http://localhost:8081/jspui/handle/123456789/2 2026-04-21T09:25:47Z 2026-04-21T09:25:47Z Yadav, Monica http://localhost:8081/jspui/handle/123456789/20459 2026-04-20T10:30:26Z 2024-04-01T00:00:00Z

Title: Characterization of milk fat globule membrane interaction with the gut microbiota Authors: Yadav, Monica Abstract: Lipids' unique composition and arrangement in the human milk fat globule membrane (hMFGM) provides infants versatile benefits, including interaction with the gut and gut microbiota. Probiotics, such as Lactobacillus, associated with breastfed infants' gut microbiota have been proven effective in regulating intestinal health. Our study is designed to assess the probiotic potential of Lactiplantibacillus plantarum (MRK3), Limosilactobacillus ferementum (MK1) isolated from infant feces, and its interaction with globule membrane during conditions mimicking infant digestive tract, which is not well elucidated. The finding reveals that both strains (MK1, MRK3) had a high tolerance to gastrointestinal conditions (87-96%) with remarkable cell surface hydrophobic properties (>90%) and significantly strong anti-pathogen activity against S. aureus with an inhibition zone of 12 mm. Notably, significant difference in membrane-bound xanthine oxidase activity, membrane roughness, and surface topography was observed along the infant digestion tract. In the presence of hMFGM, survival of MRK3 was higher than MK1 throughout in vitro digestion. Scanning Transmission Electron Microscopy (STEM) and Scanning Electron Microscopy (SEM) results revealed structural integrity, interaction and successful entrapment of MRK3 in the membrane matrix throughout digestion. Interestingly, probiotic-membrane matrix interaction showed significant synergy to alleviate oxidative stress and damage induced by cell free supernatant (CFS) of E.coli infected Caco-2 cells. In addition, pre-incubation with hMFGM before adding CFS of E. coli and P. aeruginosa significantly reduces the deleterious effect of pathogens. Our results show that a probiotic encapsulated membrane matrix could protect against oxidative stress and disruption induced by pathogens in an infant's gut, thereby opening the route for developing functional infant formula.

2024-04-01T00:00:00Z Chennakesavulu, Kanchapu http://localhost:8081/jspui/handle/123456789/20444 2026-04-20T06:32:07Z 2024-03-01T00:00:00Z

Title: UNDERSTANDING FUNCTIONS OF AP2-DOMAIN CONTAINING TRANSCRIPTION FACTORS IN RICE Authors: Chennakesavulu, Kanchapu Abstract: Plants produce various organs throughout their life cycle, and the process of forming any new organ is known as organogenesis. Plant organogenesis requires cellular reprogramming, associated with dedifferentiation, pluripotency acquisition, cell cycle reactivation, new cell fate acquisition, and re/trans-differentiation processes. The root formation in plants is one of the best suitable systems for studying cellular reprogramming in plants. In higher plants, two types of root systems can be observed: the tap and the fibrous root systems. The dicots, for example, Arabidopsis thaliana, a dicot model plant, contains the tap root system. The tap root system comprises an embryonic-derived primary root (PR) and root-borne post-embryonic lateral roots (LRs). The monocots, including cereals, contain the fibrous root system comprising shoot borne crown roots (CRs), brace roots (BRs), root-borne LRs, and an embryonic-derived PR. It is believed that the PR in cereals is short-lived, and they depend on the shoot-borne roots that make up a major root system along with LRs in lateral stages. The shoot-borne root formation that is highly relevant to cereal crop production is not naturally present in Arabidopsis, thus all of its knowledge cannot be transferable to the other plants. Rice (Oryza sativa) belongs to a grass family called Poaceae and is considered to be a perfect model for cereal crop research because of its relatively small genome size. Also, a well established and highly efficient transformation system, extensive genetic resources, and synteny with the sequences of other cereal crops strengthen rice as a model organism. Rice is a staple food crop and feeds a significant population worldwide. The rice develops a fibrous root system that contains the post-embryonic shoot-borne CRs and root-borne LRs. An embryonic PR provides anchorage, water absorption, and nutrient uptake at the early stages of rice seedlings. As PR is short-lived, rice depends on post-embryonic roots to complete its life cycle. In the rice stem base, the inner ground meristem cells close to the vascular tissues initiate the CR formation in rice. Meanwhile, the LRs initiate from the endodermal and pericycle cells located opposite to the protophloem in rice. The post-embryonic root formation in rice requires cellular reprogramming. During crown root primordium (CRP) initiation, the innermost ground meristem cells (shoot cells) dedifferentiate and enter into cell division to produce CR initials. The initial cells further divide and differentiate into root meristems that emerged in the CRP outgrowth stage. Our previous laser-captured CRP transcriptome profiling study revealed that six PLETHORA (OsPLT) genes are sharply induced during CRP initiation and outgrowth stages in rice. In rice, OsPLT genes encode AP2-domain containing transcription factors. There are ten PLT genes present in rice and categorized into two subclades: clade A (OsPLT1~6) and clade B (OsPLT7~10). Based on the expression pattern and phylogenetic analysis from the literature, I have chosen OsPLT1, OsPLT6, OsPLT7, and OsPLT9 for their functional characterization in rice root development in this study. Using loss-of-function and gain function approaches, we have identified the functional role of these PLTs in rice. OsPLT1 positively regulates both CR and LR formation in rice. While osplt1 mutants produced fewer CRs and LRs, the OsPLT-GR overexpression plants produced a robust root system in rice. Further, the ectopic root formation from leaf tissues and the induced scutellum-derived callus formation were observed in OsPLT1-GR lines. It suggests that overexpression of OsPLT1 alone can induce root cellular reprogramming in rice. Next, the osplt6 mutants produced fewer CRs and LRs than the wild type. However, in OsPLT6-GR plants, we observed that the shoot and root tissues were converted into callus-like tissues. The scutellum-derived callus formation was also promoted in OsPLT6-GR plants. Further, the CR formation was induced in the OsPLT6 GR plants. Therefore, OsPLT6 plays a key role in differentiation and cellular reprogramming, regulating CR root development in a dose-dependent manner in rice. The phytohormone auxin regulates the OsWOX11-ERF3-OsRR2 module, which plays an important role in rice crown root development. Using qRT-PCR analysis, our study found that the expression of WOX11-ERF3-OsRR2 module was affected in osplt1 and osplt6 mutants. In contrast, the expression of these genes was significantly upregulated in overexpression lines consistent with the induced CR and LR formation in rice. Further, we observed that WOX11 and ERF3 expression were upregulated in OsPLT6 overexpression lines. Although the plants have been converted into a callus, the CR formation was promoted in these lines due to induction in the ERF3 gene expression. Therefore, OsPLT1 and OsPLT6 genes regulate the expression of the WOX11-ERF3-OsRR2 module during CR development in rice. Next, through ChIP-qPCR, we identified OsYUC3, an auxin biosynthesis gene, is the direct target of OsPLT1 in rice. We also identified that OsYUC3, OsYUC6, OsYUC7, and OsYUC9 genes were significantly upregulated in OsPLT6-GR plants. Therefore, OsPLT1 and OsPLT6 function upstream of the auxin signaling and regulate auxin-mediated developmental processes, including root development in rice. Further, we observed that the CR and LR development was impaired in OsPLT7 mutants. However, further studies are required to study the functional roles of OsPLT7 and OsPLT9 in rice.

2024-03-01T00:00:00Z Vishnu, Nehul Sanketkumar http://localhost:8081/jspui/handle/123456789/20443 2026-04-20T06:31:43Z 2024-05-01T00:00:00Z

Title: Protein-protein interactions as therapeutic target in RNA viruses. Authors: Vishnu, Nehul Sanketkumar Abstract: Chapter 1 provides an overview of the existing literature on protein-protein interactions and their role in the life cycle of plus-strand RNA viruses with focus on Alphaviruses and Coronaviruses. The first part of the chapter delves into a comprehensive overview of the Chikungunya virus (CHIKV) life cycle, genomic organization, detailed functions of structural and non-structural proteins, epidemiology, and details about reported in vitro and in vivo CHIKV inhibitors. An in-depth description of the capsid protein, its structural details, multi-faceted roles, and its involvement in virus particle maturation and budding process has been done. Furthermore, the chapter also explains the essential role of protein-protein interactions in the life cycle of alphaviruses. The second part briefly gives an overview of SARS-CoV-2, and describes the life cycle of SARS-CoV-2. The vital role of RBD-ACE2 interaction in the entry of virus particles into host cells is emphasized. The essential RBD-ACE2 interaction as an ideal target for the development of therapeutics against SARS-CoV-2 has been explained in detail with previously reported entry inhibitors against coronaviruses. Chapter 2 reports identifying and evaluating the antiviral potential of thymoquinone, a natural compound targeting Chikungunya virus (CHIKV) capsid protein. The capsid protein (CP) of CHIKV is a multifunctional protein with a conserved hydrophobic pocket that plays a crucial role in the capsid assembly and virus budding process. This study demonstrates the antiviral activity of thymoquinone, a natural compound targeting the hydrophobic pocket of CP. The binding of thymoquinone to the hydrophobic pocket of CHIKV CP was analyzed using structure based molecular docking, isothermal titration calorimetry, and fluorescence spectroscopy. The binding constant KD obtained for thymoquinone was ~27 µM. Additionally, cell-based antiviral studies showed that thymoquinone diminished CHIKV replication with a half maximal concentration (EC50) value of ~4.4 µM. Reduction in viral RNA copy number and viral replication as assessed by the quantitative reverse transcription polymerase chain reaction (qRT PCR) and immunofluorescence assay (IFA), confirmed the antiviral potential of thymoquinone. Our study reveals that thymoquinone is an effective antiviral targeting the hydrophobic pocket of CHIKV CP and may serve as the basis for the development of a broad-spectrum therapy against alphavirus diseases. I Chapter 3 describes the anti-CHIKV activity of efavirenz investigated by in vitro cell culture based antiviral assay, IFA and qRT-PCR. These studies demonstrated dose-dependent robust anti-CHIKV activity of efavirenz at low micromolar concentration (EC50 = ~1.33 µM). To determine potential broad anti-alphavirus activity of efavirenz, its inhibitory activity against the Sindbis virus was detected. Interestingly, efavirenz also inhibited the replication of SINV at a low micromolar range (EC50 = ~0.7 µM). Further, several approaches were employed to investigate the possible mechanism of anti-viral activity of efavirenz. Time of addition assay, direct transfection of virus replicon RNA and viral negative sense RNA specific RT-PCR elucidated that efavirenz inhibits the early stage of viral replication after virus entry by strongly interfering with the synthesis of negative-sense viral RNA. Chapter 4 reports the important role of RBD-ACE2 interaction in the entry of SARS-CoV-2 into the cells. Viral attachment and entry are of particular interest among possible protein-protein interaction targets for therapeutics development in the life cycle of viruses because it is first step in the replication of viruses. This chapter reports the evaluation of SARS-CoV-2 entry inhibition activity of GR 128935 hydrochloride hydrate identified by structure-assisted drug design and high-throughput virtual library screening against the protein-protein interaction of RBD-ACE2. The binding of GR 128935 hydrochloride hydrate against the ACE2 were evaluated by the Surface plasmon resonance (SPR) (KD = ~3.9 µM). The inhibition of RBD-ACE2 interaction was proved by ELISA and entry inhibiting ability of GR 128935 hydrochloride hydrate was demonstrated with the help of pseudovirus entry inhibition assay (IC50 = ~0.61 µM). Finally, GR 128935 hydrochloride hydrate inhibited the SARS-CoV-2 replication in Vero cells with the EC50 value of ~0.8 µM. Chapter 5 comprehensively concludes the findings in the thesis, representing a multi-faceted strategy in addressing the challenges posed by emerging viruses of global healthcare concern, CHIKV and SARS-CoV-2. The first study evaluated the affinity of thymoquinone against the hydrophobic pocket of CHIKV CP, followed by an assessment of anti-CHIKV potential in cell culture-based studies, opening up the possibilities for further in vivo investigations. The next study focuses on repurposing HIV antiretroviral drug efavirenz against CHIKV, as robust anti CHIKV activity of efavirenz was observed in cell culture-based antiviral assays. The mechanism by which efavirenz exerts the antiviral activity was investigated and was found that it inhibited the synthesis of minus-strand viral RNA. This supports and suggests that efavirenz modulates the early stage replication processes in the virus life cycle. The third study in thesis confirms GR 128935 hydrochloride hydrate as an entry inhibitor of SARS-CoV-2, after detailed investigation II by SPR, RBD-ACE2 ELISA, pesudovirus inhibition assay and cell culture based anti-SARS CoV-2 assay. In conclusion, the present study, for the first time, identified thymoquinone and efavirenz as inhibitors of CHIKV replication. Further, in the case of SARS-CoV-2, the entry inhibition activity of GR 128935 hydrochloride hydrate was validated using various techniques. Out of these three inhibitors thymoquinone and GR 128935 hydrochloride hydrate are potential protein-protein interaction inhibitors. These identified compounds hold potential as therapeutic treatments for infections of their respective target pathogens, CHIKV and SARS-CoV-2.

2024-05-01T00:00:00Z Shroti, Gireesh Kumar http://localhost:8081/jspui/handle/123456789/20397 2026-04-13T06:27:13Z 2024-01-01T00:00:00Z

Title: PRODUCTION AND CHARACTERIZATION OF GREEN PLASTIC POLYMER POLYHYDROXYALKANOATES DERIVED FROM MICRO-ORGANISM USING WASTE RESOURCES Authors: Shroti, Gireesh Kumar Abstract: Polyhydroxyalkanoates (PHAs) are a type of biodegradable, thermoplastic polyester produced by various microorganisms as a way to store carbon and energy inside their cells. These PHAs are made up of repeating units of hydroxyalkanoic acids, which can have different lengths and branching patterns, resulting in a wide range of PHA materials with diverse physical and chemical properties. PHAs are considered promising biopolymers that could potentially replace synthetic plastics. While the production cost of biopolymers is typically higher than that of synthetic plastics, they offer a vital solution to the problem of plastic pollution. Therefore, further research and development are needed in this area, particularly to rapidly screen and isolate bacteria capable of producing PHAs with exceptional yields at low production costs. Ultimately, polyhydroxyalkanoates are materials that can degrade naturally. PHA production can be produced using different carbon sources like sugars, lipids, and waste materials including food and agricultural waste. This approach helps reduce the environmental impact of PHA manufacturing. Moreover, PHAs can be easily degraded by microorganisms in various environments such as soil, water, and marine ecosystems, preventing their accumulation in the environment. The present research aimed to identify microorganisms capable of producing PHAs and utilize them efficiently by employing agro-industrial waste as a substrate. Chapter 1 provides an introduction to the PHA polymer and presents a review of the existing literature on the topic. It discusses the mechanism involved in the production of PHA, specifically focusing on the pathway through which it is synthesized. Additionally, this section highlights the unique features of PHA that make it suitable for a wide range of applications. The chapter also provides a brief overview of relevant studies related to PHA production using different types of waste materials and the microorganisms involved in the conversion of carbon into commercially viable polymers. Chapter 2 of the thesis focuses on a detailed study of the sample procurement process for isolating the potential PHA producer, HLI13B. This involved screening bacteria using different dyes, and the methodology and results of this screening are presented. The chapter also covers an extensive examination of the experimental setup for bacterial culture to optimize growth conditions such as pH, temperature, and glucose concentration. Polymer production using the optimized conditions, as well as the extraction of the polymer, are described in this section. Furthermore, the chapter includes a comprehensive study of the physical, chemical, and thermal characterization of the polymer. Various analyses were conducted, including FTIR, TGA, DTA, DTG, XRD, 1H NMR, GPC, SEM, SPM, Nanoindentation, and Biodegradability. These analyses aimed to determine the chemical composition, mechanical strength, and thermal stability of the polymer, thereby validating its suitability for various applications in packaging and biomedical fields. Additionally, the chapter incorporates a study of the genome analysis of the PHA producer, HLI13B, with the objective of identifying the genes responsible for PHA production within the organism. Chapter 3 focuses on the production of the PHA polymer using waste resources, specifically utilizing the isolated species HLI13B. The chapter also discusses recent studies that have utilized waste for PHA production. The primary objectives of this chapter include the production of environmentally friendly plastic, namely polyhydroxyalkanoate (PHA), from beverage industrial wastewater, as well as controlling water pollution by consuming residual sugar and reducing pH levels. PHA is a biopolymer that accumulates in various bacterial species when grown in a carbon-rich medium. The novel organism HLI13B, isolated from mess sludge, demonstrates the ability to accumulate PHA and has a strong tolerance for highly alkaline conditions. Beverage industrial wastewater, which contained residual sugar (4.65 g/L) and had a pH of 11, was aseptically collected and used as a carbon source supplemented with mineral salt media (MSM) to culture HLI13B for biomass production under optimized conditions (37 ℃ and 2% glucose). The use of wastewater as a medium significantly reduced the pH from 11 to 7.5 within 24 h and eventually to pH 6.5. The polymer yield reached up to 1.38 g/L under the optimized conditions. The accumulated polymer was extracted using solvent extraction and was found to be identical to the polymer produced using standard glucose. The agricultural applicability of the treated water and wastewater was evaluated by irrigating chickpea plants. The results indicated that the alkaline wastewater had a negative effect on plant growth. Chapter 4 focuses on the detailed study of an organism called Bacillus sp. S4, which was isolated from the roots of sugarcane plants. The chapter provides information about the screening optimization, production, and characterization of the PHA polymer produced by Bacillus sp. S4. Notably, this organism is capable of efficiently utilizing xylose, an alternative, and abundant sugar i.e., xylose. Xylose is a major component present in the leftover hydrolysate of ethanol production waste, as the yeast Saccharomyces cerevisiae primarily consumes glucose during ethanol production, leaving xylose behind. The utilization of this waste significantly reduces production costs. The chapter also delves into the characterization of the PHA polymer synthesized by Bacillus sp. S4 and explores its potential application in nanofiber synthesis. The nanofibers derived from the PHA polymer are thoroughly characterized to assess their suitability for biomedical applications. Chapter 5 has detailed the structural aspects of the enzyme involved in PHA synthesis pathways PhaC. The naturally occurring polymers PHA are incredibly useful in the medical field and tissue engineering because of their unique physical and chemical characteristics. In the PHA production pathway, PHA synthase (PhaC), is one of the significantly important enzymes for synthesizing this commercially applicable polymer. The current study concentrated on one such PhaC Class I enzyme activity using an in-silico approach. The present study initializes with the phylogenetic analysis of PHA synthase class I sequences to understand their evolution over time. Further, to investigate the structural realm, the homology modeling-based approach was used followed by Molecular Dynamics (MD) simulation. Interestingly, this enzyme was reported to have open, closed, and semi-closed conformations with an immense structural difference. Hence, the molecular dynamic simulation was performed for all three conformations and a CoA complex structure to investigate the dynamic behavior. Simulation analysis includes backbone variations, residue-wise fluctuations, and compactness of the system of the PHA synthase PhaC I. In conclusion, open conformation simulation was observed to be a more structurally moving or fluctuating structure than that of closed and semi-closed conformations. Further, CoA complexed structure attained more stable behavior over the trajectory of simulation than that of only protein (open) structure. Additionally, the regions of structural importance and indicating dynamicity are identified and their in-detailed analysis is presented. This novel study sheds light on the structural insights of PHA synthase as this enzyme has the potential target of protein engineering to facilitate it to produce a variety of biopolymers for commercial use. Chapter 6 summarizes the whole study and conclude the outcomes of the study. It also includes the future copes of the study.

2024-01-01T00:00:00Z