PhD Theses (Biosciences and Bioengineering)
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Item Conversion of Methane into Polyhydroxybutyrate using Methylosinus trichosporium NCIMB 11131: Applications in Food Packaging, Bone Tissue Engineering, and Electronics(2023) Mohammed, NoorThe Industrial Revolution led to significant increases in atmospheric greenhouse gases, with methane, the second most harmful, rising from 700 ppb to 1922 ppb by 2022. Methane has a 25-fold greater infrared absorption potential than CO2, contributing significantly to global warming. At current emission rates, the planet is on track for a 2°C temperature rise by 2036, highlighting the urgency for innovative solutions. Simultaneously, plastic pollution has emerged as a critical issue, with over 450 million tons produced annually, of which only a small fraction is recycled. As both methane emissions and plastic waste continue to rise, sustainable alternatives are required. This study addresses these challenges by utilizing Methylosinus trichosporium NCIMB 11131, a Type-II methanotrophs, to convert methane into polyhydroxybutyrate (PHB), an eco-friendly, biodegradable bioplastic. PHB production is induced under nitrogen-starved conditions, using methane as a carbon source. This dual-purpose approach mitigates methane emissions while producing a sustainable alternative to traditional plastics. Optimal conditions yielded PHB content of 57.3% (w/w), with a production rate of 1.11 g.L-1.d-1 in a 5-L bioreactor, demonstrating scalability. Further process optimization included maximizing methane mass transfer using sparger and silicone oil vectors in airlift reactor. Applications of PHB were explored in three sectors: food packaging, bone tissue engineering, and electronics. PHB films exhibited excellent mechanical properties, degradability, and moisture resistance, making them suitable for sustainable packaging. In bone tissue engineering, a composite of PHB and hydroxyapatite derived from waste eggshells demonstrated biocompatibility and osteo-conductivity, promoting cell attachment and proliferation. In electronics, PHB films served as insulating covers for batteries, displaying significant electrical insulation and heat resistance. This research highlights the potential of M. trichosporium NCIMB 11131 for PHB production using methane as a carbon source, with diverse industrial applications. The study advances sustainable bioplastic production, contributing to both greenhouse gas reduction and a circular economy.Item Therapeutic Insights of Entomopathogenic Mycotoxins in Breast Cancer Cells(2024) Patra, ArupamChemotherapeutic resistance in breast cancer, especially in aggressive forms like triple-negative breast cancer (TNBC), presents a major obstacle in treatment, driving the search for more effective therapeutic agents. TNBC is marked by high mortality rates and limited treatment options, underscoring an urgent need for targeted therapies. Researchers have focused on unique fungalderived compounds with anticancer properties that may offer novel approaches to overcome drug resistance and directly targeting cancer cell survival mechanisms. Two such compounds, the ribotoxin Anisoplin and the secondary metabolite Beauvericin, have demonstrated promising effects against breast cancer, especially in TNBC, where they can potentially overcome resistance and induce cell death through unique molecular pathways.Item Understanding the defence mechanism of CRISPR-Cas subtype I-C of Leptospira interrogans(2024) Anand, VineetLeptospira interrogans is a bacterium that causes Leptospirosis. However, its pathogenicity is poorly understood due to the lack of effective conventional genetic manipulation tools. L. interrogans serovar (sv.) Copenhageni encodes two CRISPR-Cas systems - I-B and I-C in its genome. The presence of the CRISPR-Cas system in the genome of pathogenic Leptospira has been hypothesized as the reason for low success in genetic manipulation and, hence, deciphering its virulent gene function. The CRISPR-Cas I-B locus in L. interrogans has been extensively studied. However, CRISPR-Cas I-C lacks an array element, making it an interesting subject to investigate its Cas protein's role in CRISPR biology. The CRISPR-Cas I-C locus encodes all sets of proteins required for adaptation (LinCas1C, LinCas2C, and LinCas4C), maturation (LinCas5C), and interference (LinCas7C, LinCas8C, LinCas3C, and LinCas3Cʹ). Moreover, the genes encoding these proteins are transcriptionally active. The nucleotide sequence and encoded amino acids of the cas genes show low sequence similarity with subtype I-B and are phylogenetically distantly related. The adaptation Cas protein (LinCas1C, LinCas2C, and LinCas4C) exhibits metal-ion-dependent DNase and metal-ion-independent RNase activity under in vitro conditions. However, neither rLinCas1C nor LinCas2C demonstrates any activity towards small DNA oligos, except for rLinCas4C. The crystal structure analysis of rLinCas2C shows that it adopts a dimeric conformation, and each subunit exhibits the characteristic ferredoxin fold. The crystal structure of rLinCas2C indicates its existence in a catalytically inactive conformational state. The substitution mutation of active site residues (Tyr7, Asp8, Arg33, and Phe39) of rLinCas2C with Ala and deletion mutation of RNA recognition loop (Asp60, Lys62, Thr63, and Asp64) resulted in compromised DNase activity.Item Development of Cardiac Troponin-T (cTnT) aptasensors for point-of-care diagnosis of myocardial infarction(2023) Vinay, BThe main objective of this thesis is the development of aptasensors for cardiac troponin T (cTnT) for the diagnosis of myocardial infarction (MI). The thesis has been divided into six chapters, starting with a brief introduction expressing the motivation behind this work and outlining the work performed to address the primary objective defined for this thesis.Item ERα and AhR mediated genomic effects of karanjin in breast cancer cells(2023) Bhatt, GauravKaranjin, a bioactive flavonoid found in Pongamia pinnata seed oil, possesses a wide range of therapeutic properties, including antioxidant, antibacterial, anti-cancer, anti-ulcer, antihyperglycemic, and anti-inflammatory effects. In vitro, it has demonstrated potential as an anticancer agent by inhibiting cancer cell growth and promoting apoptosis. However, its effects on cell proliferation appear to be dose-dependent, with lower concentrations promoting proliferation and higher concentrations inhibiting it, particularly in breast cancer cells. The global transcriptomic footprint of MCF-7 and T47D breast cancer cells, suggests partial estrogen-like nature. Karanjin interacts with the estrogen receptor alpha (ERα), influencing gene expression and protein turnover. Interestingly, karanjin is postulated to be a novel phytoestrogen or SERM based on molecular and cellular effects produced on breast cancer cells. Additionally, it has been identified as a novel agonist of the aryl hydrocarbon receptor (AhR), similar to dioxin but without toxic effects. This research employs both in vitro and in silico methods to better understand how Karanjin regulates ERα and AhR, providing insights into its therapeutic potential.Item Understanding the expression stage of CRISPR-Cas defense system in Leptospira interrogans(2023) Prakash, AmanThe pathophysiology of Leptospirosis, caused by pathogenic Leptospira spp., is unknown mainly due to the lack of efficient genetic manipulation tools. Thus, harnessing the endogenous CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins) system of Leptospira is an attractive strategy to study its pathogenesis; however, it relies on the understanding of the CRISPR-Cas immunity process. This study characterizes the CRISPR arrays and CRISPR-associated proteins (LinCas6, LinCas5, and LinCas3) involved in the expression and interference of RNA-mediated immunity. In L. interrogans, we account for the transcriptionally active CRISPR arrays in the direction of cas-operons. The recombinant LinCas6 (rLinCas6) overexpressed and purified in this study can process the precursor-CRISPR RNA (pre-crRNA) to generate mature crRNA and remains bound with it. The rLinCas6 follows single turnover kinetics where substituting one of the predicted active site residues (His38) reduced cleavage activity on its cognate repeat RNA. Biochemical analysis of the overexpressed and purified rLinCas5 suggested that it is catalytically inactive on nucleic acids. However, rLinCas5 binds to the rLinCas6-crRNA complex essential for stabilizing the mature crRNA during interference. Similarly, the overexpressed and purified rLinCas3 nuclease activity demonstrated that it is a metaldependent nuclease. This study features insight into CRISPR transcription, crRNA biogenesis, and the onset of the effector complex formation in Leptospira, which is essential for RNA-mediated interference of invading nucleic acids. In addition, this study proposes the physiological requirements of Leptospira CRISPR-Cas I-B during interference.Item Monitoring Protein Unfolding and DNA binding using Protein Charge Transfer Spectra(2023) Priyadarshi, AnuragProteins are not expected to show electronic absorption or emission in the near UV-Visible region in the absence of aromatic amino acid residues and cofactors. However, evidence of novel protein absorbance and luminescence in the near UV-Visible region independent of the presence of aromatic amino acid residues has now been presented by multiple research groups. By employing Time-dependent density-functional theory (TDDFT) calculations, it was postulated that charge transfer involving charged atoms in the Lys/Glu sidechain and the peptide backbone gave rise to the novel absorbance observed in the 250—800 nm region for protein α3C. This previously unknown intrinsic chromophore was termed Protein Charge Transfer Spectra (ProCharTS) by Prasad et al. Characteristic features of the observed blue luminescence include low to moderate quantum yields, excitation-emission spectral overlap among the multiple proteins studied, large Stokes shifts, and similar mean lifetimes for luminescence intensity decays.Item Molecular Characterization of Begomoviruses Causing Yellow Mosaic Disease in Mungbean and Disease Management Through Biotechnological Intervention(2024) Dhobale, Kiran VilasYellow mosaic diseases (YMD), caused by Begomovirus, pose a significant threat to mungbean cultivation in the Indian subcontinent. This study investigates the epidemiology of begomovirus in three YMD hotspot regions, identifying Mungbean Yellow Mosaic Virus (MYMV) in Bihar and Mungbean Yellow Mosaic India Virus (MYMIV) in Assam and Orissa. The study explored the population structure and genetic diversity of MYMV and MYMIV isolates, revealing independent evolution of DNA-A and coevolution of DNA-B. To identify YMD-resistant mungbean genotypes, an agroinoculation-based genotype screening approach was employed. Using prepared infectious clones (MYMV and MYMIV) for screening YMD-resistant and susceptible mungbean genotypes, we identified genotypes highly susceptible to MYMV (cv. ML267) and MYMIV (cv. K851), as well as genotypes immune to MYMV (cv. PDM139, cv. SML668) and MYMIV (cv. Pusa Vishal). The study explores a non-transgenic approach for inducing resistance to YMD in mungbean. Two target regions within the viral genomes were identified for gene silencing using RNAi. We show that out of three intron hpRNAi constructs, namely hpTR-1: AC4/AC1, hpTR-2: AC2/AC3, and hpTR-1+2: AC4/AC1_AC2/AC3 (fusion construct), the hpTR-1+2 construct provided 100% protection, validated through a transient agroinfiltration assay. Subsequently, we show that in vivo synthesized hpRNA of hpTR-1+2 can persist and induce the generation of small interfering RNA (siRNA) in both local and systemic tissues for at least 12 days’ post-spray without viral inoculation, validated through semi-reverse transcription-PCR and northern blotting. Our data indicate that the naked hpRNA spray conferred resistance to MYMIV in mungbean, with the most significant inhibition of MYMIV replication observed when plants were treated on the same day, two days, and four days before viral inoculation. Furthermore, the study explored the role of the apoplast in Begomovirus infection. Importantly, we show the presence of genomic components of MYMIV in apoplastic fluid validated by molecular detection of viral genome through RCA and PCR analysis to enhance our understanding of the cell-to-cell movement of begomovirus via apoplast. Additionally, we have shown that virus infection induces elevated secretion of vesicles into the apoplast. NMR-based metabolomics analysis reveals altered metabolic profiles in both apoplast and symplast in response to MYMIV infection. Citrate downregulation and increased levels of valine, α-β-glucose, and pipecolic acid were observed in both compartments. Phenolic metabolites were absent in the apoplast and downregulated in the symplast, while proline exhibited contrasting levels in MYMIV-infected samples. Additionally, heightened aspartate levels were confined to the symplast. These findings provide insights into metabolites associated with stress and defense mechanisms triggered by MYMIV infection. In conclusion, our findings may help prevent an epidemic of YMD in Vigna species, and the study may contribute to enhancing disease management strategies in mungbean cultivation.Item Understanding the role of alpha-synuclein in Japanese encephalitis virus replication and evaluation of pyrazole derivatives as therapeutics against its infection(2024) Gupta, AnjaliIn the prognosis of Japanese encephalitis virus (JEV) infection, many host factors have been identified as being involved in the various steps of the viral life cycle. Since it is a neurotropic virus, understanding the role of neuronal-specific proteins and local cellular homeostasis in developing therapeutics against JEV is an active area of research. Alpha-synuclein (α-syn) is one of the neuronal-specific proteins regulating synaptic plasticity and has been reported to have antiviral potential in related neurotropic viruses. JEV-infected patients displaying Parkinson's disease (PD)-like symptoms have been reported to have α-syn overexpression in the brain regions. As per reports, phosphorylation at S129 position plays a major role in aggregation and α-synucleinopathy. Therefore, exploring the function of α-syn in JEV induced death of dopaminergic neurons and α-synucleinopathy is essential. To this day, the present study reports the functional role of α-syn in JEV pathogenesis as well as explores the anti-JEV therapeutic candidates. There is a significant increase in endogenous α-syn expression during JEV replication, demonstrating a substantial reduction in JEV replication, suggesting an anti-JEV effect. α-syn was found to modulate the anti-oxidative pathway by increasing the expression of superoxide dismutase 1 (SOD1). The pathological implications of α-syn phosphorylation were carried out by studying casein kinase 2 (CK2) and Polo-like kinase (PLK2) involved in α-syn phosphorylation. Detailed analyses of CK2 and PLK2 reveal a notable reduction in these kinases, particularly during the late phase of JEV replication, thereby reducing the phosphorylated α-syn (pα-synS129) protein level. The intracellular α-syn oligomerization was increased in JEV-infected cells. Pyrazole derivatives with anti-oxidative properties were found to have anti-JEV activity. Comprehensive in vitro and in vivo studies showed compounds suppressed JEV-induced reactive oxygen species (ROS) generation through NRF2-SQSTM1 signaling mechanisms. This study contributes valuable insights into the interplay between α-syn and JEV, shedding light on avenues to study further the potential role of α-syn aggregation in JEV pathogenesis and exploit it to develop broad-spectrum antiviral therapeutics.Item Process Analytical Technology (PAT) Control Tools for High Cell Density Cultivation of Glycoengineered Pichia pastoris for Human Interferon α2b Production(2024) Pavan, Allampalli Satya SaiInterferons are a group of multifunctional secreted proteins categorized as cytokines involved in intracellular signalling. Human interferon α2b (huIFN α2b) is a type I interferon and one of 13 variants of interferon α that have been reported. huIFN α2b triggers many biological activities such as antiviral, antiproliferative, and immunomodulatory functions. As a result, it is used in the treatment of hepatitis B and C, hairy cell leukaemia, melanoma, and AIDS-related Kaposi’s sarcoma. In the context of recombinant protein production, yeast expression systems, particularly Pichia pastoris, offer advantages over bacterial hosts. They facilitate the synthesis of glycosylated recombinant proteins through appropriate post-translational modifications and entail lower operational costs than mammalian cell lines. However, challenges arise in managing higher expression, leading to the accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER). Inappropriately/unfolded protein fractions in culture supernatants require extensive purification steps and are strongly discouraged from the Quality by Design (QbD) perspective. In accordance with the Process Analytical Technology (PAT) initiative, the critical process parameters (CPPs) and critical quality attributes (CQAs) of the process need to be identified and monitored in real-time to achieve improved product quality. Therefore, optimization of the protein titer from P. pastoris could be achieved by developing relationships between the various CPPs/CQAs and product titers. Cultivation of P. pastoris by manipulating the specific growth rate , a CPP, has a stronger influence at the metabolic level. Therefore, controlling in real-time leads to enhanced product output. However, deconvolution of the soft sensor/online sensor input into CPPs is at the nascent stage for most therapeutic protein production.Item Enhancing Water Quality through Adsorptive Removal of Contaminants of Emerging Concern using Bio-based Engineered Adsorbents: Modeling and Toxicological Evaluation(2024) Priyan, Vishnu VRapid industrialization and urbanization cause tremendous stress on the natural resources which leads to severe ecological impacts, amongst water is the foremost concern that can be easily polluted by contaminants of emerging concern (CECs). The persistence and/or pseudo-persistence nature of these pollutants could cause various environmental and health impacts on water ecosystems and living beings. Thereby, sequestration of these contaminants from the water systems is mandatory. So, adsorption is the prominent approach for the sequestration of these contaminants due to its efficiency at lower pollutant concentration, easy implementation, cost-effective, selectivity, and economic feasibility. The research focuses on the development and characterization of bio-based engineered adsorbents derived from bio-based precursors. These adsorbents are designed to possess high surface area and tailored surface chemistry to efficiently capture CECs from water sources. Furthermore, the thesis integrates mathematical modeling techniques to predict the adsorption behavior of CECs onto bio-based adsorbents under different scenarios. This modeling approach facilitates the optimization of adsorption processes and provides insights into the mechanisms governing pollutant removal. In addition to assessing the adsorption efficiency, the thesis evaluates the potential toxicological implications of using bio-based adsorbents for CEC removal. Toxicological evaluations encompass the analysis of adsorbent leachates and the assessment of any adverse effects on model organisms. Overall, this interdisciplinary study contributes to the advancement of sustainable water treatment technologies by offering insights into the efficacy, mechanisms, and environmental implications of using bio-based engineered adsorbents for the removal of CECs. The findings hold significant implications for water resource management and public health protection in the face of emerging contaminants.Item Mechanism of Antimicrobial Peptide Binding to Membrane-mimetic Systems: Insight from Molecular Dynamics Simulations(2023) Ghosh, SuvankarThe focus of the Ph.D. thesis was to understand the mechanism of cationic antimicrobial peptide (7 and 14 amino acid residue long) binding to membrane-mimetic systems (micelle and bilayer) using classical molecular dynamics free energy simulations. The peptides were modeled and subjected to conventional classical MD simulations in the presence and absence of micelle/bilayer [micelles: SDS/DPC and bilayers: (DOPE:DOPG and POPE:POPG)/POPC as bacterial/mammalian membrane-mimic]. The structures of the free peptides in water and in complex with the membrane-mimetic systems were predicted from the conventional MD simulations and verified by our experimental collaborators. The MD structures were used as a template for estimating the energetics of peptide: micelle/bilayer binding (ΔGbind; absolute binding affinity and ΔΔG; Binding free energy difference between two peptides to the membrane-mimetic system) by employing various popular methods: Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA), Steered Molecular Dynamics (SMD), Umbrella Sampling (US), and Alchemical free energy simulations (FEP, TI, BAR). The simulations of peptide binding to the simplest membrane-mimetic systems provide insight into the kinetics and establish a direct link between the calculated energetics and molecular structures.Item Spectroscopic, structural and functional characterization of Intrinsically Disordered Protein DHN1 from Zea mays(2023) Ahari, DileepIntrinsically Disordered proteins (IDPs) emerged as an essential class of proteins in the past two decades due to their functional relevance without having a proper 3-dimensional structure. Here, we investigated dehydrin (DHN1) protein from Zea mays, an IDP that belongs to the Late Embryogenesis Abundant (LEA) protein family. This thesis work focused on studying DHN1 structural dynamics and functional role using the novel spectroscopic tool Protein Charge Transfer Spectroscopy (ProCharTS) and other spectroscopic techniques like UV-visible and fluorescence spectroscopy. To investigate the structural change and functional role of DHN1, two mutants: DHN1 CW1 (Trp122 −Cys62) and DHN1 W3 (Trp3), were generated using sitedirected mutagenesis. The structural analysis of the DHN1 protein divulges its complete random coil conformation in the native state. ProCharTS absorption was found to be sensitive to the conformational changes in DHN1 induced by the changes in pH and temperature of aqueous medium. Next, we investigated the luminescence characteristic of this novel ProCharTS in the DHN1 protein. This study suggests that ProCharTS in DHN1 protein is luminescent in nature. We found that the observed luminescence is excitation wavelength dependent and possesses a significant presence in the UV-Visible region. ProCharTS luminescence has a low quantum yield and lower luminescence lifetime. The origin behind this luminescence could be charge recombination. Further, ProCharTS luminescence found to modulate the fluorescence of other chromophores like Trp in the same spectral region. Spectroscopic studies reveal that DHN1 and its mutant proteins are highly dynamic in nature and displays the disorder-to-order structural transitions in presence of TFE, SDS and at high temperatures. Further, the overall structural change in DHN1 protein in presence of SDS was measured using the Förster Resonance Energy Transfer (FRET) from excited Trp122 (donor) to dansyl-labelled Cys62 (acceptor). Intramolecular FRET distance between Trp—Dansyl indicated that the distance between Trp122 and Cys62 in DHN1 CW1 was reduced from 34 Å to 25 Å in the presence of SDS. Finally, the functional studies of DHN1 and its mutant protein indicate that DHN1 has significant cryoprotective and heat-protective functions via potential weak electrostatic binding to the target enzyme. Taken together, ProCharTS can serve as a label-free tool to study and detect structural transitions in the IDPs rich in charged amino acid residues and devoid of aromatic chromophores. The structural changes and the cryoprotection and heat-protection function of DHN1 protein could be attributed to its significant role in the stress tolerance mechanism in plants.Item Isolation, characterization and pharmacological application of bioactive compound from leaves of Alpinia nigra (Gaertn.) B.L. Burtt(2022) Gupta, Manish KumarThe present study is focused on investigating the potential uses of the herbaceous plant Alpinia nigra (Gaertn.) B.L. Burtt (Zingiberaceae). Traditionally this plant is used in folk remedies for curing gastritis and infectious diseases. The plant leaves are used as a food-flavoring agent by tribal people in Northeast (NE) India. However, the scientific community has not explored the plant leaf for its medicinal properties. Thus in the present study, the leaf extracts hexane (L-Hex), ethyl acetate (L-EtAc), and methanol (L-Met) were subjected to phytochemical analysis. The antibacterial, anti-biofilm, and anti-quorum sensing activities of the L-EtAc extract was determined by in-vitro analysis and was found to be potential. Further, the compound 3, 5-dihydroxy 4′,7-dimethoxy flavone (DHDM) was purified from L-EtAc, crystallized, and structural characterization was performed using multispectroscopic techniques including HRMS, FTIR, Raman, SC-XRD, 1-D NMR, and 2-D NMR. The cell viability assay showed no inhibition of DHDM at ≤200 μM concentration in THP-1 (human macrophage) and ≤80 μM in HaCaT (human keratinocyte) cell lines. Additionally, strong antioxidant properties and reduced ROSItem Evolutionary landscape of dipteran insects(2022) Kabiraj, DebojitInsects are the most successful animals on the planet, in terms of both ecological and evolutionary success. Diptera are typical insect order having two wings and accounts for one-tenth of all living creatures on the earth. Their ability to feed on a variety of foods has allowed them to thrive in a wide range of food chains and climatic niches. Our knowledge of the evolution of flies is muddled by limited and contradictory morphological and genomic data, as well as the difficulty of capturing the huge species diversity in a single complete phylogenetic analysis.Item Targeting Notch Signalling in the EMT Dynamics of Triple-Negative Breast Cancer cells(2023) Sen, PlaboniThe thesis work describes the identification and targeting the pivotal molecules of the Notch signalling pathway to combat the aggressive characteristics of TNBC cells (such as EMT, drug resistance and stemness). Initially, the identification of the nodes was carried out, which provided us with the major proteases. They were further targeted using a Polypharmacology approach by a repurposed drug, selected from extensive in silico screening. Furthermore, an effective combination therapy was established, which aimed in targeting the Notch signalling pathway along with the VEGF pathway. Finally, a combination therapeutic module was established, which aimed in increasing the effectiveness and outcome of an HDAC inhibitor (SAHA), thereby inhibiting EMT, stemness and survival of the TNBC cells. The effectiveness of combination treatment on monolayer cultures, and complex tumour spheroids illustrates the therapeutic significance of combination treatment as an attractive alternative in cancer therapeutics.Item Targeting Triple Negative Breast Cancer Using Membrane-Derived Nanocarriers for Potential Therapeutic Applications(2023) Saha, MuktashreeThe current thesis focusses on targeting metastatic triple negative breast cancer via modulation of the tumor micro environment by targeted delivery of inhibitors using a biologically derived nanosystem. Significant signaling pathways and the drug resistant potentiality of the TNBC cells were primarily targeted via the biologically derived nano-carriers which resulted in efficient suppression of metastatic breast cancer and development of an effective treatment module for targeting TNBC. Firstly, nano-vesicles were synthesised from the cell membranes of cancer cells and were evaluated for its homologous targeting efficiency with further loading of the nano-vesicles with an inhibitor. The use of cell membranes as biological nano-carrier, demonstrated impressive self-homing capabilities, thereby leading to the development of a potential nano-delivery system for cancer therapy in vivo, as they can be derived from the patient’s own cells. In the subsequent endeavor, exosomes were employed for the modulation of tumor microenvironment and MDR dynamics of metastatic triple negative breast cancer cells. The exosomes from non-invasive breast cancer cells lead to the decrement in the expression of the ABC transporters, thereby making the metastatic TNBC cells more susceptible to chemotherapeutic drugs for effective anti-cancer activities. Taking into consideration, the advantages of the nano-vesicles and exosomes, a hybrid nanosystem was synthesised by fusing the nano-vesicles and exosomes. Significant signaling networks were targeted by loading an HDAC inhibitor into the fused nanosystem in combination with a tyrosine kinase inhibitor. The study showed that the targeted co-therapy resulted in an efficient subduing of metastatic TNBC that not only resulted in apoptosis of the MDA MB-231 cells, but also affected the regulatory pathways at the genetic and proteomic levels in a synergistic manner. Thus, the study showed that targeted co-therapy via the developed biomimetic hybrid nanocarriers played a very substantial role in the site specific delivery of the drugs for a more efficacious outcome. Thus the corroborations of this research might open up new horizons for the curtailment of metastatic TNBC with further validations in in vivo system.Item Analyzing Charge Transfer Spectra arising from non-aromatic amino acids in proteins, aggregating peptides and viral capsid assemblies(2023) Alom, Shah EkramulNovel intrinsic chromophores in apo-proteins lacking aromatic amino acids have gained importance and have been a recent topic of interest. Here in this thesis, Protein Charge Transfer Spectra (ProCharTS), which is proposed to be the origin of such intrinsic absorbance and luminescence features, is being studied. Although the major contribution of Lysine towards ProCharTS phenomenon has been ascertained in highly charge rich proteins. Here we report for the first time, ProCharTS in proteins and peptides rich in Arginine, Aspartate residues, but lacking Lysine residues. Similar absorbance and luminescence properties as that in Lysine-rich protein were observed. ProCharTS can be expected to be evident as long as charge transfer takes place, irrespective of the type of charged species. Since ProCharTS and conventional chromophores like Tryptophan share a similar spectral domain, the influence of ProCharTS on the indole fluorescence in Tryptophan was studied and was found to contaminate the indole fluorescence. Moreover, the decay kinetics from the excited state population in the indole ring was found to be affected by the presence of charged residues, which may explain the multi-exponentiality of Tryptophan fluorescence intensity decay often observed in proteins. Finally, the applicability of ProCharTS on peptide aggregation and viral capsid assembly was studied. ProCharTS was able to monitor the early stages of aggregation in Aβ-derived switch peptides, where the random coil peptides interconvert into β-sheets. Similarly, ProCharTS could detect the formation of the large clusters of HBV core protein dimers into capsids with T=4 and T=3 icosahedral geometry in real-time. The increased interactions among the charged residues at close proximity in the peptide oligomers or HBV capsids are proposed to enhance ProCharTS signal. ProCharTS, being a simple, label-free technique can thus be used for rapid initial screening of drugs for amyloid-linked diseases and viral core protein allosteric modulators (CpAMs), without interfering with the aggregation or capsid assembly kinetics.Item To gain insights into the function of yeast Dnm1 in mitochondrial dynamics(2024) Banerjee, RiddhiMitochondria, 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.Item 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."