Дисертації з теми "Molecular Meidicine"

Epithelial tumour or carcinoma is the most common cause of death in individual with cancer worldwide. Molecular basis of epithelial tumor is poorly understood. To elucidate the mechanism behind the abstractness of epithelial tumor, “Molecular Signature” is the more accurate and effective than possible standard approach. Microarray data analysis has made it possible to obtain high feature molecular snapshot of genes of an organism at various disease state and experimental conditions. In this study, we discussed the uncovering of molecular signature from epithelial tumor (Brain, Stomach, Cervical cancer) on the basis of relative fold change and potential biomarker ability in cancer. To explore the molecular signature in epithelial tumor, we compared the gene expression profile of brain, stomach, cervical cancer. From microarray analysis we found 201 exclusive set of common genes in epithelial origin tumor and from 201 genes, we are able to identify 10 genes that can be used as molecular signature for all types of cancer which has epithelial origin. Selected two genes (SERPINA3, SH3GL3) were experimentally validated by qRT-PCR in HeLa cell line. qRT-PCR established that these two genes are showing their up regulation with respect to Beta-Actin, which is a housekeeping gene. The identification of molecular signature has promising application for accurate detection, promote early diagnosis and screening of cancer.

Absorption of proteins into cationic chitosan microparticles through electrostatic interaction is a common process suitable for oral delivery of proteinaceous drugs. In this research work, in order to achieve a good stability and encapsulation efficiency for an oral drug delivery system, different combinations chitosan, acetic acid Sodium tripolyphosphate and model protein bovine serum albumin were tried and formulated. Then alginate microparticles were coated with the BSA loaded chitosan. Morphological characterizations of the particles were done using zeta sizer and SEM (scanning electron microscope). It was found that Chitosan of 4mg/ml and 5mg/ml concentration had loading efficiency more than 60% and with a particle size in between 400-500 nm. Again when the particles were coated with alginate, the particle size increased from 1400-1600nm (1.4-1.6 μm), can be effectively used for oral drug delivery. In vitro release of BSA from chitosan microparticles and BSA loaded alginate coated chitosan particles were checked by taking particles at different time intervals at pH7.4 using PBS (phosphate saline buffer). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) assay shows that encapsulation with chitosan and further coating with alginate could effectively protect BSA from degradation or hydrolysis in acidic condition for at least 2 hours (using HCl pH 2.0).

The biodegradable polymers like poly lactic acid (PLA) and poly (lactide-co-glycolic acid) (PLGA) are considered as the „green‟ eco-friendly materials due their biocompatibility and non-toxic properties. Biodegradable microspheres and nanoparticles have proven to be very useful in protein and DNA delivery systems. These are easily taken up by immunocompetent cells, shows prolonged antigen release characteristics and provide a long lasting immunity. Micro and nano-particulate based protein and DNA delivery systems have its importance for various therapeutic and biomedical applications. PLA and PLGA microparticles and nanoparticles were formulated by double solvent emulsion evaporation (w/o/w) method and characterised for their surface morphology, size, loading efficiency and release profile study. The microsphere and nanosphere morphology were examined by SEM and Zeta sizer. It was found that PLA encapsulated with BSA (2.5%) showed loading efficiency more than 82% and that with plasmid DNA (Concentration: 1mg/ml), it was found to be 41%. It was also found that the particle size for PLA was varying between 162-373 nm. Similarly for PLGA particles when encapsulated with BSA, the loading efficiency became 91% whereas for encapsulated plasmid DNA, the loading efficiency was 44%, with their respective particle size between 113-335 nm. In vitro release of BSA and plasmid DNA from encapsulated PLA and PLGA nanoparticles were checked spectrophotometrically with optical density 562 nm for protein and 260 nm in case of plasmid DNA, by taking samples at different time intervals dissolved in PBS (phosphate saline buffer, at pH 7.4).

The genus Vibrio is most extensively characterized and medically important group within the family vibrionaceae and it is the most important member of the genus is Vibrio cholerae, the causative agent of cholera. Depending on the presence of O1 antigen they are devided in to two serogroups or serovars: O1 and non-O1. Further O1 is devided in to two biotypes: classical and El Tor. These two biotypes are divided into three serotypes that are inaba, ogawa and hikojima. The virulence properties vary from strain to strain (serogroups). The current study was undertaken aiming at exploring the factors involved with virulence in V. cholerae classical in comparison to V. cholerae O139 by various studies like bacterial antibiotic susceptibility test, haemolytic activity, In silico approach to find potential virulence factors and gene expression pattern study for OMPU & OMPT (Semi quantitative study). MIC of V. cholerae O139 was found to be 12.5, 50 and 25 µg/ml and MIC of V. cholerae classical was found to be 6.25, 25, 12.5 µg/ml for amphicillin, chloramphenicol and amphotericin respectively. So V. cholerae O139 showed more resistance to all the three antibiotics when compared to V. cholerae classical. Similarly, V. cholerae O139 showed higher haemolytic activity than V. cholerae classical. Gene expression pattern confirmed that OMP U gene expression was significantly higher in V. cholerae O 139 as compared V. cholerae classical biotype. But OMP T gene expression is significantly higher in V. cholerae classical as compared to V. cholerae O 139. As OMP U plays a measure role in virulence (as reported previously), V. cholerae O139 is more virulent than V. cholerae classical.

Green synthesis of nanoparticle is a novel way to synthesis nanoparticles by using biological sources. It is gaining attention due to its cost effective, ecofriendly and large scale production possibilities. In this present study five plants Hibiscus rosa sinensis, Cucurbita maxima, Moringa oleifera, Azadirachta indica and Acorus calamus were taken to investigate their potential for synthesizing silver nanoparticle. The silver nanoparticles synthesized were confirmed by their change of colour to dark brown due to the phenomenon of surface plasmon resonance. The characterization studied was done by UV-vis spectroscopy, Scanning electron microscopy (SEM), Atomic force microscopy (AFM), Dynamic light scattering (DLS) and zeta potential studies, X-Ray diffraction (XRD), Fourier Transmission infrared spectroscopy (FTIR). All the five plants synthesized silver nanoparticle show good antimicrobial activity against clinically important pathogens Staphylococcus aureus, Klebsiella pneumoniae, Vibrio cholera and Escherichia coli.

In modern science Nanotechnology is a ablaze field for the researchers.Nanoparticles having a size of 1-100 nm in one dimension, is used significantly concerning medical chemistry, atomic physics, and all other known fields. Nanoparticles are used immensely due to its small size, orientation, physical properties,which are reportedly shown to change the performance of any other material which is in contact with these tiny particles. These particles can be prepared easily by different chemical, physical, and biological approaches. But the biological approach is the most emerging approach of preparation, because, this method is easier than the other methods, ecofriendly and less time consuming. The semiconductor ZnO has gained substantial interest in the research community in part because of its large exciton binding energy 60 meV which could lead to lasing action based on exciton recombination even above room temperature. The Green synthesis was done by using the aqueous solution of Abrus precatorius seeds extract and zinc acetate. A fixed ratio of plant extract to metal ion was prepared and the color change was observed which proved the formation of nanoparticles. The nanoparticles were characterized by UV-vis Spectrophotometer, FTIR, DLS, Zeta Analysis, XRD, and SEM. The particles synthesized were of the size ranging from 90-500 nm.

Tumors mainly originate from epithelial and mesenchymal cells, majority of which are from epithelial origin. There are certain mechanism like EMT and MET which plays a critical role in malignant tumors or cancer progression. There are various other phenomenon and mechanisms occurring at the molecular and cellular level which are yet to be discovered. Success in unraveling such a mystery can give clue to understand the fatal disease like cancer. Roles of genes involved in various cancers and their expression in different forms of cancer as well as in normal condition can give an insight about cancer. This study could be carried out using microarray analysis. Present work mainly focuses to understand the similarity and dissimilarity between the tumors of epithelial and mesenchymal origin by microarray analysis of differentially expressed genes through Genespring software. Gene ontology analysis was also carried out through Genomatix software and two genes (ABCA8 and SMC4) were selected for experimental validation based upon their regulation and association with cancer. Expressions of these two genes were experimentally validated through qRT-PCR with respect to housekeeping gene beta-actin in the HeLA cell line, a cervical cancer cell line. Studies like this can help us to understand the behavior of different types of cancer at the molecular and cellular level, thereby developing an effective treatment measure for deadly disease like cancer.

In recent science Nanotechnology is a burning field for the researchers. Nanotechnology deals with the Nanoparticles having a size of 1-100 nm in one dimension used significantly concerning medical chemistry, atomic physics, and all other known fields. Nanoparticles are used immensely due to its small size, orientation, physical properties, which are reportedly shown to change the performance of any other material which is in contact with these tiny particles. These particles can be prepared easily by different chemical, physical, and biological approaches. But the biological approach is the most emerging approach of preparation, because, this method is easier than the other methods, ecofriendly and less time consuming. The Green synthesis was done by using the aqueous solution of Azadirachta indica leaf extract and AgNO3. Silver was of a particular interest for this process due to its evocative physical and chemical properties. A fixed ratio of plant extract to metal ion was prepared and the color change was observed which proved the formation of nanoparticles. The nanoparticles were characterized by UV-vis Spectrophotometer, FTIR, DLS, Zeta Analysis, XRD, and SEM. The nanoparticles were found have the size ranges from 160-180 nm.

MicroRNAs are a class of small endogenous RNA molecules that is involved in the posttranscriptional inhibition of gene expression. They directly interact with target gene transcripts and influence cellular physiology. MicroRNAs have been reported to be involved in breast cancer tumorigenesis and metastasis thus playing a vital role in cancer progression. Our study aims at identification of novel miRNA-mRNA target pairs that are hypothesized to play a role in breast cancer through a miRNA- mRNA interaction map analysis of microarray data and experimental validation of selected set of mRNAs. The target interaction map analysis revealed three novel target pairs, hsa-miR-27a–MARCKS, hsa-miR-27a–SIK1 and hsa-miR-21–BTG2 which can be potential therapeutic targets in breast cancer. Therefore, with the better understanding of the regulation of miRNAs, the gene networks and cellular pathways regulated by miRNAs, it will be of immense significance to further comprehend breast cancer pathogenesis and target interaction as a therapeutic for breast cancer.

Skin act as a major target as well as a principle barrier for transdermal drug delivery. Vesicular system is one of the most promising approaches for transdermal delivery of active substances. Liposomes are most commonly used vesicular delivery system. But it has certain limitations such as lesser stability, reduced encapsulation efficiency, etc led to formulation of ethosomes. In the present work we encapsulated various concentration of ketoconazole (an antifungal drug) within ethosome and liposome and made a comparative evaluation of their morphology, size, potential, stability and anti-fungal efficacy. Ethosomes showed better stability, encapsulation efficiency and anti-fungal activity as compared to liposome due to its ethanolic content. So ethosomal formulation may prove as a very promising option for transdermal delivery and has potential for new opportunities for topical application of ketoconazole in the fungal infections.

Rice is one of the most important grain with regard to human nutrition and caloric intake, providing more than one fifth of the calories consumed worldwide by humans. It is the most widely consumed staple food for a large part of the world’s human population, especially in Asia. It is an agricultural commodity with the third-highest worldwide production, after sugarcane and maize. A crop failure possess a real threat of starvation. Rice blast, caused by a fungus M. oryzae. It can affect all above ground parts of a rice plant: leaf, collar, node, neck, parts of panicle, and sometimes leaf sheath. Rice blast is one of the most devastating diseases of rice. A leaf blast infection can kill seedlings or plants up to the tillering stage. At later growth stages, a severe leaf blast infection reduces leaf area for grain fill thus reducing grain yield. Leaf blast can kill rice plants at seedling stage and cause yield losses in cases of severe infection. Arabidopsis is a model plant for studying NHR against several plant pathogens. Here, nonhost resistance in Arabidopsis is studied against rice blast pathogen M. oryzae. The infection in mpk6-1 mutants of Arabidopsis was higher in comparison with wild type Col-0. This was evident from confocal microscopy. Furthermore, on studying the expression of PR1, it can be concluded that the pathogen is hemibiotrophic as PR1 expression was absent.

Peanut lectin (PNA) is a plant protein isolated and purified from its natural source Arachis hypogea using biophysical technique called salting out, and analytical technique called size exclusion chromatography, respectively. The isolated lectin was characterised by SDS-PAGE followed by FT-IR study of peanut lectin, which gives the secondary structure of peanut protein. The FTIR data were further strengthened using Circular Dichroism spectropolarimeter. Although peanut lectin comprises majorly of ß-sheet, the protein gave strong negative ellipticity at ~223 nm, a signature of lectin proteins. In addition to circular dichroism study, extrinsic fluorescence study of PNA using 8-Anilino Napthalene-1- Sulphonic acid (ANS) was performed under different conditions such as a range of pH, varying concentration of SDS, and GdnHCl for protein conformational studies. Thermal profiling of PNA was accompanied to study the denaturation pattern of peanut agglutinin, and to know its melting point.

Apoptosis, autophagy and autophagy dependent cell death are important cellular processes with complex and intersecting protein networks; as such, they have been the subjects of intense investigation. Here, we highlight the crucial factors governing the crosstalk between autophagy and apoptosis to describe the mechanisms controlling cell survival and death. We identified the autophagy gene Ulk1 to induce apoptosis by inhibiting the mitochondrial MnSOD enzyme and accentuating the level of mitochondrial reactive oxygen species (ROS). Likely, we found pre-autophagy initiator ATG14 to be interacting with Ulk1 to trigger lipophagy mediated endoplasmic reticulum (ER) stress that induced mitochondrial stress to stimulate apoptosis. Further, we established that cellular stress like serum starvation facilitates anti-apoptotic cIAP1 accumulation in regulating mitophagy through ubiquitination. Moreover, cIAP1-induced mitophagy led to dysfunctional mitochondria resulting in abrogation of mitochondrial oxygen consumption rate that followed to decline in ATP level representing another crucial link between autophagy and apoptosis. We further defined nature of autophagy during cellular stress and serum starvation triggered autophagy doesn’t follow a linear trend when exposed to a prolonged time period of starvation, rather there exists two distinct peaks primary smaller one that arrives at lesser time of stress exposure while the secondary is triggered at a later stage. Protracted cellular stress beyond the secondary peak results in cellular death. Knocking down mTOR disrupts the variation pattern whereas apoptosis gene Bax silencing intensifies the phenomena. The observation highlights that when cells are exposed to stress; an initial primary autophagy peak refluxes certain amount of nutrients in the cell which results in decline of stress for an intermittent period but protracted stress results in a secondary autophagy peak, crossing which the cell is committed to undergo cellular death.

Nanotechnologies are gaining in commercial application. Nanoscale materials are currently being used in electronic, magnetic and optoelectronic, biomedical, pharmaceutical, cosmetic, energy, catalytic and materials applications. Nano particles as defined are particulate dispersions or solid particles with a size in the range of 10-1000nm. Gelatin based nanoparticle are prepared because of its biocompatibility and biodegradability. Gelatin nanoparticles are a delivery vehicle that could be used to deliver many therapeutics to the brain, they will be most effective in delivering drugs that cannot cross the bloodbrain barrier. In addition, they can be used for drugs of high-toxicity or a short half-life. Liposomes are mostly used for drug delivery to the targeted gene. Liposomes increases the effective action, availability, absorption of entrapped dietary and nutritional supplements which can be used as topical drug delivery system. Stavudine is an analog of thymidine. It is phosphorylated by cellular kinases into active triphosphate. The drug we use here is Stavudine, which inhibits the HIV reverse transcriptase by competing with natural substrate, thymidine triphosphate. It also causes termination of DNA replication by incorporating into the DNA strand. The drug is dissolved, entrapped, encapsulated or attached to a nanoparticle matrix depending upon the method of preparation. The drug Stavudine conjugated with liposomes and gelatin NPs can improve the longevity of the HIV patient,leading to a better life.

Polycyclic aromatic hydrocarbons form an active source of air pollution that affects our health and environment. In this study, we deciphered the role of benzo[a]pyrene (B[a]P) on cellular mechanism associated cell death. The particulate matter collected from an industrial area of Rourkela city found to have B[a]P and other unidentified environmental pollutants that had mutagenic and proapoptotic activity. The apoptotic potential of B[a]P was supported by ligand-protein and protein-protein interaction in silico which was validated on human keratinocyte (HaCaT) cell line. Our prediction showed that B[a]P was activated by cytochrome P450 (CYP1B1) to induce multiple cellular effects related to activation of the aryl hydrocarbon receptor (AhR) due to formation of toxic metabolites and this in turn activated caspases. Further, we showed that B[a]P induced mitochondrial mediated autophagy dependent cell death through the canonical pathway in HaCaT cells. The autophagic cell death induced by B[a]P was found to be mediated through AMPK/mTOR pathway. We showed that B[a]P abrogated ATP generation and activated reactive oxygen production to induce toxic mitophagy in HaCaT cells. In addition, we identified Bacopa monneiri (BM) plant extract as an inducer of protective autophagy, which may directly contribute to the antioxidant promoting potential of BM on B[a]P induced cell death through Beclin-1 dependent autophagy activation. The present study provided deep insight into the mechanism of B[a]P-mediated cellular toxicity and elucidated the further scope for the development of phytotherapeutics against environmental air pollutants.

In recent years, nanoparticles and nanomaterials have found their far reaching applications in various fields including the field of biology and medicine. The consequences of these nanoparticles in biological milieu need to be properly assessed. As soon as a nanoparticle enters a biological milieu, a myriad of changes take place. Proteins present in milieu get adsorbed and desorbed over the nanoparticle interface in a dynamic process, resulting in a protein corona. Despite the advances made in nanosciences, our understanding of interactions between protein and nanoparticle interface is still limited. Nanoparticle interface behaviour is anticipated to change with change in accessible surface behaviour of the proteins present in biological milieu. Hence, it becomes essential to study the impact of nanoparticle interface on conformational and amyloidogenic properties of proteins with varying surfaces present in biological milieu. Thus, the thesis discusses the observed effects of zinc oxide nanoparticle (ZnONP) interface on conformational and amyloidogenic propensities of protein models belonging to structurally different hierarchies. Initially, the thesis shows changes in conformational and amyloidogenic propensities for an intrinsically disordered polypeptide (IDP), like IAPP, with change in the length of negatively charged polymeric surfaces, i.e. heparin fragments; diffusive binding of smaller heparin fragments through IAPP sequence is anticipated to delay the fibrillation, whereas interactions with longer fragments (> heparin heptamer) stabilize N- and C- terminus charged residues and expose IAPP self-recognition element resulting in enhanced fibrillation. On the other hand, ZnONP with negative surface potential interaction with monomeric IAPP found to inhibit the fibrillation and the fibril-mediated cytotoxicity. The second part of the thesis indicates the effect of ZnONP interface on another, relatively longer, IDP, i.e. -synuclein. The results indicated that highly favorable interaction between the interface and protein forms thermodynamically stable complex resulting into amorphous aggregation, instead of fibrillation; the interaction must have raised the threshold barrier between the structures, complex and amyloid structures, resulting the complex to kinetically trap in amorphous aggregate. However, the interaction with globular protein like insulin showed opposite results, which is discussed in subsequent chapter of the thesis. The interaction of insulin with ZnONP interface results in protein conformational rearrangement into an amyloid-prone conformation. The conformation fibrillates relatively faster and causes enhanced fibril-mediated cell death on increasing the interface concentration in solution at physiological pH. Otherwise, the protein at higher concentrations only forms amorphous aggregate in physiological pH. The thesis ends with the discussion on the effects of interface interaction with quaternary protein, like Concanavalin A (ConA). Interestingly, ConA adopts different unfolding conformations upon interaction with different chaotropes, like SDS, guanidinium chloride etc. Guanidinium chloride completely unfolds the protein above 2 M, whereas sodium dodecyl sulfate took protein into all-α protein from an all- protein. Additionally, ConA interaction with ZnONP interface causes conformational rearrangement with relatively more exposed hydrophobic patches, resulting into amorphous aggregation of the protein. Thus, the thesis findings, altogether, indicate that the interacting interfaces, like ZnONP with negative interfacial potential and protein interface, predominantly determine conformational changes in protein upon interaction, and its subsequent consequences like amyloidosis, flocculation.

Among wide variety of plants only few species are able to produce milky fluid called latex. This contains a complex mixture of plant proteins like lectins, chitinases, proteinases etc. which exhibit a specific biochemical property. Jatropha curcas, a perennial plant widely found in tropical and sub tropical region is well known for its great capacity of producing latex. The plant mainly grown for biodiesel production has potential medical applications for a variety of human diseases. The latex of Jatropha contains alkaloids and lectins like Curcin, a protein which is proposed to exhibit antitumor properties. This protein has a sequence similarity with RIP family of proteins which are known for their anti cancerous nature. Preliminary studies suggested the use of Curcin as a part of antitumor treatment. Changing lifestyle and ageing in the developing world has resulted in rising number of individuals suffering from cancer. In order to assist further development in cancer related drug discovery, naturopathy and to widen the scope of research an in-silico study was performed in search for potential targets of Curcin which can be exploited to treat cancer. Homology modeling was done to predict the structure of Curcin molecule and protein-protein docking operation was carried out using EGFR and mTOR as target receptors. Interestingly the results went in the favor of Curcin efficiently proving its anti malignant property. However, the predicted model has to go for clinical trials to establish its effectiveness as an inhibitor of tumors caused by EGFR over expression.

Inside the biological milieu, nanoparticles come in myriad shape and size those upon interaction with different biomolecules form nano-biomolecular complexes. The interface formed as a result of nanoparticle and biomolecular interactions determines fate of both the nanoparticle and biomolecules inside the biological milieu. Accordingly, investigating the interaction pattern at different interfaces will help in optimizing the use of nanoparticle for relatively wider biomedical applications. Hence, the thesis intends to study the effects of different photocatalytic nanoparticle interfaces on biological membranes, like prokaryotic and eukaryotic membranes, and biomacromolecules, like nucleic acid and protein. To this end, photocatalytic nanoparticles, such as zinc oxide (ZnONP), iron oxide (IONP) and silver (AgNP) nanoparticles, were synthesized using chemical synthesis or green synthesis methods. Initially, the effects of interfacial potential and interfacial functional groups were studied against Gram-positive and Gram-negative bacteria. The studies demonstrated that the interfacial potential and surface functionality significantly affect interaction pattern at the interface, which defines anti-bacterial/cytocompatible property of nanoparticles. In addition, second part of the thesis explored the effect of nanoparticle surface defects on cytotoxic and antimicrobial propensities of nanoparticle. The study revealed that energy band gap reduction significantly enhances the oxidative stress in cells, leading viable cells into non-viable cells. The second part, unlike the first part of the thesis where the focus was cell membrane functionality, focused on the interface effects on nucleic acid. Third objective of the thesis observed photocatalytic nanoparticle interaction with antimicrobial peptide (AMP), like nisin, and its effect on the peptide conformational and functional dynamics. The interaction leading into nisin assembly onto AgNP interface enhanced the efficacy of peptide by many folds, without significant change in peptide conformation. Whereas in fourth objective, interaction with globular protein, like lysozyme, showed that the assembly onto ZnONP interface led into conformational rearrangement that hinders the amyloidogenic propensity of lysozyme in studied conditions. Nevertheless, with increase in ZnONP fraction in the conjugate mixtures, the protein attains relatively more regular conformation than partially unfolded conformation at pH 9. Insignificant conformational changes in lysozyme assembled onto ZnONP interface was observed at pH 7.4. Thus, the findings, altogether, suggested that the physico-chemical properties of photocatalytic nanoparticle interface significantly affect the fate of biomembrane and biomacromolecules inside the biological milieu.

Sarcomas are rare, and aggressive cancers of mesenchymal origin, with a survival rate ＜15% if metastasized. The molecular heterogeneity of this cancer complicates its diagnosis, prognosis, and treatment. Further, acquired chemoresistance remains a major clinical challenge, accounting for its treatment failure and tumor relapse. Therefore, it is critical to decrypt key regulators and the underlying molecular mechanisms of sarcomagenesis and drug resistance. The current study sought to identify key regulators of tumorigenesis and chemoresistance that could be modulated by PIWI-interacting RNA (piRNA), a class of small non-coding RNAs dysregulated in two types of sarcomas, soft tissue sarcoma (STS) and osteosarcoma (OS). Initially, we used omics analysis of STS clinical data, and found Ribonucleotide reductase regulatory subunit M2 (RRM2) as a potential oncogene, whose higher expression was found to be linked with STS prognosis, lower survival, and recurrence. In vitro studies revealed that overexpression of RRM2 in HT1080 fibrosarcoma cells, a type of STS induces proliferation, migration, invasion, and colony formation, whereas silencing arrests the cell cycle at the G0/G1 phase and induces apoptosis. Interestingly, we discovered that piR-39980, which is downregulated in HT1080 cells, regulates RRM2 expression by directly targeting its 3' UTR and modulates sarcomagenesis by acting as a tumor suppressor. Furthermore, we found that piR-39980 was very less expressed, while its targets, RRM2 and Cytochrome P450 Family 1 Subfamily A Member 2 (CYP1A2) are highly upregulated in doxorubicin (DOX)-resistant HT1080 (HT1080/DOX) cells compared to parental HT1080 cells. Our findings from several molecular assays revealed that RRM2 confers DOXresistance by rescuing DOX-induced DNA damage by promoting DNA repair, whereas CYP1A2 induces DOX-resistance by decreasing intracellular DOX-accumulation via its metabolism. Interestingly, overexpression of piR-39980 in HT1080/DOX cells significantly increased the DOX sensitivity by promoting intracellular DOX accumulation, DNA damage, and apoptosis, indicating that piR-39980 could reduce DOX resistance by modulating RRM2 and CYP1A2 expression. On the contrary, piR-39980 is significantly upregulated in OS and acts as an oncogene by targeting Serpin Family B Member 1 (SERPINB1), resulting in Matrix metalloproteinase-2 (MMP2) activation. In summary, this study discovered that piR 39980, through modulating key targets, play crucial roles in sarcoma oncogenesis and chemoresistance, and thus could be a promising RNA-based therapeutic agent, which needs to be studied further.

This thesis attempts to investigate the membrane parameters associated with neurodegeneration caused by zinc oxide nanoparticles (ZnONPs) and Amyloid Beta-40 (Aβ- 40) leading to neuronal cell deformation. Intrinsically disordered peptides (IDPs) like Aβ- 40 are known to cause neurodegeneration, although the exact mechanism through which they elicit this toxicity is still elusive. And lately, the extensive use of nanoparticles, specifically ZnONP, in cosmetics, coating, and pigments has increased tremendously. This leads to the possibility of unintentional exposure of NPs as a hazardous pollutant for humans. Recent studies have shown the implication of ZnONP exposure that induces neuronal damage. But the mechanism of entry of these NPs into the cells is poorly understood. The current understanding of nanoparticle–membrane interaction is drawn mostly from computational studies and lacks sufficient experimental evidence. We firstly explored the ZnONP-membrane interactions. Here, we try to investigate the lipid specificity and the role of the membrane biochemical and physical forces at play in modulating the penetration of ZnONPs. Using confocal fluorescence imaging and potentiometric dye-based fluorimetry, we first investigated the interaction of ZnONP in both multi-component and individual lipid membranes using cell-like giant unilamellar vesicles to dissect the lipid specificity; also, we measured the changes in membrane order, anisotropy and hydrophobicity. Amongst the single lipid membranes, ZnONP interacted strongly with phosphatidylinositol followed by phosphatidylcholine head-group containing lipids. We further compared the interaction of ZnONP with three physiologically relevant membrane conditions varying in composition and dipole potential. We found that ZnONP interaction leads to a photoinduced enhancement of phase separation that ranges from partial to complete depending upon the membrane composition and cholesterol content. Interestingly, while the lipid order of a partially-phase-separated membrane remained unchanged upon ZnONP crowding, a fully-phase-separated membrane showed an increase in the lipid order. Strikingly, ZnONP crowding induced a contrasting effect on the rigidity of the membrane upon binding to the two membrane conditions, which was inferred through fluorescence anisotropy, in line with the measured diffusion coefficient. ZnONP seems to preferentially penetrate through the liquid disordered areas of the membrane and the boundaries of the phase-separated regions driven by the interplay between the electrostatic forces and phase boundary conditions, which are collectively dictated by the composition and ZnONP induced lipid reorganization. The results may lead to a greater understanding of the interplay of membrane parameters and ZnONP interaction in driving passive penetration. Coming to Amyloid-beta (Aβ-40) aggregation mediated neuronal membrane deformation, although poorly understood, is implicated in Alzheimer's Disease (AD). The peptide aggregates, forming amyloid plaques which were long suspected to be toxic to the neurons. But lately, the leitmotif in the field has changed, where now the more soluble forms of the peptide are considered toxic for the neurons. Hence, we set out with a motive to find synthetic molecules, specifically the arylamines that could compete with the aggregating peptide for the binding pockets leading to retardation or complete arrest of the fibrillation of the peptide. Further, we also looked into whether these molecules had the potential to dissolve pre-formed aggregates. The screening of these molecules led to two potential molecules that retarded the Aβ-40 aggregation and one of which could also dissolve the preformed aggregates. Furthermore, We then wanted to emulate the Aβ-40 mediated myelin membrane deformation observed physiologically. For which myelin membrane mimic was incubated with Aβ-40 in vitro and temporally mapped for 24 hours using confocalmicroscopy, initial strong binding and extensive tubulated structures at longer timescales were observed. To dissect the lipid specificity of Aβ-40 in the myelin membrane, we used the single lipid models. ThT assay & fluorescence microscopy were used to check for potential modulation of the Aβ-40 aggregation and binding. Binding was observed in PI, PG, PC/BSM, and DOPC/PIP2. As observed, negatively charged lipids bound strongly, and hence electrostatic forces seem to play a role in binding. Interestingly zwitterionic PC/BSM too showed strong binding leading us to suspect that something else apart from electrostatics is also at play. Using the differences in the shapes of the individual lipids, we formulated three different conditions along with myelin membrane and estimated the packing defect densities through MD simulation and then further experimentally confirming The binding of Aβ-40 depends predominantly on the lipid packing defect densities and electrostatic interactions and results in rigidification of the myelin membrane in the early time scales. Furthermore, elongation of Aβ-40 into higher oligomeric and fibrillar species leads to eventual fluidization of the myelin membrane followed by extensive membrane tubulation observed in the late phase. \ Taken together, our results capture mechanistic insights into snapshots of temporal dynamics of Aβ-40 - myelin membrane interaction and demonstrate how short timescale, localphenomena of binding, and fibril mediated load generation manifests into long timescale, global phenomena of myelin tubulation and demonstrates the ability of Aβ-40 to demyelinate.

The weaponry possessed by Mycobacterium tuberculosis (M. tb) in the form of immunodominant antigens hijack the host defense system to give a survival advantage to this intracellular fiend, but the mechanism of this control is not entirely known. The present study was undertaken to understand the effect of mycobacterial antigens on the anti-mycobacterial effect of Calcimycin. We found significant downregulation of autophagy by purified protein derivative (PPD) 3 (PPD fraction with a molecular weight of antigens > 3 kDa) pre-treatment in Calcimycin-treated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 (dTHP-1) cells compared to PPD 10 (antigenic mol. weight > 10 kDa). This reduction in autophagy also corroborated with the enhanced survival of M. smegmatis and M. bovis BCG in macrophages. We further demonstrate that recombinant early secreted antigenic target 6 (rESAT-6), an immunodominant antigen of M. tb, is responsible for inhibiting Calcimycin-induced autophagy and enhancing intracellular survival of mycobacteria. We also show that pre-treatment with rESAT-6 upregulates microRNA (miR)-30a-3p expression and vis-à-vis downregulates miR-30a-5p expression in Calcimycin-treated dTHP-1 cells. Further, transfection studies using miR-30a-3p inhibitor or -5p mimic highlighted the contrary roles of different arms of the same miRNA in regulating autophagy and IL-18 response by rESAT-6 in Calcimycin-treated dTHP-1 cells. By using either IL-18 neutralizing antibody or inhibitors of phosphoinositide 3-kinase (PI3K)/NF-κB/phagosome-lysosome fusion in the miRNA-30a transfected background, IL-18 mediated signaling and intracellular killing of mycobacteria was reversed in the presence of rESAT-6. Overall, the results of this study conclusively prove the contrary roles of miR-30a-3p and miR-30a-5p in regulating autophagy and IL-18-mediated phagosome-lysosome fusion by rESAT-6 in dTHP-1 cells upon Calcimycin treatment that affected intracellular survival of mycobacteria.

MTP18, a novel human nuclear-encoded protein with a molecular weight of 18 kDa, localized to the inner mitochondrial membrane, has a vital role in maintaining mitochondrial morphology and cell survival. In this study, we have investigated the role of MTP18 in mitophagy activation to clear dysfunctional mitochondria and its physiological significance in cell survival of oral squamous cell carcinoma (OSCC). Our study identified MTP18 as a mitophagy receptor to target damaged mitochondria into the autophagosomes for elimination and degradation. Interestingly, MTP18 interacts with LC3 through its LC3 interacting region (LIR) to induce mitochondrial autophagy, and mutation in the LIR motif inhibits this interaction leading to suppress mitophagy. Further, we established that Parkin-mediated proteasomal degradation of the outer mitochondrial membrane is essential for the interaction of LC3 and MTP18 to successful mitophagy. In this setting, we conclude MTP18 provides a survival advantage to the OSCC in exposure to cellular stress, and inhibition of MTP18-dependent mitophagy effectively reduces cell growth and induces cell death in OSCC. In addition, we have identified a novel inhibitor for MTP18 named S28, which promotes stress-induced mitochondrial hyperfusion (SIMH) in OSCC by limiting MTP18 mediated mitochondrial fission. Mechanistically, S28-mediated SIMH triggers loss of mitochondrial membrane potential, leading to the generation of mitochondrial superoxide followed by apoptosis. Intriguingly, it showed that S28-stimulated mitochondrial superoxide enhances lysosomal membrane permeabilization, resulting in decreased lysosomal pH, which entertained impairment of autophagosome-lysosome fusion. Furthermore, S28 in the combination of FDA-approved anticancer drugs inhibits cell viability and displays enhanced apoptosis suggesting the anticancer drugs exhibit better therapeutic response in MTP18 inhibition conditions in OSCC. In addition, our study unraveled that Abrus agglutinin (AGG), a plant lectin, promotes autophagy through inhibition in the expression of MTP18 in OSCC. We found that AGG-induced autophagy triggers loss of p62 and Nrf2 expression in OSCC. It showed that Nrf2 expression is restored in the presence of 3-methyladenine and Bafilomycin-A1, establishing the role of autophagy in the modulation of Nrf2 through p62. Moreover, we found that Nrf2 inhibition by AGG results in ROS accumulation followed by apoptosis and subsequently inhibits tumor growth in DMBA-induced oral carcinogenesis. Hence, targeting MTP18 may serve as a potential therapeutic for oral cancer treatment.

Diabetes mellitus is one of the most prevalent metabolic disorders of the current century. High calorie diet having high glycemic index (GI) are the major contributors of diabetes. Due to the complex nature of this disease, it is difficult to treat and often leads to huge treatment burden. Numerous studies have tried to understand the detailed mechanism of this disease to design new and effective therapeutic approaches. The application of nanotechnology in the field of diabetes have been proven beneficial. The current study aims to investigate the effect of dietary modulation in the onset of diabetes and established the role of anti-diabetic NPs by using Drosophila melanogaster as a model system. The first objective aims to investigate the effects of dietary advanced glycated end products (AGE). Oral feeding of three types of AGE compounds (i.e. AGE glucose, AGE-fructose and AGE-ribose) was found to alter growth and development of the flies. Beside this, the larva and adults showed persistent hyperglycemic condition, excess fat deposition and micronuclei formation in gut and fat body as well as insulin resistance. The flies also showed increased ROS formation via downregulation of the antioxidant enzyme system. Behavioral defects were also evidenced in the larval and adult locomotion, suggesting neuronal damage. The second objective depicts the role of Strontium ferrite, a metallic nanoparticle as a non-toxic anti-diabetic agent. Files fed with a high fat diet (HFD) were used as a diabetic model. The toxicity profile of the nanoparticles was checked, showing no DNA damage or cytotoxicity. Feeding of the NPs to the diabetic flies demonstrated that, the NPs were able to reduce fly weight, metabolic sugar and triglyceride level, reduce the deposition of fat and also reduce ROS level and behavioral abnormalities. In the third objective, the flies were reared on a high sugar diet (HSD), which tremendously affected their growth and development. Beside this, behavioral alterations was also seen. Hyperglycemia followed by excess fat deposition in the gut, fat body and crop confirmed the diabetic phenotype. A novel polymeric nanoparticle, namely polyvinylpyrollidone-curcumin (PVP-C) was checked for antidiabetic potential. Non-cytotoxic and non-genotoxic potential of the nanoparticles were evidenced from no DNA damage, and absence of trypan blue staining, as well as no phenotypic abnormality. Treatment of PVP-C NPs to the diabetic flies showed reduction of metabolic contents and ROS level. Together the study suggests the role of diet in diabetic onset and importance of nanoparticles having potential to be used alone as an anti-diabetic agent or a combination to deliver therapeutic molecules.

The current study describes the development of span-60 based organogels using sunflower oil (SO) as the apolar solvent. The organogels were analyzed for their stability. Subsequently, the stable organogels were characterized by gel-sol transition studies, microscopic analysis, opacity measurement, FTIR spectroscopy, XRD analysis, thermal analysis (using simultaneous TGA-DTA and DSC) and pH measurement. Salicylic acid (SA), model drug, was incorporated within gels and their in vitro release behavior and antimicrobial efficiency against E. coli and B. subtilis were studied. To ascertain the biocompatibility of the gels, hemocompatibility tests were conducted. The stability tests indicated that the gels were inherently stable when stored below 25°C. The gel-sol transition study indicated that as the concentration of the gelator was increased, there was a subsequent increase in the transition temperature. This was also confirmed by the instrumental thermal analysis studied. Microscopic analysis indicated that the solid fibers, formed by the clusters of needle-shaped gelator particles, form the backbone of the organogels structure. Opacity measurements suggested that the rate of gelation of the organogels is higher in organogels with higher gelator concentration. FTIR spectroscopy indicated the presence of hydrogen bonding in both blank and SA-loaded organogels. XRD studies showed that there was a change in the crystallinity of the samples and the change was dependent on the composition of the organogels. The pH of the organogels was found to be within the normal human skin pH range. The release studies indicated that the release of SA from the organogels occurred by Higuchian kinetics and were able to restrict the growth of E. coli and B. subtilis efficiently. The organogels were found to be hemocompatible in nature. Based on the results, the developed organogels may be tried as a drug carrier for transdermal and/or topical formulations.

Small non-coding RNAs (sncRNAs) are presently being recognized as an essential player in the modulation of regulatory events and implicated in the etiology of human diseases. In the present study, we comprehensively decoded the presence of Piwi-interacting RNAs (piRNAs), a recently discovered class of sncRNAs in the normal human ovary (NO) and its cancer subtypes: endometrioid ovarian cancer (ENOCa) and serous ovarian cancer (SOCa), along with their characteristic features encoded within sequence and structures as well as target binding features. In this regard, we performed next-generation sequencing (NGS) followed by comprehensive bioinformatics analysis and identified a catalog of 219, 256, and 234 piRNAs in NO, ENOCa, and SOCa samples, respectively. Furthermore, our study revealed some key piRNAs, piR-52207 and piR-33733 involved in the modulation of the pathophysiology of ovarian carcinoma. Moreover, we have identified several sequence and structural features of piRNAs, including its precursors and piRNA target sites, that might aid in the proper identification of piRNAs and their targets, which is essential for decrypting piRNA biogenesis and functions. In summary, the present study decodes piRNA-mediated target regulation in the pathophysiology of ovarian cancer subtypes and distinct sequence and structural features of piRNAs and their target sites providing a complete picture of the piRNAs and piRNA-mediated regulations. This might pave the way for developing tools for predicting piRNAs and their targets more precisely.

SRY-related high mobility group box 9 (SOX9) has been identified as a promising transcription factor in the initiation and progression of various tumor types, including lung cancer. The present study investigates the role of SOX9 expression in EGFR-TKI (gefitinib) resistant lung cancer in vitro and ex vivo. It was found that upregulation of SOX9 was associated with gefitinib resistance with an increased rate of cell proliferation, migration and invasion, single-cell colony-forming ability, and reduced apoptosis in gefitinib resistant cells A549/GR and NCI-H522/GR. Moreover, upregulated SOX9 promoted epithelial to mesenchymal transition (EMT) via targeting β-catenin and its knockdown reversed the resistance and EMT phenotype in A549/GR and NCI-H522/GR cells. Similarly, it was found that multicellular spheroids of A549/GR and NCI-H522/GR cells showed a larger surface area with more dispersion, while SOX9 knockdown abolished these gained properties ex vivo. To date, many natural compounds have shown promising chemotherapeutic potential. Piperlongumine (PIP), a white to beige biologically active alkaloid has high pharmacological relevance as an anticancer agent. It has been found that PIP suppresses the cell proliferation, migration, and stem-cell-like phenotype by targeting SOX9, resulting in induction of apoptosis in A549 and NCI-H522 cells via attenuating EMT. PIP effectively showed cytotoxicity against A549 and NCI-H522 cells and induced reactive oxygen species (ROS) production and cell cycle arrest. Additionally, PIP induced dissipation in mitochondrial membrane potential and affected the expression of intrinsic apoptotic pathway proteins in lung cancer cells. PIP inhibited EMT and stemness of lung cancer cells. Further, ex vivo analysis revealed that PIP induced apoptosis and size reduction in multicellular spheroid of A549 and NCI-H522 cells. The emergence of resistance to gefitinib indicates that use of a single agent may not be sufficient for the effective treatment of lung cancer. Therefore, combinatorial effect of PIP with gefitinib in A549/GR and NCI-H522/GR cells was examined. The result suggested that co-treatment of PIP with gefitinib in lung cancer cells showed increased cytotoxicity as compared to gefitinib or PIP alone and reversed the resistance via inhibiting EMT. At last, the overall study delineated a deep insight into the mechanism of PIP mediated cancer inhibition and its combinatorial therapeutic effect in re-sensitization of SOX9 induced EGFR-TKI resistance in lung cancer.

Circadian clock is an endogenous time keeping system which is conserved from bacteria to human beings. Years of research in vertebrates, suggest close association of circadian clock with parameters like metabolism, physiology, aging, sleep, behavior, neurodegeneration and other metabolic disorder. Several model organisms including Drosophila melanogaster are used to establish the role of circadian rhythm in day to day activity. The current study aims to investigate the role of three key parameters on circadian rhythm. The first parameter is high-fat diet mediated diabesity and its effect on circadian rhythm. For this experiment the larva and adults of D.melanogaster were exposed to high-fat diet (HFD) through oral route. After feeding the larva and adults have excess fat and micronuclei within the gut, fat body, and crop. Larva and adults of HFD showed behavioral defects, impaired metabolite profile, and overexpression of insulin gene (Dilp2) and tribble (trbl) gene confirmed insulin resistance. Elevated ROS level, developmental delay, altered metal level, growth defects, locomotory rhythms, sleep fragmentation, and expression of circadian genes (per, tim, and clock) in HFD larva and adults. In the second objective the effect of host pathogen interaction and its co-relation between circadian rhythm was established. For this study both gram-negative bacteria, Pseudomonas aeruginosa and gram-positive bacteria Staphylococcus aureus was examined on D. melanogaster physiology, behavior and circadian clock. Infections lead to phenotype and behavioral defects, developmental delay, oxidative stress and nuclear damages in larvae as well as adults. Increased level of expression of immune genes and disrupted circadian clock related co-mordities in Drosophila melanogaster were detected. The third objective checked Drosophila clock (ClkJrkst) mutation on behavioral and molecular changes in their eye and hearing organ, defective oxidative state, and status of TRP channel genes, physiology, metabolism, and behavior in Drosophila. The current study suggests the chronobiological consideration towards treatment for type-II diabetes, infectious disease and circadian anomalies.