assessed qualitatively and quantitatively, against biofilm forming capabilities of Staphylococcus aureus, one of the key causative agents of diabetes. The alkaloids, palmatine and jatrorrhizine possessed 87.0% and 78.3% biofilm inhibition, respectively. While the steviol glycosides, rebaudioside A and stevioside demonstrated 65.8% and 70.0% reduction against biofilm formation by S. aureus. Furthermore, the study concludes with exploring anti-oxidative elucidations of protoberberine alkaloids, steviol glycosides and crude extracts in building effective future therapeutics.
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    (The) G-protein coupled estrogen receptor in breast tumors positively associates with ERalpha, and constitutes a clinically significant genomic target of estrogen in breast cancer cells
    (2023) Pal, Uttariya
    Estrogen exerts its effects on target cells, and tissues via genomic, and non-genomic pathways. The genomic effects of estrogen are mediated by the canonical estrogen receptors, namely ERα and ERβ. These are ligand-dependent transcription factors encoded by the ESR1 and ESR2 genes, respectively. The non-genomic effects of estrogen are mediated by membrane-tethered canonical estrogen receptors, ERα36, a splice variant of ERα, or the non-canonical G-protein coupled estrogen receptor (GPER).
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    Production of biodiesel and exopolysaccharides from Scenedesmus abundans cell factory in flat panel photobioreactor with autoflocculation harvesting strategy
    (2023) Mahesh, R
    Fatty acid methyl ester (FAME, biodiesel) production from microalgae has been gaining momentum in order to mitigate current energy demand but still facing major challenges such as low biomass production in open pond cultivation systems, unsuccessful closed photobioreactor (PBR) design, inappropriate media balancing under natural sunlight, inefficient harvesting technology and unsuitable transesterification process. Moreover, in order to make biodiesel economically viable, there is a need to exploit other products concomitantly apart from biodiesel and reduce downstream processing costs. Exopolysaccharides (EPS) are one of the major valuable byproducts secreted by some microalgae directly into the media. Concomitant synthesis of intracellular and extracellular product fractions in microbial cell factories would be an excellent biorefinery outlook for the researchers to reduce overall cost of their production and yield profits. Major factors that influence oleaginous microalgae growth and lipid production under natural sunlight are photobioreactor design, adequate supply of macronutrients and micronutrients, availability of sunlight intensity, CO2 supply, nitrogen starvation, mixing and mass transfer, process pH and culture temperature. Therefore, the algal biorefinery approach should focus on PBR design, media balancing for enrichment of multi-products, efficient harvesting technology for biomass recovery and desirable transesterification process for producing biodiesel. Arising from the above, the objectives of the study were formulated to develop process strategy for construction and characterization of medium scale flat panel PBR for producing biodiesel and exopolysaccharides from S. abundans. Accordingly, modulation of macronutrients and micronutrients were studied to enhance lipid content of S. abundans in single stage medium scale flat panel PBR under natural sunlight. Also, multi-objective optimization of nutrients was performed in this study to enhance biodiesel, autosedimentation of Scenedesmus abundans and exopolysaccharides production in single stage medium scale flat panel PBR under diurnal natural sunlight. Finally, free lipase and immobilized lipase mediated direct transesterification optimized strategy was developed to enhance biodiesel from microalgal biomass in presence of methanol. Parallel mini flat panel PBR and medium scale flat panel PBR were constructed for the cultivation of S. abundans. Customized unidirectional LED lighting system was developed to supply light energy to microalgae inside the laboratory. A high mass transfer efficient membrane sparger was designed and equipped at the bottom of flat panel PBR. The medium scale flat panel PBR produced overall mass transfer coefficient of CO2 (𝐾𝐿𝑎,𝐶𝑂2 ) and mixing time ( 𝑡𝑚 ) of 0.0125 s-1 and 8 sec respectively at 0.43 cm/s superficial gas velocity. Maximum biomass titer of 6.9 g/l was achieved at end of growth phase using optimized growth media in medium scale flat panel PBR at 2162 μE/m2/s, 1 VVM indoor condition. The productions were 1.53 g/l (22% of DCW) FAME with productivity of 67 mg/l/ day and 236 mg/l EPS with yield of 37 mg/g biomass under nitrogen starvation. This microalgal strain have potential to produce multi-products (biodiesel, EPS) and also has the capability of natural autoflocculation. Further to improve lipid production of S. abundans, the effect of macro and Micronutrients were investigated. The optimized lipid media was formulated for enhancing lipid content in microalgae. Comparison of single stage and two stage strategies were also carried out in this study. The single stage produced 2.5 fold higher %FAME in DCW than two stage and economical as harvesting steps could be minimized. Biomass titer of 2.79 g/l with maximum FAME content of 46% was achieved using developed media in medium scale flat panel PBR under diurnal natural sunlight. FAME concentration, FAME productivity and maximum EPS concentration of 1.28 g/l, 27 mg/l/day and 155 mg/l were obtained under sunlight study. Therefore, the exploitation of S. abundans as a co-producer of biodiesel and EPS under outdoor sunlight could be a feasible approach. In addition to that, multi-objective optimization approach has been developed to increase the production of biodiesel, secretion of EPS and enhance autoflocculation (ASF) using S. abundans under diurnal light. For this study, Plackett-Burman design (PBD) was used for screening significant process variables. Diurnal simulated light intensity was generated from pulse width modulation (PWM) and OPTO22 control system that controls LED (light emitting diode) light intensity ranging from 0 to 2200 μE/m2/s (capable of mimicking sunlight intensities in laboratory). PBD determines urea, CoCl2.6H2O, CuSO4.5H2O and Na2MoO4.2H2O as significant process variables in modified Chu-13 media that influences FAME content in dry biomass, FAME concentration, ASF and EPS concentration by S. abundans. The quadratic model equations of responses from RSM-CCD (response surface methodology-central composite design) enhance single objective at a time. Therefore, these model equations from RSM-CCD are used with composite desirability approach for formulating optimized multi-objective RSM media. The developed media increased the FAME production by 1.72 fold (70% in DCW), FAME titer by 2.3 fold (3.83 g/l), ASF by 1.08 fold (1.93) and EPS by 1.60 fold (462.50 mg/l) as compared to unoptimized media under diurnal LED light. The formulated media produced 55% of FAME content in dry biomass, 1.79 g/l FAME, ASF of 1.83 within 1 hr, 290 mg/l EPS under natural sunlight. For this study, immobilized lipase mediated direct transesterification process was developed. Methanol was used as lipid extraction solvent, acyl acceptor and reaction medium. The optimized conditions for free lipase were 16:1 methanol to biomass mass ratio, lipase amount with respect to biomass (80%) with 50 mg/ml lipase concentration, 30 ºC reaction temperature (room temperature) and works optimum without initial pH adjustments. Lipase loading of 10 mg/alginate bead (62.5 mg/ml enzyme concentration) produced highest FAME content in DCW (44%) among different enzyme loadings using calcium alginate entrapment method. Kinetic study reveals that transesterification efficiency increases with increase in reaction time for both optimized free lipase and immobilized lipase. Water behaves as a better storage medium as compared to buffers for immobilized lipase in reusability experiments. Lipase loading of 20 mg/alginate bead (125 mg/ml enzyme concentration) showed better reusability up to 5th cycle. As far as our knowledge is concerned, this is the first study of multi-objective optimization of nutrients to enhance FAME, autoflocculation and EPS concomitantly in Scenedesmus abundans. Also, very few studies have reported immobilized lipase mediated direct transesterification of algal biomass in presence of methanol to produce biodiesel. Hence, enhancement of biodiesel and EPS under sunlight without photoinhibition by natural autoflocculation in medium scale flat panel PBR paved way for biorefinery. Therefore, generation of multi-products by S. abundans, natural autoflocculation based biomass harvesting and lipase immobilization by entrapment method can make the process sustainable.
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    Molecular investigations of cellular roles of calmodulin and calcium/calmodulin-dependent kinases in stress responses, asexual and sexual developments in Neurospora crassa
    (2023) Marak, Christy Noche K
    Calcium (Ca2+) plays a primary role in regulating numerous cellular processes and adaptive responses in eukaryotes. The binding of Ca2+ changes the conformation and charge of the protein, which are the basis for signal transduction. Calmodulin (CaM) is the principal Ca2+ binding protein expressed in all eukaryotes including Neurospora crassa. When bound to Ca2+, CaM activates over 300 target proteins. Ca2+/CaM-dependent kinases (Ca2+/CaMKs) are one of the main effector proteins of CaM. Preliminary studies in N. crassa have shown the importance of CaM and Ca2+/CaMKs in growth, development, stress response, sexual development, and in regulating the circadian system. However, the detailed cellular roles and molecular mechanism by which CaM and Ca2+/CaMKs regulate the different phases of life cycle or the cell under different conditions still remain unclear in N. crassa. Therefore, this study sought to understand the cellular roles and the mechanism by which CaM and Ca2+/CaMKs mediated the cell under stress conditions, during sexual development, and in regulating the circadian clock.
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    Studies on the effect of modulatory nanoagents in protein amyloidogenesis
    (2023) Karmakar, Srijeeb
    This thesis provides a detailed account of how the aggregation behavior of two model proteins, cytochrome C and insulin, is modulated in the presence of intrinsic and extrinsic nanoagents that act as modulators. Under amyloidogenic conditions (pH 9, 75°C, and 192 hours), cytochrome C undergoes self-oxidation, resulting in extensive fragmentation of the protein. These fragments then interact with each other to form aggregates. Additionally, the prosthetic group of cytochrome C, known as heme, dissociates from the holo form and acts as an intrinsic modulator by influencing the structure of cytochrome C. The self-oxidation process leads to the conversion of ferroprotoporphyrin to ferriprotoporphyrin, which further converts to chloro-ferriprotoporphyrin in the presence of Cl- ions, resulting in the formation of fluorescent quantum dots. These quantum dots exhibit lattice planes of Fe2O3/ Fe3O4/hemin, indicating the formation of a mixed species. In the case of insulin, incubation at 60°C and pH 7.4 for 6 hours, with equimolar Zn2+ concentration, leads to the formation of a dissoluble condensate structure with increased viscosity, a macromolecular network, and shear thickening properties. Extrinsic modulatory nanoagents, including long-length cellulose nanofiber (CNF), iron functionalized CNC (MagCNC), citrate-capped gold nanoparticles (AuNP), and iron (II,III) oxide nanoparticles (IONP), are incubated with the equimolar [Zn2+]:[insulin] reaction mixture. It is observed that the condensate structure undergoes significant changes depending on the properties of the nanoagents, and the nanoparticles themselves undergo transformation. CNF is converted into considerably shorter fibers, which disperse on the condensate surface, leading to the formation of CNR-decorated insulin condensate. MagCNC resolves into ultrafine nanothreads, which form nucleated species within the condensates. These nucleating species then convert into spherical CNCs that are packed within insulin condensate microspheres, resulting in MagCNC-decorated insulin condensate. The metallic nanoparticles, AuNPs, and IONPs, modulate the condensate structure in different ways. Citrate-capped AuNPs interact with amyloidogenic residues of insulin, adsorbing them on the nanoparticle surface and forming dendritic species. These dendritic species interact with each other, creating a reversible condensed assembly presenting AUNPs on the surface. On the other hand, incubation with IONPs leads to the formation of a remarkable two-dimensional nanosheet assembly with ordered surface channels. The IONPs (av. Size 20 nm) oxidizes from Fe2O3/ Fe3O4 to complete Fe2O3 ultrasmall crystallites adhered on the surface. Importantly, all the aforementioned materials demonstrate the potential to support and enhance BHK-21 cell growth without exhibiting any cytotoxicity. Further investigations could pave the way for the development of functional biomaterials with a wide range of applications, from bioelectronics to therapeutics.
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    Bioengineered systems for the biodegradation and toxicity removal of endocrine disrupting phthalates (EDPs)
    (2023) Kanaujiya, Dipak Kumar
    The world's water resources are increasingly threatened by climate change and various organic and inorganic pollutants. Phthalic acid esters (PAEs) are one such pollutant and have emerged as an environmental threat to aquatic ecosystems. PAEs are synthetic chemicals that mimic activity of natural hormones and disrupt the endocrine system of organisms, including humans, leading to harmful effects. Due to their endocrine-disrupting nature, PAEs are also known as endocrine disrupting phthalates (EDPs). Hence, this thesis work focused to establish the efficient and low-cost treatment strategy for biodegradation and toxicity removal of EDPs. In order to find out the best treatment system, the biodegradation of different EDPs at various concentration combinations were evaluated using different bioengineered systems, viz. continuous stirred tank bioreactor (CSTB), two-phase partitioning bioreactor (TPPB) and membrane-integrated bioreactor system, under aerobic condition. The toxicity removal efficiency of different systems was evaluated by the toxicity assessment of the treated water using brine shrimp mortality and seed germination bioassays.
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    Investigating the functions of Phospholipase C-1, Ca2+/H+ exchanger, and Secretory Phospholipase A2 in growth, stress responses, and cellulose degradation in Neurospora crassa
    (2023) Baruah, Darshana
    Thesis Title: Investigating the functions of Phospholipase C-1, Ca2+/H+ exchanger, and Secretory Phospholipase A2 in growth, stress responses, and cellulose degradation in Neurospora crassa