http://localhost:8081/jspui/handle/123456789/22 2025-07-13T05:00:55Z http://localhost:8081/jspui/handle/123456789/15800 Title: AUXIN-CYTOKININ RESPONSES AND FUNCTIONAL SIGNIFICANCE OF AUXIN DURING RICE CROWN ROOT DEVELOPMENT Authors: Neogy, Ananya Abstract: Rice root system is a fibrous root system, composed of primary/seminal, adventitious/crown and lateral roots. Proper establishment of rice root system is a key determinant of productivity. Plant hormones such as auxin and cytokinin have been shown to play critical role in during crown root (CR) development but the dynamic responses and interactions between these hormones have not been extensively studied during crown root differentiation. The broad aim of this thesis was to study responses of auxin and cytokinin signaling and explore deeper insight on the functional significance of auxin during rice crown root development. The study first focused on identifying auxin and cytokinin responsive domains during various stages of CR development. Auxin and cytokinin responsive synthetic promoter::reporter constructs, DR5::erYFP and TCSn::erGFP, were generated for monitoring auxin and cytokinin responses, respectively. Stable transgenic rice lines generated raised with these constructs were used to monitor hormonal responses in rice stem base containing developing CRs. Using RNA-RNA in situ hybridization and fluorescence analysis, we observed show that in the early crown root primordia, auxin response is abundant in the early root cap cells of the root tips whereas TCSn::erGFP signals were relatively broader in the root tips. Importantly, TCSn::erGFP signals were significantly less in the auxin responsive domains in the root tip. Auxin-cytokinin cross talk is well established during root development in plants. During CR development, we also identified some narrow domains with overlapping auxin and cytokinin responses that might be a domain where auxin and cytokinin signaling directly interact with each other. Next, in order to understand the functional importance of auxin in the auxin-cytokinin interaction domain, we genetically altered active pool of endogenous auxin in the cytokinin responsive domain by expressing auxin inactivating gene, OsMGH3 using TCSn promoter. We observed that reducing active auxin resulted pleiotropic abnormalities such as stunted plant growth, loss-of apical dominance and root architecture in the transgenic plants, indicating key role of auxin and cytokinin interaction during CR development. Further, to dissect out mechanism of auxin-regulated crown root formation, we selected two plant-specific AP2-domain containing specialized transcription factors (OsAP2/ERF-40 and OsAP2/PLT3) for functional studies using reverse genetics-based approaches. RNA in situ hybridization and RT-PCR analyses showed that both genes are specifically expressed in the developing crown root primordia. Interestingly, and the expression of OsAP2/ERF-40 was induced upon exogenous auxin treatment induced whereas OsAP2/PLT3 was not affected in the rice stem base upon auxin treatment. We show x that down-regulation and ectopic over-expression of OsAP2/ERF-40 in the transgenic rice display defects in the crown root development in the transgenic rice. These, together with the regulatory gene expression analysis revealed that OsAP2/ERF-40 is sufficient to induce adventitious root and to trigger the root developmental program in a dose dependent manner. OsAP2/PLT3 is a member of PLETHORA gene family, key regulators for stem cell specification and maintenance in the root apical meristem. High genetic redundancy in the PLT family compelled us to use mis-expression approach to decipher the function of OsAP2/PLT3. We observed that ectopic over-expression of OsAP2/PLT3 causes defects in root architecture, radial growth, leaf angle and plant fertility. Overall, our studies not only reveal specific and overlapping auxin and cytokinin response domains but also provide evidence for two novel transcription factors regulating adventitious/crown root developmental program, a key agronomically important quantitative trait. 2019-07-01T00:00:00Z http://localhost:8081/jspui/handle/123456789/15799 Title: ANTIBIOFILM ACTIVITIES AND PROTEOMIC ANALYSIS OF CANDIDA TROPICALIS IN RESPONSE TO TERPENES Authors: Chatrath, Apurva Abstract: Candida albicans and the emerging threats caused by non-albicans Candida species have started to show resistance which calls for identification of new alternatives or modification of the existing antifungal agents to defy the dominant tendency of infection. Presently, available anti-Candida drugs exhibit a high frequency of resistance, low specificity and toxicity at a higher dosage. In addition, the discovery of natural or synthetic anti-Candida drugs is slow-paced and often does not pass clinical trials. Among non-albicans Candida species, Candida tropicalis is frequently emerging fungal pathogen in the Asia-Pacific region, causing a high mortality rate due to candidiasis. The preponderance of C. tropicalis in clinically isolated strains is recorded as higher as 42.1%. Also, the resistance in isolated C. tropicalis strains towards fluconazole is observed to be 38.5%. This increased rate of fluconazole resistance and frequent isolation of C. tropicalis makes it a major concern. C. tropicalis is an opportunistic human pathogen with an ability to cause superficial as well as systemic infections in immunocompromised patients. The formation of biofilm by C. tropicalis can cause dreadful and persistent infections which are difficult to treat due to acquired resistance. Azole drugs including fluconazole are enormously used in several antifungal treatments. However, C. tropicalis isolates showing significant azole drug resistance attributed to the point mutations in cytochrome P-450 lanosterol 14-α-demethylase (Erg11p) protein. Therefore, alternative drugs against Erg11p are immensely required to combat the acquired resistance. Owing to the potential of essential oils in terms of their antimicrobial activities as well as traditional usage has emphasized their applicability in various fields ranging from agriculture to food technology and as natural alternatives alongside synthetic drugs. However, more clinical studies are required to confirm and verify the true potency of these natural substances for their safe applications in human health and the environment. Essential oils are found to be rich in terpenes which are known to have good antimicrobial properties along with their explicit aroma. Various terpenes have shown multiple antifungal activities against planktonic cells in Candida species. However, their effects on the biofilm formation and its eradication are still in infancy. Previous studies have revealed antifungal activities of these terpenes at multiple levels in different organisms. Therefore, the effect of antifungal agents could not be specified by studying it over one organism or even on established targets. More studies are required to be done in respect to finding probable genes, proteins and metabolites responsible for resistance against antifungal agents. Furthermore, many terpenes have been tried as antifungal agents against Candida species. These are found as a potent anti-Candida compound with a broad ii range of antimicrobial properties. Thus, the exploitation of antifungal and antibiofilm properties could serve as a bridge between traditional uses and rational utilization of essential oils and citral. In the present thesis entitled “Antibiofilm activities and proteomic analysis of Candida tropicalis in response to terpenes”, an extensive study has been done to elaborate on the effects of terpenes on C. tropicalis biofilm. Firstly, in vitro growth and development of C. tropicalis biofilm were recognized and antifungal activities of the seven terpenes, namely, geraniol, menthol, citral, cinnamaldehyde, carvacrol, eugenol and thymol were screened. They were further checked for the abilities of biofilm inhibition and eradication. The calculated MIC50, BIC50 and BEC50 values for citral and thymol were lesser than other terpenes, therefore, observed to be more effective against C. tropicalis. The morphological changes have also shown damaged surface in the presence of these terpenes. These results clearly indicated that among these terpenes; citral and thymol were significantly effective against both planktonic and biofilm forms of C. tropicalis, compared to others. Secondly, the comparative potentials of citral and thymol against C. tropicalis were explored. The administration of citral and thymol on C. tropicalis biofilm leads to a fungicidal effect. The relative fold change in the expression of certain key genes which are involved in major pathways followed by C. tropicalis, to reduce the effect of well-known drugs, has also been investigated to get the insights in the plausible mechanism for its survival in the presence of these two components. Citral and thymol have formed indentations in the cells depicting distorted surface and decreased viability. Also, the cell membrane and DNA damage were determined in C. tropicalis biofilm cells when treated with citral and thymol as antifungal agents. The cells membrane integrity was compromised and DNA damage was observed. Quantitative real-time PCR analysis showed augmented expression of the cell membrane biosynthesis genes including ERG11/ CYT450 against citral and the cell wall-related tolerance genes involving CNB1 against thymol thus, depicting their differential mode of actions. Next, the homology model of well-known azole drug target Erg11p of C. tropicalis was employed to unravel the interaction between citral and lanosterol-14-α-demethylase (Erg11p). The molecular interactions of two isomers of citral namely neral and geranial, with CtErg11p, were evaluated. The three-dimensional structure of CtErg11p was prepared through in silico structural modelling approach. The best-generated model was validated and further used for the molecular docking studies with citral. The molecular docking studies provide insights into citral-CtErg11p interactions, which could be helpful for further optimization and development of other inhibitory analogues of citral asserting the expansion of proficient broad-spectrum antifungals. The molecular interactions between citral, heme group and participating amino iii acid residues of CtErg11p protein have been identified that could lead to understanding the protein-ligand interaction. Furthermore, insights into the changes in the biofilm cell proteome and the extracellular matrix of C. tropicalis in the presence of citral were obtained. One-dimensional polyacrylamide gel electrophoresis (1D-PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF-MS) were employed to identify the changes in the protein expression of C. tropicalis in response to the sub-lethal concentration of citral. The results revealed citral-induced proteins of C. tropicalis biofilm. In 1D-PAGE, a total of six differential proteins, involved in oxidative stress, amino acid biosynthesis, heme biosynthesis and glucose metabolism pathways were detected. However, in 2D-PAGE, the differential expression of proteome has revealed a total of twenty-five proteins in C. tropicalis biofilm which were induced in the presence of citral. Among these, amino-acid biosynthesis (Met6p, Gln1p, Pha2p); nucleotide biosynthesis (Xpt1p); carbohydrate metabolism (Eno1p, Fba1p, Gpm1p); sterol biosynthesis (Mvd1p/ Erg19p, Hem13p); energy metabolism (Dnm1p, Coa1p, Ndk1p, Atp2p, Atp4p, Hts1p); oxidative-stress (Hda2p, Gre22p, Tsa1p, Pst2p, Sod2p); and biofilm specific (Adh1p, Ape1p, Gsp1p) proteins were identified. The overexpression of oxidative-stress related proteins indicates the response of biofilm cell in combating oxidative-stress during citral treatment. Moreover, the upregulation of Adh1 is of particular interest because it subsidises the biofilm inhibition through ethanol production as a cellular response. The augmented expression of Mvd1/ Erg19 signifies the effect of citral on ergosterol biosynthesis. The overall sugar and protein moieties were diminished in the treated extracellular matrix; however, ergosterol and cell wall content e.g. hexosamine were relatively increased. Therefore, it is clearly indicated that the cellular response towards citral acts through multifactorial processes. 2019-09-01T00:00:00Z http://localhost:8081/jspui/handle/123456789/15798 Title: IMPACT OF ALTERED EXTRACELLULAR MATRIX PROTEIN DISTRIBUTION ON IMMUNE CELL FUNCTION DURING INFLAMMATION Authors: Bhan, Chandra Abstract: Extracellular matrix proteins (ECM) form the structural support for the migration of immune cells and provides multiple signals to assist in the functions of immune cells during physiological and pathological conditions. Secretion of inflammatory mediators such as cytokine and MMP (expand) s could influence the expression and distribution of ECM in the inflamed tissues, which in turn could modify cellular functions. Previous reports have shown changes in the expression of ECM proteins during local inflammatory responses such as asthma, fibrosis, chronic obstructive pulmonary disease (COPD), etc. However, limited knowledge is available on the changes occurring in the expression and distribution of important ECM proteins in multiple tissues during systemic inflammation. One part of the doctoral work was focused on investigating, the expression profile of important ECM proteins in systemic inflammation using two systemic inflammation models, i.e, Lipopolysaccharide induced endotoxemia and Cecal Ligation and Puncture (CLP) induced polymicrobial sepsis were used having a distinct pattern of events which drives the systemic inflammations. Following LPS injection and CLP surgery, lung, liver, kidney & mesentery were isolated at 6 hr, 12 hr, 24 hr, and 36 hr time point. Using real-time PCR and western blot technique, expression of important ECM protein both at transcript and protein levels were measured. RT-PCR and Western blot analysis showed changes in the expression of various ECM proteins such as collagen 4, fibrinogen and vimentin. A unique expression pattern of these prominent ECM proteins was observed in both at the site of inflammation (mesentery) as well as in the visceral organs (Lung, liver & kidney) in both models, possibly due to the difference in inflammation induction pattern. Moreover, to confirm the importance of upregulated ECM protein on immune cell function and various inflammatory and functional signaling pathways, bioinformatics analysis with STRING software was performed to predict the important signaling pathways that get activated upon cell-matrix interaction such as activation, apoptosis, cytoskeleton modulation, integrin expression and migration during the inflammatory scenario both in murine and humans. Secondly, the work also focused on assessing the effect of Fibulin7 (Fbln7), one of the newly identified adhesion matrix protein on neutrophils. Fibulin7 was recently identified as members of the fibulin family of secreted glycoproteins and was found to be expressed in developing tooth, bone, cartilage as well as immune-privileged locations such as eye and ii placenta. Previous studies from our laboratory have shown that the Fbln7 full-length and its C terminal fragment (Fbln7C) have regulatory effects on human monocytes and macrophages but its effect on neutrophils is not known. Thus, experiments were designed to further understand the effects of Fbln7, specially, its C-terminal fragment-Fbln7C on the immunological functions of neutrophils which are key cell type of innate immune system and also play vital role in driving the pathological events such as multi organ failure in infection induced systemic inflammations. In vitro cell adhesion and inhibition assay showed that the neutrophils could bind to adhesion protein fbln7 via integrin β1and could compete with other ECM proteins such as fibronectin for binding to neutrophils. Significant reduction in ROS and inflammatory cytokine production (i.e. IL-6, IL-1β) was observed including reduction in Erk1⁄2 phosphorylation in neutrophils stimulated with LPS and fMLP in presence of Fbln7C compared to untreated controls. Furthermore, treatment of Fbln7-C in mice decreased the number of cells in both blood and peritoneum, as well as reduced the PMA induced ROS production from neutrophils isolated from peritoneum and lungs of endotoxemic group treated with Fbln7C compared to controls animals. In summary, data from the ECM expression during systemic inflammation and immunomodulatory role ofFbln7-C will help in better understanding of ECM-Cell dynamics in the inflammatory microenvironment and may contribute to the development of cell adhesion based therapeutics. 2019-10-01T00:00:00Z http://localhost:8081/jspui/handle/123456789/15797 Title: INVESTIGATING HUMAN LONG INTERSPERSED ELEMENT 1 (LINE 1) ACTIVITY IN HEALTH AND DISEASES Authors: Sur, Debpali Abstract: LINE 1 (L1), is the only active autonomous retrotransposon that propagate via RNA intermediate, comprising 20% of the human genome. The ongoing activity of L1 turned to be the major instrumental force that shapes the genomic architecture but whether the effect is detrimental or beneficial is still an enigma. Although most of the L1s are “molecular fossils” (due to truncation in 5’UTR), around 80-100 human specific L1 (L1Hs) are competent to retrotranspose. Normal somatic cells have evolved their own defensive mechanism to prevent L1 mobility. It is being postulated that L1 retro-transposition event have to occur in the germ-line or during early development in-order to ensure their evolutionary success; but with what extent this process impact somatic cells is yet to reveal. Strikingly, L1 shows high activity in brain encouraging the intriguing hypothesis of L1 mediated retrotransposition might generate neuronal genomic diversity. In recent years, expression of L1 encoded proteins is indicated as a hallmark feature for most of the cancers. Malignancy and ageing are commonly associated with accumulation of deleterious mutation leading to altered function within the cell. Here in, I tried to decipher somatic L1 retrotransposon activity in normal human brain and in oral squamous cell carcinoma (OSCC) samples. An active full-length L1 is ~6 kb in length encoding two proteins designated as ORF1p (40 kDa) and ORF2p (150 kDa) that are prime essential to facilitate its transposition. My first aim was to investigate the activity of L1 in different anatomical regions of normal human brain by looking the expression of L1ORF1p with an in-house human L1ORF1p (hL1ORF1p) antibody. I have characterized the hL1ORF1p antibody and my data showed that our in-house L1ORF1p antibody is very sensitive and specific towards detecting L1ORF1p and will be very useful to study retrotransposon biology in human brain and other tissues. Next, I employed immune histological staining (IHC) to check the expression of L1-ORF1p, in different anatomical regions of normal human brain. My data elucidated significant amount of L1ORF1p expression in different anatomical regions of postmortem normal human brain; old age brain showed more expression compared to the young age group. Further investigation was conducted to detect full length L1 RNA and L1ORF2p expression in the postmortem brain samples. Cumulatively my data showed that higher L1ORF1p Abstract ii expression in different anatomical regions of aged brain compared to younger one. Overall, my data indicates that L1 might have some role with aging and age related neurodegenerative diseases. In Indian population OSCC is prevalent and having panacea is challenging due to its high mortality rate and late diagnosis. Studies regarding L1 in OSCC are scanty; hence I have investigated the expression of L1 proteins (ORF1p and ORF2p) in small cohort of operated OSCC samples obtained from Surgical Oncology Department, AIIMS, Rishikesh, India. My data showed around 50% samples expressed L1 proteins (L1ORF1p and L1ORF2p). Further investigation was conducted to find out the methylation status of L1 promoter in paired normal cancer tissues. Overall my study revealed that L1 retrotransposon is active in this particular cancer in stage independent manner and thus L1 proteins (L1ORF1p and L1ORF2p) can be used as potential biomarker. The outline of Ph.D. thesis is divided into four chapters. A brief description of each chapter is mentioned below: Chapter 1: Introduction and literature review: In this chapter, rationale behind the study will be described. The three objectives will be elaborated and the link among objectives will be presented in details. A comprehensive and up-to-date literature review related to each objective will be narrated. The chapter will also include to the point description regarding retrotransposable elements (with special preference to LINE1 retrotransposon) and their propagation mechanism. Detailed description of the following will also be included in this chapter: Activity of LINE1 in neuronal genome and its capability to shape the neuronal genome architecture, regions of brain supporting L1 activity and its possible role in age related neurological disorder, upregulation of L1 activity in different cancer that induce genomic instability, status of oral squamous cell carcinoma in India, and activation of L1 in oral squamous cell carcinoma. Chapter 2: Materials and Methods: This section consists the detailed description about the recipes for reagent, solution and experimental protocols performed to answer all the objectives. The protocol which will be described in details are animal cell culture and transfection, over expression of proteins in bacterial system, immunization protocol for antibody generation, tissue processing, SDS- Abstract iii PAGE gel electrophoresis, Western blotting, Northern blotting, retrotransposoition assay, immunofluroscence, immunoprecipitation, immunohistochemistry and semi qRT-PCR. Chapter 3: Results: The chapter includes objective wise major finding of this study: The main objectives are 1. Characterization of in house generated human L1ORF1p antibody to detect L1 encoded ORF1p in different cancer cell lines 2. Detection of L1ORF1p in different anatomical regions of human brain 3. Investigating the expression of L1 proteins (ORF1p and ORF2p) in Oral squamous cell carcinoma 1. Characterization of inhouse generated human L1ORF1p antibody to detect L1 encoded ORF1p in different cancer cell lines i. Characterisation of αhL1ORF1 antibody by western blotting: Here western blotting was performed to characterize the hL1ORF1p antibody by looking its expression in different human and murine cancer cell line (MCF7, Hela, Du145, NIH3T3, and HEK293T). The dilution to be used to perform Western blotting was calibrated and data showed that the inhouse hORF1p antibody detects ORF1p as single bands with MW ~40 kDa in human cancer cell lines (HEK293T and MCF7). ii. Application of the generated antibody for immunofluroscence (IF): Here the IF technique was employed to see endogenous and exogenous expression of ORF1p in MCF7 and HeLa respectively. The Hela cells didn't not show any endogenous expression. In MCF7 cells ORF1p was detected in cytoplasmic fraction. iii. Validation of the antibody for Immunoprecipitation (IP): Next, I purified the antibody using affinity capture mechanism by protein A agarose and the purified antibody detected L1ORF1p in HEK 293T cell line at a concentration of 1:5000 dilution indicating that the antibody is more efficient than the commercially available antibodies against L1ORF1p. Thus my data depicts that the antibody can be used for immunoprecipitation of over expressed ORF1 protein in bacterial expression system. Abstract iv 2. Detection of L1 ORF1p in different anatomical regions of human brain i. Characterisation of generated antibody for Immunohistochemistry (IHC): The generated hL1ORF1p antibody was characterised and employed for immunohistochemistry. I took commercialized L1ORF1p antibody from Merk Millipore (Cat no: MABC1152) as a positive control. Primary anti-His and primary non-immune sera was used as negative control. The data revealed that inhouse L1ORF1p antibody can significantly detect L1 ORF1p at a concentration of 1:500 in brain as compared to the commercial antibody. ii. Detection of L1 encoded ORF1p in different human tissue: Next my focus was to elucidate the expression of L1ORF1p in different anatomical region of human brain. My data indicated that expression of L1ORF1p increases significantly in older brain as compared to young brain. Non brain somatic tissue (kidney, heart, liver and lungs) shows no reactivity to L1ORF1p. The expression of ORF1p was specifically in the neurons and the protein tends to localise in the neuronal nucleus with age. Comprehensively the data indicate expression of ORF1p increase with age and the expression is more in frontal cortex, hippocampus and basal ganglia than other region of the brain. iii. Detection of L1 transcription in human brain sample: To dissect the L1 transcription level we next employed Northern blotting to detect the amount of L1RNA transcript in old and young aged brain samples. The data depicted increased expression of L1RNA in old aged (80 years) samples as compared to young age (15years). Scarcity of fresh normal brain tissue was a great limitation for the study. Hence on the same samples we tried to check expression level of both ORF1 and ORF2 by semi quantitative PCR. Together the data exhibited enhanced expression of L1 ORF1 and ORF2 in the old age sample suggesting lower transcription of L1 in younger age. Further to validate the data we performed western blotting to detect the L1ORF1p in old aged brain sample and as expected we got distinct band at 42 kDa for L1ORF1p. Abstract v 3. Investigating the expression of L1 proteins (ORF1p and ORF2p) in Oral squamous cell carcinoma i. Sample collection, Block preparation and histology: The operated OSCC samples were collected from surgical oncology department of AIIMS, Rishikesh following institutional ethical clearance and proper consent from the patient’s family. We focused mainly on samples having previous habit of tobacco addictions. Upon collection of the samples fresh tissue were further stored in RNA later solution in -200C and formalin fixed tissues were used to make paraffin blocks. In this section detailed study indicating altered histology during cancer progression is also elaborated. ii. Validation of αhL1ORF1 and αhL1ORF2 by immunohistochemistry in collected OSCC samples. In this section the antibody concentration for L1ORF1p and L1ORF2p was optimised for performing IHC in cancer tissues. The positive and negative controls were also established to screen OSCC samples. iii. Detection of L1 encoded ORF1p and ORF2p in OSCC samples: On a small cohort of 27 samples, the expression of both the L1 proteins (ORF1p and ORF2p) was investigated. Data revealed more than 50 % of the samples were positive for both the proteins. We took Ki67 and CK19 as cellular proliferation marker and squamous cell carcinoma marker respectively. We divided the pattern of expression in three different group i.e high, moderate and low. The OSCC samples showing high expression of L1 proteins was further used for methylation studies to check activation of L1 5’UTR. Our data indicate that expression of L1 proteins are elevated in OSCC that might be used as a marker protein. iv. Patho-clinical significance of ORF1p and ORF2p This section we will describing the stage wise expression of both the L1 proteins. For a deeper insight we tried to correlate the expression of L1 with the TNM and grade of squamous cell carcinoma. Samples showing higher expressions are well differentiated carcinoma but distinct stage dependent variation was not found. The percentage of expression throughout all the samples screened was quite significant but in a stage independent manner. Expression in early stage OSCC indicates that both the L1 proteins could be exploited as a potential biomarker. Abstract vi Chapter 4: Discussion This chapter includes the discussion part of the thesis which concludes the inferences obtained from the results. Further conclusion and future prospective of the work has been discussed. Overview of the drawn conclusion is as follows: 1. The in house generated novel αhL1ORF1p antibody can be used for western blotting, immunofluorescence, and immunohistochemistry. 2. L1 ORF1p is expressed in a significant amount in different anatomical region of the brain. 3. Frontal cortex among the other entire brain exhibited robust expression. 4. Expression of L1ORF1p and L1 RNA increases as age progress in human. 5. L1 encoded ORF1p and ORF2p are overexpressed in oral squamous cell carcinoma in a stage independent manner. Both the proteins ORF1p and ORF2p can be exploited as OSCC biomarker. 2019-12-01T00:00:00Z