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Kerala Veterinary and Animal Sciences University, Wayanad

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20234
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Subject: Biochemistry × End date: 2023 × Start date: 2020 ×
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Now showing 1 - 4 of 4
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    ThesisItemOpen Access
    EXPRESSION PROFILING OF MICRORNA IN LIPOPOLYSACCHARIDE CHALLENGED PERIPHERAL BLOOD MONONUCLEAR CELLS OF CATTLE
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) AKSHARA B. VENU; Dr. Divya P.D
    MicroRNAs are endogenous, small non-coding RNAs approximately 20-22 nucleotides long which play a key role in gene regulation by affecting translation and transcription mechanisms. The present study was undertaken to analyse the differences in the microRNA (miRNA) expression in (LPS) challenged peripheral blood mononuclear cells (PBMCs) of cattle. The current research work was carried out using the isolated blood samples from apparently healthy female crossbred cattle maintained at the University Livestock Farm and Fodder Research and Development Scheme, Mannuthy. Isolation of PBMCs were done by density gradient centrifugation using HisepTM lymphocyte separation medium 1077.Expression of miRNAs viz; bta-miR-451, bta-miR-107, bta-miR-93 and bta-miR-125a, whose expression was found to be varying in the LPS treated and untreated bovine PBMCs by miRNAome analysis of previous studies was validated in the present study by qRT-PCR assay. Insilico analysis was done using various online tools for the prediction of target genes of real time PCR validated miRNAs, gene ontology analysis and also to study the cellular pathways associated to the target genes. Targets of each miRNAs were predicted using the online target prediction programme TargetScan Human 8.0. Through the use of the online DAVID bioinformatics tool, gene ontology analysis of the real time PCR validated miRNAs was carried out. Predicted targets for each miRNAs were annotated to biological process, molecular function and cellular component categories. The pathway analysis programme (KEGG) of the DAVID database was utilized to analyze and interpret the pathways associated with the predicted targets of real time PCR verified miRNAs. The study also included the assessment of cytokines associated to the cellular pathways influenced by the target genes of real time PCR validated miRNAs in the supernatants of PBMC cultures from both treatment and control groups by ELISA. The expression of miRNAs; bta-miR-451and bta-miR-107 was found to up regulated while bta-miR-93 and bta-miR-125a showed decrease in their expression in LPS treated PBMCs when compared to control cells. The results of real time PCR validation of aforementioned miRNAs expression were in accordance with the findings of miRNAome analysis. Besides, significant enrichment of target genes of the real time validated miRNAs was noticed in many immune related GO terms as well as in critical immune associated cellular pathways. Measurement of cytokine, TNF α revealed a significantly higher level in LPS treated PBMCs when compared to LPS untreated cells. The findings of the current study will help in understanding the role of miRNAs in LPS mediated immune responses in cattle.
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    ThesisItemOpen Access
    CHARACTERISATION OF ACID SOLUBLE COLLAGEN FROM DAGGERTOOTH PIKE CONGER (MURAENESOX CINEREUS) AND ITS APPLICATION IN WOUND HEALING
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) NIMNA AJAY; Dr. Varuna P. Panicker
    Collagen, the abundant structural protein in the extracellular matrix has wide application in medical, tissue engineering and pharmaceutical industry. The present study was conducted to characterize and evaluate the wound healing potential of isolated ASC from Daggertooth pike conger (Muraenesox cinereus).Conger eels were collected from Kochi harbour and pre-treatment for removal of fats and non-collagenous proteins. ASC was isolated by treating with 0.5 M acetic acid for 3 days and supernatants were collected. Protein precipitation was carried out by adding NaCl to final concentration of 2.3 M. Precipitated samples were dissolved in 0.5 M acetic acid and dialysed against 0.1 M acetic acid for one day and in distilled water until neutral pH was obtained. Yield of ASC was 12.78 and 31.95 per cent on wet and drymatter basis respectively. ASC on Lowry’s test showed a concentration of 8.8µg/ml. On electrophoretic separation revealed the presence of characteristic α (2 α1 and α2), β, γ subunits similar to that of type I calf collagen. An absorption peak of 221 nm characteristic to triple helix was observed on UV-visible spectroscopy. FTIR spectrum of ASC showed Amide bands similar to that of collagen. Analysis for secondary structure, by CD spectroscopy revealed a CD spectra with positive absorption maximum at 222 nm, and negative at 214 nm. Denaturation temperature of the extract was obtained at 38.2℃ which in turn confirm that collagen can withstand very high temperature. Solubility assay revealed a maximum solubility at low salt concentration and acidic pH. Hydroxyproline concentration in the extract was 9.9g. Invivo wound healing study showed that group treated with paste has increased wound healing compared to gel and control group. Results were confirmed by histopathology, immunohistochemistry and relative quantification of selected growth factors and cytokines. On histopathological evaluation increased fibroblast proliferation, collagen deposit and re-epithelisation were observed on collagen treated group. Difference in the collagen deposition in response to treatment was studied using staining like Picrosirus and Masson’s trichome. Angiogenesis was studied by immunohistochemistry using CD31 antibody. Observations on histopathology and immunohistochemistry could correlate with the gene expression study of TGFβ-1, VEGF, IL-10 and TNF-α. Upregulation of TGFβ-1, VEGF and IL-10 and downregulation of TNF-α was observed in collagen treated groups which might be due to the wound healing potential of collagen.
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    ThesisItemOpen Access
    ANALYSIS OF DIFFERENTIAL EXPRESSION OF microRNA IN LIPOPOLYSACCHARIDE CHALLENGED LYMPHOCYTES OF VECHUR AND CROSSBRED CATTLE
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) DIVYA P. D; Dr. Shynu M.
    The present study was carried out to analyse the differences in the expression of miRNAs in response to the in vitro LPS stimulation of PBMC cultures of crossbred and Vechur cattle. The study was performed in adult, apparently healthy, female crossbred and Vechur cattle maintained at University Livestock Farm and Fodder Research and Development Scheme, Mannuthy, and Vechur Conservation Unit, Kerala Veterinary and Animal Sciences University, respectively. The known/ novel miRNAs and the differential expression of miRNAs in response to bacterial endotoxin, LPS in PBMCs of the two genetic groups were identified by Short-read Illumina Next Generation Sequencing. Validation of NGS data of selected differentially expressed miRNAs was carried out by qRT-PCR assay. Prediction of target genes of differentially expressed miRNAs, functional gene enrichment analysis, analysis of cellular pathways involved and protein-protein interaction (PPI) network evaluation of the predicted target genes were also studied using various online bioinformatics tools. Cytokines associated with the immune related pathways of targets of differentially expressed miRNAs were analysed by ELISA. The differences in cytokine expression were also measured after overexpression of selected miRNA with miRNA mimics in the PBMC cultures of Vechur cattle. A total of 0.47 and 0.64 million clean reads, with an average Phred score of 34.92 and 34.75 corresponding 55.3 and 62.1 per cent of the adapter trimmed reads, respectively, for crossbred and Vechur samples were retrieved by NGS. Analysis of miRNAome identified 979 and 853 known miRNAs, and 393 and 139 novel miRNAs in samples from Vechur and crossbred cattle, respectively. Differential expression studies of NGS data revealed significant variation in the expression of miRNAs in LPS challenged PBMCs cultures of Vechur cows with respect to crossbred cattle. The results of real time validation of the expression of selected miRNAs by qRT-PCR assay were also consistent with the results of NGS. Functional gene enrichment analysis, analysis of pathways associated to the targets of differentially expressed miRNAs also revealed significant enrichment of targets of differentially expressed miRNAs in many immune related GO terms, immune associated cellular pathways as well as major cell signalling pathways. The PPI network analysis also showed active involvement of proteins encoded by these target genes in many of the important immune mechanisms. The study could identify differences in the immune related pathways associated to target genes of both up regulated and down regulated miRNAs though some pathways were found to be identical among both. Assessment of cytokines associated to the pathways regulated by the target genes of differentially expressed miRNAs in the supernatants from LPS treated PBMC cultures of also showed significant variations in the level of cytokines viz; TNF α, IL-4 and IFN γ among the crossbred and Vechur samples when compared to the control groups within the breed as well as between the breed. A significantly higher level of TNF α was noticed in LPS treated PBMCs of crossbred cattle whereas, IL-4 level was found to be significantly increased in LPS stimulated PBMCs of Vechur cattle compared to the LPS untreated cells from both groups. However, the present study could not detect any significant difference in IFN γ level among the LPS treated and untreated cells of both crossbred and Vechur cattle. The overexpression studies of miRNAs in Vechur PBMCs by transfecting with selected miRNA mimic could identify significant differences in IL-4 level while the changes were negligible with respect to other cytokines. The findings of the current research work suggest that both Vechur and crossbred cattle are having differences in their potential to tackle the immunological changes in response to an acute inflammation caused by the bacterial endotoxin; LPS. These differences might be contributing to the alleged immunological sturdiness to Vechur cattle compared to crossbred animals although, the specifics needs to be further validated.
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    ThesisItemOpen Access
    EVALUATION OF ANTICANCER PROPERTY OF RECOMBINANT GOAT LACTOFERRIN
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-02-08) VASUDHAR BHAT S. V.; Dr. Uma R.
    The present study was conducted with the objective to express and purifyrecombinant Malabari goat lactoferrin in Escherichia coli (E. coli). The study was carried out in three phases. In the phase I, a portion of lactoferrin (Lf) gene encoding the N lobe of Malabari goat lactoferrin (cLf-N) was cloned into pJET1.2/blunt cloning vector and transformed into E. coli strain JM109 and was sequenced which revealed a 793 bp fragment with 100 per cent similarity to goat Lf in the database. This ampliconwas subcloned into pET28a(+) expression vector and transformed into BL-21 (DE3) pLysS bacterial host. The transformed E. coli BL21 (DE3) pLysS containing the recombinant plasmid expressed maximum recombinant protein (rcLf-N) upon induction with 1mM IPTG at 37°C for five hours. The recombinant protein, rcLf-N, containing polyhistidine tag was purified using Ni-NTA affinity column chromatography and confirmed as a derivative of Lf by Western blotting. In phase II, rcLf-N was analysed for its in vitro anticancer activity in Daltons Lymphoma Ascites (DLA) cell lines. In MTT assay there was a significant (p<0.05) reduction in the per cent inhibition of cell proliferation in a concentration-dependent manner from 800μg/mL to 8.75μg/mL, indicating the cytotoxic effect of rcLf-N in a dose-dependent manner upon DLA cell lines. The half-maximal inhibitory concentration (IC50) value was found to be 263.5 μg/mL using Graph pad prism. Further, upon Acridine orange/ Ethidium bromide (AO/EB) and Hoechst staining of the treated cells, the apoptotic changes produced by rcLf-N and standard drug Cisplatin in DLA cells were highlighted. During phase III trial, the in vivo anticancer activity of rcLf-N was analysed on Swiss albino mice bearing DLA induced solid tumour. Based on preliminary studies the concentrations 50μg and 75μg of rcLf-N per animal were selected for comparison with the standard drug Cisplatin. A significant (p<0.05) reduction in tumour weight, tumour volume and tumour weight to body weight ratio was observed in the rcLf-N and cisplatin treated groups compared to the control group. Maximum reduction was observed in group treated with rcLf-N @75μg intratumourally for three days. Histopathological examination of tumour tissue in all the groups treated with rcLf-N and cisplatin showed the presence of apoptotic changes with decreased spread of neoplastic cells into surrounding tissues and decreased neovasculature.Relative expression of VEGF and Caspase-3 genes was analysed in both in vivo and in vitro studies with GAPDH as the reference gene via quantitative real-time PCR. A dose-dependent downregulation of VEGF and upregulation of Caspase-3 was revealed in the cells/tissues treated with IC50 and double IC50 doses of rcLf-N both in vitro and in vivo. Similar results were observed with IC50 Cisplatin. On comparison between intratumoural and intraperitoneal routes of treatment in vivo, the intratumoural route of treatment was better in downregulating VEGF and activating Caspase-3 on day 7 and 14, although the fold change was non-significant. From the present study, it could beconcluded that the novel recombinant protein produced antineoplastic activity through apoptosis and rcLf-N @75μg exhibited most potent anticancer activity against DLA cells.