Ph.D Theses

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Browsing Ph.D Theses by Subject "Biotechnology"

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    A study on Neuroprotective potential of in vitro and field tissues of Withania somnifera using Caenorhabditis elegans model
    (Avinashilingam, 2023-03) Krishnapriya C; Dr. Kalaiselvi Senthil
    Withania somnifera is a prevalent medicinal herb used all over the world as a domestic remedy for addressing several age-related ailments. The plant is also one of 32 medicinal plants that have been ranked as priority medicinal plants by the National Medicinal Plant Board (NMPB). Ayurveda refers the field grown W. somnifera roots as a Rasayana medication (Rejuvenator). It has been used as the major ingredient in a variety of formulations to help slow down the aging process, cope with stress, and be an excellent neuroprotectant. However, the quality and quantity of traditionally cultivated plants present a significant obstacle to their utilization in herbal formulations. This study aims to demonstrate that in vitro shoot tissues of W. somnifera could be used as an alternative and be as bioactive as roots grown in the field. The HPTLC quantification of major withanolides and GC-MS profiling of metabolites revealed that the pharmacological actives of IS (in vitro shoot) showed the overall similar metabolite profile as in FR (field grown roots). As measured by DPPH radical scavenging activity, the antioxidant potential of in vitro shoots (IS) was also higher than that of field grown tissues (FR & FS) and in vitro roots (IR). The animal model study in Caenorhabditis elegans presented numerous lines of evidence regarding the effectiveness of the IS on the health and life expectancy over the FR, IR and FS. Along with this, the study compares the molecular level mechanisms underlying the beneficial effects of FR, IR, FS and IS supplementation by using gene-specific mutants. The efficacy of W. somnifera extracts to prevent α-synuclein aggregation, its associated pathologies, and its capability for neuroprotection were studied in Parkinson’s disease-modeled worms. The finding of this study highlighted that IS is equally bioactive as traditionally used FR. Moreover, the IS extracts efficiently prolongs the lifespan, heath span and stress resistance via insulin/insulin- like growth factor-1 (IGF-1) signaling (IIS) and mitochondrial electron transport chain complexes (mETC). The IS extract is more effectual for suppressing oxidative stress, a remarkable neuroprotectant in Parkinson’s disease modeled worms. As the first study to investigate the bioactivity of W. somnifera shoots cultivated in vitro, these results could contribute to the scaling up of IS culture systems and in vitro shoot tissues for treating neurological and age-related ailments, extending patients' lives, and improving their quality of life.
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    Epidemiological profiling of population-specific risk factors and validation of novel genetic variants and deep learning-driven mitosis detection in breast cancer patients
    (Avinashilingam, 2024-05) Suganya K; Dr. S. Sumathi
    Breast cancer is a major global health issue and one of the most common malignancy affecting women globally. Addressing the population-specific risk factors, rare genetic alterations, and disease progression is challenging, and it is crucial for developing targeted therapy and reducing the impact of breast cancer treatment on affected individuals. The comprehensive four-phase study aimed to analyse breast cancer patient's risk factors, genetic alterations, and disease progression. The investigation commenced with an epidemiological survey to identify risk factors prevalent in breast cancer patients residing in Tamil Nadu, followed by whole exome sequencing to explore if there are any rare genetic variants in the patient's primary breast tumor, which may be population-specific. Sanger sequencing was performed to validate the reliability of the identified novel variants. With the advent of artificial intelligence (AI) in all fields, especially the healthcare sector, we tried to use AI to detect breast cancer progression accurately using histopathology images. The convolutional neural network (CNN) was applied to histopathology images to assess the mitotic index for evaluating proliferation in breast cancer patients. The results of an epidemiological study of 517 patients highlighted the significance of demographic characteristics, lifestyle choices, and treatment modalities for breast cancer. Our investigations ascertained that women aged 41-50 were the most affected, having a mean age of 47.40 (SD 11). The increased incidence rate was observed in women with lower levels of education and who were unemployed. Among reproductive factors, menopausal status was found to be a significant determinant, with premenopausal women facing a higher risk (p<0.001). Lifestyle factors showed that less physical exercise/activity (p<0.01), water intake (p<0.05), and sleep duration (p<0.01) displayed significant associations with breast cancer incidence. We also identified significant associations between comorbidities (p<0.01), tumor location (p<0.001), treatment modalities (p<0.001), and breast cancer. These findings add valuable insights into the complex web of factors influencing breast cancer incidence and progression within the specific patient population. The second phase involved whole exome sequencing (WES) of 6 primary breast cancer patient samples (5-Female, 1-Male), which covers all the subtypes of breast cancer. We identified 857 rare genetic variants and found that the common associated disease among five patients is cardiomyopathy. The results indicate that complete cardiac evaluation is necessary before breast cancer therapy. Functional enrichment analysis exhibited that the rare variant candidate genes are mostly involved in biological processes that include signal transduction, G-protein coupled receptor, gene expression regulation, synapse assembly, and the cellular process of the genes occurred in the plasma membrane, cytosol, and cytoplasm, and the molecular function of the genes mainly involved in protein-protein binding. Dysregulation of these functional processes leads to oncogenic properties of cancer cells. The third phase validated the novel genetic variants MYBPC3: c.2816G>A and PTCH1: c.1889G>A for the first time through Sanger sequencing. Interactome network mapping and KEGG pathway analysis showed that the interacted genes of MYBPC3 and PTCH1 are mainly involved in the hedgehog signaling pathway, p53 signaling pathway, circadian rhythm, and cardiomyopathy. Mutation in MYBPC3 and PTCH1 leads to a mutation in the interacted genes, which alters the pathways and may cause both breast cancer and cardiomyopathy. Epidemiological factors such as high blood pressure, diabetes and disturbed sleep patterns of cancer patients may be one of the driving forces causing these changes in health conditions in the specific population. In the fourth phase, the convolutional neural network achieved high accuracy (0.966) in detecting mitosis in histopathology images, offering a promising method for identifying breast cancer progression at early stages which is usually missed out or neglected. We are the first to identify mitosis in real-time patient histology images. Integrating epidemiological, genetic, and image analysis approach opens the door for innovative strategies for prevention, treatment, and comprehensive care in the battle against breast cancer. The outcome of the study highlights personalized treatment options based on an individuals' risk factors and genetic alterations thereby offering hope for improved patient outcomes and a better quality of life.
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    In vitro, in vivo and in silico approaches to assess the protective effect of Rosa indica petals on male infertility
    (Avinashilingam, 2023-03) Nithya S; Dr. N. Santhi
    Male infertility is a growing concern in India, with an estimated 12-18% of couples facing infertility issues. The present study was carried out with the aim of analysing the impact of lifestyle and environment on semen parameters and the protective effect of Rosa indica petal extracts on heat stress induced male infertility. The study was structured into five phases. In the first phase of the study, retrospective data from 299 male participants seeking medical assistance for fertility issues at a private hospital was analysed. The results of this phase showed a significant association between prolonged sitting (more than 4 hours per day) and semen parameters. It was observed that individuals who sat for extended periods showed a significant decrease in semen volume (p-value 0.004), sperm count (p-value 0.01), and normal forms (p-value 0.039), increase in non-progressive (p-value <0.001) and nonmotile (p-value 0.037) sperms in comparison to those who sat for less than 4 hours. While there are notable variations in sperm parameters across seasons, whereas, the observed values align with the standards set by the World Health Organization (WHO). However, the study did not establish statistically significant correlations between male infertility and addictions such as smoking and alcohol consumption. The antioxidant, anti-inflammatory and cytoprotective potential of the fresh and dry petal extracts of R. indica was assessed in phase II. The ethanolic extract from dry petals exhibited the highest radical scavenging and anti-inflammatory activity with low IC50 value compared to aqueous petal extract. Additionally, the cytoprotective test conducted with the MTT assay demonstrated the viability of TM3 cell lines even at the maximum concentration of 200 μg/ml. Therefore, a dosage of 200 mg/kg was selected for in vivo studies in phase III. In this study, the male Wistar rats were exposed to heat stress by immersing hind legs, tail and scrotum in 43⁰C thermostatically controlled water bath for 30 min for 6 consecutive days from day 8 treated with a dosage of 200 mg/kg of equal ratio of fresh and dry petals of aqueous and ethanolic extracts, a positive control with quercetin (50 mg/kg), a negative control without treatment and a normal control to evaluate the alleviating effect of the extract on heat stress induced sperm abnormalities. The results showed a significant difference in the weight of the testis between the negative control (0.25±0.12 g) and different treatment groups (0.45±0.12 g in ethanolic extract and 0.43±0.11g in aqueous treated groups). The decline in body weight observed in the negative control group of animals from 301.5 ± 2.15 g to 280.83 ± 14.97 g could be attributed to factors such as heat stress. There was a substantial contrast in semen parameters between animals subjected with heat stress in negative control and those treated with extract, quercetin and control groups. The ethanolic extract-treated group showed an increased level of serum testosterone and the antioxidant levels (CAT,, GPx, GSH and SOD) compared to negative control group (p-value 0.05). The histopathology of the testis showed that the negative control group had germ cell aplasia, mild hyperplasia of Leydig cells, and maturation arrest. In contrast, the positive control group and the ethanolic extract treated groups showed normal spermatogenesis (90% and 80% respectively).. The Phase IV studies focused on the identification of phytocompounds in both the extracts by GC- MS and HPTLC analysis. 194 compounds identified in phase IV were subjected to molecular docking studies using GLIDE (Schrodinger’s module) in phase V to understand their interactions with key target proteins such as COX2, AKT1, StAR, and AR. Among 194 compounds, the Kaempferol a flavonoid and the phenolic compound 2,4-DTBP present in all the extracts showed interaction with all the target proteins studied. Kaempferol, alongside the presence of Vitamin E specifically in the ethanolic extract, could be a contributing factor in the restoration of normal spermatogenesis in the mice induced with heat stress and treated with ethanolic extract. Therefore, the present study clearly demonstrates the antioxidant and anti-inflammatory properties and the presence of kaempferol, Vit E and the phenolic compounds of the ethanolic extract of R. indica petals and its efficacy in alleviating heat stress-induced semen abnormalities in male Wistar rats.