PhD Theses (Biosciences and Bioengineering)

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(A) Study on the Role of Lipogenic Enzymes in Oral cancer
(2022) Devi, Khwairakpam Amrita
Oral cancer is the sixth most prevalent cancer of the world and is the second highest cancer type in India. The increase rise in the consumption both smoking and smokeless tobacco is one of the major reasons behind the rise in oral cancer cases in India. Moreover, in spite of the different therapies available such as surgery, chemotherapy and radiotherapy, there are always problems associated with it such as chemoresistance, side effects and tumor recurrence. Several studies have shown that ATP citrate lyase, the first enzyme involved in the first step of de novo lipogenesis (accountable for the production of oxaloacetate and acetyl-CoA in the cytosol) played an important role in the tumorigenesis of various types of cancer. ACLY is also known to form crossroads between glucose metabolism and fatty acid synthesis pathway. It has been evidenced that upregulated expression of ACLY enhanced tumor growth and proliferation in different cancers such as breast cancer, brain cancer, bladder cancer, lung cancer, prostate cancer and ovarian cancer. However, the role of ACLY is still unknown in oral cancer. Therefore, understanding the role of ACLY in oral carcinogenesis might prove significant for the early detection and prevention of oral malignancy. Here, in our study an attempt has been made to investigate the role of ACLY in oral cancer. In a study conducted in the cbioportal of cancer genomics, we found that alterations in ACLY gene of patients with HNSCC had a lesser median month survival rate as compared to patients without alterations indicating that ACLY might play an important role in the development and progression of oral cancer. We observed upregulated expression of ACLY both in the mRNA as well as the protein level of ACLY in oral cancer cells as compared to normal cells suggesting its important role in the tumorigenesis of oral cancer. IHC analysis using tissue micro array slides showed upregulation in the expression of both ACLY and p-ACLY in oral cancer tissues as compared to the normal tissues of the oral cavity. Several studies have also shown increased expression of ACLY in various types of cancer. This is the first report showing ACLY is upregulated ‘in oral cancer. In addition, tobacco and its components also enhanced the expression of ACLY thereby leading to the progression of tumorigenesis in oral cancer. The IHC analysis using tissue microarray slide also showed that the increase expression of ACLY is correlated with the different stages of development of oral cancer. Moreover, increased expression of the protein was also observed with the increase in stages and grades of oral cancer as compared to normal. Further, knockout of ACLY using CRISPR/Cas 9 system of gene editing in oral cancer cells inhibited the survival, proliferation and migration of the tumor cells. Moreover, downregulation in the expression of the important signalling molecules involved in the cancer metabolism such as phospho-S6, Akt1, Akt2, mTOR was observed in ACLY knockout HSC-3 cells as compared to the scrambled control suggesting the involvement of Akt/mTOR/s6 pathway. Therefore, further in vivo studies are requisite to confirm and strengthen our findings and provide valuable information thereby validating the effect of ACLY in the development and progression of oral cancer.
Ammonium rich wastewater treatment and value addition using microalgae-bacterial consortia in photo-activated systems.
(2021) Arun, S
The conventional activated sludge process (ASP) for ammonium removal relies on mechanical aeration. In addition to very high operating cost due to mechanical aeration, ASP requires addition of external carbon source for nitrification and denitrification. On the other hand, microalgae-bacteria consortia are not only able to grow autotrophically on ammonium but also offer advantages, such as high N affinity, anoxic zone for denitrification, algal photosynthesis, shortcut nitrogen removal, biomass with good settling characteristics and energy production. Hence, this work was aimed at treatment and value addition to ammonium rich wastewater by using microalgae-bacterial consortia. The microalgae-bacterial biomass was initially studied for its ability to remove ammonium at a high concentration of 200 mg/L. Addition of organic carbon source in a photosequencing batch reactor (PSBR) during the dark period enhanced the activity of denitrifying bacteria (DNB) in the microalgae-bacterial consortium, which resulted in nitrogen as the main end product. Detailed bio-kinetics of ammonium removal by the microalgae-bacterial consortium revealed an active role played by microalgae, ammonium oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and DNB for achieving an efficient removal of ammonium. Both the ammonium and nitrogen gas production were best described by using the microalgae-AOB-methanol utilizing denitrifier (MUD) based bio-kinetic models. Also, the profiles of ammonium, nitrite, nitrate and DO in this study were accurately predicted by the microalgae-AOB-MUD bio-kinetic models. Estimated values of the bio-kinetic model parameters further supported the shortcut nitrogen removal without nitrate formation by microalgae, AOB and DNB in the consortium. Light intensity significantly affected the ammonium removal by microalgae-AOB-NOB consortium. Empirical model as mentioned earlier showed that light intensity below 40 μmol photons m−2 s−1 was ineffective towards nitrification due to oxygen limitation condition. Light intensity in the range 40-160 μmol photons m−2 s−1 was found suitable for complete nitrification, whereas a light intensity above 100 μmol photons m−2 s−1 caused inhibition of microalgal-AOB-NOB consortium, thereby resulting in a low nitrification efficiency. The effect of different nitrogen source competition using microalgae-bacterial consortia on ammonium removal was further studied and the NH4 + removal rate values were similar at 50 and 100 mg NH4+-N/L respectively, indicating enhancement in ammonium removal by both nitrification and microalgae uptake. By comparing the results from this work with a previous study, O2 supplied by microalgae through NH4 + uptake was found to strongly influence the overall NH4 + removal by consortium. Finally, ammonium removal by algae-AOB-DNB consortium was integrated in a microbial fuel cell (MFC) and the system was referred as an integrated shortcut nitrogen removal-microbial fuel cell. This study provided direct evidence of shortcut nitrogen removal in membrane photosynthetic microbial fuel cell (MPMFC) by integrating nitritation into the cathodic compartment, which demonstrated a very good ability to overcome issues related to high aeration cost and NOB activity.
(An) Investigation of the Role of Lipocalin Receptor in the Development of Lung Cancer
(2021) Sailo, Bethsebie Lalduhsaki
Lung cancer is the most prevalent malignancy globally. Tobacco smoking in all forms is the predominant risk factor for lung carcinogenesis. This aggressive disease has a very low overall 5-year survival rate with approximately 15% in developed countries and 5% in developing countries which can be attributed to its late-stage diagnosis due to lack of suitable biomarkers and efficacious therapeutic strategies. Increasing lines of evidence suggested that neutrophil gelatinase-associated lipocalin receptor (NGALR) is strongly implicated in the development and progression of various human malignancies including glioma, colon, oesophagus, endometrial, liver cancer, etc. However, the expression and role of NGALR in the pathogenesis of lung cancer have not been elucidated thus far. Interestingly, our study showed for the first time that NGALR is significantly upregulated in lung cancer tissues compared to the normal adjacent tissues. It was also found to be upregulated in different stages and grades of lung cancer tissues, thus indicating its involvement in the positive regulation of lung carcinogenesis. In addition, this is the first report to show that knockout of NGALR decreased the proliferation, survival, and migration of lung cancer cells via downregulation of a wide range of intracellular signals including Akt/mTOR, NF-κB, JAK/STAT-3 and EGFR/MAPK signaling pathways. Furthermore, this lipocalin receptor was also found to be involved in the positive regulation of NNK (nicotine-derived nitrosamine ketone), tumor necrosis factor (TNF)-α and TNF-β induced proliferation, survival and migration of lung cancer cells through modulation of proteins involved in different cellular processes. This is the first study that shows the invaluable role of NGALR in the pathogenesis of lung cancer along with its involvement in tobacco as well as TNF-mediated lung carcinogenesis. Our findings not only increase the understanding of this important lipocalin receptor but also provide a potential therapeutic target for developing novel and efficacious therapeutic interventions for lung cancer patients.
(An) Investigation on the role of tumor necrosis factor-α induced protein 8 (TNFAIP8) family in the development and progression of oral cancer
(2019) Padmavathi, G
Cancers of oral cavity is emerging as a major health concern worldwide with highest incidence in India. Despite the huge medical advancements in cancer therapy and the easy accessibility of tumor, the 5-year survival rate of oral cancer remains as low as 50–55% and with increase in disease progression survival rate tend to decrease drastically. Unfortunately, in India, almost 2/3 of the oral cancer cases are diagnosed only in the advanced stage of the disease making it difficult to treat and decreasing the chances of survival. The remarkable decrease in the survival of advanced disease is mainly attributable to the lack of effective diagnostic, therapeutic and prognostic biomarkers. Therefore, it is essential to develop novel biomarkers for the better management of this deadly disease. In the current study, a novel tumor necrosis factor alpha induced protein 8 (TNFAIP8 or TIPE) protein family comprising of four proteins namely, TNFAIP8 (TIPE), TNFAIP8L1 (TIPE1), TNFAIP8L2 (TIPE2) and TNFAIP8L3 (TIPE3) was explored for its therapeutic and prognostic potential against oral cancer. Immunohistochemical analysis of tissue micro array (TMA) containing samples from normal, hyperplasic and neoplastic patients revealed that expression of TIPE, TIPE2 and TIPE3 were upregulated and levels of TIPE1 were downregulated in squamous cell carcinoma (SCC) tissues compared to the normal tissues and correlated with disease progression from hyperplasia to SCC. Further assuring the involvement of TIPE proteins in the development and progression of oral cancer, treatment of oral cancer cells with the major risk factor of oral cancer (i.e. tobacco and related carcinogens) resulted in a significant upregulation of TIPE, TIPE2 and TIPE3 and downregulation of TIPE1 protein. Furthermore, knockout of TIPE proteins by CRISPR/Cas9-mediated gene editing was found to significantly modulate the different cancer hallmarks associated with oral cancer. In brief, silencing of TIPE or TIPE2 or TIPE3 significantly reduced the survival, proliferation, colony formation and migration of oral tongue squamous carcinoma cells (SAS) whereas knockout of TIPE1 had an opposite effect on OSCC cells. Further, the TIPE, TIPE2 and TIPE3 knockouts mediated inhibition of proliferation was found to be facilitated through inhibition of cell cycle progression at S or G2/M phases and downregulation of proteins involved in cell survival, proliferation, migration, invasion and angiogenesis such as Cox-2, survivin, Bcl-2, cIAP1, XIAP, CXCR4, MMP-9 and VEGF-A.
Analysis of genes encoding outer membrane protein of leptospira interrogans and Its modulation due to host factors: an approach for understanding host-pathogen crosstalk
(2019) Ghosh, Karukriti Kaushik
The pathogens like Leptospira interrogans upon infection to human or animals and to sustain itself in the diverse host defense system have evolved sensor mechanisms to discern different host factors such as catecholamines (stress hormones), temperature, osmolarity, oxidative stress, etc. These pathogens exploit these host factors as an environmental cue to alter their growth and virulence for its survivability. The leptospiral outer membrane proteins are thought to be important for understanding the host-pathogen interactions due to their location on the bacterial surface. The present study aimed at understanding the function of genes in L. interrogans predicted to encode membrane proteins and is experimentally modulated on exposure to host factors under in vitro condition. Such membrane proteins are considered to be an interface for the host and pathogen interaction and may be critical in the leptospiral pathogenesis. The molecular characterization of selective genes (LA0616, LA3961, LB186, LB047, LA3307, LB191, and LA3263) encoding hypothetical membrane proteins and that are modulated by host factors revealed that these are outer membrane adhesins and can be implemented as novel serodiagnostic candidates for the diagnosis of leptospirosis across a wide range of hosts. This research work elucidates the possible means of leptospiral-host crosstalk after mimicking host environment under in vitro condition. In this study, by mimicking a single host factor environment, under the given in vitro growth condition, we could identify novel antigen markers and predict its possible role during the Leptospira natural infection. As a future perspective, the intervention of the function of identified proteins essential for host-pathogen crosstalk during leptospirosis may aid in treatment and reduction of Leptospira virulence.
Analyzing Charge Transfer Spectra arising from non-aromatic amino acids in proteins, aggregating peptides and viral capsid assemblies
(2023) Alom, Shah Ekramul
Novel intrinsic chromophores in apo-proteins lacking aromatic amino acids have gained importance and have been a recent topic of interest. Here in this thesis, Protein Charge Transfer Spectra (ProCharTS), which is proposed to be the origin of such intrinsic absorbance and luminescence features, is being studied. Although the major contribution of Lysine towards ProCharTS phenomenon has been ascertained in highly charge rich proteins. Here we report for the first time, ProCharTS in proteins and peptides rich in Arginine, Aspartate residues, but lacking Lysine residues. Similar absorbance and luminescence properties as that in Lysine-rich protein were observed. ProCharTS can be expected to be evident as long as charge transfer takes place, irrespective of the type of charged species. Since ProCharTS and conventional chromophores like Tryptophan share a similar spectral domain, the influence of ProCharTS on the indole fluorescence in Tryptophan was studied and was found to contaminate the indole fluorescence. Moreover, the decay kinetics from the excited state population in the indole ring was found to be affected by the presence of charged residues, which may explain the multi-exponentiality of Tryptophan fluorescence intensity decay often observed in proteins. Finally, the applicability of ProCharTS on peptide aggregation and viral capsid assembly was studied. ProCharTS was able to monitor the early stages of aggregation in Aβ-derived switch peptides, where the random coil peptides interconvert into β-sheets. Similarly, ProCharTS could detect the formation of the large clusters of HBV core protein dimers into capsids with T=4 and T=3 icosahedral geometry in real-time. The increased interactions among the charged residues at close proximity in the peptide oligomers or HBV capsids are proposed to enhance ProCharTS signal. ProCharTS, being a simple, label-free technique can thus be used for rapid initial screening of drugs for amyloid-linked diseases and viral core protein allosteric modulators (CpAMs), without interfering with the aggregation or capsid assembly kinetics.
Antibacterial Activity and Potential Therapeutic Applications of Pyridine-based Synthetic Amphiphiles
(2015) Goswami, Sudeep
The present study on pyridine-based synthetic ampliphiles argues well in addressing a very important and contempoaray global healthcare problem. The study is an illustration of the use of prudent synthetic drug design and medicinal chemistry to generate synthetic ampliphiles with high bacterial activity and therapeutic potential. The significant leads, which emerged from the study and the future prospect of the findings is discussed...
Antidiabetic Evaluation of Medicinal Plants Dillenia indica, Solanum indicum and Solanum torvum from North East Region of India
(2017) Gadewar, Manoj
We reported for the first time antidiabetic activity of medicinal plants, Dillenia indica, Solanum indicum and Solanum torvum on normoglycaemic and STZ-induced Wistar rats through the estimation of serum glucose level in the presence of the plant extract and compared with the hypoglycaemic effect of standard oral antidiabetic agent, glibenclamide. Phytochemical analysis of the three plant species were carried out to determine the presence of alkaloids, glycosides, saponins etc and involvement of these phytoconstituents in their pharmacological activities. The acute toxicity studies and in vitro cytotoxicity studies were carried out to establish the safety profile of the plant species. Furthermore, the antioxidant activities of the plant species were measured on the basis of the scavenging ability of stable DPPH free radical so as to determine their radical scavenging effects and their relationship of these effects to the pharmacological effects of plant species. Biochemical analyses of the plant extracts were also done to determine their effects on various biomarker enzymes, serum triglycerides and cholesterol level and their involvement in the pharmacological activities of the plant species. Here, we reported for the first time antidiabetic activity of methanolic fruit extract of Dillenia indica, Solanum indicum and leaf extract of Solanum torvum. The plant species have radical scavenging effects which may be important in the reducing oxidative stress associated with diabetes. The plant species caused significant reduction in the serum triglycerides and low density lipoprotein cholesterol which are the risk factors in the pathology of diabetes. The methanolic fruit extract of Dillenia indica, Solanum indicum and leaf extract of Solanum torvum have been shown to contain many bioactive constituents including phenolics, flavonoids, alkaloids, glycosides and saponins which have been linked to free radical scavenging and antidiabetic effect of the plant species. The plant species have also been shown to lower the elevated level of hepatic biomarker enzymes such as aspartate aminotransferase and alanine aminotransferase, suggesting their protective role in the hepatic damage caused by the action of STZ.
Antimicrobial peptides from membrane-interacting stretches of bacterial proteins
(2019) Saikia, Karabi
In the present day scenario, tackling multi-drug resistant organisms has become quite challenging. Escalating rate of resistance and slow discovery of antibiotics has brought us back to pre-antibiotic period. Therefore, it has become utmost important to design new antimicrobials that can efficiently kill microbes and has less chances of acquiring resistance. Antimicrobial peptides (AMPs) hold promise to be one such molecules that can ward-off a broad range of invading microrganisms without instigating any toxic effect to the host cells. AMPs possess distinctive characteristics such as amphipathicity and cationicity, which helps them to interact with the microbial membrane. We explored the shorter peptide fragments from membrane-binding stretches of bacterial proteins. Studies were conducted on peptides derived from E. coli membrane binding protein MreB, and FtsA. In addition to that, we investigated the activity of another shorter peptide LCI22-47 from the C-terminal region of B. subtilis AMP LCI. All the peptides were found to be very effective against both Gram-positive bacteria, Gram-negative bacteria, M. smegmatis, and fungus as well. Moreover, membrane binding studies have also shown peptides ability to interact and permeabilize the bacterial outer and inner membranes. Majority of the peptides retained their activity in the presence of salts and divalent cations without instigating any toxicity to mammalian erythrocytes. Therefore, development of membrane-perturbing AMPs from the membrane-binding stretches of microbial proteins, could be an excellent strategy to combat microbes.
Aptamers for breast cancer protein markers
(2016) Sett, Arghya
In the present thesis, specific DNA aptamers were screened against breast cancer protein biomarkers Her2 and ER alpha. The aptamers were screened by in-vitro SELEX process from a pool of 1014 number of oligonucleotides. The sensitivity of the candidates were evaluated by isothermal calorimetry and other in-vitro binding studies. To assess the biocompatibility of the selected aptamers, MTT assay was performed in marker positive and negative cancer cell lines and normal cell lines also. Specificity of the aptamers toward the marker positive cell lines were tested by flow cytometry technique. Immunocytochemistry of ER_Apt1 and ECD_Apt1 candidates suggest the specific binding of screened aptamers to ER alpha and Her2 positive cell lines respectively. Immunohistochemistry data with biotinylated aptamers showed the specific staining of breast cancer tissue specimens with negligible cross reactivity. Cellular and histochemical studies revealed that these DNA aptamers could be used as a theranostic agent for Her2 and ER positive carcinomas and could provide a novel cost effective alternate to conventional antibody in solid and solution based immunoassays for cancer diagnosis and related applications. Furthermore, these aptamers were modified with methylene blue at one end and thiol at another end to suite them for development of aptamer based sensor applications. The aptamers were immobilized on gold electrode surfaces and various electrochemical measurements (CV, SWV) were recorded with and without the protein biomarkers in solution phase by a standard 3-electrode system. The characteristic peaks indicate that, the aptamer based electrochemical sensors can detect the analytes efficiently. Thus, this study explores the potential of aptamer based biosensors for detection and prognosis of marker positive breast carcinoma and related disorders.
Artificial Scaffolds for Neural Tissue Engineering
(2015) Das, Suradip
The present study demonstrates the utility of nanocomposite based scaffolds as potential biomaterials for fabricating nerve conduits. Although silk fibroin is one of the most used biomaterial as an implant (sutures), its highly insulating properties render it unsuitable for fabricating scaffolds for mimicking the electrically conductive tissues of the body like nerve and cardiac muscles. We hereby demonstrate how silk based nanocomposites consisting of metallic nanoparticles and conducting polymer enhances functional and morphological regeneration of nerve in a rat sciatic nerve injury model. The novel sheet rolling method utilized for fabricating conduits from nanofibers enables multiple conduit development of varied dimensions from a single electrospun sheet. Pre-seeding the conduits with Schwann cells also enhanced and accelerated neural regeneration through the nerve gap as evident from the extensive electrophysiological and histological studies. Further the long duration studies undertaken herein establish the safety and neuro-regenerative potential of such nanocomposite conduits.
Attaining protein thermostability : A rationalized approach
(2016) Chakravorty, Debamitra
In proteins natural mutation occurs such that it tries to maintain maximum stability while retaining functionality indicating that nature may be rationalizing the way of attaining such mutations. Thermophilic proteins are examples of natural mutations that lead to stability of proteins at extreme of temperatures. Chapter I of the dissertation work unravels that the mechanism of thermostability has been attributed to the cumulative effect of numerous factors. However a guided approach to attain thermostabilizing mutations was still elusive. The intention of this work was to fill this caveat of attaining thermostabilizing mutations, by developing a rational approach of predicting thermostabilizing mutations. Chapter II of the thesis is related to data collection and creation of a database for thermostable proteins which can be accessed through www.extreme-stabledb.in. Feature collection and analysis was accomplished and it was observed that majority of the thermostable proteins were hydrolases. In Chapter III, therefore, thermostable lipases which are hydrolases were analysed for the features that render them thermostable. For the first time it was observed that -turn increases not only in thermostable lipases but in all thermostable proteins also. It was also realized that structural features were more important in understanding thermostability. Chapter IV deals with prioritizing these features according to their role in contributing towards thermostability and a model has been generated which can predict multiple mutations leading to thermostability. The ranking was developed based on an elaborate analysis of a set of 17 quantitative structural feat ures on a final dataset of 127 pairs of thermostable and mesostable protein structures. Ionic interaction and main-chain to main-chain hydrogen bonds were the features showing the highest priority vectors for thermostability.
Bacillus subtilis as an expression host for the production of glutaminase free recombinant L-asparaginase II
(2017) Chityala, Sushma
The bacterial L-asparaginase has been widely used as a therapeutic agent in the treatment of ALL (acute lymphoblastic leukemia). Moreover, it is used in food industry for the production of acrylamide free starchy/baked foods, L-asparagine biosensor for diagnosis of leukemia and as a model enzyme for the development of new drug delivery systems. The various side effects of L-asparaginases are mainly due to the presence of partial glutaminase activity. Hence, we made an attempt to produce glutaminase-free L-asparaginase II, which is highly desirable for its successful application.The gene encoding glutaminase-free L-asparaginase II (ans B2) from Pectobacterium carotovorum MTCC 1428 was cloned into pHT43 vector and transformed in Bacillus subtilis WB800N. It was further optimized to maximize the expression levels of recombinant enzyme (rL-asp II). A three-fold higher enzyme production was observed with an efficient transformant as compared to native strain. Enzyme localization studies revealed that > 90 % of recombinant enzyme is secreted extracellularly. The expression of recombinant L-asparaginase II was confirmed by SDS-PAGE, IMAC (Immobilized metal ion affinity chromatography) purification followed by Western blotting. Process parameter optimization with OFAT (one factor at a time) revealed that an agitation (120 rpm), temperature (37 ºC), Isopropyl b-D-1-thiogalactopyranoside (IPTG) concentration (1 mM) and time of induction (at 0.8 OD600nm) plays a vital role in achieving a maximum of 55 IU/ml. Furthermore, consecutive induction by IPTG improved the enzyme production up to 105 IU/ml of protein.
Bacillus subtilis: a microbial cell factory for human interferon gamma production and process development
(2019) Kumar, Nitin
The B. subtilis WB800N microbial cell factory was tackled at three levels for enhanced extracellular human interferon gamma production namely: 1) genetic level 2) substrate, medium components and extracellular cultivation environment level and 3) process operation level. The genetic level modulation of the B. subtilis WB800N physiology was performed by designing three human interferon gamma genes (IFNγ) with different optimum codon usage, RNA free energy and secondary structures. At substrate level complex and completely defined medium were assessed for modulation of the B. subtilis physiology along with various carbon and nitrogen sources by applying metabolic modelling and the stoichiometric modelling approaches for higher IFNγ production. Various amino acids were screened for their positive enhancing effects on IFNγ production using stoichiometric modelling and demand. The concentration level of carbon and nitrogen sources along with other medium components were optimized using statistical Design of Experiments (DoE) approach and machine learning based artificial neural network (ANN) based evolutionary programing using genetic (GA) and simulated annealing (SA) algorithms. At process operation level, the batch process was established with physical and environmental condition optimization of the B. subtilis WB800N culture for an antifoaming agent, optimum dissolved oxygen mass transfer coefficient and agitation rate. Fed-batch mode of operation was established with organic acid and amino acid feeding for positive stimulatory effect on IFNγ production. The high cell density cultivation of the B. subtilis WB800N with glycerol-amino acid-organic acid feeding was established which resulted in further enhancement in IFNγ production.
Bactericidal potency and extended plasma half-life of stereochemically diversified polypeptide constructs
(2018) Hazam, Prakash Kishore
Conventional peptide design only guarantees functional group constitution by opting specific amino acid sequence, and not their spatial orientation, because peptide molecules are very fluxional and its conformation is subjected to external flux. This is principally due to the isotactic stereochemistry of the peptide chain. Stereo-chemical engineering of peptide main chain offers expansion of design space of a typical sequence, possibly offering greater conformational rigidity by avoiding the ‘roughness’ of the folding funnel. In this thesis, we experiment with this conceptual possibility by designing amphipathic peptide systems of diversified stereochemistry, which can potentially act as bactericidal agents. We have designed three series of peptides, stepwise investigating the possibilities of such an enquiry, eventually resulting in at least half a dozen peptide molecules, qualified for future development as therapeutic agents. The principal objective of our first series of peptides was to verify, whether we can use the gramicidin helix as a template for AMP design. Gramicidin is a class of penta-decapeptides isolated from soil bacteria Bacillus brevis, but their utility as an antibiotic was limited to topical use due to high levels of hemo-toxicity. Activity profiles of the four de novo designed peptide variants designed in the first phase, show better efficiency in treating Gram-positive bacteria than Gram-negative variants. Significantly, our hemolytic assay confirms very low levels of hemo-toxicity for the re-designed peptides, unlike gramicidin. In the next series of peptides in the second phase, we put to test, this conceptual possibility already established in theoretical models, by designing amphipathic peptide systems and experimenting them on Gram-positive, Gram-negative and antibiotic-resistant bacteria. The unusual conformational rigidity and stability of syndiotactic peptides enable them to retain the designed electrostatic environment, while they encounter membrane surface. All the six designed systems exhibited bactericidal activity, pointing to the utility and specificity of stereo-engineered peptide systems for therapeutic applications. This phase of the work provided us important insights and useful directives in designing novel peptide systems with antimicrobial activity, by expanding the design space, incorporating D-amino acid as an additional design variable. We employed this knowledge-base in the design of next series of eight peptides, with varied electrostatic fingerprints. The phenomenal levels of anti-bacterial potency exhibited by half a dozen peptides in this series, high on specificity and less on toxicity, qualify them for next level of development as an effective therapeutic agent.
Bactericidal potential of a dual-target synthetic amphiphile
(2017) Thiyagarajan, D
The growing menace of drug-resistant pathogenic bacteria underscores the need to develop efficient bactericidal agents that can act on irrefutable targets and defy the resistance mechanism. In this context, the present investigation reports the bactericidal activity and therapeutic potential of a rationally designed dual-target pyridiniumbased synthetic amphiphile. The amphiphile referred to as C1 consisted of (i) a cationic pyridinium head group to initiate electrostatic interactions with anionic bacterial cells and DNA, (ii) a hydrophobic tail (12 carbon chain length) for membrane insertion and (iii) a fluorogenic pyrene group to facilitate intercalation with DNA and spectroscopic probing of interactions. Antibacterial screening experiments indicated that C1 exhibited broad-spectrum bactericidal activity, while the presence of a fluorogenic pyrene in C1 enabled probing of amphiphile-bacteria interactions and membrane-insertion. Florescence-based assays in conjunction with spectroscopic and molecular techniques demonstrated the membrane-directed bactericidal activity, intracellular transit, cellular DNA binding and intracellular plasmid DNA cleavage activity of C1. The bactericidal activity of C1 was retained in simulated gastric fluid (SGF), simulated intestinal fluid (SIF) and simulated body fluid (SBF). In a combinatorial regime, C1 rendered a substantial reduction of the minimal inhibitory concentration (MIC) of therapeutic antibiotics gentamicin and erythromycin against the target bacteria. An albumin-based nanocarrier loaded with C1 (C1-HNC) rendered facile release of the payload in S. aureus MTCC 96 biofilm matrix, which could subsequently cleave the extracellular DNA (eDNA) barrier and target the underlying cells resulting in dramatic annihilation of biofilm. Interestingly, C1-HNC could eradicate S. aureus biofilm from the surface of a model catheter and was non-toxic to HEK 293 cells in an in vitro assay. A C1-loaded Poly (lactic-co-glycolic acid) (PLGA) nanocarrier (C1-PNC) was developed, which could render significant reduction of the minimum biofilm eradication concentration (MBEC90) of gentamicin and ciprofloxacin against a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA).
Bacteriocin, antioxidant and novel glucan production from probiotic Lactobacillus plantarum DM5 isolated from Marcha of Sikkim
(2014) Das, Deeplina
Abstract not available
Biochemical and system biology approach to characterize novel fresh water microalgal isolate directed towards biodiesel
(2015) Muthusiv, Aramapandian M
Microalgae have gained significant interest as one of the most promising alternative and renewable sources for biodiesel production attributed to their intrinsic ability to accumulate large amounts of neutral lipids...
Biochemical characterization Transformation, Mass Production and Formulation of Beauveria Bassiana and Metarhizium Anisooliae Isolates
(2011) Dhar, Priyanka
The overall objective of the work done in this dissertation was to investigate thirty one isolates of entomopathogenic fungi, including 17 B. bassiana and 14 M. anisopliae isolates for their biochemical characteristics at the enzyme and molecular level and transformation, mass production and formulation aspects were studied as well. The first aspect of the thesis describes the biochemical characterization of the thirty one isolates and selection of isolates based on virulence candidate enzymes. The extracellular enzyme activity of cuticle hydrolyzing enzymes such as protease, chitinase and lipase was investigated. The induction and repression mechanisms of these enzymes were studied using different medium constituents. Pr1 activity was induced by supplemented protein in the media whereas chitinase activity was repressed by glucose. In-Gel enzyme activity was studied and a predominant chitinase of 58 & 14.3 kDa was observed in activity gels for B. bassiana and M. anisopliae isolates respectively whereas a 97 and 66 kDa protease was frequent for both B. bassiana and M. anisopliae. Two isolates were finally screened out (B. bassiana (UB9) and M. anisopliae (UM10) isolates) and used for further studies. The protease and chitinase enzymes from these two isolates were partially purified and characterized. The purified fraction was characterized on the basis of temperature, pH and effect of inhibitors. Molecular weight of the enzymes was detected on SDS-PAGE. A conventional 33 kDa chitinase was purified from B. bassiana and 23 kDa chitinase was purified from M. anisopliae. 47 & 43 kDa protease was purified from B. bassiana and M. anisopliae respectively. The genes of virulence determinant enzymes viz. protease and chitinase were studied as well. Chitinase and protease specific primers (two primer pairs for each gene and each isolate) were designed based on the conserved sequences. PCR amplified products were sequenced (Xcelris Labs) and sequences were analyzed by BLAST (BAST N & BLAST X) and conserved domains in the amplified sequence were detected by CD finder. 351 & 312 bp and 434 & 438 bp amplified fragments were observed for B.bassiana and M. anisopliae chitinase gene respectively. A 504 & 517 bp and 535 & 551 bp amplified fragments were observed for B. bassiana and M. anisopliae protease gene respectively. Sequence alignments showed conserved sequences are present in the amplifications. Pathogenicity potential of B. bassiana (UB9) and M. anisopliae (UM10) isolates against cottonbollworm Helicoverpa armigera was investigated. Bioassay was performed using Diet Surface Technique and 5 replicates were taken for each treatment. Fungus was reisolated from the mycosed cadaver of H. armigera and several virulent factors of indigenous conidia and insect passaged conidia were investigated. M. anisopliae (UM10) was more effective against 1st and 2nd instar larvae of H. armigera than B. bassiana (UB9). The second aspect deals with the studies optimizing protoplast yield from M. anisopliae (UM10) and B. bassiana (UB9) and transformation of these strains to herbicide resistance. Several factors were investigated influencing protoplast release from mycelium (B. bassiana (UB9) and M. anisopliae (UM10)) including effective cell wall lysing enzyme, osmotic stabilizer, mycelium age and enzyme incubation time. 10 mg/....
Bioengineered Silk Based Small Diameter Vascular Grafts
(2022) Gupta, Prerak
Blood vessels are the main component of the circulatory system which essentially carry blood to the distal parts of our body. An obstructed blood flow leads to oxygen scarcity and hypoxia in the affected tissue. One of the most common examples is myocardial infarction (commonly known as ‘heart attack’), which results from coronary artery occlusion due to various reasons. Current treatment modalities attempt to correct it by replacing the diseased portion with artificial plastic-based counterparts (e.g., Gore-Tex, ePTFE, etc.). While these grafts are an apt choice for larger vessels (>6mm diameter), they fail under low flow, high-pressure conditions in smaller vessels (<6mm diameter) due to compliance mismatch. Recent progress in the field suggests that bioengineered strategies stand strong in providing a pragmatic solution. The current thesis delineates various potential strategies to progressively develop bioresorbable tissue-engineered vascular grafts that employ natural silk biomaterial (mulberry- Bombyx mori; non-mulberry- Antheraea assama and Philosamia ricini). Investigation of cell-material interaction revealed biocompatibility of vascular cells with silk biomaterials. Nanoengineered silk film substrates effectively induced the unidirectional alignment of vascular cells via contact guidance. Leveraging the former capability, a multilayered biomimetic vascular graft based on primary porcine vascular cells is developed, exhibiting an adequate burst strength comparable to native human blood vessels. While patient-specific primary vascular cells are immunocompetent, limited in vitro proliferation ability restricts their clinical implementation. To overcome former setbacks, a novel bi-layered vascular scaffold is further designed and seeded with human adipose stem cells (stromal vascular fraction). In vivo studies suggested the clinical feasibility of grafts that was validated in terms of ease of handling during interposition grafting in rat abdominal aorta, adequate mechanical properties (suture retention, dynamic compliance, etc.), patency, and constructive remodeling. Further efforts were inclined towards developing cell-free grafts to improve upon the clinical feasibility of tissue engineered vascular grafts bypassing the cell seeding. Leveraging the notion that stem cells facilitate graft remodeling via paracrine signaling, two rational approaches were adopted to confer bioactivity in acellular grafts. The bi-layered scaffold system is functionalized with human Wharton’s jelly-derived matrix presumed to preserve stem cell-secreted factors and with monocyte chemoattractant protein-1 (MCP-1). Both approaches were effective and improved in vivo acute patency by immunomodulation. In this thesis, various designs of vascular grafts are progressively developed and tested in pre-clinical animal models The outcomes of this thesis open new avenues in the field of vascular regeneration.

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